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Fasting-based differential stress resistance to enhance cancer treatment: a novel strategy to protect normal cells and sensitize cancer cells to chemotherapy

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Fasting-based differential stress resistance to enhance cancer treatment: a novel strategy to protect normal cells and sensitize cancer cells to chemotherapy

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Content FASTING-BASED DIFFERENTIAL STRESS RESISTANCE TO ENHANCE
CANCER TREATMENT: A NOVEL STRATEGY TO PROTECT NORMAL CELLS
AND SENSITIZE CANCER CELLS TO CHEMOTHERAPY

by

Changhan Lee

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)

May 2010

Copyright 2010 Changhan Lee
ii

ACKNOWLEDGMENTS

Differential stress resistance (DSR) became a larger project than initially
anticipated, and I would like to thank the many people who gave their time, sweat,
expertise, and other various forms of support to bring the project to what it is
today. I truly believe that we have all contributed to laying the foundations for a
novel approach to battle cancer.
I would like to begin by thanking my PhD committee, professors Valter Longo,
Caleb Finch (Tuck), and Louis Dubeau. I have been very fortunate to be in the
hands of such a supportive and sincere group. Valter has a contagious degree of
energy, enthusiasm, and a creative mind for innovation. He has a remarkable
ability to extract the best out of me through a combination of encouragement and
constructive criticism. I have learned so much on aging research and also on
being an academic investigator. It has been a privilege to have an advisor who is
like a friend, mentor, and colleague. Tuck has an unexplainable aura of an
accomplished scholar; one learns just by listening to him (and maybe over a
glass of good wine one day). I am very glad our paths have crossed. Dr. Dubeau
is without a doubt a sharp yet gentle professor. In addition to being a fantastic
pathologist, he is a gifted educator, with more patience than most of us can
imagine. I also know that traveling between campuses to attend a meeting is not
too exciting, and thank him for never complaining, but always welcoming.
iii

The Longo lab has been my second home. I have probably spent as much time,
if not more, with them than I have with my family. Special thanks to Drs. Min Wei,
Fernando Safdie, and Federica Madia for working with me on numerous projects.
Min, thanks for all the daily discussions and help, I’m going to miss writing at one
in the morning with you. Fer, it would have been a very different experience
without you, thanks for always being there for me and for the unconditional
support as a friend and colleague. And finally Fede, I admire your genuinely
positive mentality and your high standards for management and attention to
detail. Thanks for all the supportive conversations and discussions.
I thank Dr. Pinchas Cohen at UCLA for providing us with transgenic mice and
various materials, but most of all, for being an excellent mentor, colleague, and
friend. You have enriched me as a scholar and a person. I also thank Dr. Amy
Lee for her expertise on ER stress and for being so supportive at all times.
Also, I must mention our collaborator Dr. Lizzia Raffaghello, who is in Genova,
Italy. We have done so much from such a distance; just imagine what we can
accomplish under one roof.
Finally, I wholeheartedly thank my family for being my fortress and source of life.
My adorable wife Yejee, for the perpetual encouragement and support, and for all
she is. I would never have made it this far without my loving parents, who have
always stood by my side under all circumstances.

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS ..................................................................................... ii

LIST OF TABLES ............................................................................................... vii

LIST OF FIGURES ............................................................................................. viii

ABSTRACT ........................................................................................................ xii

CHAPTER ONE:
Introduction: Differential Stress Resistance in Cancer Therapy.......................... 1
Overview of cancer and its treatment ................................................................. 1
Conserved regulation of longevity and stress resistance: Dietary and genetic
interventions ....................................................................................................... 4
Fasting and cancer metabolism: metabolic reprogramming .............................. 12
‘Differential’ Stress Resistance to Enhance Chemotherapy .............................. 15
Fasting: History, Clinical application, and Stress Resistance ............................ 18
The GH/IGF-I axis on growth and metabolism................................................... 22

CHAPTER TWO:
Fasting-dependent Differential Stress Resistance Protects Normal
but Not Cancer Cells Against High Dose Chemotherapy ................................. 25
Chapter Two Abstract ........................................................................................ 25
Introduction ........................................................................................................ 26
Results .............................................................................................................. 27
Discussion ......................................................................................................... 48
Materials and Methods ...................................................................................... 51

CHAPTER THREE:
Fasting and Cancer Treatment in Humans: A Case Series Report ................... 56
Chapter Three Abstract ..................................................................................... 56
v

Introduction ........................................................................................................ 57
Results .............................................................................................................. 62
Discussion ......................................................................................................... 87
Methods ............................................................................................................. 93

CHAPTER FOUR:
Reduced Levels of IGF-I Mediate Differential Protection of Normal and
Cancer Cells in Response to Fasting and Improve Chemotherapeutic Index .... 94
Chapter Four Abstract ....................................................................................... 94
Introduction ........................................................................................................ 95
Results .............................................................................................................. 97
Discussion ........................................................................................................ 123
Materials and Methods ..................................................................................... 126

CHAPTER FIVE:
Fasting Selectively Sensitizes Cancer Cells to Chemotherapy by Modulating
Stress Resistance Via the IGF-I/PI3K/Akt Pathway .......................................... 137
Chapter Five Abstract ....................................................................................... 137
Introduction ....................................................................................................... 138
Results ............................................................................................................. 141
Discussion ........................................................................................................ 163
Materials and Methods ..................................................................................... 168

CHAPTER SIX:
Essential Amino Acid Restricted Diet Enhances Stress Resistance
against Chemotherapy Toxicity in Mice ............................................................ 172
Chapter Six Abstract ......................................................................................... 172
Introduction ....................................................................................................... 173
Results ............................................................................................................. 175
Discussion ........................................................................................................ 183
Materials and Methods ..................................................................................... 184
vi

CHAPTER SEVEN:
Conclusion and Future Directions ..................................................................... 186

BIBLIOGRAPHY ............................................................................................... 193

vii

LIST OF TABLES

Table 1.1 Comparison between CR and GH/IGF-I deficient mice ...................... 9
Table 2.1 The effect of STS on metastases pattern in a neuroblastoma
mouse model ......................................................................................... 47

Table 2.2 Yeast strains used in chapter 2 ....................................................... 51
Table 3.1 Toxicity side-effect survey form ....................................................... 59
Table 3.2 Treatment summary of all patients................................................... 60
Table 3.3 Main demographics and clinical characteristics of patients ............. 61
Table 3.4 Summary of case 1 .......................................................................... 79
Table 3.5 Summary of case 6 .......................................................................... 80
Table 6.1 Purified amino acid diet formula; control diet for the methionine
and tryptophan restricted diet .............................................................. 176

Table 6.2 Purified methionine restriction diet formula. .................................... 177
Table 6.3 Purified tryptophan restriction diet formula. .................................... 178
viii

LIST OF FIGURES

Figure 1.1 The conserved effect of CR on increasing lifespan in various
organisms. ........................................................................................ 6

Figure 1.2 The conserved role of IGF-I in lifespan regulation and stress
resistance ........................................................................................ 11

Figure 1.3 Metabolic reprogramming of a cancer cells ..................................... 14
Figure 1.4 The hallmarks of cancer: acquiring stress resistance ...................... 16
Figure 1.5 A model for DSR in response to short-term starvation (STS). ......... 17
Figure 1.6 Conserved pathways regulate longevity and resistance to stress
in yeast and mice ........................................................................... 21

Figure 2.1 DSR to oxidative stress in yeast ..................................................... 31

Figure 2.2 DSR against chronic CP treatment in yeast ................................... 32
Figure 2.3 DSR to H
2
O
2
by glucose restriction in primary rat glia and
glioma cells ..................................................................................... 34

Figure 2.4 DSR to menadione by glucose restriction in primary rat glia
and glioma cells ............................................................................. 35

Figure 2.5 DSR to cyclophosphamide by glucose restriction in primary
rat glia and glioma cells .................................................................. 38

Figure 2.6 DSR to cyclophosphamide by glucose restriction in
sub-confluent primary rat glia and glioma cells ............................... 39

Figure 2.7 STS protects against high dose chemotherapy in vivo .................... 42
Figure 2.8 STS reduces toxicity following etoposide injection .......................... 43
Figure 2.9 DSR to etoposide in an allograft mouse model of neuroblastoma ... 46
Figure 3.1 Laboratory values of blood cell counts for case 1 ............................ 64
Figure 3.2 Self-reported side-effects after chemotherapy for case 2 ................ 67
ix

Figure 3.3 Laboratory values of blood cell counts for case 3 ............................ 70
Figure 3.4 Laboratory values of blood cell counts for case 4 ............................ 73
Figure 3.5 Self-reported side-effects after chemotherapy for case 5 .............. 75
Figure 3.6 Laboratory values of blood cell counts for case 6 .......................... 78
Figure 3.7 Laboratory values of blood cell counts for case 7 .......................... 82
Figure 3.8 Self-reported side-effects after chemotherapy for case 8 .............. 83
Figure 3.9 Self-reported side-effects after chemotherapy for case 9 .............. 85
Figure 3.10 Laboratory values of blood cell counts for case 10 ........................ 86
Figure 3.11 Self-reported side-effects after chemotherapy with or
without fasting ................................................................................ 89

Figure 3.12 Self ‐reported side ‐effects after chemotherapy with or
without fasting ................................................................................ 90

Figure 4.1 The effect of 72 hour fasting on glucose levels, IGF-I, and
IGFBP-1/3 .................................................................................... 100

Figure 4.2 IGF-I restoration during STS reverses STS-dependent
protection ...................................................................................... 101

Figure 4.3 in vitro DSR to CP treatments by reducing IGF-I .......................... 104
Figure 4.4 The role IGF-I signaling in protection against DXR in mouse
embryonic fibroblasts (MEF) ......................................................... 106

Figure 4.5 The conserved regulatory pathways of stress resistance in
response to starvation/calorie restriction ..................................... 109

Figure 4.6 The effect of Sch9-/Ras2-deficiencies on DSR against DXR
in S. cerevisiae. ............................................................................ 110

Figure 4.7 The effect of Octreotide (OCT) on short-term starvation (STS)
based DSR against etoposide (Eto) .............................................. 113

Figure 4.8 Stress resistance testing in LID mice with various high-dose
chemotherapeutic drugs ............................................................... 117
x

Figure 4.9 Weight loss after high-dose chemotherapy in LID mice ................. 118
Figure 4.10 LID mice show no signs of cyclophosphamide-dependent
toxicity ........................................................................................... 119

Figure 4.11 Differential stress resistance (DSR) against 2 cycles of
high-dose DXR in melanoma bearing LID mice ............................ 121

Figure 4.12 LID mice death is due to metastasis and not cardiotoxicity .......... 122
Figure 4.13 Weight of LID and control mice .................................................... 122
Figure 5.1 Fasting sensitizes breast cancer allografts to the nutrient
deprivation itself and to CP ........................................................... 143

Figure 5.2 Fasting sensitized cancer cells to CP treatment in a mouse
model of metastatic breast cancer ................................................ 144

Figure 5.3 Fasting sensitizes glioma allografts to the nutrient deprivation
Itself and to DXR........................................................................... 145

Figure 5.4 Fasting sensitizes cancer cells to DXR in a metastatic mouse
model of melanoma ...................................................................... 146

Figure 5.5 Fasting enhances DXR treatment and causes increased
survival in a metastatic mouse model of melanoma ..................... 147

Figure 5.6 Fasting inhibits the progression of subcutaneous melanoma
allografts ....................................................................................... 148

Figure 5.7 Fasting sensitizes human neuroblastoma to chemotherapy ......... 149
Figure 5.8 Fasting sensitizes murine neuroblastoma to chemotherapy ......... 150
Figure 5.9 Fasting is as effective as irradiation and increases its
effectiveness in reducing tumor growth ........................................ 152
Figure 5.10 Glucose restriction interferes with DNA repair following
irradiation in breast cancer cells ................................................... 153

Figure 5.11 Low glucose sensitizes murine cancer cells to chemotherapy ..... 155
Figure 5.12 Serum from fasted rats sensitize cancer cells to chemotherapy ... 156
xi

Figure 5.13 The effect of growth factors on cellular resistance to
chemotherapy in cancer cells ....................................................... 158

Figure 5.14 Differential response of Akt and ERK to fasting in liver and
breast cancer allografts in mice .................................................... 160

Figure 5.15 The activation status of Akt and ERK in murine cancer cells ........ 161
Figure 5.16 Cell lines with constitutive activation of the PI3K/Akt pathway
are less responsive to fasting ....................................................... 162

Figure 5.17 VEGF measurements from mice with glioma allografts after
a 24-72 hour fast .......................................................................... 164

Figure 5.18 The effect of GRP78 on stress resistance to DXR in MEFs ......... 166
Figure 5.19 GRP78 levels in normal organs and tumor allograft in fasted
mice ............................................................................................. 167

Figure 6.1 Methionine restriction leads to enhanced protection against
DXR ................................................................................................ 180

Figure 6.2 Tryptophan restriction leads to enhanced protection against
DXR ................................................................................................ 182

Figure 7.1 Potential targets of DSR ................................................................. 192
xii

ABSTRACT

One of the leading problems in modern cancer treatment is the toxic side
effects secondary to chemotherapy and radiotherapy. In fact, toxicity is the major
limiting factor of chemotherapy, shadowing its full therapeutic potential. Currently,
there are no established interventions or drugs to adequately reduce the toxicity
and protect cancer patients. This thesis provides a revolutionary approach to
chemotherapy by selectively protecting normal cells and sensitizing cancer cells
simultaneously by fasting. The theory behind fasting-dependent stress resistance
was built on strong experiment-based scientific studies on aging and cancer
research. Briefly, the compiled data from dietary, genetic, and pharmacological
studies have greatly contributed to our understanding of the conserved pathways
that regulate lifespan and stress resistance in organisms spanning from the
simple yeast, worms, flies, rodents, to non-human primates.
This thesis progressively describes how fasting can differentially modulate
the stress response of normal and malignant cells to chemotherapy. First, I will
demonstrate that briefly fasting prior to chemotherapy can selectively protect
normal cells, and mice, but not allografted tumors. Second, reports on 10 cancer
patients - some in advanced stages - who have voluntarily fasted in combination
with chemotherapy will be provided. Fasting was safe and feasible for cancer
patients, and was reported to have reduced common side effects of
chemotherapy. Third, I will provide data showing that the insulin-like growth
xiii

factor 1 (IGF-I), and its downstream signaling, is a major mediator of the
beneficial effects of fasting. Fourth, I will show that fasting not only protects the
host, but also sensitizes certain cancer cells to chemotherapy. Last, data on
substitution diets that enhance the protection to chemotherapy in mice will be
presented. The diets were also based on aging studies where a single amino
acid restriction prolonged lifespan and also increased stress resistance.
Collectively, differential manipulation of cellular stress resistance, by means of
dietary interventions and targeted molecular pathways, can selectively protect
normal cells, or can be exploited to sensitize malignant cells to cytotoxic
treatments.

1

CHAPTER ONE

Introduction: Differential Stress Resistance in Cancer Therapy

Overview of cancer and its treatment
Cancer has become a leading global health issue and a cause of mortality,
only second to heart disease. In the United States, 1 out of every 4 deaths will be
due to cancer (Jemal, Siegel et al. 2008). However, compared to the
improvement in the treatment of heart disease, cancer mortality has been
relatively steady over the past 30 years (Jemal, Siegel et al. 2008). Traditional
cytotoxic therapies, chemotherapy and radiotherapy, are still the choice of
treatment in many cancers. Although they are effective in the treatment of certain
cancers, its full clinical potential is greatly limited by the toxic side effects.
Recently, new and less toxic drugs are slowly replacing or being added to the
widely used chemotherapeutics, interventions to reduce toxicity are not
established.
Chemotherapy is a major arm of current cancer treatment. The first
chemotherapy drug was a toxic mustard nitrogen gas tested by Goodman and
Gillman in 1942, commissioned by the US Department of Defense in search for
therapeutic properties of chemicals developed for chemical warfare during World
War II (Chabner and Roberts 2005). Since then, it has been over 30 years since
the legislation by the US Congress created the first federal program for
2

anticancer drug discovery (Chabner and Roberts 2005), which has contributed to
the discovery of various chemo drugs with multiple mechanisms. Yet, the
strategy of chemotherapy is still based on targeted killing of rapidly proliferating
cells, by means of genotoxicity as well as the production of reactive oxygen
species (ROS) (Sangeetha, Das et al. 1990; Look and Musch 1994; Faber,
Coudray et al. 1995; Conklin 2004). The US Food and Drug Administration (FDA)
has approved 132 cancer chemotherapy drugs, of which 56 have been reported
to cause oxidative stress, which lead to collateral damage to normal cells (Chen,
Jungsuwadee et al. 2007). Although they were first believed to be quite selective,
we now know that normal cells also experience severe toxicity, leading to dose-
limiting side effects.
The other major arm of modern cancer treatment is radiation-based
cancer therapy. Irradiating (IR) biological materials lead to a rapid burst of ROS,
which is generated primarily from the ionization of water molecules and direct
ionization of target molecules (Fischer-Nielsen, Jeding et al. 1994; Riley 1994). It
is estimated that 60% of the damage is caused by ROS (Barcellos-Hoff, Park et
al. 2005). In addition, IRs such as X-rays and γ-rays cause direct
macromolecular damage by energy deposition. Despite its therapeutic benefits,
ROS significantly damages normal cells and is responsible for various side-
effects (Wilson, Taffe et al. 1993; Bialkowski, Kowara et al. 1996; Olinski,
Zastawny et al. 1996; Zastawny, Czerwinska et al. 1996). Direct macromolecular
damage may be quickly dealt with by tagging proteins for proteosome-dependent
degradation (Barcellos-Hoff, Park et al. 2005). It has been reported that even
3

localized small field radiotherapy of the head and neck patients can cause
oxidative damage at the organismal level, and may lead to secondary mutations
(Roszkowski, Gackowski et al. 2008). Currently, fractionation strategies are
considered an effective approach to improve tumor control rates without
increasing late toxicity, but they often cause enhanced acute toxicity
(Andreassen, Grau et al. 2003). Studies indicate that administering antioxidants
with radiotherapy reduces the toxic side-effects. For instance, the free-radical
scavenger amifostine has been shown to reduce side effects in certain types of
cancer (Bohuslavizki, Klutmann et al. 1998; Buntzel, Kuttner et al. 1998; Brenner,
Kampen et al. 2001), and in another study, pharmacologically targeting the toll-
like receptor 5 (TLR5) which activates nuclear factor-kappaB (NFκB) signaling, in
turn increasing superoxide dismutase 2 (SOD2) expression, selectively
protected mice and rhesus monkeys to radiation (Burdelya, Krivokrysenko et al.
2008).
As different as they are, the strategies used by both therapies converge
on cytotoxicity, in which the goal is to preferentially kill malignant cells. However,
the selectivity is far from ideal, and collateral damage to normal cells is inevitable
leading to severe side-effects such as myelosuppression, fatigue, vomiting,
diarrhea, and in some cases even death. Today the same fundamentals still
apply to modern chemo/radiotherapy, and its clinical potential is greatly
shadowed by its side effects. Despite the focused efforts on drug development
designed to target certain cancer markers, cytotoxic drugs will accompany side
effects unless a novel strategy-interventions or strategies that selectively protect
4

normal cells and simultaneously sensitize cancer cells-is adopted. Currently,
chemoprotectants such as amifostine, glutathione, mesna, and dexrazoxane
have been investigated and shown to provide drug-dependent protection to
specific tissues, but the use of these compounds has not been shown to increase

disease-free or overall survival (Links and Lewis 1999).
In this thesis, I will progressively describe novel approaches to cancer
treatment by integrating the fields of biogerontology and oncology, based on well
accepted dietary and genetic interventions that extend lifespan and/or enhance
stress resistance. The accumulation of data from yeast, mammalian cells, mice,
and humans would hopefully be convincing enough for further investigation and
clinical application. As this thesis is being written, a clinical trial testing the effect
of fasting in patients is being conducted at the USC Norris Cancer Center
involving approximately 100 cancer patients. This trial will test the translational
potential of fasting from basic science to everyday clinical practice. In this thesis,
I will also present evidence that fasting can also sensitize cancer cells to
chemotherapy, further separating normal and malignant cells, resulting in a
greater increase in therapeutic index (Chapter 5). In the future, it would be ideal
for chemo- and radiotherapy to distinguish normal and cancer cells at the level
that antibiotics distinguish host cells and microbes.
Conserved regulation of longevity and stress resistance: Dietary and
genetic interventions

Dietary, genetic, and pharmacological interventions have all contributed
greatly to our understanding of lifespan and stress resistance regulation. The
5

most powerful and reproducible dietary intervention that increases lifespan and
stress resistance across species is calorie restriction (CR). Also, genetic studies
show that increased lifespan and stress resistance converge on the GH/IGF-I
axis and its homologs. In fact, it is suggested that the GH/IGF-I axis is a major
mediator of the beneficial effects of CR (Bonkowski, Dominici et al. 2009).
Pharmacological manipulations have also shown to increase lifespan, as shown
in the rapamycin fed mice (Harrison, Strong et al. 2009). Rapamycin inhibits the
mammalian target of rapamycin (mTOR), which is downstream of IGF-I and acts
as a major regulator of cellular proliferation, metabolism, and stress (Reiling and
Sabatini 2006; Wullschleger, Loewith et al. 2006).
Calorie restriction (CR) is the most effective and reproducible intervention
to decelerate the rate of aging and increase healthy lifespan, as studied in
various model organisms ranging from the simple yeast to worms, flies, mice,
rats, and recently even non-human primates (Fig 1.1) (Guarente and Kenyon
2000; Kenyon 2001; Longo and Finch 2003; Colman, Anderson et al. 2009). In
1934, Mary Crowell and Clive McCay of Cornell University reported that white
rats fed a calorie restricted diet with sufficient nutrients from the time of weaning
resulted in life spans nearly doubling (McCay, Crowell et al. 1989). Following this
seminal discovery, using 2 different strains of laboratory mice, Roy Walford and
Richard Weindruch of UCLA reported that „adult-initiated‟ dietary restriction
(“undernutrition without malnutrition”) which began at the 12 months of age not
only retarded growth, increased life-span, but also reduced spontaneous cancer
incidence by more than 50% (Weindruch, Gottesman et al. 1982; Weindruch and
6

Walford 1982). Weindruch et al. also reported that calorie restriction initiated at
the time weaning also increased lifespan, reduced tumor incidence, and delayed
immunologic aging in laboratory mice (Weindruch, Walford et al. 1986),
confirming the CR studies in rats, and also recently published a 20-year
longitudinal adult-onset CR study in rhesus monkeys showing that CR (30%)
delayed disease onset and mortality, with a 50% decrease in cancer incidence
(Colman, Anderson et al. 2009). Lower eukaryotes also reap the benefits of CR.

Figure 1.1 The conserved effect of CR on increasing lifespan in various
organisms. Adapted from Mair and Dillin, 2008 (Mair and Dillin
2008).
7

Although the precise mechanism of CR is not clearly understood, energy
allocation appears to be fundamental. In other words, given a certain amount of
energy at any given time, the cellular energetic network must economically
balance the finite amount of energy between reproduction/growth and
repair/maintenance (Kirkwood, Kapahi et al. 2000). However, under starvation or
chronic calorie restriction, the favored survival strategy is to discourage
reproduction/growth and invest the remaining energy in repair/maintenance
(Kirkwood, Kapahi et al. 2000). This switch of energy allocation will be referred to
as entering a „maintenance mode‟, which could explain why calorie restricted
mice have reduced size and fertility, and increased lifespan and stress resistance
(Shanley and Kirkwood 2000). Notably, mice enrolled in chronic calorie restricted
diets show parallel characteristics with long-lived growth hormone (GH) deficient
mice (Table 1.1), which are resistant to stress and also have smaller body size
(dwarf), dampened fertility, reduced plasma GH/IGF-I, insulin, and glucose
(Longo and Finch 2003). All things considered, the insulin and GH/IGF-I axis
appear to be a major mediators/regulators of aging and stress resistance
(Bonkowski, Dominici et al. 2009).
In addition to CR, restricting a single essential amino acid in a normal diet
can also increase lifespan and stress resistance (Segall 1977; De Marte and
Enesco 1986; Ooka, Segall et al. 1988; Richie, Leutzinger et al. 1994;
Zimmerman, Malloy et al. 2003). Amino acids are central to CR-dependent
lifespan regulation. In flies, adding back essential amino acids to the CR diet
decreased lifespan to that of the normally fed group (Grandison, Piper et al.
8

2009). Laboratory rodents fed a methionine or tryptophan restricted diet (MR and
TR, respectively) showed extended lifespan with decreased age-dependent
diseases and increased resistance to oxidative stress, in part due to increased
antioxidant capacity (Richie, Leutzinger et al. 1994). Furthermore, methionine
restricted diet can slow down tumor growth in laboratory animals (Breillout,
Hadida et al. 1987; Tan, Xu et al. 1996), and has also been tested in patients
with advanced cancers with promising results (Epner 2001). This may be due to
the high requirement of methionine in cancer cells in response to elevated
protein synthesis and transmethylation (Hoffman 1985) in part owing to
epigenetic alterations, whereas normal cells are relatively resistant to methionine
restriction (Cellarier, Durando et al. 2003). In addition to its potential protective
benefits to the patients which I will present in this thesis, MR has been shown to
sensitize cancer cells to chemotherapy drugs (Goseki, Yamazaki et al. 1992;
Hoshiya, Kubota et al. 1996; Yoshioka, Wada et al. 1998; Tan, Sun et al. 1999;
Kokkinakis, Hoffman et al. 2001), further enhancing cancer treatment. TR also
provides longevity and reduced age-dependent deterioration (Segall and Timiras
1976; De Marte and Enesco 1986; Ooka, Segall et al. 1988).

9

Table 1.1 Comparison between CR and GH/IGF-I deficient mice.
Many physiological characteristics of GH/IGF-I deficiency are shared by CR
(Longo and Finch 2003).

Genetics has also contributed greatly to our understanding of the
pathways involved in regulating aging. Studies in S. cerevisiae, C. elegans, D.
melanogaster, and mice have demonstrated that the insulin and GH/IGF-I axis
are major regulators of lifespan and stress resistance (Fig 1.2) (Guarente and
Kenyon 2000; Kenyon 2001; Longo and Finch 2003). In yeast, we have shown
that deleting human homologs of Ras (RAS2) and/or Akt (SCH9/S6K) increased
lifespan to more than 200%, while providing increased stress resistance against
oxidants, genotoxins, and heat-shock (Longo and Finch 2003). Similarly, in
C.elegans, mutations in the human homologs of insulin/IGF-I receptor (daf-2) and
10

PI3K (age-1) extended lifespan to 200% and showed increased resistance to
thermal and oxidative stress (Kleemann and Murphy 2009). In D.melanogaster,
mutations in the insulin receptor substrate (chico) led to a 150% lifespan
extension (Giannakou and Partridge 2007). Mice with mutations in the insulin,
GH/IGF-I axis were able to increase lifespan 150% (Murakami 2006).
Conversely, mice overexpressing GH had a shortened lifespan (Bartke,
Chandrashekar et al. 2002). In addition, in vitro stress resistance studies with cell
cultures from long-lived mice with deficiencies in the GH/IGF-I axis have shown
to be resistant against oxidative stress (H
2
O
2
, paraquat), UV, genotoxins
(methylmethanesulfonate, MMS), heat, and cadmium (Salmon, Murakami et al.
2005; Murakami 2006), suggesting that enhanced stress resistance is partially
responsible for longevity, and the possibility to enhance protection by
interventions such as CR or downregulation of the GH/IGF-I axis.

11

Figure 1.2 The conserved role of IGF-I in lifespan regulation and stress
resistance

(A) Conserved IGF-I signaling pathways regulate lifespan and stress resistance.
(B-D) Wild type (left) and long-lived (right) yeast, flies, and mice are smaller due
to reduce growth signaling, but have increased lifespan and stress resistance.

A
B
C D
12

Fasting and cancer metabolism: metabolic reprogramming
Uncontrolled hyper-proliferation is characteristic of a cancer cell. Equally
important is the rewiring of cellular metabolism to support the vicious replication
both energetically and biosynthetically. One of the hallmarks of metabolic
reprogramming in malignant cells is known as the „Warburg effect‟. Observed by
Otto Warburg in the 1920s, cancer cells prefer to metabolize glucose by
glycolysis rather than oxidative phosphorylation even under ample oxygen, which
is the basis for tumor imaging by positron emission tomography (PET) using the
glucose analog
18
F-2-deoxyglucose (FDG). A tumor cell has evolved through
natural selection for maximum survival, which suggests that „aerobic glycolysis‟
must confer advantages to the rapidly growing cell. It has been suggested that In
most proliferating cells, ATP is not the rate-limiting step, and therefore glycolysis
may sufficiently generate the required ATP(Vander Heiden, Cantley et al. 2009),
as exemplified by rapidly dividing lymphocytes(Sariban-Sohraby, Magrath et al.
1983) and fermenting microbes. Aerobic glycolysis metabolizes glucose and
secretes lactate, which can be used as a major source of energy via oxidative
phosphorylation by aerobic cells, thus allowing „metabolic symbiosis‟ between
hypoxic and aerobic cells (Sonveaux, Vegran et al. 2008). Also the acidification
of the microenvironment promotes tumor invasion and immune surveillance
evasion(Luo, Solimini et al. 2009). In addition, aerobic glycolysis has been
suggested to be able to minimize oxidative stress during rapid biosynthesis and
cell division(Brand and Hermfisse 1997).
13

Although oxidative phosphorylation is far superior to glycolysis in terms of
ATP production, glycolysis provides biosynthetic precursors which are essential
to rapidly dividing cells. The Warburg effect does not only apply to cancer cells
but also to rapidly dividing normal cells such as lymphocytes, thymocytes, and
enterocytes(Newsholme, Crabtree et al. 1985). During glycolysis, glucose-6-
phosphate can be fed into the pentose shunt pathway, providing reducing power
and substrate for nucleotide synthesis, and glycerol is processed into
phospholipids that provide the cell wall (Alberts, Johnson et al. 2002).
Most mammalian cells are within 100-200µm from blood vessels and
receive adequate oxygen and nutrients (Carmeliet and Jain 2000). However, due
to the lack of or abnormal vasculature, tumor cells often find themselves deprived
of oxygen and nutrients and must adapt accordingly (Jain 2005). In a previous
study, SV40 ST antigen transformed cells upregulated autophagy via the
AMPK/mTOR pathway, providing the malignant cell with survival advantage
during glucose deprivation(Kumar and Rangarajan 2009). However, during
fasting, nutrients would be limited even after successful vascularization, failing to
meet the requirements for rapid growth of the already nutrient exhausted cancer
cells.
As discussed above, cancer cells have deranged cell proliferation and
altered metabolism, which are absolutely essential for uncontrolled proliferation.
Thus, the growing evidence of the dual roles of a proto-oncogene as a regulator
of both cell proliferation and metabolism, which often intersect the AKT pathway,
is not too surprising(Testa and Tsichlis 2005). For instance, the PI3K is
14

frequently found mutated in human cancers(Yuan and Cantley 2008), which
regulates growth and glucose metabolism via its downstream AKT
pathway(Vander Heiden, Cantley et al. 2009). The mammalian target of
rapamycin (mTOR) is another well established master regulator, which integrates
3 major components - growth factors, nutrients, and cellular energy level (ATP) -
to regulate cellular growth and metabolism.
Figure 1.3 Metabolic reprogramming of a cancer cells.
Cancer cells evolve through natural selection to gain growth advantage.
Metabolic rewiring is characteristic of a cancer cell, providing cells with
rapid energy and biosynthetic precursors (Vander Heiden, Cantley et al.
2009).

15

‘Differential’ Stress Resistance to Enhance Chemotherapy
From the discussion above, it is clear that normal and cancer cells differ in
many ways. In general, normal cells obey the regulatory signals for growth,
differentiation, and apoptosis. In contrast, cellular transformation, as a result of
mutations that collectively empower a cell with self sufficiency in growth signals
and insensitivity to growth inhibitory signals (Fig 1.4) (Hanahan and Weinberg
2000; Luo, Solimini et al. 2009), uncouples the cancerous cells from the
organism. More specifically, self sufficiency in growth signals is enabled by gain-
of-function mutations in oncogenes (e.g. Ras, Akt, mTor, etc) that enable
constitutive activation of proliferation pathways regardless of conditions. Notably,
the Ras/Raf/MAPK and the PTEN/PI3K/Akt pathways can be down-regulated by
CR and starvation (Xie, Jiang et al. 2007). On the contrary, insensitivity to growth
inhibitory signals is due to loss-of-function mutations in tumor-suppressor genes
(e.g. Rb, p53, PTEN, etc), enabling cancer cells to disregard anti-proliferation
signals (Hanahan and Weinberg 2000; Vogelstein and Kinzler 2004). This distinct
response to growth regulation between normal and cancer cells stand as the
foundation of the differential stress resistance stratagem against chemotherapy.
A short-term starvation (STS) can trigger normal cells to hold
reproduction/growth and shift into a „maintenance mode‟, while cancer cells
would disregard host signals to do so (Fig 1.5). In Chapter 2, I show that STS
increases stress resistance against chemotherapy agents in a „differential‟
manner, protecting normal but not cancer cells.
16

Figure 1.4 The hallmarks of cancer: acquiring stress resistance.
In the original 6 hallmarks of cancer proposed by Hanahan and Weinberg (top
half, white symbols), stress resistance was not discussed. In this figure, cancer
stress phenotypes (lower half, colored symbols), including metabolic stress,
proteotoxic stress, mitotic stress, oxidative stress, and DNA damage stress are
also included as hallmarks of cancer (Luo, Solimini et al. 2009).

17

Figure 1.5 A model for DSR in response to short-term starvation (STS).
In normal cells, downstream elements of the IGF-I and other growth factors
pathways, including the Akt, Ras and other proto-oncogenes, are down-regulated
in response to the reduction in growth factors caused by starvation. This down-
regulation blocks/reduces growth and promotes protection to chemotherapy. By
contrast, oncogenic mutations render tumor cells less responsive to STS due to
their independence from growth signals. Therefore, cancer cells fail to or only
partially respond to starvation conditions and continue to promote growth instead
of protection against oxidative stress and high dose chemotherapy.
18

Fasting: History, Clinical application, and Stress Resistance
Most animals live in situations of fluctuating food availability. From an
evolutionary point, the ability to withstand food deprivation would naturally be a
favored trait for survival. In mammals, there are 3 metabolic stages during food
deprivation (Wang, Hung et al. 2006). First, the postabsorptive phase, which can
last for hours following ingestion, involves the use of glycogen as the main stored
energy source. When the liver glycogen storage has been depleted, it is followed
by the second phase in which amino acids serve as the substrate for
gluconeogenesis. Eventually the proteins are spared by glycerol and fatty acid
chains released from adipose tissues, which take over and fuel the body for
several weeks. During the last phase of prolonged food deprivation, fat storage is
eventually exhausted and rapid muscle degradation occurs for gluconeogenesis
(Wang, Hung et al. 2006). The weight loss is rapid initially and tapers off as
shown in a human study where an average of 0.9 kg/day was lost daily during
the first week, subsiding to 0.3 kg/day by the third week of fasting. The study also
shows approximately a 20% body weight loss after 30~35 days of fasting (Kerndt,
Naughton et al. 1982), but it is estimated that a 70 kg person can sustain basal
caloric requirements from fat reserves during two to three months of fasting
(Cahill and Owen 1968; Cahill, Owen et al. 1968; Saudek and Felig 1976).
Prolonged fasting is feasible and generally well tolerated but does come with light
side-effects, such as headaches, light-headednenss, nausea, weakness, edema,
anemia, amenorrhea (Bloom 1959; Drenick, Swendseid et al. 1964; Thomson,
Runcie et al. 1966), and, in some rare cases, fatal complications such as renal
19

failure, heart failure, and lactic acidosis (Cubberley, Polster et al. 1965; Spencer
1968; Garnett, Barnard et al. 1969; Runcie and Thomson 1970).
Historically, fasting has been performed for medical, cosmetic, religious,
and political purposes (Kerndt, Naughton et al. 1982; Michalsen, Hoffmann et al.
2005; Johnstone 2007). Much has been learned through involuntary fasting such
as the victims of famine and war (Scrimshaw 1987; Kalm and Semba 2005), or
voluntary fasting such as the biblical 40 days of food abstinence (Kerndt,
Naughton et al. 1982). Beyond its traditional practice, fasting has also been
demonstrated to have clinical benefits. Notably, clinical studies have shown that
water-only fasting for 10-14 days significantly improved hypertension by reducing
systolic blood pressure points more than 2-fold compared to that of a combined
vegan, low-fat, low salt diet and exercise (Goldhamer, Lisle et al. 2001;
Goldhamer 2002). This study is important and holds clinical potential considering
that the leading cause of death in the US is heart disease (Jemal, Siegel et al.
2008). Moreover, the safety of fasting in patients with chronic disease has been
studied in a large cohort study with over 2000 participants (Michalsen, Hoffmann
et al. 2005). The authors determined that fasting (350kcal/day) was safe and
promising to be incorporated into an integrative medicine ward, and was also
evaluated by the vast majority of the participating patients to be beneficial to their
chronic disease (Michalsen, Hoffmann et al. 2005). Fasting has also been
proposed and used in clinics to protect the patient from ischemic reperfusion
damage, in which oxidative stress is largely responsible for the damage (Mitchell,
Verweij et al. 2009; van Ginhoven, Mitchell et al. 2009).
20

How does fasting provide protection to oxidative stress or chemotherapy? Under
nutritionally challenging conditions, survival would be maximized by reducing
growth and reproduction and reprogramming the system towards repair and
maintenance. IGF-I, being one of the more potent growth factor, will be reduced
which in turn downregulates many of the intracellular growth signals such as AKT,
Ras, and upregulate stress resistance pathways such as FoxO, GRP78 and DNA
repair genes (Chapters 4 and 5). Therefore, most scientists would agree on the
existence of continuous cross-communication between metabolic/stress-
response/cellular-growth pathways.
Although CR can increase stress resistance, possibly by inducing the
entry into a „maintenance mode‟, it requires months to be effective. Therefore we
determined it unsuitable for clinical chemotherapy settings. Instead, we
hypothesized that a short-term starvation (STS) could also induce the switch to a
„maintenance mode‟ and increase stress resistance (Fig 1.6). STS and CR both
lower circulating IGF-I, which is regulated by GH and nutrition (Clemmons and
Underwood 1991; Underwood, Thissen et al. 1994). Under normal conditions,
GH directly regulates the production of IGF-I, but during starvation, several
changes in the GH/IGF-I axis occur as a result of physiological adaptation to the
new environment. In humans, IGF-I levels decrease dramatically in response to a
short-term starvation (36-72 hours) despite increased GH secretion, which is
highly lipolytic (Merimee, Zapf et al. 1982; Thissen, Underwood et al. 1999;
Maccario, Aimaretti et al. 2001; Norrelund 2005; Moller and Jorgensen 2009).
GH levels eventually level off and drop in non-obese persons from three to ten
21

days of fasting (Cahill, Herrera et al. 1966; Merimee and Fineberg 1974;
Palmblad, Levi et al. 1977). In mice, a short-term starvation (24-72 hours)
decreases both GH and IGF-I production (Tannenbaum, Rorstad et al. 1979;
Frystyk, Delhanty et al. 1999).

Figure 1.6. Conserved pathways regulate longevity and resistance to stress in
yeast and mice. (Longo and Fontana 2010)

Studies from long-lived organisms that are deficient in that IGF-I pathway
have also been shown to be resistance to multiple types of stress (Holzenberger,
22

Dupont et al. 2003; Murakami 2006; Ayyadevara, Alla et al. 2008). In agreement,
we have obtained similar results from our preliminary studies with significant
protection against chemotherapy drugs in mice that have a 75% reduction in
circulating IGF-I. It is likely that STS could induce the switch to a „maintenance
mode‟ by decreasing the pro-growth IGF-I levels. However, since fasting cancer
patients would be challenging, it is important to understand the mechanisms
behind this protection and develop STS mimetics.
Recently, owing to the leap of technological and research advances, the
pathways that control the cellular adaptation to fasting have started to be
revealed.

The GH/IGF-I axis on growth and metabolism
The GH/IGF-I axis is of particular interest to aging researchers because,
as mentioned above, many genes that regulate lifespan converge on its
downstream effectors (Yang, Anzo et al. 2005). Physiologically, the GH/IGF-I
axis is a major regulator of primarily two important processes: growth and
metabolism.
GH has a central role in growth as indicated in animal studies showing
that deficiency in GH production or the deletion of the GH receptor gene cause
retardation. There are many mouse models showing the role of GH in growth,
including the Snell and Ames dwarf mice, in which the Pit-1 (Li, Crenshaw et al.
1990) (pituitary-specific transcription factor-1) or its upstream Prop-1 (Andersen,
23

Pearse et al. 1995) gene have point mutations, respectively. The dwarf mice do
not produce GH and weigh only a third of their WT littermates during their
adulthood, but live about 50% longer than their littermates (Cheng, Beamer et al.
1983; Brown-Borg, Borg et al. 1996). In addition, GH receptor/binding protein
(GHR/BP) knockout mice, which were initially developed to model the human
Laron dwarfism syndrome, have undetectable levels of hepatic GH receptor
expression and serum GHBP, leading to a significant decrease in body size and
approximately 50% increase in longevity(Zhou, Xu et al. 1997; Coschigano,
Clemmons et al. 2000). These GHR/BPKO mice have elevated GH levels but a
90% reduction in IGF-I levels, similar to GH resistance as observed in humans
during fasting (REF). Interestingly, unlike the GHR/BPKO mice, mice expressing
a GH antagonist (GHA) eventually reached normal body weight and the lifespan
remained similar to that of WT littermates (Coschigano, Holland et al. 2003). The
difference in GH signaling level, i.e. complete deletion of the receptor compared
to receptor antagonism may account for the differences and suggests the degree
of GH signaling inhibition to be central to growth and lifespan regulation.
Pathways downstream of GH have always shown to be involved in
lifespan regulation. In particular, genetically altered IGF-IR knockout mouse
demonstrates the significance of the GH/IGF-I signaling pathway in lifespan
extension. Although homozygous deletion of the IGF-Ir gene is lethal in mice,
female heterozygotes display a 33% increase in lifespan, whereas male
heterozygotes show a modest 16% increase in lifespan which is not statistically
significant (Holzenberger, Dupont et al. 2003). Remarkably, the heterozyotes
24

were also better protected against oxidative stress as determined by increased
survival following paraquat (oxidant) treatment (Holzenberger, Dupont et al.
2003). Embryonic fibroblasts cultured from these IGF-IR
+/-
mice showed reduced
activation of its major intracellular effectors including Akt and p66shc
(Holzenberger, Dupont et al. 2003). p66shc is a cytoplasmic signal transducer
delivering mitogenic signals from activated receptors to Ras (Pelicci,
Lanfrancone et al. 1992). Before the publication of the IGF-IR
+/-
mice, the
p66shc-/- mice were reported to have increased resistance to oxidative stress
and enhanced lifespan (Migliaccio, Giorgio et al. 1999). These studies and other
mice studies with deficiencies in the downstream effectors of IGF-IR signaling,
including mTOR (Harrison, Strong et al. 2009) and S6K1 (Selman, Tullet et al.
2009), demonstrate the central role of intracellular mitogenic pathways
downstream of IGF-I in regulating lifespan and stress resistance.
GH is also a critical metabolic switch, acting as the primary protein
anabolic hormone during stress and fasting by predominantly stimulating the
release and the oxidation of free fatty acids from lipids (lipolysis). This allows the
organism to decrease glucose and protein oxidation and consequently preserve
lean body mass (Moller and Jorgensen 2009). In fasting humans, GH levels
initially increase whereas IGF-I levels diminish, a phenomenon known as GH
resistance, clearly demonstrating the body to switch fuel sources, discourage
growth, and enter into a „maintenance mode‟.
In this thesis, I will describe how reduction in circulating IGF-I can
selectively protect mice but not cancer cells from chemo toxicity.
25

CHAPTER TWO

Fasting-dependent Differential Stress Resistance against
Chemotherapy Drugs
Chapter Two Abstract
Strategies to treat cancer have focused primarily on the killing of tumor
cells. Here, we describe a novel differential stress resistance (DSR) method that
focuses instead on protecting the organism but not cancer cells against
chemotherapy. Short-term starved (STS) S. cerevisiae or cells lacking proto-
oncogene homologs were up to 1,000 times better protected against oxidative
stress or chemotherapy drugs than cells expressing the oncogene homolog
Ras2
val19
. Low glucose or low serum media also protected primary glial cells but
not six different rat and human glioma and neuroblastoma cancer cell lines
against hydrogen peroxide or the chemotherapy drug/pro-oxidant
cyclophosphamide. Finally, STS provided complete protection to mice but not to
injected neuroblastoma cells against a high dose of the chemotherapy drug/pro-
oxidant etoposide. These studies describe a novel starvation-based DSR
strategy to enhance the efficacy of chemotherapy and suggest that specific
agents among those that promote oxidative stress and DNA damage have the
potential to maximize the differential toxicity to normal and cancer cells. This
study has been published in the Proceedings of the National Academy of
Sciences (PNAS) (Raffaghello, Lee et al. 2008).
26

Introduction
Our studies in S. cerevisiae and those of others in worms, flies, and mice
have uncovered a strong association between life span extension and resistance
to oxidative stress (Lithgow, White et al. 1994; Longo, Gralla et al. 1996;
Migliaccio, Giorgio et al. 1999; Fabrizio, Pozza et al. 2001; Holzenberger, Dupont
et al. 2003; Longo and Finch 2003). This resistance is observed in long-lived
yeast cells lacking RAS2 and SCH9, the orthologs of components of the human
Ras and Akt/S6K pathways (Fabrizio, Pozza et al. 2001; Fabrizio, Liou et al.
2003; Longo and Finch 2003) and in long-lived worms and mice with reduced
activity of homologs of the IGF-I receptor (IGF-IR), implicated in many human
cancers (Pollak, Schernhammer et al. 2004). Notably, the IGF-IR functions
upstream of Ras and Akt in mammalian cells (Lithgow, White et al. 1994;
Migliaccio, Giorgio et al. 1999; Holzenberger, Dupont et al. 2003; Longo and
Finch 2003). Stress resistance is also observed in model systems in which
calorie intake is reduced by at least 30 % (Harper, Salmon et al. 2006). This
reduced food intake, also known as calorie restriction (CR) or dietary restriction
(DR) has been studied for many years and is known to extend life span in
organisms ranging from yeast to mice (Weindruch and and Walford 1988). CR
also protects against spontaneous cancers and against carcinogen-induced
cancers (Weindruch and and Walford 1988; Dunn, Kari et al. 1997; Kritchevsky
2003), raising the possibility that CR and reduced IGF-I may increase stress
resistance by similar mechanisms.
27

Our discovery of the role of Ras2 and Sch9 in the negative regulation of
antioxidant and other protective systems together with the association between
mutations that activate IGF-IR, Ras or Akt and many human cancers prompted
our hypothesis that normal but not cancer cells would respond to starvation or
down-regulation of Ras/Akt signaling by entering a stress resistance mode. In
fact, one of the major “hallmarks of cancer cells” is the self-sufficiency for growth
signals (Hanahan and Weinberg 2000). In the majority of cancers, this ability to
grow or remain in a growth mode even in the absence of growth factors is
provided by the hyperactivation of one or several components of the IGF-IR, Ras,
Akt and mTor pathways.
Here we tested the hypothesis that short-term starvation or low
glucose/low serum can protect mammalian cells but not or less cancer cells
against high doses of oxidative damage or chemotherapy.

Results
Short-Term Starvation Induces Differential Stress Resistance against
Oxidative Stress in Yeast

To test the hypothesis that constitutively active oncogenes or oncogene
homologs can prevent the switch to a protective maintenance mode in response
to starvation, we first determined whether acute starvation would be as effective
in increasing oxidative stress resistance as it has been shown for long-term
calorie restriction (CR) (Bruce-Keller, Umberger et al. 1999). We first performed
DSR studies in S. cerevisiae. We selected a short-term starvation (STS)
28

paradigm as well as the deletion of the SCH9 and/or RAS2 genes, each of which
mimics in part calorie restriction and was shown in our previous studies to cause
high resistance to oxidative stress (Lin, Defossez et al. 2000; Fabrizio 2005;
Kaeberlein, Powers et al. 2005). Our hypothesis was that the combination of
these genetic manipulations with starvation would maximize DSR. Cells were
treated with either H
2
O
2
or the superoxide-generating agent menadione, which
can also cause DNA alkylation (Ross, Thor et al. 1986). The combination of STS
(switch from glucose medium to water at day 1 and incubation in water for 24-48
hours) with the deletion of SCH9 or both SCH9 and RAS2 caused resistance to a
30-60 minute treatment with hydrogen peroxide or menadione that was up to a
1,000-fold higher than that of cells expressing the constitutively active oncogene
homolog RAS2
val19
or cells

lacking SCH9 (sch9Δ) but expressing RAS2
val19
(sch9ΔRAS2
val19
) (Fig. 2.1A). The rationale for this experiment was to model in a
simple system the effect of the combination of short-term starvation and a
genetic approach on the differential protection of normal and cancer cells. The
results show that the expression of the oncogene-like RAS2
val19
prevents the
1,000-fold protection caused by the combination of STS and inhibition of Sch9
activity. Notably, under these conditions yeast cells are not dividing.
We also tested the effect of increased activity of Sch9 on resistance to
oxidants. As with RAS2
val19
, overexpression of SCH9 sensitized yeast cells to
both H
2
O
2
and the superoxide-generating agent menadione (Fig. 2.1B). Similar
to the effect of the deletion of RAS2 and SCH9, the deletion of the homolog of
TOR, another gene implicated in oncogenesis, slightly increased the resistance
29

to hydrogen peroxide. Whereas the expression of RAS2
val19
completely

reversed
the protective effect of the deletion of SCH9, it only had a minor effect on the
reversal of the protective effect of tor1Δ (Fig. 2.1B). This is an important
difference because it suggests that it may be risky to achieve DSR by inhibiting
intracellular targets such as Tor, which may be equally effective in protecting
cancer cells.

Short-Term Starvation Induces Differential Stress Resistance in Yeast.
We also tested whether DSR would also be effective against a high
concentration of drugs used in chemotherapy. We studied the effect of SCH9
mutations on the toxicity caused by alkylating agents methylmethane sulfonate
(MMS) and cyclophosphamide (CP, widely used in cancer treatment)(Poole, Earl
et al. 2006). Cyclophosphamide is a prodrug, which must be metabolically
activated, mainly in the liver, into its DNA alkylating cytotoxic form.
Cyclophosphamide treatments have also been shown to increase the generation
of ROS and oxidative DNA damage (8-hydroxyguanosine) in human granulosa
cells (Tsai-Turton, Luong et al. 2007) and to induce oxidative stress and lipid
peroxidation as well as GSH reduction (Manda and Bhatia 2003). As a very
simple model system to understand the differential effect of STS on the mixture
of normal and cancer cells observed in mammals with metastatic cancer, we
mixed in the same flask mutants lacking SCH9 with mutants lacking SCH9 but
also expressing RAS2
val19
at a 25:1 ratio and exposed them to chronic treatment
30

with CP or MMS. This ratio was selected to be able to start with 10 million
RAS2
val19
expressing cells while maintaining a relatively high ratio of normal vs
oncogene homolog expressing cells. The monitoring of the viability of the two
mixed populations was possible since each population could be distinguished by
the ability to grow on plates containing different selective media. Of the
approximately 10 million sch9ΔRAS2
val19
cells mixed with 250 million sch9Δ, less
than 5% of the sch9ΔRAS2
val19
cells survived a 48-hour treatment with 0.01%
MMS (Fig. 2.1C). By contrast, the great majority of sch9Δ cells survived this
treatment (Fig. 2.1C). Similar results were obtained when mixed cultures of
sch9ΔRAS2
val19
/sch9Δ were treated with cyclophosphamide (Fig. 2.1D). We also
performed an experiment in which each cell type was treated with CP separately
and observed a similar DSR between cells expressing RAS2
val19
and the cells
lacking SCH9 (Fig. 2.2). Again, in all the experiments above the yeast cells are
maintained under non-dividing conditions which rules out a role for differential
cell division in the difference in stress resistance between the various strains.
Taken together, these results confirm that the overexpression/constitutive
activation of oncogene homologs prevents the up to 1,000-fold increase in
resistance to oxidative stress or chemotherapy drugs induced by starvation
and/or mutations.
31

Figure 2.1 DSR to oxidative stress in yeast.
A, B) Survival of non-dividing (day 3) STS-treated yeast cells deficient in Sch9
and/or Ras2 (sch9Δ and sch9Δras2Δ), and cells overexpressing Sch9 or
expressing constitutive active RAS2
val19
(SCH9, RAS2
val19
,

sch9/aktΔRAS2
val19
,
and tor1ΔRAS2
val19
) after treatment with H
2
O
2
(30 min) or menadione (60 min).
At day 3, cells were treated with either H
2
O
2
for 30 min, or menadione for 60 min.
Serial dilution (10, 10
2
, 10
3
-fold dilutions, respectively, in the spots from left to
right) of the treated cultures were spotted onto YPD plates and incubated for 2-3
days at 30°C. This experiment was repeated at least 3 times with similar results.
A representative experiment is shown. C, D) Differential stress resistance (DSR)
to chronic cyclophosphamide and methylmethane sulfonate (MMS) treatments in
mixed yeast cultures. sch9Δ and sch9Δ RAS2
val19
. To model the mixture of
normal and tumor cells in mammalian cancer, sch9Δ and sch9ΔRAS2
val19
were
mixed in the same flask and incubated for 2 hours at 30
o
C with shaking. The
initial sch9Δ:sch9Δ RAS2
val19
ratio, measured by growth on selective media, was
25:1. Mixed cultures were then treated with either CP (0.1M) or MMS (0.01%).
Viability was measured after 24-48 hours by plating onto appropriate selective
media that allows the distinction of the 2 strains. Data from 3 independent
experiments are shown as mean ± SD.
32

Glucose Restriction Protects Primary Glia but Not Cancer Cells against
Oxidative Damage
Next, we tested whether short-term starvation could also induce
differential stress resistance against oxidative stress in mammalian cells. We
tested primary rat mixed glial cells (astrocytes + 5-10% microglia), four different
rat (C6, RG2, A10-85 and 9L), one human glioma (LN229), and one human
neuroblastoma (SH-SY5Y) cell line. The concentration of glucose in the media
was reduced to mimic short-term starvation. The normal physiological blood
Figure 2.2 DSR against chronic CP treatment in yeast.
Wild type (DBY746), RAS2
val19
, and RAS2
val19
strains were
inoculated at OD = 0.1, grown separately in glucose media, and treated with CP
(0.1M) 24 hours after initial inoculation. Viability was measured as colony forming
units (CFU) at 24 and 48 hours.
33

glucose level for both mice and humans is approximately 1.0 g/L but it can reach
0.5 g/L after starvation. Therefore, we tested the effect of normal glucose (1.0
g/L), low glucose (0.5 g/L) and high glucose (3.0 g/L) on oxidative stress. All cell
lines were grown until confluence to minimize proliferation and differences in
proliferation between the primary and cancer cells and then switched to medium
containing different glucose concentrations with 1% serum. Low serum was used
to minimize the addition of serum glucose, which is approximately 1.0 g/L. After a
24-hour glucose treatment, cells were challenged with 2 different oxidants: H
2
O
2

and menadione for 24 hours. In primary glial cells, STS enhanced resistance
against H
2
O
2
(0-625 µM), although the effect was more pronounced at 375 µM
H
2
O
2
where 80% of the cells pre-treated with normal and low glucose were
resistant while less than 10% of cells pre-treated with high glucose survived
(p<0.001). On the other hand, cytotoxicity of H
2
O
2
toward

cancer cells was
unaffected by varying glucose concentrations (Fig. 2.3). Whereas a reduction in
glucose concentration only partially protected primary glial cells treated with
menadione, it increased the toxicity of menadione to most cancer cell lines. Thus,
STS was still effective in generating DSR to menadione although the differential
resistance was created by a small protection of normal cells but a sensitization of
cancer cells (Fig. 2.4).
34

Figure 2.3 DSR to H
2
O
2
by glucose restriction in primary rat glia and glioma
cells.
In vitro differential stress response (DSR) to H
2
O
2
treatment. Primary rat glial cells, rat
glioma cell lines (C6, A10-85, RG2 and 9L), human glioma (LN229) and human
neuroblastoma (SH-SY5Y) cell lines were tested for glucose restriction-induced
differential stress resistance. Cells were incubated in either low glucose (0.5 g/L,STS),
normal glucose (1.0 g/L) or high glucose (3.0g/L) concentration, supplemented with
1% serum for 24 hours. Viability (MTT assay) was determined following a 24 hour
treatment with 0-1000 µM H
2
O
2
. All data are presented as mean ± SD. p-values were
calculated by Student‟s t-test (* p<0.05, ** p<0.01, *** p<0.001 ; of 0.5 and 1.0 g/L
versus 3.0 g/L glucose).
35

Figure 2.4 DSR to menadione by glucose restriction in primary rat glia and
glioma cells.
In vitro differential stress response (DSR) to menadione treatments. Primary rat
glial cells, rat glioma cell lines (C6, A10-85, RG2 and 9L), human glioma (LN229)
and human neuroblastoma (SH-SY5Y) cell lines were tested for glucose
restriction induced differential stress resistance. Cells were incubated in either
low glucose (0.5 g/L) (STS), normal glucose concentration (1.0 g/L) or default
media glucose (3.0g/L), supplemented with 1% serum for 24 hours. Viability
(MTT assay) was determined following a 24 hour treatment with 15-120 µM
menadione. All data presented as mean ± SD. p-values were calculated by one-
way ANOVA (* p<0.05, ** p<0.01, *** p<0.001).
36

Glucose Starvation Protects Primary Glia but Not Cancer Cells against
Cyclophosphamide

To test the efficacy of the starvation-based DSR method against a
chemotherapy drug/pro-oxidant in mammalian cells, we incubated primary rat
mixed glial cells (astrocytes + 5-10% microglia), three different rat, one human
glioma, and one human neuroblastoma cell line in medium containing low serum
and either normal (1.0 g/L) or low (0.5 g/L) glucose and then treated them with
cyclophosphamide for 10 hours. All cell lines were grown until confluence to
minimize proliferation and differences in proliferation. Whereas 80% of glial cells
were resistant to 12 mg/ml CP in the presence of 0.5 g/L glucose, only 20% of
the cells survived this treatment in 1.0 g/L glucose (Fig. 2.5A). The increased
stress resistance at the lower concentration of glucose (0.5 g/L) was observed
starting at 6 mg/ml CP but became much more pronounced at 12 mg/ml CP (Fig.
2.5A). By contrast, the lower glucose concentration did not increase the
resistance of cancer cell lines including C6, A10-85, RG2 rat glioma, LN229
human glioma or human SH-SY5Y neuroblastoma cells to 12-14 mg/ml CP (Fig.
2.5A). The lower glucose concentration actually decreased the resistance of RG2
glioma cells to CP at 6 and 8 mg/ml doses (Fig. 2.5A). To determine whether the
DSR is affected by the high cell density we also repeated this experiment with
cells that were only 70% confluent and obtained similar results (Fig. 2.6).
The experiments above were performed in medium containing 1% serum
and different concentrations of glucose. We also tested the effect of only
reducing the level of serum from the standard 10% to 1% on the toxicity of high
37

dose cyclophosphamide. Treatment with 15 mg/ml CP was toxic to primary glial
cells in 10% serum but the switch to 1% serum caused a reduction in toxicity (Fig.
2.5B). By contrast, the same concentration of CP was as toxic to C6 glioma cells
in 10% serum as it was in 1% serum (Fig. 2.5B).
These results strongly suggest that STS achieved by lowering the
concentration of glucose or other nutrients/factors contained in serum can be
very effective in protecting normal but not cancer cells against chemotherapy. In
some cases, low glucose/serum even increased toxicity to cancer cells.
38

Figure 2.5 DSR to cyclophosphamide by glucose restriction in primary rat
glia and glioma cells.
In vitro differential stress response (DSR) to cyclophosphamide treatments.
Primary rat glial cells, rat glioma cell lines (C6, A10-85, and RG2), human glioma
(LN229) and human neuroblastoma (SH-SY5Y) cell lines were tested. (A) Glucose
restriction-induced DSR. Cells were incubated in either low glucose (0.5 g/L, STS)
or normal glucose media (1.0 g/L), supplemented with 1% serum for 24 hours.
Cells were then treated with cyclophosphamide (6-12mg/ml) for 10 hours and
viability was determined (MTT assay) (n=9). (B) Serum restriction induced DSR.
Cells were incubated in medium containing either 1% (STS) or 10% serum for 24
hours, followed by a single cyclophosphamide treatment (15mg/ml) for 10 hours.
Cytotoxicity was determined by the LDH assay (n=12). All data presented as mean
± SD. p-values were calculated by Student‟s t-test (* p<0.05, ** p<0.01, ***
p<0.001).
39

Short-Term Starvation Induces Differential Stress Resistance Against
Oxidative Stress/Chemotherapy in Mice
We examined if STS could also enhance resistance of mice against
etoposide, a widely used chemotherapy drug which damages DNA by multiple
mechanisms and displays a generalized toxicity profile ranging from
myelosuppression to liver and neurologic damage (Vinolas, Graus et al. 1997;
Grunberg 1999; Mistry, Felix et al. 2005). Furthermore, etoposide has been
reported to increase the production of ROS in human glioblastoma cells, leading

Figure 2.6 DSR to cyclophosphamide by glucose restriction in sub-
confluent primary rat glia and glioma cells.
In vitro STS effect on differential stress response (DSR) to cyclophosphamide
treatments. Primary rat glial cells and the C6 rat glioma cells were grown to
70% confluency and then incubated in either low glucose (0.5g/L)(STS) or
normal glucose (1.0g/L), supplemented with 1% serum for 24 hours followed by
cyclophosphamide (12mg/ml) treatment. Cytotoxicity was measured by LDH
release. Data represented as mean ± SD. p-values were calculated using
Student‟s t-test (** p<0.005).
40

to cellular apoptosis possibly mediated by p53 (Sawada, Nakashima et al. 2001)
and to increase the production of ROS and MnSOD expression in myeloid
leukemia cells (Mantymaa, Siitonen et al. 2000). We administered an unusually
high dose of etoposide (80 mg/kg) to A/J mice that had been starved for 48 hours.
In humans, a third of this concentration of etoposide (30-45 mg/kg) is considered
to be a high dose and therefore in the maximum allowable range (Kroger,
Hoffknecht et al. 1998). Whereas 80 mg/kg etoposide killed 43% of control by
day 10 (Eto, n=23, 2 experiments), only one of the mice that were pre-starved
(STS/Eto, n = 17) died after etoposide treatment (Fig. 2.7A, p < 0.05). A/J mice
were considered to be survivors if they were alive at day 20. Remarkably, STS
pre-treated mice, which lost 20% of their weight during the 48 hours of starvation,
regained most of the weight in the four days after chemotherapy (Fig. 2.7B)
whereas in the same period the control mice lost approximately 20% of their
weight (Fig. 2.7B). Control mice treated with etoposide showed signs of toxicity
including reduced mobility, ruffled hair and hunched back posture (Fig. 2.8B)
whereas STS pre-treated mice showed no visible signs of stress or pain following
etoposide treatment (Fig. 2.8A).
We also tested the effect of STS on the protection of mice of another
genetic background (CD-1). To determine whether an extended STS strategy
can be effective against a higher dose of etoposide, we administered 110 mg/kg
etoposide and also increased the starvation period to 60 hours. Based on our
previous experiments with resistance to oxidative stress, we determined that this
period is the maximum STS that provides protection to mice. Longer starvation
41

periods can weaken the animals and have the opposite effect (data not shown).
This concentration of etoposide killed all the control mice (Eto) by day 5 but none
of the STS pre-treated mice (STS/Eto, n =5) (Fig. 2.7C, p< 0.01). CD-1 mice
were considered to be survivors if they were alive at day 20. As with the A/J mice,
pre-starved CD-1 mice lost 40% of the weight during the 60 hours of starvation
but regained nearly all the weight in the week after the etoposide treatment and
showed no visible signs of toxicity (Fig. 2.7D).
The effect of our STS-based method was similar in athymic (Nude-nu)
mice, which are widely used in cancer research because they allow the study of
human tumors in the mouse model. Whereas 100 mg/kg etoposide killed 56% of
the nude mice by day 5 (n = 9), none of the STS/Eto treated mice (48-hour
starvation) died (n = 6) (Fig. 2.7E, p < 0.05). Nude mice were considered to be
survivors if they were alive at day 10. As observed with the other two genetic
backgrounds, the pre-starved mice gained weight during the period in which the
Eto-treated mice lost weight (Fig. 2.7F).
In summary, out of 28 mice from three genetic backgrounds that were
starved for 48-60 hours before etoposide treatment, only one mouse died (Fig.
2.7G). By contrast, out of the 37 mice treated with etoposide alone, 20 died of
toxicity (Fig. 2.7G). These results are consistent with our yeast and glia/glioma
data showing increased resistance to oxidative damage and chemotherapy
toxicity in response to starvation.
42

Figure 2.7 STS protects against high dose chemotherapy in vivo.
(A) Percent survival and (B) weight profile of A/J mice were treated (i.v.) with 80
mg/kg etoposide with (STS/Eto, n=17) or without (Eto, n=23) a prior 48-hour
starvation (STS). (C) Percent survival and (D) weight profile of CD-1 mice were
treated (i.v.) with 110 mg/kg etoposide with (STS/Eto, n=5) or without (Eto, n=5)
a 60-hour prior starvation. (E) Percent survival and (F) weight profile of Athymic
(Nude-nu) mice were treated (i.v.) with 100 mg/kg etoposide with (STS/Eto, n=6)
or without (Eto, n=9) a 48-hour prior starvation. (G) The survival of all STS
treated (n=28) and untreated (n=37) mice from all genetic backgrounds above
(A/J, CD-1, Nude-nu) have been averaged (*** p<0.05).
43

Figure2.8 STS reduces toxicity following etoposide injection.
A) NXS2/STS/Eto group and B) NXS2/Eto group shown after etoposide
treatment.

Short-Term Starvation Prevents the Death of Mice but Not of Injected
Cancer Cells Treated with High Dose Etoposide

To determine whether the differential stress resistance observed in yeast
and mammalian cells would also occur in vivo, we followed the survival of mice
injected with cancer cells and treated with etoposide. We selected a particularly
aggressive tumor line (NXS2) that models neuroblastoma (NB), the most
44

common extracranial solid tumor, and the first cause of lethality in pre-school age
children. Advanced NB patients, who represent approximately 50% of the cases,
show metastatic dissemination at diagnosis, and have a long-term survival rate of
only 20% in spite of aggressive chemotherapy with autologous hematopoietic
stem cell support (Matthay, Villablanca et al. 1999; De Bernardi, Nicolas et al.
2003).
The NXS2 neuroblastoma line in mice induces consistent and
reproducible metastases in a pattern which resembles the clinical scenario
observed in neuroblastoma patients at advanced stages of the disease (Lode,
Xiang et al. 1997). Experimental metastases in the liver, kidneys, adrenal gland,
and ovaries were observed after 25-30 days of the inoculation with 200,000
NXS2 cells (Table 2.1) as previously described (Lode, Xiang et al. 1997). The
tumor development and survival of the NXS2/STS/Eto group was significantly
different from that of the NXS2 group (p < 0.001) (Fig. 2.9A; Table 2.1) indicating
that STS was highly effective in protecting the mice but only provided partial
protection to cancer cells against etoposide. In fact, at least 50% of the
NXS2/STS/ETO mice lived 10-20 days longer compared to the NXS2 mice (p <
0.05) (Fig. 2.9A). Considering that it takes the cells less than 30 days to go from
the injected 200,000 to the metastasis that kill the mouse, this 10-20 day longer
survival indicates that many and possibly the majority of the cancer cells have
died. As also shown in figure 4, approximately 50% of the mice treated with
etoposide in the absence of STS died of chemotherapy toxicity but the few mice
that survived died of cancer between day 80 and 140, confirming that STS also
45

partially protects cancer cells (Fig. 2.9B). Naturally, the high initial toxicity in the
Eto alone group would prevent the use of high dose etoposide in the absence of
STS.
In summary, these results suggest that STS greatly improves early
survival by ameliorating chemotherapy toxicity but reduces the effect of a highly
toxic dose of etoposide on metastases and cancer-dependent death by partially
protecting NXS2 cells. However, the improved survival of the NXS2/STS/ETO
compared to the NXS2 group suggests that STS allows the etoposide to kill a
major portion of the cancer cells or slows their growth and ability to form lethal
metastases. Notably, the increased survival observed in the NXS2/STS/ETO
group is unlikely to be due to slower cancer growth because STS is only
performed for the initial 48 hours, whereas it takes 35-60 days for metastasis to
cause mortality.
Since a significant survival extension was obtained with a single treatment
with high dose etoposide following STS and considering that the STS pre-treated
mice did not show signs of toxicity during the initial chemotherapy treatment,
these results suggest that multiple treatments with high dose chemotherapy in
combination with STS have the potential to kill most or all cancer cells without
causing significant toxicity to the host. Our attempts to perform weekly injections
of etoposide in combination with STS were discontinued due to tail damages
caused by the multiple intravenous injections. Thus, future experiments will be
necessary to develop a paradigm that allows the testing of the effect of multiple
STS/Eto cycles on metastatic cancer.
46

Figure 2.9 DSR to etoposide in an allograft mouse model of
neuroblastoma.
A-B) Survival of neuroblastoma (NXS2)-bearing mice. All mice were inoculated
(i.v.) with 200,000 NXS2 cells/mouse. The survival period of the NXS2 (control)
and NXS2/STS/Eto groups were significantly different (p<0.001) while that of the
NXS2 (control) and Eto groups were not (p=0.20). Also, the survival periods of
the NXS2/STS/Eto and NXS2/Eto groups were not significantly different (p=0.12).
C) The procedure for the in vivo experiment. D) A model for DSR in response to
short-term starvation (STS). In normal cells, downstream elements of the IGF-1
and other growth factors pathways, including the Akt, Ras and other proto-
oncogenes, are down-regulated in response to the reduction in growth factors
caused by starvation. This down-regulation blocks/reduces growth and promotes
protection to chemotherapy. By contrast, oncogenic mutations render tumor cells
less responsive to STS due to their independence from growth signals.
Therefore, cancer cells fail to or only partially respond to starvation conditions
and continue to promote growth instead of protection against oxidative stress and
high dose chemotherapy.
47

48

Discussion
The data above indicate that short-term starvation protects normal cells
and mice but not a variety of cancer cells treated with reactive oxygen species or
certain chemotherapy drugs that are also implicated in the generation of ROS. In
yeast, worms, and mice starvation or the genetic manipulation of starvation
response pathways causes a major increase in life span and protection against
multiple stresses including heat shock and oxidative damage. In mammals,
starvation causes a reduction in IGF-I signaling, which is associated with
increased stress resistance (Longo and Finch 2003). For example, calorie
restriction protects mice against liver cell death caused by acetaminophen
(Harper, Salmon et al. 2006) and against carcinogen-induced cancer (Dunn, Kari
et al. 1997). Furthermore, CR protects against the development of spontaneous
tumors in mice (Dirx, Zeegers et al. 2003; Kritchevsky 2003).
Here we show that yeast Ras and Sch9, orthologs of components of two
of the major oncogenic pathways activated by IGF-I, regulate starvation-
dependent resistance to oxidants or alkylating agents. As anticipated, based on
the constitutive activation of pathways that included homologs of yeast Ras and
Sch9 in cancer cells, starvation (STS) was highly effective in protecting
mammalian cells and mice but not cancer cells against the toxicity of
chemothrapy drugs including oxidants and alkylating agents. Although we have
not investigated the role of IGF-I in the mediation of DSR in mammalian cells and
mice, others have shown a 40% decrease in IGF-I in CD-1 mice that were
starved for 36 hours (O'Sullivan, Gluckman et al. 1989), raising the possibility
49

that decreasing IGF-I signaling may mediate in part the protective effect of
starvation. One of the most surprising findings of this study is the ability of mice
of 3 different genetic backgrounds that have been starved for 48-60 hours to
show no visible signs of toxicity in response to doses of chemotherapy highly
toxic to control animals and gain back the 20-40% weight that was lost during
starvation even in the presence of doses of etoposide that caused a 20-30%
weight loss and killed over 40% of the control mice. This high resistance to a
drug that damages the DNA of dividing cells, particularly blood cells, would be
consistent with the entry of most or all of the normally dividing cells into a high
protection/cell cycle arrested mode in response to the 48-60 hour starvation (Fig.
2.9D). Since etoposide is rapidly excreted (up to 90% within 48 hours in humans),
such “protective mode” may only need to last for a few days. Our recent results in
S. cerevisiae, indicate that the lack of SCH9 and to a lesser extent starvation,
protected against DNA damage in cells lacking the RecQ helicase SGS1, which
forms a DNA repair complex with topoisomerase III, by reducing errors during
DNA repair (Madia et al, unpublished results). It will be important to establish
whether STS or reduction of IGF-I/Akt/S6K signaling can protect mammalian
cells against the topoisomerase II inhibitor etoposide by similar mechanisms.
Chemotherapy treatment often relies on the combination of several DNA
damaging agents such as etoposide, cyclophosphamide, and doxorubicin.
Although these agents are supposedly much more toxic to cancer cells than to
normal cells, our in vitro studies show that cyclophosphamide, for example, can
be as or more toxic to primary glial cells as it is to glioma cancer cells. This
50

implies that the combination of multiple chemotherapy drugs cause massive
damage not only to blood cells but also other tissues, especially at high doses.
Notably, the differential stress resistance of mammalian cells to the alkylating
agent cyclophosphamide by our starvation-response methods was less than 10-
fold whereas starved yeast lacking SCH9 reached a 1,000-fold higher resistance
to menadione and hydrogen peroxide compared to RAS2
val19
expressing yeast
cells (Fig. 2.1). Furthermore, the 1,000-fold differential toxicity in yeast was
obtained after only 30 minutes with hydrogen peroxide compared to the several
days required for the differential toxicity of MMS or cyclophosphamide. Although
toxic molecules such as hydrogen peroxide are not suitable for human cancer
treatments, these results suggest that the identification of novel chemotherapy
drugs and possibly agents that generate a high level of reactive oxygen species
in combination with DSR has the potential to result in an even more rapid and
effective toxicity to cancer cells.
The ability to reach a 1,000-fold or a much more modest differential
toxicity between cancer cells and normal human cells would lead to improved
therapies for many cancers. Naturally, we don‟t know whether such an elevated
differential stress resistance can be achieved in cancer patients, but considering
the results obtained with a single treatment with etoposide in mice bearing
metastasis of the aggressive NXS2 neuroblastoma line that we injected in mice,
we are optimistic about the potential efficacy of multiple cycles of STS/etoposide
treatment against different types of cancers.

51

Materials and Methods

Yeast Strains and Growth Conditions
All the experiments were performed using yeast of DBY746 background.
Knockout strains were prepared with standard PCR-mediated gene disruption
protocol. Strains used in this study:
Strain Genotype
DBY746 MATα, leu2-3, 112, his3Δ1, trp1-289, ura3-52, GAL
+

sch9/aktΔ DBY746 sch9::URA3
ras2Δ DBY746 ras2::LEU2
tor1Δ DBY746 tor1::HIS3
RAS2
val19
DBY746 RAS2
val19
(CEN, URA3)
*

tor1ΔRAS2
val19
DBY746 tor1::HIS3 RAS2
val19
(CEN, URA3)
*

sch9/aktΔRAS2
val19
DBY746 sch9::TRP RAS2
val19
(CEN, URA3)
*

SCH9/AKT DBY746 SCH9 (CEN, URA3)
*

Table 2.2 Yeast strains used in chapter 2

* pRS416-RAS2
val19
was constructed by inserting 1.9 kb ClaI-HindIII fragment
from pMF100 (provided by Dr. Broach) into pRS416. Plasmid overexpressing
SCH9 was provided by Dr. Morano.

Yeast Viability Assay
Overnight SDC cultures were diluted to OD
600
0.1 into fresh SDC medium.
After 24 hours (day 1), sch9ΔRAS2
val19
and sch9Δ were mixed at an initial ratio
of 1:25 (10 million:250 million cells), and incubated for 2 hours at 30°C with
shaking. The mixed cultures were then treated with either cyclophosphamide (CP,
52

0.1M) or methyl methanesulfonate (MMS, 0.01%, Sigma). MMS was prepared in
ddH
2
0 from stock solution and was diluted directly into the mixed culture to a final
concentration of (v/v) 0.01%. However, due to the high concentration of CP
(0.1M) required, CP crystals were dissolved directly into the medium. To do so,
mixed cultures were centrifuged for 5 minutes at 2,500 rpm and the spent media
was collected, in which CP crystals were dissolved to a concentration of 0.1M.
The mixed culture was then resuspended in the CP-containing spent medium.
Viability was measured as colony-forming units (CFUs) every 24 hours by plating
onto appropriate selective media. Viability of individual strains was measured
using the same method as for the mixed cultures. Relative survival shown was
determined by the percentage of the ratio between the treated and untreated
(control) cells.

in vitro Drug Treatments
Briefly, primary glia, glioma or neuroblastoma cells were seeded into 96-
well microtiter plates at 20,000-30,000 cells/well and incubated for 2 days.
Glucose restriction was done by incubating cells in glucose free DMEM
(Invitrogen) supplemented with either low glucose (0.5 g/L) or normal glucose
(1.0 g/L) for 24 hours in 1% serum. Serum restriction was done by incubating
cells in DMEM/F12 with either 10% or 1% FBS for 24 hours. Following STS
treatments, cells were treated with H
2
O
2
or menadione for 24 hours.
Cyclophosphamide (CP, Sigma) was used for in vitro chemotherapy studies.
53

Following STS treatments, cells were incubated with varying concentrations of
cyclophosphamide (6-15mg/ml) for 10 hours in DMEM/F12 with 1% FBS. Glial
cells have been reported to express cytochrome P450 and thus are capable of
metabolizing the prodrug cyclophosphamide (Kempermann, Knoth et al. 1994;
Geng and Strobel 1998). Survival was determined by the MTT/LDH assay
(supplementary materials) and presented as percent ratio of treated to control.
in vitro Cytotoxicity Assays
Cytotoxicity was measured by either lactate dehydrogenase released
using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) or the
ability to reduce methylthiazolyldiphenyl-tetrazolium bromide (MTT). LDH
released into the medium by lysed cells were measured with a 10-minute
enzymatic assay that converts a tetrazolium salt (INT) into a red formazan
product. A 96-well based colorimetric assay measured the amount of the red
formazan formed, which is proportional to the number of dead cells. % LDH
release was determined with reference to the maximum and background LDH
release of control cells. MTT is reduced in the mitochondria (metabolically active
cells) by mitochondrial reductase enzymes to form insoluble purple formazan
crystals, which are solubilized by the addition of a detergent (1). Briefly, MTT was
prepared at 5mg/ml in PBS and was diluted in DMEM/F12 1% FBS media to a
final concentration of 0.5mg/ml for assays. Following experimental treatments,
media was replaced with 100μl of MTT and was incubated for 3~4 hours at 37
o
C.
Formazan crystals were dissolved overnight (16hours) at 37
o
C with 100 μl lysis
buffer ((w/v) 15% SDS, (v/v) 50% dimethylformamide, pH 4.7). MTT assay
54

results were presented as percentage of MTT reduction level of treated cells to
control cells. Absorbance was read at 490nm and 570nm for LDH and MTT
assays respectively using a microplate reader SpectraMax 250 (Molecular
Devices) and SoftMax Pro 3.0 software (Molecular Devices). p-values were
calculated by the Student‟s t-test using GraphPad Prism 4 software (GraphPad
Software).
in vivo Studies in Mice
Briefly, to evaluate resistance to high dose etoposide, three different
genetic backgrounds i.e. A/J, CD-1 and Nude/nude mice, were used.. Six week
old female A/J mice (Harlan, S. Pietro al Natisone, Italy), weighing 15-18 g., and
four week old female athymic (Nude-nu) mice (Harlan), weighing 20-22 g, were
starved for 48 hours and then i.v. injected with 80 mg/kg and 100 mg/kg
etoposide (Teva Pharma B.V., Mijdrecht, Holland), respectively. Four week old
female CD-1 mice, weighing 18-20 g, were starved for 60 hours and then i.v.
injected with 110 mg/kg etoposide. In all experiments the mice were offered food
after chemotherapy and were monitored daily for weight loss and general
behaviour. Survival time was used as the main criterion for determining the
differential stress resistance.
For in vivo cancer studies, 6-7 week old female A/J mice, weighing 15-18
g were injected intravenously with murine neuroblastoma NXS2 cell line
(200,000/mouse), as previously described (Lode, Xiang et al. 1997). After tumor
cell injection, some groups of animals were starved for 48 hours and then i.v.
55

treated with etoposide, administered as a single dose. Control groups (NXS2
group) of mice without diet starvation were also investigated.
-Time 0: 200,000 NXS2/mouse
- Time 0 to 48 hours: STS
- 48 hours: Etoposide (80 mg/kg), followed by feeding

To determine toxicity and efficacy, mice were monitored routinely for
weight loss and general behavior.

Statistical Analyses
The significance of the differences between groups in mouse experiments
was determined by Kaplan-Meier curves and Peto‟s log-rank test using StatDirect
(CamCode, Ashwell, UK). The differences were considered significant if the p-
value was less than 0.05.
Comparisons between groups in the in vitro mammalian DSR experiments
were done by Student‟s t-test using GraphPad Prism v.4.00 (GraphPad Software,
San Diego). Comparisons were between different glucose treatment groups for a
specific drug concentration. All statistical analyses were two-sided and p-values
less than 0.05 were considered significant.

56

CHAPTER THREE
Fasting and Cancer Treatment in Humans: A Case Series Report

Chapter Three Abstract

Short-term fasting (48 hours) was shown to be effective in protecting
normal cells and mice but not cancer cells against high dose chemotherapy
(Differential Stress Resistance; DSR). However, the safety and feasibility of
fasting in cancer patients undergoing chemotherapy is unknown. Here we
describe 10 cases in which patients diagnosed with a variety of malignancies had
voluntarily fasted prior to (48-140 hours) and/or following (5-56 hours)
chemotherapy. Patients received an average of 4 cycles of various
chemotherapy drugs in combination with fasting and reported no significant side
effects caused by the fasting itself other than hunger and lightheadedness.
Chemotherapy associated toxicity was graded according to the Common
Terminology Criteria for Adverse Events (CTCAE) of the National Cancer
Institute (NCI). The six patients who underwent chemotherapy with or without
fasting reported a reduction in fatigue, weakness, and gastrointestinal side
effects while fasting. In those patients whose cancer progression could be
assessed, fasting did not prevent the chemotherapy-induced reduction of tumor
volume or tumor markers. Although the 10 cases presented here suggest that
fasting in combination with chemotherapy is feasible, safe, and has the potential
57

to ameliorate side effects caused by chemotherapies, they are not meant to
establish practice guidelines for patients undergoing chemotherapy. Only
controlled-randomized clinical trials will determine the effect of fasting on clinical
outcomes including quality of life and therapeutic index.

Introduction
Chemotherapy can extend survival in patients diagnosed with a wide
range of malignancies. However, side effects caused by toxicity to normal cells
and tissues limit chemotherapy dose density and intensity, which may
compromise efficacy. For instance, the cardiotoxicity and nephrotoxicity
associated with the widely prescribed anti-cancer drugs, doxorubicin and
cisplatin, respectively; limit their full therapeutic potential (Dobyan, Levi et al.
1980; Rajagopalan, Politi et al. 1988; Fillastre and Raguenez-Viotte 1989; Hale
and Lewis 1994). Thus, reduction of undesired toxicity by selective protection of
normal cells without compromising the killing of malignant cells represents a
promising strategy to enhance cancer treatment.
Calorie restriction (CR) is the only effective and reproducible intervention
for increasing life span, reducing oxidative damage, enhancing stress resistance
and delaying/preventing aging and age-associated diseases such as cancer in
various species, including mammals (mice, rats, and non-human primates)
(Weindruch, Walford et al. 1986; McCay, Crowell et al. 1989; Masoro 1995;
Colman, Anderson et al. 2009). In chapter 2, I discussed a fasting-based
58

intervention capable of differentially protecting normal and cancer cells against
high-dose chemotherapy in cell culture and in neuroblastoma-bearing mice was
reported (Raffaghello, Lee et al. 2008). In the neuroblastoma allograft mouse
model, animals were allowed to consume only water for 48 hours prior to
etoposide treatment. Whereas high dose etoposide led to 50% lethality in ad
libitum fed mice, fasting protected against the chemotoxicity associated with the
drug but did not prevent the killing of neuroblastoma cells (Raffaghello, Lee et al.
2008).
Previous human studies have shown that alternate day dietary restriction
and short-term fasting (5 days) are well tolerated and safe (Isley, Underwood et
al. 1983; Maccario, Aimaretti et al. 2001; Johnson, Summer et al. 2007). In fact,
children ranging from 6 months to 15 years of age were able to complete 14 to
40 hours of fasting in a clinical study carried out at the Children‟s hospital of
Philadelphia (Katz, DeLeon et al. 2002). Furthermore, alternate day calorie
restriction caused clinical improvements and reduced markers of inflammation
and oxidative stress in obese asthmatic patients (Fontana, Meyer et al. 2004;
Johnson, Summer et al. 2007).
Here, we report 10 cases of patients diagnosed with various types of
cancers, who have voluntarily fasted prior to and following chemotherapy. The
results presented here, which are based on self-assessed health outcomes and
laboratory reports, suggest that fasting is safe and raise the possibility that it can
reduce chemotherapy side effects. However, only a randomized controlled
clinical trial can establish its efficacy and therapeutic index.
59

Table 3.1 Toxicity side-effect survey form.
60

Table 3.2 Treatment summary of all patients
* also utilized low glycemic diet for 24 hours prior to fast. ** also utilized liquid diet for 24
hours after fast. n/a = not applicable, due to chemotherapy being administered in the
adjuvant setting.
61

Gender Age
Primary
Neoplasia
Stage at
Diagnosis
Case 1 Female 51 Breast IIA
Case 2 Male 68 Esophagus IVB
Case 3 Male 74 Prostate II
Case 4 Female 61 Lung (NSCLC) IV
Case 5 Female 74 Uterus IV
Case 6 Female 44 Ovary IA
Case 7 Male 66 Prostate IV/DI
Case 8 Female 51 Breast IIA
Case 9 Female 48 Breast IIA
Case 10 Female 78 Breast IIA

Table 3.3 Main demographics and clinical characteristics of patients.

62

Results
Ten cancer patients receiving chemotherapy, 7 females and 3 males with
a median age of 61 years (range 44-78 yrs), are presented in this case series
report. Four suffered from breast cancer, two from prostate cancer, and one each
from ovarian, uterine, non small cell carcinoma of the lung, and esophageal
adenocarcinoma. All patients voluntarily fasted for a total of 48 to 140 hours prior
to and/or 5 to 56 hours following chemotherapy administered by their treating
oncologists (Table 3.2 and 3.3).

Case 1
This is a 51-year-old Caucasian woman diagnosed with stage IIA breast
cancer receiving adjuvant chemotherapy consisting of docetaxel (TAX) and
cyclophosphamide (CTX). She fasted prior to her first chemotherapy
administration. The fasting regimen consisted of a complete caloric deprivation
for 140 hours prior and 40 hours after chemotherapy (180 hours total), during
which she consumed only water and vitamins. The patient completed this
prolonged fasting without major inconvenience and lost 7 pounds, which were
recovered by the end of the treatment (Fig 3.1H). After the fasting-chemotherapy
cycle, the patient experienced mild fatigue, dry mouth and hiccups (Fig 3.1I);
nevertheless she was able to carry out her daily activities (working up to 12 hours
a day). By contrast, in the subsequent second and third treatment, she received
chemotherapy accompanied by a normal diet and complained of moderate to
severe fatigue, weakness, nausea, abdominal cramps and diarrhea (Fig 3.1I).
This time the side effects forced her to withdraw from her regular work schedule.
63

For the 4
th
and last cycle, she opted to fast again, although with a different
regimen which consisted of fasting 120 hours prior to and 24 hours post
chemotherapy. Notably, her self-reported side effects were lower despite the
expected cumulative toxicity from previous cycles. Total white blood cell (WBC)
and absolute neutrophil counts (ANC) were slightly better at nadir when
chemotherapy was preceded by fasting (Fig 3.1A, C; Table 3.1). Furthermore,
platelets level decreased by 7-19% during cycles 2 and 3 (normal diet) but did
not drop during the 1
st
and 4
th
cycles (fasting), (Fig 3.1D). After the 4
th

chemotherapy cycle combined with 144-hour fast her ANC, WBC, and platelet
counts reached their highest level since the start of chemotherapy 80 days earlier
(Fig 3.1A, C and D).
64

Figure 3.1 Laboratory values of blood cell counts for case 1.
(A) Neutrophils; (B) Lymphocytes; (C) White blood cells, WBC; (D) Platelets; (E)
Red blood cells, RBC (F) Hemoglobin, Hgb; (G) Hematocrit, Hct; (H) Body
weight. Filled triangle indicates day of chemotherapy; open square indicates
fasting. Normal ranges of laboratory values are indicate by dash lines; (I) Self-
reported side-effects after chemotherapy for case 1. Data represent the average
of 2 cycles of chemo-alone vs the average of 2 cycles of chemo-fasting
treatments.

65

Case 2
This is a 68-year-old Caucasian male diagnosed in February 2008 with
esophageal adenocarcinoma metastasic to the left adrenal gland. The initial
treatment consisted of 5-fluorouracil (5-FU) combined with cisplatin (CDDP)
concurrent with radiation for the first two cycles. Throughout these first two cycles,
the patient experienced multiple side effects including severe weakness, fatigue,
mucositis, vomits and grade 2-3 peripheral neuropathy (Fig 3.2). During the third
cycle, 5-FU administration had to be interrupted due to severe nausea and
refractory vomiting (Fig 3.2). In spite of the aggressive approach with
chemotherapy and radiation, his disease progressed with new metastases to the
right adrenal gland, lung nodules, left sacrum, and coracoid process documented
by computed tomography - positron emission tomography (CT-PET) performed in
August 2008. These prompted a change in his chemotherapy regimen for the
fourth cycle to carboplatin (CBDCA) in combination with TAX and 5-FU (96 hour
infusion) (Table 3.2). During the fourth cycle, the patient incorporated a 72-hour
fast prior to chemotherapy and continued the fast for 51 hours afterward,
consuming only water. The rationale for the 51 hour post-chemotherapy fasting
was to cover the period of continuous infusion of 5-FU. The patient lost
approximately 7 pounds, 4 of which were regained during the first few days after
resuming normal diet (data not shown). Although a combination of three
chemotherapeutic agents were used during this cycle, self-reported side effects
were consistently less severe than during cycles in which calories were
consumed ad lib (Fig 3.2). Prior to his fifth cycle the patient opted to fast again.
66

Instead of receiving the 5-FU infusion for 96 hours, as he did previously, the
same dose of the drug was administered within 48 hours, and the fasting
regimen was also modified to 48 hours prior to and 56 hours post chemotherapy
delivery. Self-reported side effects were again less severe than those in
association with the normal diet and the restaging CT-PET scan indicated
objective tumor response, with decreased standard uptake values (SUV) in the
esophageal mass, the adrenal gland metastases, and the lung nodule. From the
sixth to eighth cycle, the patient fasted prior to and following chemotherapy
treatments (Table 3.2). Fasting was well tolerated in all cycles and
chemotherapy-dependent side effects were reduced except for mild diarrhea and
abdominal cramps that were developed during the seventh cycle (Fig 3.2).
Ultimately, the patient‟s disease progressed and the patient died in February
2009.

67

Figure 3.2 Self-reported side-effects after chemotherapy for case 2.
68

Case 3
This is a 74-year-old Caucasian man who was diagnosed in July 2000
with stage II prostate adenocarcinoma, Gleason score 7 and baseline PSA level
of 5.8 ng/ml. He achieved an undetectable PSA nadir after radical prostatectomy
performed in September of 2000, but experienced biochemical recurrence in
January 2003 when PSA rose to 1.4 ng/ml. Leuprolide acetate together with
bicalutamide and finasteride were prescribed. However, administration of these
drugs had to be suspended in April 2004 due to severe side effects related to
testosterone deprivation. Additional therapies including triptorelin pamoate,
nilutamide, thalidomide, CTX and ketoconazole failed to control the disease. In
2007 the patient‟s PSA level reached 9 ng/ml and new metastases were detected
on bone scan. TAX at 25mg/m
2
on weekly basis was administered, but the PSA
level continued to increase, reaching 40.6 ng/ml (data not shown). bevacizumab
was added into the treatment and only then did the PSA drop significantly (data
not shown). Throughout the cycles with chemotherapy the patient experienced
significant side effects including fatigue, weakness, metallic taste, dizziness,
forgetfulness, short-term memory impairment and peripheral neuropathy (Fig
3.3I). After discontinuing the chemotherapy, his PSA rose rapidly. TAX was
resumed at 75mg/m
2
every 21 days, and was complemented with granulocytic
colony stimulating factor (G-CSF). Once again the patient suffered significant
side effects (Fig 3.3I). In June 2008, chemotherapy was halted. The patient was
enrolled in a phase III clinical trial with abiraterone acetate, a drug that can
selectively block CYP17, a microsomal enzyme that

catalyzes a series of
reactions critical to nongonadal androgen

biosynthesis(Raghavan and Klein
69

2008). During the trial, the patient‟s PSA levels increased to 20.9ng/dl (Fig 3.3H),
prompting resumption of chemotherapy and G-CSF. This time the patient opted
to fast prior to chemotherapy. His fasting schedule consisted of 60 hours prior to
and 24 post drug administration (Table 3.2). Upon restarting chemotherapy with
fasting the PSA level dropped, and notably, the patient reported considerably
lower side effects than in previous cycles during which he consumed calories ad-
lib (Figure 3.3I). He also experienced reduced myelosuppression (Figs 3.3A-G).
During the last three cycles, in addition to fasting, the patient applied
testosterone (cream, 1%) for five days prior to chemotherapy. As a consequence
the PSA level along with the testosterone level increased dramatically.
Nonetheless, 3 cycles of chemotherapy combined with fasting reduced PSA from
34.2 to 6.43 ng/ml (Fig 3.3H). These results imply that the cytotoxic activity of
TAX to cancer cells was not blocked by fasting.

70

Figure 3.3 Laboratory values of blood cell counts for case 3.
(A) Neutrophils; (B) Lymphocytes; (C) White blood cells, WBC; (D) Platelets; (E)
Red blood cells, RBC (F) Hemoglobin, Hgb; (G) Hematocrit, Hct; (H) Prostate
specific antigen (PSA) level. The patient was enrolled in abiraterone acetate
(CYP17 inhibitor) trial for 90 days indicated by vertical dash lines. The patient
also received G-CSF (Neulasta) on the day of chemotherapy except during the
treatment with abiraterone acetate. Filled triangle indicates day of chemotherapy;
open square indicates fasting, arrow indicates testosterone application (cream
1%). Normal ranges of laboratory values are indicated by horizontal dash lines;
(I) Self-reported side-effects after chemotherapy for case 3. Data represent the
average of 5 cycles of chemo-alone vs the average of 7 cycles of chemo-fasting
treatments.
71

Case 4
This is a 61-year-old Caucasian female who was diagnosed in June 2008
with poorly differentiated non-small cell lung carcinoma (NSCLC). A staging PET
scan documented a hypermetabolic lung mass, multiple involved mediastinal and
left perihilar lymph nodes, and widespread metastatic disease to the bones, liver,
spleen, and pancreas. The initial treatment commenced with the administration of
TAX 75 mg/m
2
and CBDCA 540mg every 21 days. Although she was on a
regular diet, during the first 5 cycles she lost an average of 4 pounds after each
treatment, most likely due to chemotherapy-induced anorexia. The patient
reported that she did return to her original weight but only after three weeks of
the drug administration, just before a new cycle. Additional side effects included
severe muscle spasms, peripheral neuropathy, significant fatigue, mucositis,
easy bruising and bowel discomfort (Fig 3.4H). During the sixth

cycle,

which
consisted of the same drugs and dosages, the patient fasted for 48-hours-prior
and 24-hours-post chemotherapy. She lost approximately 6 pounds during the
fasting period, which she recovered within 10 days (data not shown). Besides
mild fatigue and weakness, the patient did not complain of any other side effect
that she had experienced during the five previous cycles (Fig 3.4H). Cumulative
side effects such as peripheral neuropathy, hair loss and cognitive impairment
were not reversed. By contrast self-reported acute toxic side effects were
consistently reduced when chemotherapy was administered in association with
fasting (Fig 3.4H). In the sixth and last cycle, the patient reported that her
strength returned more quickly after the chemotherapy so that she was able to
72

walk 3 miles three days after the drug administration, whereas in previous cycles
she had experienced severe weakness and fatigue which limited any physical
activity. No significant differences were observed in the patient‟s blood work
(Figs 3.4A-G). The last PET scan performed on February 2009 showed stable
disease in the lung mass and decreased uptake in the spleen and liver when
compared to the baseline study.
73

Figure 3.4 Laboratory values of blood cell counts for case 4.
(A) Neutrophils; (B) Lymphocytes; (C) White blood cells, WBC; (D) Platelets; (E)
Red blood cells, RBC (F) Hemoglobin, Hgb; (G) Hematocrit, Hct; Filled triangle
indicates day of chemotherapy; open square indicates fasting. Normal ranges of
laboratory values are indicated by dash lines; (H) Self-reported side-effects after
chemotherapy for case 4. Data represent the average of 5 cycles of chemo-alone
vs 1 cycle of chemo-fasting treatment.

74

Case 5
This is a 74 year-old woman diagnosed in 2008 with stage IV uterine
papillary serous carcinoma. Surgery (Total Abdominal Hysterectomy-Bilateral
Salpingo-Oopherectomy, TAH-BSO, with lymph node dissection) followed by
adjuvant chemotherapy were recommended. Due to a significant enlargement of
the right ureter, a right nephrectomy was also performed. Post-operatively, six
cycles of CBDCA (480mg) and paclitaxel (280mg) were administered every 21-
days. During the first treatment the patient maintained her regular diet and
experienced fatigue, weakness, hair loss, headaches and gastrointestinal
discomfort (Fig 3.5). By contrast, during cycles 2-6, the patient fasted before and
after chemotherapy, and reported a reduction in the severity of chemotherapy
side effects (Table 3.2, Fig 3.5). Fasting did not appear to interfere with
chemotherapy efficacy, as indicated by the 87% reduction in the tumor marker
CA-125 after the 4
th
cycle (data not shown).
75

Figure 3.5 Self-reported side-effects after chemotherapy for case 5.
Data represent 1 cycle of chemotherapy-alone (1
st
cycle) vs the average of 5
cycles of chemo-fasting treatments.

76

Case 6
This is a 44-year-old white female diagnosed with a right ovarian mass
(10x12 cm.) in July 2007. Surgery (TAH-BSO) revealed stage IA carcinosarcoma
of the ovary with no lymph node involvement. Adjuvant treatment consisted of six
cycles of ifosfamide and CDDP, administered from July to November of 2007.
She remained free of disease until an MRI revealed multiple new pulmonary
nodules in August 2008. Consequently chemotherapy with taxol, carboplatin and
bevacizumab was initiated. By November, however, a CT scan showed
progression of the cancer. Treatment was changed to gemcitabine plus TAX
complemented with G-CSF (Neulasta) (Table 3.2, 3.5). After the first dose of
gemcitabine (900 mg/m
2
), the patient experienced prolonged neutropenia (Fig
3.6A) and thrombocytopenia (Fig 3.6D), this forced a delay of day 8 dosing.
During the second cycle the patient received a reduced dose of gemcitabine (720
mg/m
2
), but again developed prolonged neutropenia and thrombocytopenia,
causing dose delays. For the third and subsequent cycles, the patient fasted for
62 hours prior to and 24 hours after chemotherapy. The patient not only did not
find hardship on carrying out the fasting but she also experienced a faster
recovery of her blood cell counts, allowing the completion of the regimen with
chemotherapies (gemcitabine 720mg/m
2
on day 1 plus gemcitabine 720mg/m
2

and TAX 80mg/m
2
on day 8). During the fifth cycle, she fasted under the same
regimen and received a full dose of gemcitabine (900mg/m
2
) and TAX (Table
3.4). Her complete blood count showed consistent improvement during the
cycles in which chemotherapy was combined with fasting. A trend in which nadirs
were slightly less pronounced and the zeniths were considerably higher in ANC,
77

lymphocyte and WBC counts was observed (Fig 3.6A, B, C, respectively; Table
3.4). During the first and second cycle (no fasting) gemcitabine alone induced
prolonged thrombocytopenia, which took 11 and 12 days to recover, respectively
(Fig 3.6D; Table 3.4) but following the first combined fasting-gemcitabine
treatment (3
rd
and subsequent cycles), the duration of thrombocytopenia was
significantly shorter (Fig 3.6D; Table 3.4).
78

Figure 3.6 Laboratory values of blood cell counts for case 6.
(A) Neutrophils; (B) Lymphocytes; (C) White blood cells, WBC; (D) Platelets; (E)
Red blood cells, RBC (F) Hemoglobin, Hgb; (G) Hematocrit, Hct; Filled triangle
indicates day of chemotherapy; open square indicates fasting. Normal ranges of
laboratory values are indicated by dash lines. (H) Self-reported side-effects after
chemotherapy for case 6.The patient received red blood cell transfusion (3 units)
on day 71 and also received G-CSF (Neulasta) as indicated.
79

Table 3.4 Summary of case 1.
* Time (in days) to reach the lowest blood cell count after chemotherapy.
** Time (in days) to reach normal level of blood cell count from nadir day.
80

Table 3.5 Summary of case 6.

* Time (in days) to reach the lowest blood cell count after chemotherapy.
** Time (in days) to reach normal level of blood cell count from nadir day.

81

Case 7
This is a 66-year-old Caucasian male who was diagnosed in July 1998
with prostate adenocarcinoma, Gleason score 8. A Prosta Scint study performed
in the same year displayed positive uptake of the radiotracer in the right iliac
nodes, consistent with stage D1 disease. The patient was treated with leuprolide
acetate and bicalutamide for one year, followed by finasteride maintenance
beginning in September 1999. In December 2000, the diseases progressed. He
started on a second cycle with leuprolide acetate and also received High Dose
Rate (HDR) brachytherapy and external beam radiation with Intensity Modulated
Radiation Therapy (IMRT) to the prostate and pelvis. As additional cycles of
androgen deprivation bicalutamide and triptorelin pamoate were administered,
however, PSA levels increased quickly during each off-treatment phase. In April
2008, a Combidex scan revealed a 3 x 5 cm pelvic mass and left hydronephrosis
prompting initiation of TAX chemotherapy supplemented with G-CSF. The patient
decided to fast for 60-66 hours prior to and 8-24 hours following chemotherapy
(Table 3.2). While fasting, the patient experienced grade one lightheadedness
(accordingly CTCAE 3.0) and a drop in blood pressure, none of which interfered
with his daily routine. Self-reported side effects included grade one sensory
neuropathy, (paresthesias and tingling) in his feet after seven consecutive cycles
of TAX. (Fig 3.7I). The patient‟s ANC, WBC, platelet and lymphocyte levels
remained in the normal range throughout treatment, although he did develop
anemia (Figs 3.7A-G). PSA levels consistently decreased, suggesting that
82

fasting did not interfere with the therapeutic benefit of the chemo-treatment (Fig
3.7H).
Figure 3.7 Laboratory values of blood cell counts for case 7.
(A) Neutrophils; (B) Lymphocytes; (C) White blood cells, WBC; (D) Platelets; (E)
Red blood cells, RBC (F) Hemoglobin, Hgb; (G) Hematocrit, Hct; (H) Prostate
specific antigen (PSA) level. Filled triangle indicates day of chemotherapy; open
square indicates fasting, arrow indicates abiraterone administration. Normal
ranges of laboratory values are indicate by dash lines. The patient also received
G-CSF (Neulasta) as indicated; (I) Self-reported side-effects after chemotherapy
for case 7. Data represent the average of 8 cycles of chemo-fasting treatments.
83

Case 8
This is a 53-year-old Caucasian female who was diagnosed with stage IIA
breast cancer (HER2+) in 2008. After a lumpectomy procedure, she received 4
cycles of adjuvant chemotherapy with TAX (75mg/m
2
) and CTX (600mg/m
2
)
every 21 days. For all 4 cycles the patient fasted 64 hours prior to and 24 hours
post the chemotherapy administration. Side effects reported included mild
weakness and short-term memory impairment (Fig 3.8).
Figure 3.8 Self-reported side-effects after chemotherapy for case 8.
Data represent the average of 4 cycles of chemo-fasting treatments.

84

Case 9
This is a 48 year-old Caucasian female diagnosed with breast cancer. Her
adjuvant chemotherapy consisted of 4 cycles of doxorubicin (DXR, 110mg)
combined with CTX (1100mg) followed by weekly paclitaxel and trastuzumab for
12 weeks. Prior to her first chemotherapy treatment, the patient fasted for 48
hours and reported no adverse effects. During the second and subsequent
cycles the patient fasted for 60 hours prior to the chemotherapy followed by 5
hours post drug administration (Table 3.2). She reported no difficulties in
completing the fasting. Although she experienced alopecia and mild weakness,
the patient did not suffer from other commonly reported side effects associated
with these chemotherapy drugs (Fig 3.9).
85

Figure 3.9 Self-reported side-effects after chemotherapy for case 9.
Data represent the average of 4 cycles of chemo-fasting treatments.

Case 10
This is a 78 year-old Caucasian female diagnosed with HER2 positive
breast cancer. After mastectomy, six cycles of adjuvant chemotherapy were
prescribed with CBDCA 400 mg (AUC= 6), TAX (75mg/m
2
) complemented with
G-CSF (Neulasta), followed by 6 months of trastuzumab (Table 3.2). For all the
chemotherapy treatments the patient fasted prior and after the drug
administration. Although the patient adopted a fasting regimen of variable periods,
no severe side effects were reported (Fig 3.10H, Table 3.2). Her WBC, ANC,
86

platelet and lymphocyte counts remained within normal levels (Figs 3.10A-D)
throughout the treatment, but she developed anemia (Figs 3.10E-G).

Figure 3.10 Laboratory values of blood cell counts for case 10.
(A) Neutrophils; (B) Lymphocytes; (C) White blood cells, WBC; (D) Platelets; (E)
Red blood cells, RBC (F) Hemoglobin, Hgb; (G) Hematocrit, Hct. Filled triangle
indicates day of chemotherapy; open square indicates fasting. Normal ranges of
laboratory values are indicated by dash lines. The patient also received G-CSF
(Neulasta) as indicated. (H) Self-reported side-effects after chemotherapy for
case 10. Data represent the average of 6 cycles of chemo-fasting treatments.
87

Discussion
Dietary recommendations during cancer treatment are based on the
prevention or reversal of nutrient deficiencies to preserve lean body mass and
minimize nutrition-related side effects, such as decreased appetite, nausea, taste
changes, or bowel changes (Doyle, Kushi et al. 2006). Consequently, for cancer
patients who have been weakened by prior chemotherapy cycles or are
emaciated, many oncologists could consider a fasting-based strategy to be
potentially harmful. Nevertheless using cell culture and animal models we
recently reported that fasting can reduce chemotherapy side effects by
selectively protecting normal cells(Raffaghello, Lee et al. 2008). Following this
study, several patients, diagnosed with a wide variety of cancers, elected to
undertake fasting prior to chemotherapy and shared their experiences with us. In
this heterogeneous group of men and women fasting was safely repeated in
multiple cycles for as long as 180 hours prior and/or following chemotherapy.
Minor complaints that arose during fasting included dizziness, hunger, and
headaches at a level that did not interfere with daily activities. Weight lost during
fasting was rapidly recovered in most of the patients and did not lead to any
detectable harm.
We obtained self-reported assessments of toxicity from all 10 patients who
incorporated fasting with their chemotherapy treatments. Since many of the
chemotherapy side effects are cumulative, we evaluated serial data including all
the combined fasting- and non-fasting associated chemotherapy cycles (Fig
3.11). Toxicity was graded utilizing a questionnaire based on the Common
88

Toxicity Criteria for Adverse Events of National Cancer Institute, version 3.0.
Although the lack of prospective collection of toxicity data and grading are a
significant limitation, this series provide an early insight into the feasibility and
potential benefit of combining fasting with chemotherapy. Fewer and less severe
chemotherapy-induced toxicity in combination with fasting was reported by all the
patients, even though fasting cycles were often carried out in the later portion of
the therapy (Fig 3.11). Nausea, vomiting, diarrhea, abdominal cramps, and
mucositis were virtually absent from the reports of all 10 patients in the cycles in
which fasting was undertaken prior to and/or following chemotherapy, whereas at
least one of these symptoms was reported by 5 out of the 6 patients during
cycles in which they ate ad libitum (Fig 3.11). The four patients that fasted
throughout their treatments reported low severity for the majority of the side
effects, in contrast to the typical experience of cancer patients receiving the
same chemotherapy regimens (Figs 3.7I, 3.8, 3.9, 3.10H). For the 6 patients who
received chemotherapy during /after fasting or in association with their normal
diet, we compared the severity of the self-reported side effects in the 2 closest
fasting/non-fasting cycles in which the patient received the same chemotherapy
drugs at the same dose. There was a general and substantial reduction in the
self-reported side effects in combination with fasting (Fig 3.12). Symptoms such
as fatigue and weakness were reported to be significantly reduced (p< 0.001 and
p< 0.00193, respectively), whereas vomiting and diarrhea were reported to be
essentially absent in combination with fasting (Fig 3.12). In addition, there was
89

no side effect whose average severity was reported to be increased during
fasting-chemotherapy cycles (Figs 3.11, 3.12).
Figure 3.11 Self-reported side-effects after chemotherapy with or without
fasting.
Data represent average of CTCAE grade reported by all the patients in this
study. 18 chemotherapy cycles under ad-lib diet were compared to 46 chemo-
fasting cycles.

90

Figure 3.12 Self ‐reported side ‐effects after chemotherapy with or without
fasting.
Data represent average of CTCAE grade from matching fasting and non ‐fasting
cycles (Ad Lib). 6 patients received either chemotherapy ‐alone or chemo ‐fasting
treatments. Self ‐reported side effects from the closest two cycles were compared
one another. Statistic analysis was performed only from matching cycles. Data
presented as standard error of the mean (SEM). P value was calculated with
unpaired, two tail t test. (*, P<0.05).
91

Challenging conditions such as fasting or severe CR stimulate organisms
to suppress growth and reproduction, and divert energy towards cellular
maintenance and repair to maximize the chance of survival (Longo, Ellerby et al.
1997; Longo and Finch 2003; Longo, Lieber et al. 2008). In simple organisms
such as yeast, resistance to oxidants and chemotherapy drugs can be increased
by up to 10 fold in response to fasting/starvation and up to 1,000 fold in those
cells lacking homologs of Ras, AKT and S6 kinase (Raffaghello, Lee et al. 2008).
Nevertheless, resistance to oxidative stress is completely reversed and even
decreased below wild type level in cells expressing oncogene-like genes (Longo
and Finch 2003; Raffaghello, Lee et al. 2008). In mammals the mechanism(s)
responsible for the protective effect of fasting/glucose starvation against
chemotherapy induced-toxic side effects is not completely understood. It may
involve reduction in anabolic hormones and growth factors such as insulin and
IGF-1 as well as up-regulation of several stress resistance proteins (Thissen,
Ketelslegers et al. 1994; Mote, Tillman et al. 1998; Blagosklonny and Pardee
2001; Reddy, Mao et al. 2003; Fontana and Klein 2007; Spindler and Dhahbi
2007). In fact, mice with Liver Specific IGF-I gene-deletion (LID) which have ~75-
80% reduction of circulating IGF-I and mice with genetic disruptions in the IGF-IR
(heterozygous knockout IGF-IR +/-) or its downstream elements have been
shown to be more resistant against multiple chemotherapy agents and oxidative
stress, respectively (Holzenberger, Dupont et al. 2003; Longo 2009). An
alternative explanation for DSR is that downregulation of mitogenic intracellular
pathways downstream of the IGF-I could selectively induce cell cycle arrest in
92

normal cells whereas transformed cells continue to proliferate becoming more
vulnerable to anticancer drugs (Blagosklonny and Pardee 2001; Blagosklonny
and Darzynkiewicz 2002).
Although mutations driving cancer progression are heterogeneous across
tumor types, the majority of the described oncogenic mutations render cancer
cells independent of growth signals (Hanahan and Weinberg 2000; Blagosklonny
and Darzynkiewicz 2002), which we hypothesize prevents cancer cells from
responding to the fasting-induced switch to a protected mode. Therefore, DSR
would have the potential to be applied independently of the cancer type.
In summary, in this small and heterogeneous group of cancer patients,
fasting was well-tolerated and was associated with a self-reported reduction in
multiple chemotherapy-induced side effects. Although bias could affect the
estimation of the side effects by the patients, the case reports presented here
provide preliminary data indicating that fasting is feasible, safe and has the
potential to differentially protect normal and cancer cells against chemotherapy in
humans. Nevertheless, only a formal clinical trial such as the randomized
controlled clinical trial currently carried out at the USC Norris Cancer Center, can
establish whether fasting protects normal cells and increases the therapeutic
index.

93

Methods
From April 2008 to August 2009, ten unrelated patients diagnosed with a
variety of cancer volunteered to incorporate fasting with their chemo-treatments.
We invited these patients to complete a self-assessment survey based on the
Common Terminology Criteria for Adverse Events (CTCAE) of The National
Cancer Institute (NCI) version 3.0. For the purpose of this study only, we
developed a questionnaire that contained 16 easy identifiable and commonly
reported side effects; the seriousness of the symptoms was graded from 0 to 4
with each consecutive number corresponding to no side
effect/mild/moderate/severe and life threatening. Adverse effects were further
divided into 3 major categories including, general, gastrointestinal and
central/peripheral nervous system side effects, (Table 3.1, original questionnaire).
The survey was delivered to patients by mail, e mail or fax and every patient was
instructed to complete it 7 days after each treatment cycle. Explanation and
assistance to patient‟s concern were offer throughout the study. The eligibility
criterion to participate was subjected to those patients that had voluntarily fasted
prior and/or post chemotherapy. Medical records including basic demographical
information, diagnosis, treatments, imaging studies and laboratory analysis were
also retrospectively reviewed (Table 3.2 and 3.3). All the aforementioned
procedures were in compliance with the Internal review Board of the University of
Southern California (USC).
94

CHAPTER FOUR
Reduced Levels of IGF-I Mediate Differential Protection of
Normal and Cancer Cells in Response to Fasting and Improve
Chemotherapeutic Index

Chapter Four Abstract
Inhibitors of the insulin-like growth factor-1 receptor (IGF-IR) have been
widely studied for their ability to enhance the killing of a variety of malignant cells,
but the role of IGF-I and its receptor in the differential protection of host and
cancer cells against chemotherapy is unknown. We previously showed that
starvation protects mice but not cancer cells against high dose chemotherapy
(Differential Stress Resistance, DSR). Here we provide evidence for the role of
IGF-I reduction in mediating the effect of starvation in DSR. A 72-hour fast
reduced circulating IGF-I by 70% and increased the level of the IGF-I inhibitor
IGFBP-1 by 11-fold in mice. LID mice, with a 70-80% reduction in circulating IGF-
I levels, were protected against 3 out of 4 chemotherapy drugs tested.
Restoration of IGF-I during fasting was sufficient to reverse its protective effect.
60% of melanoma-bearing LID mice treated with doxorubicin reached long-term
survival whereas all control mice died of either metastases or chemo toxicity.
Reduction of IGF-I/IGF-I signaling protected primary glia, but not glioma cells
against cyclophosphamide and protected mouse embryonic fibroblasts (MEFs)
against doxorubicin-induced DNA damage. Similarly, S. cerevisiae lacking
95

homologues of IGF-I signaling proteins displayed protection against
chemotherapy-dependent DNA damage, which was reversed by expression of an
oncogene homolog. We conclude that reducing circulating IGF-I protects normal
cells and mice against chemotherapy-dependent DNA damage by a mechanism
that involves down-regulation of proto-oncoproteins.

Introduction
Most chemotherapy agents cause considerable damage to normal cells,
leading to toxicity which is dose limiting and causes both short- and long-term
side effects in patients. Although drug development has reduced these side
effects with a succession of selective anti-tumor agents such as antibodies that
target specific antigens on cancer or agents with a wider therapeutic index,
toxicity continues to limit cancer treatment. Thus, interventions that reduce the
undesired toxic side-effects could increase the efficacy of many chemotherapy
drugs. Chemoprotectants such as amifostine, glutathione, mesna, and
dexrazoxane have been investigated and shown to provide drug-dependent
protection to specific tissues, but the use of these compounds has not been
shown to increase

disease-free or overall survival (Links and Lewis 1999).
Recently, we reported that short-term starvation (STS) differentially protects
normal but not, or much less, malignant cells, leading to improved survival
(Raffaghello, Lee et al. 2008). Here we present evidence that reduced IGF-I is a
major mediator of STS-dependent differential protection.
96

Biogerontologists have long known that calorie restriction and/or
deficiencies in the pro-growth GH/IGF-I axis increase stress resistance and
lifespan, and also share many physiological characteristics in various model
organisms (Longo and Finch 2003). These beneficial effects can be explained, in
part, by the active diversion of energy utilization in starved or IGF-I deficient
organisms. The finite energy source of an organism is finely balanced between
growth and maintenance under normal conditions (Kirkwood and Shanley 2005).
However, under challenging conditions such as starvation, the energy is diverted
from growth to maintenance, thereby enhancing protection and survival at the
price of growth (Shanley and Kirkwood 2000).
During starvation, several changes in the GH/IGF-I axis occur as a result
of physiological adaptation to the new environment. Generally, growth hormone
(GH) directly regulates the production of IGF-I, which is the major mediator of the
growth effects of GH (Bartke 2005). In humans, IGF-I levels decrease
dramatically in response to short-term starvation (36-120 hours) despite
increased GH secretion (Merimee, Zapf et al. 1982; Thissen, Underwood et al.
1999). Long-lived organisms that are deficient in IGF-I signaling have been
shown to be resistant to multiple types of stress (Holzenberger, Dupont et al.
2003; Murakami 2006). However, unlike normal cells, cancer cells are self-
sufficient in growth signals and insensitive to growth inhibitory signals (Hanahan
and Weinberg 2000). Self-sufficiency in growth signals is often enabled by gain-
of-function mutations in oncogenes (e.g. IGF-IR or its downstream PI3K, and Ras,
etc) that result in constitutive activation of proliferation pathways independently or
97

partially independently of external growth factor level. Notably, in normal cells,
the RAS/RAF/MAPK and the PTEN/PI3K/AKT pathways can be down-regulated
by CR or starvation (Xie, Jiang et al. 2007), possibly mediated by reduced IGF-I.
On the other hand, insensitivity to growth inhibitory signals is due to loss-of-
function mutations in tumor-suppressor genes (e.g. Rb, p53, PTEN, etc),
enabling cancer cells to disregard anti-proliferation signals (Hanahan and
Weinberg 2000; Vogelstein and Kinzler 2004). Here we test the hypothesis that
the reduction of circulating IGF-I and its signaling mediates the protection of
normal cells and mice against chemotherapy toxicity, whereas oncogene-bearing
cancer cells do not respond to reduced IGF-I.

Results

Short-term starvation regulates components of the pro-growth GH/IGF-I
axis

To investigate the role of the GH/IGF-I axis in the beneficial effects of
short-term starvation (STS) on differential stress resistance (DSR), we started by
measuring the level of circulating GH, IGF-I and its binding proteins IGFBP-1 and
-3 in mice undergoing STS. CD-1 mice were fasted for 72 hours and blood was
collected to measure glucose levels and plasma GH, IGF-I, and IGFBP-1 and -3
levels. After a 72-hour STS, mice had lost approximately 20% of body weight,
glucose levels were reduced by 41%, GH levels were slightly increased, IGF-I
levels decreased 70% (Fig 4.1A-D). The bioavailability of IGF-I, which can
98

activate IGF-I receptors (IGF-IR), is regulated by IGF binding proteins. In fasted
mice, the level of IGFBP-1, which normally reduces IGF-I signaling, increased
11.4-fold (Fig 4.1E). These results are in agreement with the reports showing
that IGFBP-I increases in response to fasting in humans and rats (Cotterill, Holly
et al. 1993; Katz, Satin-Smith et al. 1998; Frystyk, Delhanty et al. 1999), and also
that its overexpression in mice effectively retards growth by sequestering IGF-I
(Murphy 2000). Furthermore, the 72-hour fast decreased IGFBP-3 levels by 42%
(Fig 4.1F) in agreement with reports in short-term fasted humans and rats
(Frystyk, Delhanty et al. 1999; Norrelund, Frystyk et al. 2003). However, the
mechanistic explanation for the decrease in IGFBP-3 is not clear.
To test if restoring the level of IGF-I during STS reverses the protection
against chemotherapy toxicity, CD-1 mice underwent a 48-hour STS with IGF-I
(200 µg/kg) administration every 12 hours. The level of injected IGF-I was
determined from prior serum IGF-I measurements of ad lib fed mice. Following
the STS/IGF-I treatments, mice were intravenously injected with 16 mg/kg
doxorubicin (DXR), a widely used chemotherapy drug acting as an intercalating
agent and topoisomerase II inhibitor (Zeman, Phillips et al. 1998). Indeed, the
restoration of IGF-I during STS abolished the protective effect of STS on DXR
toxicity, resulting in a 100% vs. 38% survival in the STS and STS/IGF-I group,
respectively (Fig 4.2).
Previously, we showed that primary glia but not glioma cell lines pre-
incubated with low glucose (50 mg/dl compared to the normal 100 mg/dl) and low
serum (1% fetal bovine serum with the consequent reduction of several growth
99

factors including IGF-I) showed enhanced protection against the alkylating
chemotherapy agent cyclophosphamide (Raffaghello, Lee et al. 2008). The
glucose levels of fasted mice were reduced to a similar level, along with a
dramatic decrease in IGF-I levels (Fig 4.1A, D). Therefore, the reduction of IGF-I,
a potent growth factor, may mediate part of the effect of fasting on DSR.

100

Figure 4.1 The effect of 72 hour fasting on glucose levels, IGF-I, and
IGFBP-1/3.
30 week old CD-1 mice were fasted for 72 hours and sacrificed. Blood was
collected via cardiac puncture under anesthesia, and blood glucose was
measured immediately. Plasma GH, IGF-I and IGFBP-1/3 levels were measured
by a mouse-specific in-house ELISA. All P values were calculated by Student‟s t-
test except for IGFBP-1 which was done by the Mann-Whitney U test.
101

Figure 4.2 IGF-I restoration during STS reverses STS-dependent
protection.
16-20 week-old female CD-1 mice were fasted for 48-hours (STS). During STS,
IGF-I (200 µg/kg) was intraperitoneally injected every 12 hours to restore IGF-I
levels. Immediately following STS/IGF-I treatment, all mice were intravenously
injected with 16 mg/kg DXR. For STS/IGF-I treatments, mice were transferred to
clean new cages to eliminate residual food pellets and excretion (reduce
coprophagy). Food was withheld completely during STS (48 hours), but water
was given ad lib.

102

Reduced IGF-I signaling protects primary glia but not glioma cells against
high-dose cyclophosphamide

IGF-I-like signaling pathways are implicated in regulating life span and
stress resistance in organisms ranging from the simple yeast to worms, flies, and
mice (Brown-Borg, Rakoczy et al. 2002; Longo and Finch 2003; Yan, Vatner et al.
2007; Suh, Atzmon et al. 2008). To test the role of IGF-I signaling in DSR against
chemotherapeutic drugs in vitro, we incubated normal and the equivalent cancer
cell lines with either an IGF-I receptor (IGF-IR) blocking antibody, low serum
concentrations, or excess IGF-I prior to treatment with cyclophosphamide (CP), a
commonly used chemo drug based on its DNA alkylating properties (de Jonge,
Huitema et al. 2005). Primary mixed rat glia (astrocytes + 5-10% microglia) and 3
different rat glioma cell lines (C6, A10-85 and 9L) were tested. All cells were
grown to confluence to minimize differences in proliferation rate. Pre-incubation
with an antagonistic IGF-IR antibody (αIR3) protected primary glia but not the
three glioma cell lines against CP toxicity (Fig. 4.3A). Reduction of serum level
from the standard 10% to 1%, with consequent reduction of growth factors
including IGF-I, decreased the toxicity of 15 mg/ml CP to primary glia but not to
C6 glioma cells (Fig. 4.3B). On the other hand, pre-incubation with 100ng/ml
IGF-I (in the low normal range for adult human serum) (Manetta, Brun et al.
2002) caused a 3-fold increase in the toxicity of CP to primary mixed glia but did
not increase the toxicity of CP to C6 glioma cells (Fig. 4.3C). Similar results were
obtained with primary neurons and neuron-like pheochromocytoma cells (PC12)
treated with a combination of IGF-I and the oxidative stress agent paraquat (Fig.
103

4.3D). These results are consistent with our previous studies on fasting and DSR
(Raffaghello, Lee et al. 2008) and support the hypothesis that down-regulation of
IGF-I signaling can protect normal but not cancer cells against cytotoxic agents.

104

Figure 4.3 in vitro DSR to CP treatments by reducing IGF-I
Primary rat glial cells and rat glioma cell lines (C6, 9L, and A10-85) cell lines
were tested. (A) Cells were pre-incubated in DMEM/F12 with 1% serum and
neutralizing anti-IGF-IR monoclonal antibody alpha-IR3 (1 ug/ml) for 24 hours (15
mg/ml ; n=12). (B) Cells were pre-incubated in medium with either 1% (STS) or
10% FBS for 24 hours (15 mg/ml ; n=12). (C) Cells were pre-incubated in
medium with 1% serum with or without rhIGF-I (100ng/ml) for 48 hours (12
mg/ml ; n=21). (D) The effect of IGF-I on DSR against oxidative stress.
Chemotherapy drugs such as etoposide, cyclophosphamide, 5-fluorouracil, and
DXR have been shown to increase reactive oxygen species (ROS) and cause
oxidative stress. Primary neurons and PC12 pheochromocytoma cells were pre-
treated with IGF-I (100ng/ml) for 30 minutes, followed by paraquat treatments for
24 hours. Cytotoxicity (LDH assay) was determined following treatment. * P
<0.05, ** P <0.005, *** P<0.0001 by Student‟s t test.
105

Effect of IGF-IR deletion or overexpression on stress resistance in mouse
embryonic fibroblast cells

To begin to investigate the mechanism responsible for differential stress
resistance, we treated mouse embryonic fibroblasts (MEF) bearing an IGF-Ir
deletion (R
-
cells) or overexpressing IGF-IR (R
+
cells) with DXR (Drakas, Tu et al.
2004). All cells were grown to confluence to minimize the difference in
proliferation rate and were treated with DXR for 24 or 48 hours. After a 24 hour
DXR treatment, R
-
cells showed greater survival compared to R
+
cells. The effect
was most pronounced at 25µM where more than 80% of R
-
cells were viable,
whereas only 30% of R
+
cells were alive (Fig. 4.4A, P <0.0005). Similar results
were observed when cells were treated for 48 hours, with 50% vs. 12% survival
rate for R
-
and R
+
cells, respectively, at 25µM (Fig. 4.4B, P <0.02).
To further investigate how deficiency in IGF-I signaling protects against
chemotoxicity we measured DNA damage using the comet assay. DXR induced
DNA damage was significantly higher in R
+
cells compared to R
-
cells, with more
than a 3-fold difference as assessed by the comet assay (Fig. 4.4C,D; P <0.001).
These results support our hypothesis that reduced IGF-I signaling protects
normal cells by reducing chemotherapy-dependent DNA damage (Longo, Lieber
et al. 2008). Notably, R
-
cells have been shown to be resistant against
transformation by the SV40 large T-antigen, which is remarkable considering that
fibroblasts frequently transform in culture spontaneously (Baserga 1999).

106

Figure 4.4 The role IGF-I signaling in protection against DXR in mouse
embryonic fibroblasts (MEF).
R
+
and R
-
cells were grown to confluence and treated with DXR (0-500μM) in
DMEM/F12 supplemented with 10% FBS for (A) 24 hours or (B) 48 hours.
Viability was determined by the relative degree of MTT reduction compared to
untreated; mean ± SD. * P <0.05, ** P <0.01, *** P <0.001 by Student‟s t test; R
+

vs. R
-
cells at same DXR concentration. (C) Comet assay. Cells overexpressing
IGF-IR or with IGF-IR deficiency (R
+
and R
-
) were treated with 50µM DXR for 1
hour. Significant DNA damage was observed in the DXR treated R+ cells,
whereas R- cells showed enhanced protection against DXR induced DNA
damage. (D) Tail olive moment analysis of the comet assay. *** P <0.001 by
Student‟s t test; R
+
DXR vs. R
-
DXR. Similar results were obtained from two
independent experiments. Representative experiment is shown.

107

The role of yeast homologs of downstream effectors of the IGF-IR in DSR
To investigate the mechanisms by which down-regulation of the IGF-IR
signaling protects against chemotoxicity and its effect on DNA damage, we
turned to the simple model system S. cerevisiae. The rationale for utilizing yeast
is based on the role of Ras2 and Sch9, homologs of the mammalian Ras and
Akt/S6K, respectively, in modulating cellular defense against oxidative stress,
DNA alkylation, and thermal stress demonstrated in our previous studies (Madia,
Gattazzo et al. 2008; Raffaghello, Lee et al. 2008; Wei, Fabrizio et al. 2008), and
on the central signaling role of homologs of SCH9 and RAS2 downstream of IGF-
IR (Fig 4.5A). We tested the effect of the deletion of RAS2 and SCH9 on the
resistance against DXR. To further investigate DSR, we also studied cells
transformed with a gene expressing a constitutively active RAS2 (RAS2
Val19
) that
models human oncogenic Ras mutations. The deletion of SCH9 (sch9Δ) or both
SCH9 and RAS2 (sch9Δras2Δ) provided remarkable protection against DXR
compared to its wild-type (WT) strains (Fig 4.5B). However, analogously to what
we observed in mammalian cells, the expression of the oncogene-like RAS2
Val19
reversed the protection provided by RAS2 and SCH9 deficiency. Following 48
hours of DXR treatment, 50% of WT and RAS2
val19
expressing cells survived,
whereas 70% of sch9Δ and more than 90% of sch9Δras2Δ survived (Fig 4.5B).
The protection was more pronounced after 72 hours of DXR treatment where
sch9Δras2Δ and sch9Δ were highly protected (88% and 70% survival
respectively) but the protection was reversed by the expression of RAS2
val19

(sch9ΔRAS2
val19
; 27% survival). To study the molecular mechanisms of
108

differential resistance to DXR, we monitored DNA mutation frequency, measured
as mutations in the CAN1 gene (Can
r
colonies/10
6
cells) (Madia, Gattazzo et al.
2007). DXR treatments increased mutation frequency in all strains. In agreement
with the survival analysis, sch9Δ and sch9Δras2 Δ exhibited the lowest mutation
frequency, whereas RAS2
val19
expression increased mutation frequency (Fig
4.5C). The expression of RAS2
val19
in sch9Δ (sch9ΔRAS2
val19
) completely
reversed the protection provided by the Sch9 deficiency resulting in a 3-fold
increase in mutation frequency (Fig 4.5B,C). These data suggest that lowered
Ras2 and Sch9 signaling has a beneficial effect that could be explained, at least
in part, due to the enhanced protection against DNA damage in the cell, which
can be reversed by the expression of oncogenes.
109

Figure 4.5 The conserved regulatory pathways of stress resistance in
response to starvation/calorie restriction.
In yeast, nutrient-sensing pathways controlled by Sch9, Tor, and Ras converge
on the protein kinase Rim15. In turn, the stress response transcription factors
Msn2, Msn4, and Gis1 transactivate stress response genes and enhance cellular
protection, which leads to life span extension. In mice and humans, short-term
starvation leads to a significant reduction in circulating IGF-I levels. The partially
conserved IGF-I signaling pathways negatively regulate the FoxO family
transcription factors through Akt. Ras and Tor also function downstream of IGF-I,
although their roles in the regulation of stress resistance and aging are poorly
understood. Mice deficient in type 5 adenylyl cyclase (AC) are stress resistant,
analogous to the adenylate cyclase deficient yeast. Notably, oncogenic mutations
that cause the hyperactivation of IGF-I, Akt, Ras, Tor and PKA are among the
most common in human cancers (Hanahan and Weinberg 2000).

110

Figure 4.6 The effect of Sch9-/Ras2-deficiencies on DSR against DXR in S.
cerevisiae.
(A) Wild type (DBY746), sch9Δ, sch9Δras2Δ, RAS2
val19
, and sch9ΔRAS2
val19

strains were inoculated at OD
600
= 0.1, grown separately in glucose media, and
treated with DXR (200μM) 24 hours after initial inoculation. Viability was
measured as colony forming units (CFU) onto appropriate selective media. Data
from 3 independent experiments are shown as mean ± SE. * P <0.05 by
Student‟s t test, sch9Δras2Δ vs. sch9ΔRAS2
val19
. (B) Mutation frequency over
time, measured as Can
r
mutants/10
6
cells. Strains shown are wild type (WT),
cells lacking Sch9 and/or Ras2, and cells overexpressing constitutively active
Ras2
val19
. Mean ± SEM (n= 3-5 experiments). Cells were treated with DXR
(200µM) on day 1. Mutation frequency of wild type untreated cells was reported
as control. * P < 0.05 by Student‟s t test, sch9Δras2Δ vs. sch9ΔRAS2
val19
.
111

Octreotide sensitizes NXS2 neuroblastoma cells but does not protect mice
against high-dose etoposide

Since reduction of IGF-I provided differential chemotherapy protection in
mammalian cell culture, we tested if pharmacological manipulation of the
GH/IGF-I axis could induce DSR in vivo. The somatostatin analogue octreotide is
used in clinics to reduce GH secretion and IGF-I production primarily in
acromegaly patients. Also, octreotide was selected because somatostatin
increases in response to fasting (Ishikawa, Mizobuchi et al. 1997). Interestingly,
octreotide and other somatostatin analogs have been shown to have therapeutic
effects in a number of cancers (Hejna, Schmidinger et al. 2002) through two
distinct effects: direct actions on tumors mediated by somatostatin receptors
(Zalatnai and Schally 1989; Susini and Buscail 2006), and indirect effects
through inhibition of growth hormone release and the lowering of IGF-I (Zalatnai
and Schally 1989; Pollak, Schernhammer et al. 2004; Susini and Buscail 2006).
In a previous report, we showed that short-term starvation (STS) provides DSR
against high-dose etoposide (Raffaghello, Lee et al. 2008), a widely used
chemotherapy drug that inhibits topoisomerase II (Hande 1998). Here we tested
if inhibiting the GH/IGF-I axis with octreotide could increase the protection
against etoposide. We selected a particularly aggressive tumor line (NXS2) that
models neuroblastoma (NB) (Lode, Xiang et al. 1997). Intravenous injection of
NXS2 cells results in a consistent formation of metastasis in multiple organs
including the liver, kidneys, adrenal gland, and ovaries (Lode, Xiang et al. 1997).
A single injection of high-dose etoposide (80 mg/kg) extended the lifespan of
112

tumor-bearing mice, which otherwise would have succumbed to metastasis
within 40 days. In our previous study, STS caused a remarkable reduction in
acute chemotoxicity-related deaths, but also provided partial protection to the
cancer cells (Raffaghello, Lee et al. 2008). Our present results indicate that
octreotide is not sufficient to protect the animals against chemotherapy but its
combination with STS sensitizes the NXS2 cancer cells to etoposide (Fig. 4.7A-
C, Gr. 4 vs. Gr.7, P <0.01). However, octreotide had a minimal effect on lowering
IGF-I levels in mice (Fig. 4.7D), which could explain its inability to protect the
animal. It is possible that homeostatic mechanisms counteract the effect of
somatostatin and lead to tachyphylaxis to octreotide treatment, thus failing to
reduce IGF-I levels significantly.
To test if octreotide exerted its sensitizing effect on NXS2 cells directly or
indirectly, we treated NXS2 cells with octreotide and etoposide in vitro (Fig. 4.7E).
Octreotide did not alter the toxicity of etoposide to NXS2 cells in cell culture,
suggesting the sensitizing effect of octreotide in mice is indirect. Together, this
implies that octreotide alone does not provide starvation-like host protection, but
may reverse the partial protection provided by STS to cancer cells by sensitizing
them. Further studies are necessary to investigate the possibility that octreotide
may sensitize other tumors against chemotherapy.

113

Figure 4.7 The effect of Octreotide (OCT) on short-term starvation (STS)
based DSR against etoposide (Eto)
(A) Stress resistance in A/J mice after a 48 hours STS and 4 days of octreotide
treatment prior to and 4 days following Eto injection. (B) The weight profile of A/J
mice after high-dose etoposide (80mg/kg) injection is shown. (C) Survival rate of
neuroblastoma (NXS2) bearing mice after a single high-dose Eto (80 mg/kg)
treatment. All mice were inoculated intravenously with 200,000 NXS2
cells/mouse on day 4. Octreotide was administered for 4 days prior to tumor
inoculation and 4 days following chemotherapy. Survival of mice was monitored
daily. The different groups were treated as following: (Gr. 1) Control - I.V.
inoculation with NSX2 tumor cells on day 4 (n=16); (Gr. 2) OCT - pre-treatment
with 1 mg/kg/day octreotide for 4 days before and after tumor inoculum (n=8); (Gr.
3) OCT/STS/OCT - pre-treatment with 1 mg/kg/day OCT before tumor cell
inoculum + 48-hour STS on days 4-6 + post-treatment with 1 mg/kg/day OCT on
days 8-11 (n=8); (Gr. 4) OCT/STS/Eto/OCT - pre-treatment with 1 mg/kg/day
OCT for 4 days before tumor cell inoculum + 48-hour STS on day 4-6 + I.V.
injection of 80 mg/kg Eto on day 7 + post-treatment with 1 mg/kg/day OCT on
days 8-11 (n=13); (Gr. 5) OCT/Eto/OCT - pre-treatment with 1 mg/kg/day OCT
for 4 days before tumor cell inoculum + I.V. injection of 80 mg/kg Eto on day 7 +
post-treatment with 1 mg/kg/day OCT on days 8-11 (n=17); (Gr. 6) STS - 48-hour
STS on days 4-6 (n=8); (Gr. 7) STS/Eto - 48-hour STS on days 4-6 + I.V.
injection of 80 mg/kg Eto on day 7 (n=15); (Gr. 8) Eto - I.V. injection of 80 mg/kg
Eto on day 7 (n=6). Statistics: P Gr. 4 vs. Gr.1 < 0.0001, P Gr.5 vs. Gr.1 < 0.005,
114

Figure 4.7 Continued
P Gr.7 vs. Gr.1 = 0.001, P Gr.6 vs. Gr.1 > 0.999, P Gr.4 vs. Gr.5 = 0.34, P Gr.4
vs. Gr.7 = 0.009, P Gr.5 vs. Gr.8 = 0.46, P Gr.7 vs. Gr.8 = 0.8. P values by Peto‟s
log rank test. (D) IGF-I levels were measured by a mouse-specific ELISA from
mice treated I.P. with 1 mg/kg/day or 20 mg/kg/day octreotide for 7 days. (E)
NXS2 cells were incubated with different concentrations of Eto (1-3 μM) ±
octreotide (10 and 50 μM) for 72 hours. Cell viability was determined by trypan
blue exclusion. Data shown as percentage of dead cells (i.e. trypan blue positive),
mean ± SE (n= 3 independent experiments).

115

Enhanced stress resistance in LID mice against high-dose chemotherapy
Mice with genetic disruptions in the IGF-IR or its downstream effectors are
more resistant to oxidative stress (Migliaccio, Giorgio et al. 1999; Holzenberger,
Dupont et al. 2003). To determine whether reducing IGF-I signaling protects from
chemotoxicity, we tested a transgenic mouse model with a conditional liver IGF-I
gene deletion (LID)(Yakar, Liu et al. 1999), resulting in a post-natal 70-80%
reduction of circulating IGF-I (Anzo, Cobb et al. 2008), which is similar to that of a
72-hour fasted mice (Fig. 4.1B). The LID mice provides a model for investigating
the mechanistic relationship between IGF-I and fasting in chemotherapy
resistance (Patel, Nunez et al. 2004). A single administration of high-dose
etoposide led to 50% vs. 88% survival rate respectively in the LID and control
mice (Fig. 4.8A; Fig. 4.9A). Based on our in vitro results, we tested CP in LID
mice. LID mice treated with 500 mg/kg CP showed significantly higher resistance,
with 70% vs. 30% survival rate for LID and control mice respectively (Fig. 4.8B).
Furthermore, while LID mice only lost an average of 10% of their weight, control
mice lost 20% (Fig. 4.9B). The surviving LID mice also did not show signs of
toxicity (Fig. 4.10). To determine the range of protection by reduced IGF-I, we
tested two additional drugs, 5-fluorouracil (5-FU) and doxorubicin (DXR), which
represent different classes of chemotherapy drugs. 5-FU is an anti-metabolite
(Longley, Harkin et al. 2003). Survival after a treatment with high-dose 5-FU was
improved, with a 55% vs. 10% survival rate in LID and controls respectively,
although the difference was not statistically significant (Fig. 4.8C). Similar but
more pronounced effects were observed with DXR. Unlike etoposide and other
drugs that can cause irreversible damage to the tail vein of rodents after a single
116

high-dose injection, DXR can be injected for up to 2-3 cycles (data not shown). In
order to test the effect of multiple cycles of chemotherapy, we challenged LID
mice with 2 cycles of high-dose DXR. The first DXR injection (20 mg/kg) did not
result in any toxicity related deaths, but led to considerable weight loss in all mice
(Fig. 4.8D; Fig. 4.9C). Weight loss was more evident in LID mice during the first
5 days following DXR injection, but unlike controls who continued to lose weight
and showed signs of toxicity, LID mice regained their weight during the following
3 weeks. The second DXR injection (28 mg/kg) caused a considerable amount of
DXR-related deaths in the control (25% survival) but not in the LID mice (100%
survival) (Fig. 4.8D).

117

Figure 4.8 Stress resistance testing in LID mice with various high-dose
chemotherapeutic drugs.

LID and control mice received (A) a single injection of 100 mg/kg etoposide
(n=10/LID, n=9/control, P=0.064), (B) a single injection of 500 mg/kg CP
(n=20/group, P=0.001), (C) a single injection of 400 mg/kg 5-fluorouracil
(n=11/LID, n=10/control, P=0.148), (D) two injections of doxorubicin (DXR). The
first injection of 20 mg/kg was given on day 0, and the second injection of 28
mg/kg was given on day 22 (n=5/LID, n=4/control, P=0.022). Toxicity evaluated
by percent survival is shown. P values by Peto‟s log rank test.

118

Figure 4.9 Weight loss after high-dose chemotherapy in LID mice.
LID and its control mice received a single high-dose injection of either (A)
etoposide (100 mg/kg) or (B) cyclophosphamide (500 mg/kg). (C) DXR was
given twice, first on day 0 at 20 mg/kg, and the second on day 22 at 28 mg/kg.

119

Figure 4.10 LID mice show no signs of cyclophosphamide-dependent
toxicity.
(A) A mouse from the control group and (B) Liver IGF-I gene deleted (LID)
group shown after high-dose cyclophosphamide (500mg/kg) treatment.

120

Differential stress resistance in melanoma bearing LID mice against high-
dose doxorubicin

Next, we tested DSR in vivo by monitoring LID mice inoculated with a
highly aggressive melanoma cell line (B16Fluc) that metastasizes primarily to the
lungs (Craft, Bruhn et al. 2005) and treating them with DXR. B16Fluc is a
luminescent derivative of the B16 mouse melanoma cell line. Therefore tumor
progression and regression can be visualized and quantified in vivo using
bioluminescence imaging technology (BLI) (Craft, Bruhn et al. 2005). LID and its
control mice were intravenously injected with B16Fluc (2x10
5
cells/mouse)
melanoma cells and treated with 2 cycles of high-dose DXR (Fig 4.11A).
Although IGF-I plays a major role in tumor growth and metastasis (Samani,
Yakar et al. 2007), both LID and its control mice started to succumb to
metastasis as early as 25 days following cancer inoculation. The 2 cycles of high-
dose DXR extended survival time by delaying metastasis in all mice (Fig 4.11C).
43% of control mice died with signs of DXR-induced cardiac myopathy, whereas
none of the LID mice died from DXR toxicity (Fig 4.11D, 4.12A). In addition, LID
mice showed a slight advantage in weight maintenance (Fig 4.13). 90 days after
cancer inoculation, all control mice that received chemotherapy had died from
either cancer metastases or DXR toxicity, but 60% of LID mice that received 2
cycles of high-dose DXR treatment were cancer-free with no apparent toxic side-
effects (Fig 4.11B,C, Fig 4.12). All the LID mice deaths were caused by cancer
metastases. The progression of cancer between control and LID mice was
similar after DXR treatments (Fig 4.12B), suggesting that the reduction of
121

circulating IGF-I protects the host but not cancer cells against high dose
chemotherapy.
Figure 4.11 Differential stress resistance (DSR) against 2 cycles of high-
dose DXR in melanoma bearing LID mice.
(A) Timeline of experimental procedures (n=4/LID-B16, n=5/LID-B16-DXR,
n=8/Control-B16, n=7/Control-B16-DXR). (B) Bioluminesence imaging of
B16Fluc melanoma bearing LID mice and control mice treated with 2 cycles of
high-dose DXR. Five mice were randomly selected and followed throughout the
experiment to monitor tumor progression or regression. (C) Survival rate
comparison between B16Fluc melanoma bearing LID and control mice treated
with 2 cycles of high-dose DXR (P<0.05). (D) DXR induced cardiomyopathy in
control and LID mice. Heart failure is a major outcome of acute DXR toxicity.
Histological slides of the heart from DXR treated control mice showed loss of
myofibrils and infiltration of immune cells, whereas DXR dependent cardiac
myopathy was not observed in LID mice. Hematoxylin and eosin staining (H&E).
Representative slide shown. Bar, 100µm.
122

Figure 4.12 LID mice death is due to metastasis and not cardiotoxicity.
(A) LID mice are protected against DXR toxicity and (B) show slightly reduced
metastasis related deaths. Survival recorded at Day 90 following cancer
inoculation.

Figure 4.13 Weight of LID and control mice.
B16Fluc cells were injected intravenously on Day 0. DXR injections were given
intravenously on Days 3 and 17.
123

Discussion
In a previous report, we described a STS-based DSR method to protect
the host but not cancer cells against high-dose chemotherapy. The basis for this
appears to be the existence of a non-dividing state, which normal cells enter in
response to starvation for the purpose of investing the remaining energy
resources in cellular protection against various insults. Here we show that a
major reduction in circulating IGF-I can protect the host but not cancer cells
against chemotherapy. Low levels of IGF-I can reduce intracellular mitogenic
signaling pathways, including those regulated by Ras and Akt, two of the major
pathways downstream of the IGF-IR. We believe that the reduction of mitogenic
stimuli may protect normal cells in part by inducing cell cycle arrest
(Blagosklonny and Pardee 2001; Blagosklonny and Darzynkiewicz 2002;
Keyomarsi and Pardee 2003) and in part by shifting the energy towards repair by
mechanisms regulated by proteins including Akt, Ras/ERK, FOXO, SirT1, SODs,
and DNA repair genes (Murakami 2006; Li, Xu et al. 2008; Longo, Lieber et al.
2008), thereby entering a highly protected „maintenance mode‟ (Blagosklonny
and Pardee 2001; Raffaghello, Lee et al. 2008). In yeast, we have previously
shown that protection can be increased in non-dividing cells by up to 1,000-fold,
suggesting that a major component of the protective mechanisms is independent
of the switch from a dividing to a non-dividing state, at least in this simple
organism (Raffaghello, Lee et al. 2008). This is also in agreement with the effect
of IGF-IR overexpression in sensitizing fibroblasts grown to confluence to
doxorubicin (Fig 4.4). On the other hand, cancer cells are self sufficient in growth
124

signals, less or not responsive to physiological anti-growth signals, and in many
cases do not undergo cell cycle arrest due to check point dysregulation
(Hanahan and Weinberg 2000; Blagosklonny and Darzynkiewicz 2002; Longo,
Lieber et al. 2008). In fact, it has been shown that pre-treatment with non-toxic
doses of cell cycle arresting drugs (e.g. DXR) or growth factor inhibitors
(inhibitors of MEK or EGF receptor) protect normal cells but not cancer cells
against chemotherapy (Blagosklonny, Bishop et al. 2000; Blagosklonny, Robey
et al. 2000; Demidenko, Halicka et al. 2005).
In support of our hypothesis, our yeast experiments show that the deletion
of the homologs of RAS and/or SCH9 (AKT/S6K) promotes protection against
DXR, but the expression of the oncogenic RAS2
Val19
reverses this cellular and
DNA protection independently of cell division. These results raise the possibility
that oncogenic mutations that activate pathways such as Ras, AKT or PKA may
reverse the protective effect of reduced IGF-I signaling in malignant cells, thus
allowing differential protection of host and various cancers. Notably, inhibition of
the pathway downstream of oncogenic mutations could have either a positive or
negative effect on the protection of cancer cells. Preclinical studies show that
IGF-IR targeting strategies can be effective in the treatment of multiple myelomas,
prostate, breast and colon cancer in addition to other cancers (Pollak,
Schernhammer et al. ; Tao, Pinzi et al. 2007). The antitumor effect seen with
such agents is thought to be dependent on apoptosis resulting from IGF-IR
inactivation (Tao, Pinzi et al. 2007). However, it must be noted that IGF-IR
blockade could also trigger apoptosis in normal cells, and may not protect
125

against high dose chemotherapy by interfering with the growth/recovery of
certain types of cells (e.g. bone marrow cells). As observed with our LID mice,
reduced IGF-I, unlike IGF-IR blockade, does not cause cancer cell death but can
selectively enhance the resistance of normal cells against chemotoxicity and may
sensitize cancer cells to chemotherapy. This is in agreement with the normal
development of prostatic carcinoma in the LID-TRAMP model (Anzo, Cobb et al.
2008). Based on our results from etoposide treated LID mice, strategies that
reduce circulating IGF-I may also increase the toxicity of certain chemotherapy
drugs. Therefore, the compatibility between each drug and IGF-I
reduction/blockade therapy should be carefully tested in pre-clinical studies
before being considered as a candidate. Although it appears to be central, IGF-I
may represent simply one of a number of growth factors that can activate Ras,
Akt etc in normal cells and promote cell death in cancer cells and therefore only
one of the factors that can be down-regulated to provide differential stress
resistance (Blagosklonny and Darzynkiewicz 2002).
In summary, our studies in mice indicate that a major reduction in
circulating IGF-I and in intracellular IGF-I signalling enhances resistance of the
host, but not cancer cells against chemotherapy, thus providing the foundation
for a method to augment cancer treatment without the need to fast. However, the
combination of fasting and IGF-I reduction could result in an even more
pronounced effect.
126

Materials and Methods

Cell lines
Primary mixed glial cells were obtained from the cerebral cortex of 1 to 3
day old Sprague Dawley rat pups (Charles River) as described before (McCarthy
and de Vellis 1980). Cells cultured for 10-14 days in DMEM/F12 medium with
10% fetal bovine serum (FBS) were used in assays. C6, A10-85, and 9L rat
glioma cell lines, kindly provided by Dr. Chen (University of Southern California)
and R
+
and R
-
cells, kindly provided by Dr. Baserga (Thomas Jefferson
University), were maintained in DMEM/F12 with 10% FBS at 37
o
C under 5% CO
2.

R
+
and R
-
cells are mouse embryonic fibroblast (MEF) that overexpress human
IGF-IR or have IGF-IR deletion, respectively, and were generated as previously
described (Drakas, Tu et al. 2004). R
-
cells are 3T3-like cells originating from

mouse embryos with a targeted disruption of the IGF-Ir genes (Drakas, Tu et al.
2004).

The R
+
cell line was

derived from R- cells, and express the human IGF-Ir
cDNA under the control

of the cytomegalovirus (CMV) promoter (Drakas, Tu et al.
2004). Primary neurons from embryonic day 18 Sprague-Dawley rat cerebral
cortices were dissociated in neurobasal medium (Invitrogen) supplemented with
0.5 mM L-glutamine, 25 µM L-glutamic acid and 2% B-27 and plated at 640
cells/mm
2
in 96-well plates which were pre-coated with 10 µg/ml poly-D-lysine
dissolved in Borax buffer (0.15 M, pH 8.4). Neurons were maintained at 37°C in
5% CO
2
in neurobasal medium supplemented with B-27 and 0.5 mM L-glutamine
for 4 days. PC12 rat pheochromocytoma cell line (ATCC) was maintained in
127

F12K medium supplemented with 15% horse serum and 2.5% fetal bovine serum
at 37
o
C under 5% CO
2.

in vitro IGF-I modulation
All cells were grown to confluence prior to treatments. The inhibition of
IGF-IR activation was achieved with monoclonal anti-IGF-IR antibody (αIR3,
1μg/ml; Calbiochem) in DMEM/F12 1% FBS for 24 hours. Serum restriction was
performed by incubating cells in DMEM/F12 with either 10% or 1% FBS for 24
hours. IGF-I treatment was carried out by incubating cells for 48 hours in
DMEM/F12 with 1% FBS and rhIGF-I (100ng/ml, ProSpec-Tany TechnoGene,
Rehovot, Israel), which is shown to be within the IGF-I level range for middle age
humans (Manetta, Brun et al. 2002).
in vitro drug treatments
Primary glia and C6, A10-85, and 9L rat glioma cells were seeded at
2x10
4
cells/well and incubated for 48 hours in 96 well plates prior to treatments to
reach confluence and minimize differences in proliferation. Various IGF-I
modulating pretreatments were followed by cyclophosphamide (CP, Sigma)
treatments. Glial cells have been reported to express cytochrome P450 and thus
are capable of metabolizing the prodrug CP (Kempermann, Knoth et al. 1994;
Geng and Strobel 1998). CP was prepared in DMEM/F12 with 1% FBS at 40
mg/ml and was filter sterilized. The stock solution was stored at 4
o
C for no longer
than 2 weeks. Cells were incubated with varying concentrations of
cyclophosphamide (0-15 mg/ml) for 10 hours in DMEM/F12 with 1% FBS. R
+
and
R
-
cells were seeded at 2x10
4
cells/well and incubated in 96 well plates and were
128

also grown to confluence (2 days) prior to doxorubicin (DXR) treatments. DXR
was prepared at 5mg/ml in sterile saline. Cells were treated with DXR for 24
hours and 48 hours prior to survival analysis by MTT reduction. NXS2
neuroblastoma cells treated with different concentrations of etoposide (1-3 µM)
in the presence or absence of octreotide (10 and 50 µM) for 72 hours were
harvested by scraping, washed with complete medium, and incubated with trypan
blue (0.04%; Sigma; St. Louis, MO) for 1 minute at 37 °C. The cells were then
placed in a Burker chamber (Tecnovetro, Monza Milan, Italy) and counted with a
contrast phase microscope (Olympus Optical Co LTD, Tokyo, Japan). Trypan
blue-positive cells (i.e. dead cells), trypan blue-negative cells (i.e. living cells),
and total cells were counted per microscope field (four fields were counted for
each treatment). The proportion of dead (or living) cells was calculated by
dividing the number of dead (or living) cells by the total number of cells per field.
Primary rat neurons and PC12 cells were treated with IGF-I and paraquat to
determine the effect of IGF-I on oxidative stress. Cortical neurons were treated
for 24 hours in Eagle‟s minimal essential medium (Invitrogen) supplemented with
21 mM glucose and 1% horse serum. PC12 cells were plated at 5 x10
4
cells/well
onto poly-D-lysine coated 96-well plates and were grown for 24 hours in F12K
1% HS. Both types of cells were then treated with either 100 µM of paraquat
alone or followed 30 minutes later by IGF-I (100 ng/ml) or IGF-I (100 ng/ml) alone
in appropriate media. Survival was determined by the MTT reduction assay and
presented as percentage of treated to control.

129

in vitro viability assays
Cytotoxicity was measured by either lactate dehydrogenase (LDH)
released using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega)
or the ability to reduce methylthiazolyldiphenyl-tetrazolium bromide (MTT). MTT
is reduced in the mitochondria (metabolically active cells) by mitochondrial
reductase enzymes to form insoluble purple formazan crystals, which are
solubilized by the addition of a detergent (1). Briefly, MTT was prepared at 5
mg/ml in PBS and was diluted in DMEM/F12 1% FBS media to a final
concentration of 0.5 mg/ml for assays. Following experimental treatments, media
was replaced with 100 μl of MTT and cells were incubated for 3~4 hours at 37
o
C.
Formazan crystals were dissolved overnight (16 hours) at 37
o
C with 100 μl lysis
buffer ((w/v) 15% SDS, (v/v) 50% dimethylformamide, pH 4.7). Survival was
presented as percentage of MTT reduction level of treated cells to control cells.
Absorbance was read at 570nm using a microplate reader SpectraMax 250
(Molecular Devices) and SoftMax Pro 3.0 software (Molecular Devices).

STS and IGF-I treatments in mice
Mice were fasted for 48-72 hours. Food was completely withdrawn, but
water was given ad lib. All mice were single caged in a new clean cage to
eliminate the exposure to residual food and to reduce coprophagy. Mice were
monitored thrice and weighed once daily. rhIGF-I (Prospec, Rehovot, Israel) was
injected intraperitoneally every 12 hours during the 48-hour STS. Control mice
received bolus injections of the solvent (ddH
2
O).
130

Stress resistance against chemotherapy treatments in LID mice
LID mice of 75-100 weeks of age were used to model human cancer.
Since liver is the major source of IGF-I production, mice with a conditional
hepatic IGF-I gene knockout have reduced circulating IGF-I levels by 80% (Anzo,
Cobb et al. 2008). Because albumin is expressed in the liver after 10 days of
birth, resulting in liver IGF-I gene deletion, LID mice do not experience early
death, growth retardation, or developmental defects like the IGF-I gene knock-out
(IGF-I-/-) mice (Yakar, Liu et al. 1999). LID and its control mice were given 100
mg/kg etoposide intravenously. CP was given at 500 mg/kg. CP was dissolved in
saline at 40 mg/ml and injected intraperitoneally. 5-Fluorouracil (5-FU, Sigma)
was injected at 400 mg/kg intraperitoneally. Doxorubicin (DXR, Sigma) was
prepared at 5 mg/ml in saline and injected intravenously first at 20 mg/kg and 22
days later at 28 mg/kg. All drugs have been selected from different categories. All
mice were monitored daily for weight loss and signs of pain and stress. Mice
determined terminally moribund were euthanized by CO
2
narcosis and necropsy
was performed. Experiments were performed in accordance with Institutional
Animal

Care and Use Committee (University of Southern California, Los Angeles,
CA) and the National Institutes of Health

guidelines.

Differential stress resistance against DXR in LID mice
In order to study differential stress resistance, mice were injected with
highly metastatic melanoma cells. LID and its control mice of ages 75-100 weeks
131

were used. B16Fluc melanoma cells were a generous gift of Dr. Noah Craft at
UCLA. B16Fluc cells are derivatives of B16 cells but produce light by stable
transfection of the Firefly luciferase gene driven by the CMV promoter (Craft,
Bruhn et al. 2005). Prior to injection, cells were washed and resuspended in
sterile saline. Each mouse received 2x10
5
cells in 100 µl saline, followed by
another 100 µl of sterile saline to wash off remaining cells in the tails. 3 days after
tumor inoculation, the first DXR (Bedford Laboratories) injections were given at
16 mg/kg. 2 weeks following the initial DXR administration, the second DXR
injection was given at 12 mg/kg. Mice were observed daily for signs of stress or
pain and body weight was recorded. Mice determined terminally moribund were
sacrificed by CO
2
narcosis and necropsy was performed. The heart was collected
for further histological examination.

Comet assay protocol
Cells were diluted to 10
5
/ml in culture medium (DMEM/F12 with 10% FBS),
and treated with 50 µM DXR for 1 hour at 37°C. Cells were then washed once
with ice cold PBS and subject to CometAssay (Trevigen, Inc, Gaithersburg, MD)
according to the manufacturer‟s recommended procedure. Comet images were
acquired with a Nikon Eclipse TE300 fluorescent microscope and analyzed with
the Comet Score software (TriTek Corp., ver1.5). 100-300 cells were scored for
each genotype/treatment group.

132

Plasma mGH, mIGF-I, and mIGFBP-1 and -3 measurements
Plasma mIGF-I and mIGFBP-1 and -3 assays were performed as
previously described by in-house ELISA assay using recombinant mouse IGF-I
protein and monoclonal antibodies from R&D systems (Minneapolis, MN) (Hwang,
Lee et al. 2008). mGH levels were measured by rat/mouse GH ELISA kit
(ALPCO Diagnostics).

Blood glucose measurements
Following a 72 hour fast, mice were anesthetized with 2% inhalant
isoflurane and blood was collected by left ventricular cardiac puncture. Blood
glucose was measured using the Precision Xtra blood glucose monitoring system
(Abbott Laboratories, USA).

STS/Octreotide treatments in mice
All mice received an I.V. injection of 80 mg/kg Eto on day 7. The different
groups were treated as follows: (Eto) treatment with 80 mg/kg Eto on day 7
(n=23); (OCT/Eto/OCT) pre-treatment with 1 mg/kg/day octreotide for 4 days +
treatment with 80 mg/kg Eto on day 7 + post-treatment with 1 mg/kg/day
octreotide on days 8-11 (n=17); (STS/Eto) 48-hour STS on days 4-6 + treatment
with 80 mg/kg Eto on day 7 (n=16); (OCT/STS/Eto/OCT) pre-treatment with 1
mg/kg/day octreotide for 4 days + 48-hour STS (day 4-6) + treatment with 80
mg/kg Eto on day 7 + post-treatment with 1 mg/kg/day octreotide on days 8-11
(n=35). P =0.0002 by Peto‟s log rank test.
133

STS/Octreotide treatments in NXS-2-bearing mice
The murine NX3IT28 cell line was generated by hybridization of the GD2-
negative C1300 murine neuroblastoma cell line (A/J background) with murine
dorsal root ganglional cells from C57BL/6J mice, as previously described
(Greene, Shain et al. 1975). The NXS2 subline was then created by the selection
of NX3IT28 cells with high GD2 expression (Lode, Xiang et al. 1997). Female A/J
mice, weighing 15-18 g were purchased from Harlan Laboratories (Harlan Italy, S.
Pietro al Natisone, Italy) and housed in sterile cages under specific virus and
antigen-free conditions. All procedures were reviewed and approved by licensing
and ethical committee of the National Cancer Research Institute, Genoa, Italy,
and by the Italian Ministry of Health. A/J mice were pretreated with 1 mg/kg/day
doses of octreotide (OCT, ProSpec-Tany TechnoGene, Rehovot, Israel) for 4
days given slowly through the tail vein in a volume of 100 µl. Following the 4 days
of octreotide treatment, mice were intravenously injected with NXS2 cells
(200,000 cells/mouse), as previously described (Lode, Xiang et al. 1997). After
tumor cell injection, some animals were starved for 48 hours and then I.V.
injected with 80 mg/kg of etoposide (Teva Pharma B.V., Mijdrecht, Holland),
administered as a single dose. Additional daily doses of OCT were administered
for 4 days after chemotherapy. Control groups without dietary intervention and
OCT treatment were also investigated.
Octreotide pre-treatment: 4 days 1mg/kg/day on days 1-4
NXS2: 200,000/mouse on day 4
STS: from day 4 to day 6 (after tumor cell injection)
Etoposide: 80 mg/kg on day 7
134

Octreotide post-treatment: days 8-11

To determine toxicity and efficacy, mice were monitored routinely for
weight loss and general behavior. The animals were killed by cervical dislocation
after being anesthetized with xilezine (Xilor 2%, Bio98 Srl, Milan, Italy) when they
showed signs of poor health, such as adbominal dilatation, dehydration, or
paraplegia.

Histology
The heart was collected for histological examinations of melanoma
bearing LID and its control mice after 2 cycles of high-dose DXR. Heart failure
has been documented as the major cause of acute toxicity after receiving DXR
and therefore we examined the heart at the tissue level (Rajagopalan, Politi et al.
1988). The organs were collected and washed in ice cold PBS and stored in 10%
neutral buffered formalin (VWR). Samples were paraffin embedded and
sectioned at 5µm and H&E stained. Samples were examined and analyzed with
Dr. Dubeau, professor of pathology at USC Keck School of medicine.

Bioluminescence imaging
For bioluminescence imaging (BLI), 5 mice were randomly selected from
LID and control groups and followed throughout the experiment. All BLI imaging
procedures were performed at the University of Southern California (USC) Small
Animal Imaging core facility. Prior to imaging, mice were anesthetized using
inhalant isoflurane (2%) and injected with 60 µl of 50mg/kg of the luciferase
135

substrate luciferin (Xenogen Corp.). 10 minutes later, mice were imaged in the
supine position and scanned for 2 minutes using the IVIS 200 optical imaging
system (Xenogen Corp.). Signal intensity was quantified as photon count rate per
unit body area per unit solid angle subtended by the detector (units of
photon/s/cm
2
/steridian). Images were analyzed with the IVIS 200 and LIVING
IMAGE 3D (Xenogen Corp.) software.
Yeast strains
All experiments were performed with the strain DBY746 (MATα,leu2-
3,112,his3Δ1,trp1-289,ura3-52,GAL
+
), provided by D. Botstein, Massachusetts
Institute of Technology, Cambridge, MA. The sch9Δ mutant has been described
previously (Fabrizio, Pozza et al. 2001). All the mutant strains were originated in
the DBY746 background by one-step gene replacement (Brachmann, Davies et
al. 1998).
Yeast growth conditions
Yeast chronological life span was monitored in expired SDC medium by
measuring colony forming-units (CFUs) every 48 hours. The number of CFUs at
day 1 was considered to be the initial survival (100%) and was used to determine
the age-dependent mortality (Fabrizio and Longo 2003). Cultures were treated
once with 200 µM DXR on day 1.
Mutation frequency measurements
To characterize the type of mutations occurring in wild type and mutant
strains, we measured the frequency of mutations of the CAN1 (YEL063) gene
136

(Fabrizio, Battistella et al. 2004; Fabrizio, Gattazzo et al. 2005). Can
r
mutations
are mostly caused by point mutations as well as other DNA mutations including
small insertion/deletion, complex events and gross chromosomal rearrangements
(Madia, Gattazzo et al. 2007). Cells from chronological aging cultures were
plated them onto selective media every two days. The mutation frequency was
calculated based on the number of viable cells as described previously (Madia,
Gattazzo et al. 2007; Madia, Gattazzo et al. 2008).

Acknowledgments
We thank T. Chen of USC for providing glioma cell lines, R. Baserga of
Thomas Jefferson University for providing R
+
and R
-
cells, N. Craft of UCLA for
providing B16Fluc cells, and D. LeRoith of Mt. Sinai School of Medicine for
providing LID and its control mice. We also thank L. Dubeau of USC for histology
expertise. Lizzia Raffaghello is a recipient of a Fondazione Italiana per la Lotta al
Neuroblastoma fellowship and a MFAG (My First AIRC Grant). This study was
also funded in part by NIH/NIA AG20642, AG025135, Ted Bakewell (The
Bakewell Foundation), the V Foundation for Cancer Research, and a USC Norris
Cancer Center pilot grant to VDL.
137

CHAPTER FIVE

Fasting Selectively Sensitizes Cancer Cells to Chemotherapy by
Modulating Stress Resistance Via the IGF-I/PI3K/Akt Pathway

Chapter Five Abstract
The beneficial effect of dietary restriction (DR) on increasing lifespan and
stress resistance, as well as reducing age-dependent diseases, such as cancer,
is well documented in various organisms including laboratory rodents and non-
human primates (Weindruch, Walford et al. 1986; McCay, Crowell et al. 1989;
Colman, Anderson et al. 2009). However, considering that DR requires several
weeks to months to be effective, the prolonged regimen would prove difficult for
cancer patients. In this report, we show that short-term fasting, an intensive but
brief form of DR, can suppress tumor growth concurrently with its sensitization to
chemotherapy treatments. Using mouse models of cancer, briefly fasting before
and after chemotherapy administration sensitized breast, glioma, melanoma, and
neuroblastoma cells to doses of chemotherapy that would otherwise be less
effective in normally fed mice. Fasting was able to reduce tumor progression as a
single treatment modality, and showed additive effects when combined with
chemotherapy. In cell culture, restricting glucose to the level of fasted mice
sensitized more than half of the tested cancer cell lines to chemotherapy
treatments. The fasting-dependent sensitization of cancer cells was mediated by
the down regulation of the extracellular growth factors including IGF-I, VEGF,
138

and the intracellular PI3K/Akt pathway. In addition, ER stress response was
partially responsible for the fasting-dependent sensitization. Along with clinical
trials being conducted to test fasting as a protective dietary intervention, our
findings indicate further separation between normal and malignant cells during
chemotherapy.

Introduction
Dietary restriction (DR) is a well established intervention to increase
lifespan and reduce the rate of cancer (Tannenbaum 1945; McCay, Crowell et al.
1989). In addition, DR increases stress resistance in normal cells and
organisms(Yu and Chung 2001). The effect of DR on cancer has been largely
focused on preventing and reducing tumor growth, whereas its effect on stress
resistance has been poorly explored. Since the efficacy of DR as a single
treatment modality is limited in curing cancer, and considering that several weeks
to months is required to be effective, it would be challenging to incorporate DR
into the lifestyle of cancer patients. Recently, fasting, an intensive but brief form
of DR, has been reported to cause protective effects in 48-60 hours by
selectively enhancing stress resistance in mice but not in the allografted cancer
cells(Raffaghello, Lee et al. 2008). Furthermore, fasting is feasible and safe to be
practiced by cancer patients receiving chemotherapy(Safdie, Dorff et al. 2009).
It is estimated that more than 50% of cancer patients receive
chemotherapy annually in the US (Halpern and Yabroff 2008). However, despite
the fact that chemotherapy is effective in several types of cancer, such as
139

testicular and ovarian germ-cell tumor(Savage, Stebbing et al. 2009) as well as
Hodgkin‟s and non-Hodgkin‟s lympohoma(DeVita and Chu 2008), its use in the
cure for many metastatic malignancies still remains elusive. This may be largely
due to the toxic side-effects, which is the major limiting factor in chemotherapy,
and results in sub-optimal treatment regimens. Therefore, protecting normal cells
and sensitizing cancer cells to chemo drugs will increase the therapeutic index
and enhance clinical outcome.
Modern chemotherapy is largely a cytotoxic treatment that takes
advantage of the rate of cellular replication, therefore comes with significant side-
effects from collateral damage to rapidly dividing normal cells. Another signature
cellular characteristic of a transformed cell is its altered metabolism, which is
coupled to its inappropriate cellular growth and proliferation(DeBerardinis, Lum et
al. 2008). Unlike normal cells, many malignant cells support its metabolic
demands through glycolysis rather than oxidative phosphorylation. Glycolysis is
an inferior method of ATP production compared to mitochondrial respiration.
However, relying on glycolysis may benefit a rapidly dividing cell by providing
macromolecular building blocks such as lipids, amino acids, and nucleotides
which are essential for cellular replication(DeBerardinis, Lum et al. 2008).
Molecular reprogramming of these metabolic pathways is often under the control
of known oncogenes and/or tumor suppressors, which are also known to regulate
cellular protection and survival(Vander Heiden, Cantley et al. 2009).
Accumulating evidence shows the PI3K/AKT and Ras/ERK pathways, which are
among the most frequently found mutated in human cancers, to be in the center
140

of metabolic reprogramming in transformed cells. Notably, aging studies have
demonstrated the conserved role of these pathways in stress-resistance in
various organisms (Fabrizio, Pozza et al. 2001; Holzenberger, Dupont et al.
2003; Murakami, Salmon et al. 2003), which would not be activated in cancer
cells due to the oncogenic mutations. For instance, reduction of insulin-like
growth factor 1 (IGF-I) signaling and the consequent down-regulation of
PI3K/AKT and RAS/ERK, mediate the fasting-dependent selective protection of
normal but not malignant cells to chemotherapy (Raffaghello, Lee et al. 2008;
Lee, Safdie et al. 2010).
Furthermore, the response to chemotherapy in malignant cells is affected
by the PI3K/AKT pathway(Martelli, Tazzari et al. 2003; Kalaany and Sabatini
2009; Pei, Li et al. 2009), whose activation also contributes to chemotherapy
resistance(Hennessy, Smith et al. 2005). Together with the fact that fasting
regulates somatic stress resistance by down-regulating IGF-I and consequently
the PI3K/Akt and Ras/ERK pathways, and that these pathways are often found
constitutivey activated in human cancers, fasting has the potential to selectively
sensitize certain cancer cells to chemo drugs in addition to its protective effect on
normal cells. Notably, here we also show that in addition to sensitizing malignant
cell to chemodrugs, fasting itself exerts a growth-static effect on already
established tumors, further contributing to tumor control.

141

Results

Fasting retards tumor progression and sensitizes cancer cells to
chemotherapy and radiotherapy in mice

To examine the effect of fasting on tumor response to chemotherapy, we
injected three different murine cancer cell lines derived from the skin (B16-fluc),
brain (GL26-luc), and breast (4T1-luc) into syngeneic mice. Once tumor was
palpable, mice were fasted for a total of 48 hours prior to receiving chemotherapy
by completely depriving them of food but providing free access to water. Cages
were freshly changed to minimize coprophagy and residual chow. In the
subcutaneous model of breast cancer (4T1-luc), two cycles of fasting and
cyclophosphamide were given. Fasting itself significantly suppressed tumor
growth in both cycles of fasting and maintained the tumor volume below that of
normally fed mice (Fig 5.1A). Remarkably, a 48-hour fast inhibited tumor growth
at a comparable extent as chemotherapy (CP), but tumor retardation was
maximized when fasting was combined with chemotherapy. Body weight lost
during fasting was typically recovered within 3 days of refeeding (Fig 5.1B),
suggesting fasting to be safe and feasible in tumor-bearing mice. To test if
fasting/CP treatments prolong survival in a metastatic model of breast cancer, we
intravenously injected 4T1 cells via the lateral tail vein, which leads to
reproducible lung metastasis. Indeed, fasting enhanced the survival of CP
treated metastatic model of breast cancer (Fig 5.2). Similar results were
obtained in a subcutaneous mouse model of glioma (GL26-luc) (Fig 5.3). Without
fasting and/or chemotherapy (DXR), the glioma mass progressed exceptionally
rapidly, and therefore the experiment had to be terminated only after one cycle of
142

fasting/chemotherapy due to the massive tumor volume of the control (ad lib, no
chemotherapy) group. Fasting was able to halt tumor progression in a
subcutaneous mouse model of melanoma (B16-luc) (Fig 5.6), and also increased
the survival of a metastatic model (B16-luc) (Fig 5.5A), in part by sensitizing the
cancer cells to DXR (Fig 5.4). Interestingly, the metastatic pattern of melanoma
(B16-luc) differed between fasted and normally fed mice (Fig 5.5B). For instance,
lung metastasis was evident in 100% vs. 65% of normally fed and fasted mice,
respectively. Also, unlike mice fed ad lib, fasted mice did not have metastasis in
the liver and spleen. Further, fasting also sensitized cancer cells to a cocktail of
chemotherapy in a metastatic model of neuroblastoma (Fig 5.7). Studies done by
our collaborator, Dr. Raffaghello and Giovanna Bianchi, the human Neuro-2A
cells were intravenously injected into Nude mice, and fasting was done 36 hours
before and 24 hours after a cocktail of chemotherapy (DXR and CDDP). While
the chemotherapy cocktail was toxic to normally fed mice, causing death in 70%
of the population (Fig 5.7A) and also irreversible weight loss (Fig 5.7B), none of
the fasted mice succumbed to chemotoxicity. In addition, a murine NXS2 cell line
was also injected into A/J mice, which causes 100% death from metastasis in
roughly 30 days (Fig 5.8). Notably, in the NXS2 mouse model, whereas 2 rounds
of high dose DXR (16 mg/kg) treatment alone increased the survival time upto 80
days, when combined with fasting 40% of the mice were still alive at day 160 (Fig
5.8).

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Figure 5.1 Fasting sensitizes breast cancer allografts to the nutrient
deprivation itself and to CP.
Female BALB/c mice were subcutaneously injected with breast cancer cells
(4T1). Once tumor was palpable, fasting proceeded for 48 hours, followed by CP
injections (150 mg/kg). (A) Tumor volume and (B) body weight shown.
N=10/group. *Control vs CP: P=0.002, Control vs STS: P=0.03, Control vs
STS/CP: P=0.003, CP vs STS: P=0.176, CP vs STS/CP: P=0.755, STS vs
STS/CP: P=0.148. P-values by Student‟s t test (unpaired, two-tailed).
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Figure 5.2 Fasting sensitized cancer cells to CP treatment in a mouse model of
metastatic breast cancer.
Female BALB/c mice were intravenously injected with 4T1 breast cancer cells,
which readily metastasize primarily in the lungs. Fasting was done for 48 hours,
immediately followed by CP (150 mg/kg) treatment. Control (n=18) STS/CP
(n=16). P-value by Peto‟s log-rank test.

145

Figure 5.3 Fasting sensitizes glioma allografts to the nutrient deprivation
itself and to DXR.
Male C57BL/6 mice were subcutaneously injected with glioma cells (GL26;
300,000/mouse). Once tumor was palpable, fasting proceeded for 48 hours,
followed by DXR injections (12 mg/kg). (A) Tumor volume and (B) body weight
shown. (C) The retardation of tumor progression during the 48 hour fast.
N=10/group. *Control vs DXR: P=0.027, Control vs STS: P=0.169, Control vs
STS/ DXR: P=0.021, DXR vs STS: P=0.173, DXR vs STS/DXR: P=0.105, STS
vs STS/DXR: P=0.049. *P-values Student‟s t test (unpaired, two-tailed). **P-
values by Mann-Whitney test.
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Figure 5.4 Fasting sensitizes cancer cells to DXR in a metastatic mouse
model of melanoma.
Mice (60-70 wk-old) were intravenously injected with a metastatic melanoma
(B16-flucA1, luciferase-tagged). On day 15, tumor progression was detected and
the STS/ DXR (48 h and 10 mg/kg, i.v., respectively) treatment was
administered. Tumor progression/regression was quantified on day 22. C#2
(control #2) is not present in the imaging due to death from metastases. STS#1
did not show metastasis by bioluminescence imaging and was confirmed to be
cancer-free upon necropsy.

147

Figure 5.5 Fasting enhances DXR treatment and causes increased survival
in a metastatic mouse model of melanoma.
To establish a metastatic model of melanoma, C57BL/6 mice were intravenously
injected with melanoma (B16) cells. One cycle of chemotherapy (DXR) combined
with or without fasting was given. Mice that fasted prior to DXR showed (A)
enhanced survival and (B) less organs affected by metastasis. DXR (n=8),
STS/DXR (n=9). P-value by Peto‟s log-rank test.

148

Figure 5.6 Fasting inhibits the progression of subcutaneous melanoma
allografts.
C57BL/6 mice were subcutaneously injected with melanoma cells (B16). Once
tumor was palpable, fasting proceeded for 48 hours. Tumor growth was largely
halted during the fast. Control (n=6), STS (n=9) * P=0.015 by Student‟s t test.

149

Figure 5.7 Fasting sensitizes human neuroblastoma to chemotherapy.
Neuro-2A cells were intravenously injected into Nude mice to establish a
metastatic model of neuroblastoma. Treatment began 7 days following
intravenous tumor injection. Fasting was done 36 hours before and 24 hours
after chemotherapy. A cocktail of DXR (10 mg/kg) and cisplatin (8 mg/kg) was
given intravenously. (A) Survival rate and (B) body weight is shown. Data
provided by our collaborator Dr. Lizzia Raffaghello. Control (n=6), Chemo
Cocktail (n=12), STS/Chemo Cocktail (n=12). P-value by Peto‟s log-rank test.

150

Figure 5.8 Fasting sensitizes murine neuroblastoma to chemotherapy.
NXS-2 cells were intravenously injected into A/J mice to establish a metastatic
model of neuroblastoma. Treatment began 2 days following intravenous tumor
injection. Fasting was done for 48 hours prior to chemotherapy. 2 cycles of DXR
(16 mg/kg/cycle) was given intravenously in combination with fasting. Data
provided by our collaborator Dr. Lizzia Raffaghello. Control (n=5), DXR (n=8),
STS/DXR (n=12). P-value by Peto‟s log-rank test.

Apart from chemotherapy, radiotherapy is another major arm of modern
cancer treatment. It is estimated that about 50% of patients with solid malignant
tumors receive radiotherapy(Bentzen 2006). Irradiation in living organisms cause
considerable amount of DNA damage by direct energy deposit, but
approximately 60% of its damage is via reactive oxygen species
(ROS)(Barcellos-Hoff, Park et al. 2005). Therefore, as with chemotherapy,
radiotherapy is a cytotoxic treatment and is accompanied by varying degrees of
side-effects which are the major limiting factor(Bentzen 2006). Therefore, the
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ability to sensitize cancer cells to chemotherapy would allow efficient killing of
cancer cells at a lower dose, causing less damage to the organism. Fasting for
48 hours sensitized breast cancer (4T1) and glioma (GL26) allografts to
irradiation, and showed significantly reduced tumor load (Fig 5.9). There are
several noteworthy points to these experiments. First, the breast cancer model
was fasted 48 hours prior to irradiation, and then fasted for an additional three
times without irradiation upon body weight recovery; the latter fasting regimens
were reduced to 24 hours. This led to a considerable reduction of tumor mass,
nearing a third of the non-treated controls and less than half of the irradiated
group (Fig 5.9A). Second, the glioma model received two cycles of fasting/IR,
which suppressed the growth of tumor to 40% of non-treated controls. Notably,
the effect of fasting alone was comparable with IR alone, suggesting a large
impact by fasting on the tumor (Fig 5.9B).
To determine the degree of DNA damage following fasting and irradiation,
we incubated the breast cancer (4T1) cells under low (0.5 g/L) and normal
glucose (2.0 g/L) for 24 hours before and 24 hours after irradiation and
performed the Comet assay at two different time points. Since the major lethal
DNA damage is repaired within hours following IR(Bentzen 2006), cells were
assayed within 2 hours (early) or after 24 hours(delayed) following gamma-IR (10
Gy). Unlike the cells cultured in normal glucose which showed decreased DNA
damage during the first 24 hours after IR, glucose restricted cells showed no
change (Fig 5.10). This suggests that impaired DNA repair, in part, mediates the
sensitization to IR by fasting (Fig 5.9).
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Figure 5.9 Fasting is as effective as irradiation and increases its
effectiveness in reducing tumor growth.
(A) BALB/c mice with subcutaneous 4T1 breast cancer allografts were initially
fasted for 48 hours and irradiated (3 Gy). Tumor was then managed by multiple
fasting cycles, in which upon weight recovery mice were fasted. N=10/group.
Control vs IR: P=0.299, Control vs STS: P=0.710, Control vs STS/IR: P=0.042,
IR vs STS: P=0.681, IR vs STS/IR: P=0.034, STS vs STS/IR: P=0.230. *P-values
by Mann-Whitney test. (B) C57BL/6 mice with subcutaneous GL26 glioma
allografts were fasted for 48 hours and irradiated with 5 Gy for the first cycle and
2.5 Gy for the second cycle. N=8/group.
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Figure 5.10 Glucose restriction interferes with DNA repair following
irradiation in breast cancer cells.
4T1 breast cancer cells were incubated in 0.5 g/L or 2.0 g/L glucose for 24 hours
prior to γ-IR (10Gy) and 24 hour post-IR. DNA damage was determined by the
Comet assay and quantified by tail olive moment. (A) Comet assay done within
the first 8 hours following IR, in which major lethal DNA repair has not completed,
and (B) 24 hours following IR, in which major DNA repair has been completed.

Glucose and growth factors regulate stress resistance to chemotherapy in
vitro

Glucose is the main energy source to metazoans, particularly so to rapidly
dividing cells such as lymphocytes and malignant cells(Newsholme, Crabtree et
al. 1985). In fact, many cancers are addicted to glucose, a phenomenon known
as the Warburg effect, which is exploited to image tumors by PET(Vander Heiden,
Cantley et al. 2009). Also, elevated blood glucose levels are highly associated
with increased cancer rate and are thought to be a major risk factor (Jee, Ohrr et
al. 2005). To mimic the cellular environment during fasting in cell culture, we
incubated cells in different glucose concentrations based on blood glucose
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measurements from normally fed and fasted mice (Fig 4.2). Cells were incubated
in 0.5 g/L or 2.0 g/L for 24 hours prior to challenging with chemo drugs and were
kept under identical conditions for 24 hours following drug treatment, based on
our observation that mice did not eat immediately after high-dose chemotherapy
and also the digestive lag due to fasting induced small bowel atrophy in
mice(Song, Wolf et al. 2009). In agreement with the in vivo studies (Figs 5.1-5.6),
glucose restriction sensitized murine melanoma (B16-flucD3), glioma (GL26-
flucD1), and breast cancer (4T1-luc) cells to 2 widely used chemo drugs,
doxorubicin and cyclophosphamide (Fig 5.11); these cell lines were also
sensitive to the glucose restriction itself (data not shown). Further, 4T1 breast
cancer cells were sensitized to DXR when incubated in medium supplemented
with serum from fasted or normally fed rats (Fig 5.12).

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Figure 5.11 Low glucose sensitizes murine cancer cells to chemotherapy.
Murine cancer cell lines were incubated in low (0.5g/L) or normal glucose
(2.0g/L) for 24 hours before and 24 hours after DXR treatments in (A) breast
cancer (4T1), (B) melanoma (B16), and (C) glioma (GL26) cells, or CP
treatments in (D) 4T1, (E) B16, and (F) GL26 cells. These cells were also tested
in vivo as described above. N=6/group. ***P<0.0001 by Student‟s t test.

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Figure 5.12 Serum from fasted rats sensitize cancer cells to
chemotherapy.
4T1 breast cancer cells were incubated in DMEM supplemented with 1.0 g/L
glucose and either serum from fasted or normally fed (ad lib) rats for 24 hours
before and 24 hours after (A) DXR (n=3) or (B) CP treatment (n=3). *P<0.05,
**P<0.01 by Student‟s t test.

Another key response to fasting is the reduction of growth factors, both at
the systemic and cellular level, e.g., the potent endocrine growth factor IGF-I
decreases 75% (Raffaghello, Lee et al. 2008) and the cell-autonomous mitogenic
signal ERK is reduced (data not shown). This is evolutionarily favorable since
growth would be a liability during fasting, and the remaining energy would better
serve the organism if invested in repair and maintenance. However, malignant
cells have „oncogene addiction‟ (Weinstein and Joe 2008), supporting its
independence from external survival cues. We have previously reported that
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reduced IGF-I signaling is a major mediator of the protective effects of fasting,
and that IGF-I infusion can reverse the protection (Fig 4.2) (Lee, Safdie et al.
2010). Interestingly, DR is also tumor-static in a mouse model of bladder cancer,
which was abolished by the infusion of IGF-I (Dunn, Kari et al. 1997).
The addition of IGF-I, which in turn stimulates the PI3K/Akt and Ras/ERK
pathways, also reversed the sensitization to chemotherapy by glucose restriction
in the murine breast cancer (4T1) and melanoma (B16) cell lines (Fig 5.13 A,B).
Further, culture medium with 1% serum rendered 4T1 cells more sensitive to CP
compared that with 10% serum (Fig 5.13 C), suggesting that reducing growth
factors contribute to the fasting-dependent sensitization to chemotherapy.

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Figure 5.13 The effect of growth factors on cellular resistance to
chemotherapy in cancer cells.
(A,B) Murine breast cancer (4T1) and melanoma (B16) cells were glucose
restricted for 24 hours before and 24 hours after DXR treatment, with or without
IGF-I (200 ng/ml) (n=12). (C) 4T1 cells were incubated in medium supplemented
with either 10% or 1% FBS for 24 hours before and 24 hours after CP treatment
(n=12). P- values by Student‟s t-test.

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Western blot analysis from the tumor allografts that were sensitive to
fasting show that the cancer cells have normal PI3K/Akt signaling, but Ras/ERK
signaling seemed to be constitutively active (Fig 5.1, 5.9A, 5.14, and 5.15). This
is agreement with the studies that show cancer cells with constitutive activation
of the PI3K/Akt pathway are resistant to DR(Kalaany and Sabatini 2009). To test
if the PI3K/Akt pathway is crucial to fasting-dependent sensitization to the
nutrient-deprivation itself and chemotherapy, we tested cell lines with known
mutations that cause constitutive activation of the PI3K/Akt pathway. Fasting was
less effective in sensitizing human glioblastoma (U87-MG), breast cancer (MCF-
7), and prostate cancer (PC-3) cells (Fig 5.16).
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Figure 5.14 Differential response of Akt and ERK to fasting in liver and
breast cancer allografts in mice
Mice with subcutaneous breast cancer (4T1) allografts were fasted for 48 hours
(Fig 5.1). The activation level of (A) Akt and (B) ERK was determined by
immunoblotting.
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Figure 5.15 The activation status of Akt and ERK in murine cancer cells.
Cancer cells have normal PI3K/Akt signaling, but Ras/ERK acitivty is less
responsive to serum stimulation. Murine breast cancer (4T1), melanoma (B16),
and glioma (GL26) cells were either cultured in normal media (C), serum-free
media for 24 hours (-), or serum-free media for 24 hours with 10% FBS
stimulation 30 minutes prior to lysis (+).

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Figure 5.16 Cell lines with constitutive activation of the PI3K/Akt pathway
are less responsive to fasting.
Human glioblastoma (U87-MG), breast cancer (MCF-7), and prostate cancer
(PC-3) cells were incubated in low (0.5 g/L) or normal (2.0 g/L) glucose 24 hours
before and 24 hours after (A-C) DXR or (D-F) CP treatment. (G) The known
mutation status of PTEN, PI3K, and Ras. N=6/group. ***P<0.0001 by Student‟s t
test.

163

Discussion

Fasting is a simple, cost-effective, and feasible dietary intervention to
increase cellular protection. We have previously reported its ability to selectively
protect the host, but not malignant cells, to chemotherapy (Raffaghello, Lee et al.
2008). Here we provide evidence of fasting-dependent sensitization of a number
of cancer cells to the nutrient deprivation itself, or to chemotherapy, resulting in
further increase in chemotherapeutic index. The beneficial effect of fasting does
not reside in the field of cancer, but extends to renal (Mitchell, Verweij et al.
2009), hepatic (Domenicali, Caraceni et al. 2001), cerebral (Go, Prenen et al.
1988), and cardiac (Varela, Marina Prendes et al. 2002) ischemic injury, which
cause considerable cellular damage by glucose restriction, hypoxia, and
oxidative stress(Loor and Schumacker 2008).
Modulating cellular stress resistance by fasting involves an orchestration
of nutrient availability and growth factors. In addition to IGF-I reduction (Lee,
Safdie et al. 2010), several other growth factors such as VEGF may significantly
contribute to the fasting-dependent tumor sensitization. Pathological
angiogenesis enables tumors to recruit new blood vessels and support their
oxygen and nutrient. This process is largely well coordinated via pro-angiogenic
factors such as the vascular endothelial growth factor (VEGF) and inhibitors
(Folkman 2007; Heath and Bicknell 2009). In our animal studies, a 48 hour fast
retarded the growth of glioma (GL26) allografts (Fig 5.3), and reduced VEGF
levels in both normal tissues and the glioma allograft following a 48-72 hour fast
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(Fig 5.17), suggesting anti-angiogenesis as a partial mediator of the fasting-
dependent growth inhibition and sensitization.

Figure 5.17 VEGF measurements from mice with glioma allografts after a
24-72 hour fast.
C57BL/6 mice with (tu) or without subcutaneous tumors were sacrificed following
a 24-72 hour water only fast. The (A) kidney, (B) cortex, and (C) tumor mass
were collected, and mVEGF levels were measured by ELISA.
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A cascade of intracellular stress response events occur following fasting,
including DNA repair, detoxification, and ER stress response. For example, in a
microarray, the most significant increase was seen in detoxification proteins such
as cytochrome and DNA repair pathways (Bauer, Hamm et al. 2004). Notably,
nutrient deprivation triggers the unfolded protein response (UPR), which is
conserved from yeast to human, and activates multiple pathways involved in
stress response. The glucose regulated protein 78 (GRP78), is a major
endoplasmic reticulum (ER) chaperone required for protein folding, and due to its
anti-apoptotic property, stress induction of GRP78 represents an important pro-
survival component of the UPR. As a master regulator of ER function, GRP78
plays a major role in regulating the balance between cell death and survival, as
well as modulating the sensitivity of cancer cells to chemotherapeutic agents.
Furthermore, Grp78+/- mouse embryonic fibroblasts (MEF) are highly sensitive to
DXR (Fig 5.18). Also, fasting greatly reduced GRP78 expression in the tumor
(Fig 5.19), and inhibited the growth of breast cancer (4T1) allografts (Fig 5.1). In
addition, following B16 melanoma injections, Grp78+/- mice showed significantly
reduced tumor size and foci compared to Grp78+/+

mice (Amy Lee, unpublished
data).
Fasting elicits a complex physiological response that tunes the organism,
and normal cell, into a protective mode by diverting the energy from
growth/reproduction to maintenance/repair (Kirkwood 2005; Lee, Safdie et al.
2010). On the contrary, fasting-induced energetic stress may cause sensitization
of cancer cells, which is an actively studied topic. Evidence from our lab and
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others point to the downstream effectors of IGF-I, with focus on the PI3K/Akt and
Ras/ERK pathways and their converging effect on the master regulator mTOR
which in turn regulates UPR, DNA repair, autophagy, and apoptosis pathways.
Collectively, fasting provides evidence and grounds to explore interventions and
drug targets to simultaneously protect normal cells and sensitize cancer cells,
leading to increased therapeutic index and thereby enhancing cancer treatment.

Figure 5.18 The effect of GRP78 on stress resistance to DXR in MEFs.
GRP78+/- fibroblasts were treated with DXR for 1 hour. DNA damage was
determined using the comet assay with quantification by calculating the tail olive
moment. Cell count was 120-220/group. **P<0.01 by Student‟s t test.

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Figure 5.19 GRP78 levels in normal organs and tumor allograft in fasted
mice.
Female BALB/c mice with subcutaneous breast cancer (4T1) allografts were
fasted for 48 hours prior to organ and tumor collection. Samples were processed
for immunohistochemistry (IHC) and stained for GRP78 lightly counter stained
with hematoxylin. The brown stainings indicate GRP78. This study was done in
collaboration with Dr. Amy Lee.

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Methods

Mouse model of cancer
To establish a breast cancer mouse model, 12 week-old female BALB/c
mice were injected subcutaneously with syngeneic 4T1-luc cells. Melanoma and
glioma models were established in male and female C57BL/6 mice. 4T1, GL26,
and B16 cells in log phase of growth were harvested and suspended in PBS at a
density of 2x10
6
cells/ml, and 100 uL (2x10
5
cells/mouse) were injected
subcutaneously in the lower back region of the mouse. All mice were shaved
prior to subcutaneous tumor injection.INTRAVENOUS NEUROBLATOMA! Body
weights were determined periodically and tumor size was measured using a
digital vernier caliper. Tumor volume was calculated using the following equation:
tumor volume (mm
3
) = (length × width × height) x π/6, where the length, width
and height are in mm.
Cell culture
Murine melanoma (B16-flucD3), glioma (GL26-luc), neuroblastoma
(NXS2-luc), and breast cancer (4T1-luc) cells were used. The cells lines were
stably transfected with firefly luciferase for non-invasive post-allograft imaging
using Xenogen IVIS 200 (Caliper, USA) to ensure successful tumor seeding.
Human neuroblastoma (Neuro-2A) cells were also stably transfected with firefly
luciferase for imaging purposes. All cell lines were cultured in DMEM 10% FBS,
except for the 4T1-luc murine breast cancer cell line which was cultured in RPMI
1640 with 10% FBS.
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Fasting
Animals were fasted for up to 48 hours total. The definition of fasting is
complete deprivation of food but free access to water. Mice were single caged to
reduced cannibalism and transferred to a clean new cage to reduce coprophagy
and residual chow. Body weight was measured immediately before and after
fasting.
Cellular fasting was done by glucose restriction which was based on blood
glucose measurements in fasted and normally fed mice; the lower level
approximated to 0.5 g/L and the upper level to 2.0 g/L. Cells were washed twice
with PBS before changing to fasted or control medium (DMEM no glucose).
Serum levels were kept to 1% to minimize additional serum glucose and
excessive growth factors.
in vitro survival
Cytotoxicity was measured by the ability to reduce
methylthiazolyldiphenyl-tetrazolium bromide (MTT). MTT is reduced in the
mitochondria (metabolically active cells) by mitochondrial reductase enzymes to
form insoluble purple formazan crystals, which are solubilized by the addition of a
detergent (1). Briefly, MTT was prepared at 5 mg/ml in PBS and was diluted in
DMEM/F12 1% FBS media to a final concentration of 0.5 mg/ml for assays.
Following experimental treatments, media was replaced with 100 μl of MTT and
cells were incubated for 3~4 hours at 37
o
C. Formazan crystals were dissolved
overnight (16hours) at 37
o
C with 100 μl lysis buffer ((w/v) 15% SDS, (v/v) 50%
dimethylformamide, pH 4.7). Survival was presented as percentage of MTT
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reduction level of treated cells to control cells. Absorbance was read at 570nm
using a microplate reader SpectraMax 250 (Molecular Devices) and SoftMax Pro
3.0 software (Molecular Devices).
Immunoblotting assay
Cells were rinsed once in ice-cold PBS and harvested in RIPA lysis buffer
containing protease inhibitors (Roche) and a cocktail of phosphatase inhibitors
(Sigma). Tumour tissues were homogenized in RIPA lysis buffer supplemented
with the same protease and phosphatase inhibitors. Proteins from total lysates
were resolved by 8–12% SDS-PAGE, and analyzed by immunoblotting using
antibodies for phospho-Thr308 Akt, Akt and phopho-ERK1/2, ERK1/2 (1:1000,
Cell Signaling Technology).
Immunohistochemistry
The immunohistochemical staining was performed using the Vectastain
elite ABC kit (Vector Laboratories, Burlingame, CA) on paraffin-fixed mouse
tissue sections. The primary antibody was mouse monoclonal anti-Grp78
antibody (BD Biosciences Pharmingen, San Diego, CA) at 1:200 dilution.

Comet assay protocol
Cells were diluted to 10
5
/ml in culture medium (DMEM/F12 with 10% FBS),
and treated with 50 µM DXR for 1 hour at 37°C. Cells were then washed once
with ice cold PBS and subject to CometAssay (Trevigen, Inc, Gaithersburg, MD)
according to the manufacturer‟s recommended procedure. Comet images were
acquired with a Nikon Eclipse TE300 fluorescent microscope and analyzed with
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the Comet Score software (TriTek Corp., ver1.5). 100-300 cells were scored for
each genotype/treatment group.

Plasma mGH, mIGF-I, and mIGFBP-1 and -3 measurements

Plasma mIGF-I and mIGFBP-1 and -3 assays were performed as
previously described by in-house ELISA assay using recombinant mouse IGF-I
protein and monoclonal antibodies from R&D systems (Minneapolis, MN) (Hwang,
Lee et al. 2008). mGH levels were measured by rat/mouse GH ELISA kit
(ALPCO Diagnostics).

Blood glucose measurements
Following a 72 hour fast, mice were anesthetized with 2% inhalant
isoflurane and blood was collected by left ventricular cardiac puncture. Blood
glucose was measured using the Precision Xtra blood glucose monitoring system
(Abbott Laboratories, USA).

Statistical Analysis
Data are presented as means ± s.e.m. In comparing two groups, two-
tailed non-paired Student‟s t test was performed and P ≤ 0.05 (unless otherwise
stated) was considered significant. For survival studies, Peto‟s log-rank test was
performed.

172

CHAPTER SIX

Essential Amino Acid Restricted Diet Enhances Stress
Resistance against Chemotherapy Toxicity in Mice

Chapter Six Abstract
With the advancement of medical care and hygiene, the average lifespan
has greatly improved resulting in a global rise of the elderly population over 65
years of age. According to the U.S. Census Bureau projections, a substantial
increase in the number of older people will occur during the 2010 to 2030 period,
after the first Baby Boomers turn 65 in 2011. The older population in 2030 is
projected to be twice as large as that in 2000, growing from 35 million to 72
million and representing nearly 20 percent of the total U.S. population at the latter
date. Therefore, there is an urgent need for developing interventions that can
reduce the incidence of age-dependent diseases, including cancer, but also
interventions that can protect older individuals who are much more sensitive than
younger patients to procedures like chemotherapy and surgery.
In previous studies, we have shown that fasting prior to chemotherapy
provides differential protection to chemo toxicity. However, even if fasting is
feasible for cancer patients, it would undoubtedly be accompanied by challenges.
Therefore, a substitution diet that can deliver the protective effects of fasting, but
without complete withdrawal of food, would be more approachable. Interestingly,
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restricting essential amino acids (EAA) in a normal diet has been shown to
increase lifespan and stress resistance to oxidative stress. Here we show
evidence that restriction of all EAA, or even a single EAA can enhance the
protection to chemotherapy toxicity in mice.

Introduction
Essential amino acids cannot be synthesized de novo by humans, and
therefore must be supplied in the diet. There are 8 essential amino acids;
tryptophan, lysine, phenylalanine, methionine, threonine, isoleucine, leucine,
valine (Young 1994). Amino acids are central to growth and are a major regulator
of IGF-I and the mammalian target of rapamycin (mTOR), which are both potent
regulators of cellular growth (Takenaka, Oki et al. 2000; Yang, Anzo et al. 2005;
Ma and Blenis 2009; Nicklin, Bergman et al. 2009). Therefore, we hypothesized
that an essential amino acid deficient diet would enhance stress resistance by
discouraging growth by reducing circulating IGF-I levels and its signaling, thereby
entering a „maintenance mode‟. A diet restricted of just a single EAA may trigger
the animal to enter a „maintenance mode‟ in response to sensing the absence of
an essential nutrient. This is in agreement with previous studies where
methionine restriction increases lifespan by about 45% in rats, decreases certain
age-related diseases such as cancer, delay the deterioration of the lens, reduces
visceral fat, and also increases the major antioxidant glutathione (GSH)
(Orentreich, Matias et al. 1993; Richie, Leutzinger et al. 1994; Zimmerman,
174

Malloy et al. 2003; Miller, Buehner et al. 2005). Notably, several human cancer
cell lines and primary tumors have strong dependency on methionine
(methionine auxothrophy), whereas normal cells tolerate methionine restriction
quite well (Lu, Hoestje et al. 2002; Cellarier, Durando et al. 2003). Thus, a
methionine restricted diet combined with chemotherapy may protect normal cells
and further sensitize cancer cells. In addition, tryptophan restriction also
increases lifespan in mice and rats, delays cancer incidence, significantly
extends reproductive capability, slows age-related hair deterioration, enhances
hair regrowth, retards weakening age-related temperature homeostasis, and also
enhances short-term memory (Segall and Timiras 1976; Segall 1977; De Marte
and Enesco 1986; Ooka, Segall et al. 1988). In addition, a thorough histological
study on rats fed a low-tryptophan diet shows reduced age-dependent
deterioration and absence of lesions in various tissues, including liver, heart,
uterus, orvary, adrenal and spleen (Ooka, Segall et al. 1988). Moreover,
tryptophan restricted diet increases locomotor activity and mobility (Uchida,
Kitamoto et al. 2005). Notably, most human tumors overexpress the tryptophan
catabolic enzyme indoleamine 2,3-dioxygenase (IDO), which is implicated in
suppressing T-cell immunity in normal and pathological settings including cancer,
and facilitate tumoral immune escape (Uyttenhove, Pilotte et al. 2003; Hou,
Muller et al. 2007; Prendergast 2008). Consequently, cancer patients show
grossly elevated tryptophan use (Rose 1967). Therefore, tryptophan restriction
may allow enhanced tumor immuno-surveillance, and may play a role in the
reduced/absent cancer and hyperplasia in the tryptophan restricted rodents.
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Since normal hepatocytes endure tryptophan depletion well (Harp, Goldstein et al.
1991), a tryptophan restricted diet combined with chemotherapy may further
enhance clinical outcomes by protecting normal cells and sensitizing cancer cells.

Results

Methionine restriction enhances protection against chemotoxicity in mice.
Methionine restriction has been shown to prolong lifespan and reduce
age-dependent deterioration in laboratory rodents (Miller, Buehner et al. 2005).
To test the effect of reduced methionine intake on stress resistance to
chemotherapy toxicity, outbred mice (CD-1) were fed with a purified amino acid
diet or the same diet in which methionine was reduced 80% for 5 days prior to
chemotherapy treatment. In addition, to keep total amino acid intake constant, we
compensated the amount of amino acid lost from methionine reduction by
increasing the most abundant amino acid L-glutamate (Newsholme, Crabtree et
al. 1985; Vander Heiden, Cantley et al. 2009). Also, we removed cysteine as it is
an intermediate metabolite of methionine and can partially compensate for the
low methionine intake. The methionine restricted (MR) diet (Table 6.2) was
based on the Baker‟s amino acid formula, which was designed for efficient
growth of animals on a synthetic purified amino acid diet. It consists of 15.4%
protein, 64.8% carbohydrates, and 8% fat by weight, with a defined amino acid
profile (Table 6.1). The rodent diet recommended by the American Institute of
176

Nutrition (AIN-93G) consists of approximately 3.9 Kcal/g, and was used as the
standard reference for the MR diet (Reeves 1997). We have also observed that
mice undergoing a brief methionine restricted diet (5 days) experience enhanced
stress resistance against the widely used chemotherapy drug doxorubicin (Fig.
6.1).

Table 6.1 Purified amino acid diet formula; control diet for the methionine
and tryptophan restricted diet.

177

Table 6.2 Purified methionine restriction diet formula.
178

Table 6.3 Purified tryptophan restriction diet formula.

6 week old female CD-1 mice were fed the methionine restricted diet for 5 days
prior to receiving doxorubicin (DXR). Regular diet was resumed immediately
upon intravenous DXR administration (24 mg/kg). Notably, the brief (5 days)
methionine restricted diet did not affect normal growth of mice compared to its
controls, capable of maintaining normal body weight (Fig. 6.1B). Furthermore,
mice showed enhanced protection against DXR, with improved survival (Fig.
6.1A). All mice were equally healthy, active, and responsive at the end of the 5-
day methionine restricted diet, compared to the normally fed mice, suggesting
179

the diet itself did not cause significant side effects, at least for 5 days. Notably,
the protective effect of the methionine restricted feeding was much lower than
that of fasting suggesting that the combination of amino acid restriction with a
hypocaloric diet described here can maximize the enhancement of the protective
effects.
180

Figure 6.1 Methionine restriction leads to enhanced protection against
DXR.
6-week old female CD-1 mice were fed a methionine restricted diet for 5 days
prior to DXR administration (24 mg/kg). (A) Survival following DXR injection is
shown as Kaplan Meier survival curve. P=0.117 by Peto‟s log-rank test (B) Body
weight and (C) food intake was recorded daily during the methionine restricted
diet. (D) Body weight and (E) food intake was also measured after DXR injection,
at which time all animals were under regular diet. Control (n=15), Methionine
(n=8).
181

Tryptophan restriction enhances protection against chemotoxicity in mice.
We have also observed that mice undergoing a brief tryptophan restricted
diet (10 days) experience halted growth and enhanced stress resistance against
the widely used chemotherapy drug doxorubicin (Fig 6.2). 6 week old female CD-
1 mice were fed the tryptophan restricted diet (Table 6.3) for 10 days prior to
receiving doxorubicin (DXR), a widely used chemotherapy agent to treat various
types of cancer, owing to its DNA intercalating properties and generation of
oxidative stress (Singal, Li et al. 2000). Regular diet was resumed immediately
after DXR administration (26 mg/kg). Notably, the brief (10 days) tryptophan
restricted diet suspended growth (Fig 6.2B). This is consistent with the
„maintenance mode‟ theory, in which suboptimal conditions, such as starvation
and tryptophan deficiency, prompt the diversion of energy from growth to
maintenance/repair and maximize the organism‟s chance of survival (Kirkwood
2005). Furthermore, mice showed enhanced protection against DXR, with
improved survival (Fig 6.2A,D). All mice were equally healthy, active, and
responsive at the end of the 5-day tryptophan restricted diet, compared to the
normally fed mice, suggesting the diet itself did not cause significant health
issues.

182

Figure 6.2 Tryptophan restriction leads to enhanced protection against
DXR.

6-week old female CD-1 mice were fed a tryptophan restricted diet for 10 days
prior to DXR administration (26 mg/kg). (A) Survival following DXR injection is
shown as Kaplan Meier survival curve. P=0.024 by Peto‟s log-rank test. (B) Body
weight and (C) food intake was recorded daily during the methionine restricted
diet. (D) Body weight and (E) food intake was also measured after DXR injection,
at which time all animals were under regular diet. N=7/group.
183

Discussion

Previously, we showed that fasting can differentially protect mice but not
the tumor allograft to chemotherapy. What is more, fasting itself was growth-
static, i.e., inhibited tumor progression, and also sensitized cancer cells to
chemotherapy. Despite the fact that fasting is safe, economical, feasible, and
effective in reducing chemotherapy-related side-effects in cancer patients at
advanced stages(Safdie, Dorff et al. 2009), it is still challenging to even the
healthiest individual. Therefore, understanding the major dietary components that
mediate the beneficial effects of fasting may introduce the use of substitution
diets to selectively protect the host against chemotherapy toxicity.
Amino acids and proteins are potent inducers of the IGF-I pathway
(Clemmons and Underwood 1991). In fact, a 4% protein diet has been shown to
reduce IGF-I levels 80%, which is similar to that of the genetically engineered LID
mouse (Naranjo, Yakar et al. 2002). The master regulator mTOR is also known
to be activated by amino acids, independently of the canonical PI3K/Akt and the
Ras/ERK pathway, thereby signaling the cell to proliferate (Ma and Blenis 2009).
We have shown that restricting only one EAA, methionine or tryptophan, can
induce the animal into a „maintenance mode‟. Since these are essential amino
acids, which by definition cannot be synthesized de novo, the animal will sense
the deficit and reduce mitogenic signals. The energy invested in
growth/reproduction will shift to maintenance/repair, thus enhancing cellular
protection (Kirkwood 2005; Raffaghello, Lee et al. 2008). EAA restricted diets
have also shown to reduce tumor growth (Theuer 1971; Lowry, Goodgame et al.
184

1979; Kamath, Conrad et al. 1988). Together with its protective effects against
chemotherapy, EAA restricted diets hold potential to enhance current
chemotherapy by allowing dose and frequency increase from what would have
been considered maximum otherwise. All EAA should be tested to determine its
role in differential stress resistance. It may be possible to maintain an organism
in a protective mode by continuous rotation of single EAA restriction throughout
the entire course of cancer treatment.

Methods and Materials
Mice
5-6 week old female CD-1 (ICR; Harlan) were single caged for accurate
food and water intake during the diet.

Amino acid deficient diet
All diets were purchased from Teklad (WI, USA) as ½” pellets; control diet
(TD.07788), methionine restricted diet (TD.07789), tryptophan restricted diet
(TD.07790). Food and water intake was recorded daily with compensation for
wasted food by collecting pellet remainings in the cage. Bedding was replaced
prior to diet initiation to eliminate remaining regular chow,and minimize the effect
of coprophagy. The safety of the amino acid restricted diet was determined by
monitoring body weight (maximum of a 20% decrease, with rapid recovery within
12 hours of ad lib feeding).

185

Chemotherapy
DXR (Sigma, USA) was intravenously injected, using a home-made
catheter system, into the lateral veins of the tail and was immediately followed by
a 100µL saline wash to reduce the irreversible damage to the tail by DXR.
186

CHAPTER SEVEN

Conclusion and Future Directions

According to the American Cancer Society (ACS), dietary
recommendation for cancer patients receiving chemotherapy is to increase
calories and protein (Doyle, Kushi et al. 2006). This is well accepted and
practiced, as it is common wisdom to eat well when one is sick. Although this
may be true for certain diseases, such as infections, the studies in this thesis
suggest a shift in the bona fide canonical dietary recommendation for cancer
patients with the intention to reduce toxicity and further sensitize cancer cells to
chemo/radiotherapy. In chapter 2, fasting was able to selectively protect mice but
not the tumor allograft from chemotherapy. In chapter 3, a case report on cancer
patients, some at advanced stages, shows that fasting is safe, feasible, and
effective in reducing chemotherapy-related side effects. In chapter 4, IGF-I was
greatly reduced in response to fasting, and was shown to be a major mediator of
the fasting-dependent differential stress resistance. In chapter 5, the effect of
fasting is extended beyond selectively protecting normal cells to sensitizing
certain cancer cells to chemo/radiotherapy and to also directly affect their growth.
This study is important since it provides a basis to study mechanisms of stress
resistance through metabolic manipulations, with the potential of discovering
drug targets that can weaken the cellular defense of malignant cells. Finally, in
chapter 6, a substitution diet is discussed, since despite of its beneficial effects,
fasting still remains a challenging lifestyle. A normal diet with the restriction of
187

only a single amino acid, methionine or tryptophan, was able deliver the
protective effect of fasting without complete food deprivation. Notably, circulating
IGF-I is regulated by protein intake and total energy (Clemmons and Underwood
1991; Thissen, Ketelslegers et al. 1994); low-protein and low-calorie diets have a
strong effect on circulating concentrations of several pro-growth factors, including
IGF-I, and clinical biomarkers (Fontana, Klein et al. 2006; Fontana, Weiss et al.
2008). Considering the fact that the average American consumes more protein
than needed (Fulgoni 2008), which has been shown to be a major risk factor for
tumor progression, there is an urgent need to reevaluate the current dietary
recommendation for cancer patients.

Future directions: converging pathways of metabolism, growth, and stress
resistance.

Upon his seminal discovery that cancer cells undergo cell autonomous
aerobic glycolysis, Otto Warburg suggested defective metabolism as the origin of
cancer (Warburg 1956; Garber 2004). Since then, with the discovery of
oncogenes and tumor-suppressors, cancer has been accepted as a genetic
disease. Together, we now understand that these oncogenic mutations not only
rewire the cellular proliferation machinery, but also reprogram metabolic
pathways. Although the process of transformation is heterogeneous among
different cancers with multiple steps involved, the Warburg effect is considered a
metabolic hallmark of cancer (Hsu and Sabatini 2008). Despite the focused
efforts on drug development, the main strategy of modern cancer treatment still
188

lies on cytotoxic therapy which takes advantage of one of the main hallmarks of
cancer, rapid proliferation. Together with aging research where the converging
pathways of cellular metabolism, growth and stress resistance are well studied,
revisiting the Warburg effect may provide a novel approach to cancer treatment
based on metabolism. Nonetheless, starving a cancer cell to death may sound
far-fetched at this point, but as shown in this thesis, it is possible to manipulate
both normal and cancer cells to our favor by fasting. Mammalian cells are non-
autonomous, therefore cellular policies depend on extracellular nutrient
availability and growth signals, which are concurrently adjusted during fasting.
Hence, future studies will need to understand the major dietary components to
develop sophisticated diets and the molecular pathways to develop drugs at both
extra- and intracellular levels to deliver the beneficial effects of fasting.

Dietary interventions
Macronutrients are bio-fuels and precursors of biosynthesis, but also act
as signaling molecules. For instance, mTOR, which is a master regulator of
growth, metabolism, and stress resistance, can be directly activated by amino
acids, or inactivated by an overall decline in energy (ATP) (Avruch, Hara et al.
2006). Also, as shown in chapter 6 and studies by others, the absence of an
essential amino acid (EAA) can signal a cell to discourage growth/reproduction
and activate maintenance/repair mechanisms. However, prolonged restriction of
an EAA is toxic and thus should be carefully designed. Notably, a protein-
restricted diet reduced DNA synthesis (Grube and Gamelli 1988) and tumor
189

growth (Daly, Reynolds et al. 1980) in tumor allografts. In a recent meeting, a
study from the laboratory of Dr. Douglas Spitz at the University of Iowa showed
that a ketogenic diet (high fat, low carbohydrate) suppressed the growth of
glioma cells in a mouse model (unpublished data). Added, the use of the glucose
analog 2-deoxyglucose (2-DG) was successful in sensitizing cancer cells,
including drug resistant cell lines, to chemo/radiotherapy (Kaplan, Navon et al.
1990; Aft, Zhang et al. 2002).

Pharmacological interventions
Another approach to mimic fasting is targeting specific signal transduction
pathways. A thorough examination of literature suggests multiple candidate
targets including IGF-I (extracellular) and its downstream effectors such as the
PI3K/Akt and Ras/ERK pathways (intracellular) which have been discussed in
this thesis. Nonetheless, metabolic reprogramming in cancer cells is believed to
be a well orchestrated dysregulation of multiple pathways including apoptosis,
autophagy, and mitochondrial function, which can be manipulated by both extra-
and intracellular strategies. As briefly mentioned in chapter 4, fasting decreases
extracellular growth factors such as IGF-I, VEGF, EGF, and G-CSF. A
proteomics approach would provide holistic insight into the type of growth factors
involved and its degree of response to fasting. Notably, many chemotherapy
drugs are also strong oxidants, and therefore the investigation can be extended
190

to the protection against oxidative stress with intentions to prevent cancer and
other age-dependent diseases.
However, intracellular pathways are more complex and challenging due to
the finely tuned cross-communication within multiple signaling effectors. For that
reason, an evolutionary approach may provide a blueprint of a cancer cell and
point to effective targets. A cell must be within 100-200 um from blood vessels to
be adequately supplied with oxygen, nutrients, and growth factors. A malignant
cell frequently encounters an „ischemic‟ condition due to premature, or the lack of,
vessel formation resulting from its uncontrolled growth. Therefore, the force of
natural selection favors acquired mutations that reprogram cellular metabolism to
be more self-sufficient. First, tumors adapt to hypoxia or anoxia by upregulating
the HIF-1α transcription factor, which in turn induces the production of VEGF to
recruit blood vessels. Second, to support the nutritional needs under low oxygen,
cells undergo aerobic glycolysis (fermentation), also known as the Warburg effect.
Further, cancer cells have evolved to efficiently utilize lactate, a byproduct of
aerobic glycolysis, as a major metabolic substrate. Third, autophagy, a process
of self-degradation of cellular components into macromolecules which are
recycled back to the cytoplasm for reuse during starvation (Yorimitsu and
Klionsky 2005), is upregulated in cancer cells (He and Klionsky 2009). Finally,
mutations are frequently found in genes that promote growth such as the
PI3K/PTEN (Jiang and Liu 2008), and Ras (Bos 1989). As a result, whereas
normal cells undergo apoptosis under growth factor starvation, cancer cells are
able to evade apoptosis, which can contribute to resistance to cancer therapy.
191

Interestingly, recent studies suggest autophagy-dependent programmed cell
death as a potential target to induce cancer cell death (Amaravadi, Yu et al.
2007; Apel, Herr et al. 2008).
Therefore, future studies should test the above mentioned candidates in
modulating stress resistance in cancer cells (Fig 7.1). Dietary and
pharmacological interventions may complement each other, for instance, drugs
targeting the cellular bioenergetic pathways may allow the fasting-resistant
cancer cells to be susceptible again to dietary interventions. The controlled
clinical trial currently being conducted at the USC Norris Cancer Center will
provide information on the safety and efficacy of fasting in cancer patients under
chemotherapy. I believe this is the beginning of a novel cancer treatment where
patient protection and sensitization is achieved by a single intervention.

192

Figure 7.1 Potential targets of DSR

Dietary interventions (fasting and amino acid restricted diets) and the modulation
of IGF-I pathways have been shown to induce DSR. The effect of differential
manipulation of stress resistance (DSR) can be two-fold, in which we can protect
normal cells and sensitize cancer cells. This figure presents potential targets of
either dietary or pharmacological targets to mimic fasting. „X‟ represents targets
that were tested in studies (yeast, mammalian cells, mice and humans) included
in this thesis.
193

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Abstract (if available)

Abstract One of the leading problems in modern cancer treatment is the toxic side effects secondary to chemotherapy and radiotherapy. In fact, toxicity is the major limiting factor of chemotherapy, shadowing its full therapeutic potential. Currently, there are no established interventions or drugs to adequately reduce the toxicity and protect cancer patients. This thesis provides a revolutionary approach to chemotherapy by selectively protecting normal cells and sensitizing cancer cells simultaneously by fasting. The theory behind fasting-dependent stress resistance was built on strong experiment-based scientific studies on aging and cancer research. Briefly, the compiled data from dietary, genetic, and pharmacological studies have greatly contributed to our understanding of the conserved pathways that regulate lifespan and stress resistance in organisms spanning from the simple yeast, worms, flies, rodents, to non-human primates.

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Asset Metadata

Creator Lee, Changhan (author)

Core Title Fasting-based differential stress resistance to enhance cancer treatment: a novel strategy to protect normal cells and sensitize cancer cells to chemotherapy

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 05/17/2010

Defense Date 02/26/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cancer,chemotherapy,differential stress resistance,fasting,insulin-like growth factor 1 (IGF-I),OAI-PMH Harvest,Radiotherapy,sensitization

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Longo, Valter D. (committee chair), Dubeau, Louis (committee member), Finch, Caleb E. (committee member)

Creator Email changhal@usc.edu,changhan.lee@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3075

Unique identifier UC1299782

Identifier etd-Lee-3206 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-345793 (legacy record id),usctheses-m3075 (legacy record id)

Legacy Identifier etd-Lee-3206.pdf

Dmrecord 345793

Document Type Dissertation

Rights Lee, Changhan

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu