The differential effects of genistein on cellular effects in T47D tumorigenic and MCF10A nontumorigenic breast epithelial cells: role of metabolism

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Content THE DIFFERENTIAL EFFECTS OF GENISTEIN ON CELLULAR EFFECTS IN
T47D TUMORIGENIC AND MCF10A NONTUMORIGENIC BREAST
EPITHELIAL CELLS: ROLE OF METABOLISM

by

Dominique Truong-Giang Nguyen

_____________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2007

Copyright 2007 Dominique Truong-Giang Nguyen
ii
Dedication

This dissertation is dedicated to my late mother whose dedication and
devotion inspired a boat refugee child like me to dream big, and to my devoted
husband, Robert A. Wascher for his patience, love, and support during years of my
challenging graduate school training.
To my beautiful children, Aaron and Alexis, who have shown me the
meaning of unconditional love.
iii
Acknowledgments

I would like to acknowledge the support from the US Department of Defense
for their funding 17-99-1-9375. I am indebted to Dr. Enrique Cadenas for his
mentorship and guidance throughout my three years of training in his laboratory. I
would like to thank my collaborators Dr. Jeremy Spencer and Dr. Catherine Rice-
Evans whose expertise and guidance in flavanoid metabolism was a great asset to my
work. I would like to thank my committee members Dr. Axel Schoenthal, and Dr.
Alex Sevanian for their expert guidance and excellent teaching.
In addition, I would like to thank Drs. Sandra Johnson and Deborah Johnson
for supplying the MCF10A and RAT1A cell lines. Finally, I would like to thank my
friends and colleagues, especially, Drs. Jerome Garcia, Daniel Rettori and Siranoush
Sharzhrad for their friendship, encouragement, and support. They have made my
time at USC very memorable.
iv
Table of Contents

Dedication ii

Acknowledgements iii

List of Figures vi

Abbreviations xv

Abstract xvii

Chapter 1. Introduction 1
1.1 Phytoestrogens: isoflavones and lignans 1
1.2 Health benefits associated with dietary phytoestrogen 5
1.3 In vivo chemopreventive effects 6
1.4 Cell cycle control and phytoestrogens 7
1.5 Metabolism of genistein 8
1.6 Cell cycle effects of genistein in cancer cells 9
1.7 Study Objectives 11
1.8 Hypothesis 13

Chapter 2
The intracellular genistein metabolite
5,7,3,4-tetrahydroxyisoflavone mediates G2-M cell cycle
arrest in breast cancer cells via two distinct signaling pathways 14

Chapter 3
Contrasting the effects of genistein on cell proliferation and
cell cycle arrest in nontumorigenic human breast epithelial
cells and human breast cancer cells: Involvement of α-cdk2
kinase, cyclin B1 kinase and cyclin dependent kinase
inhibitor, p27 59

Chapter 4
The effects of genistein on cellular transformation in
RAT1A and c-myc RAT1A fibroblast cells 80

v
Chapter 5
Conclusions 99

References 101
vi
List of Figures

Figure 1. Major isoflavone phytoestrogens 2

Figure 2. The molecular and cellular actions of phytoestrogens
and their contributions to beneficial health effects 3

Figure 3. Cell cycle phases and regulation: Progression through
the cell cycle is tightly controlled by phase-specific
cyclin-dependent kinases (CDK) and cyclins. 11

Figure 4 Anti-proliferative effects of genistein on the
proliferation of T47D and MCF10A cells.
(A) % Growth inhibition induced by genistein
(0.1-30 µM) in T47D cells (light gray), and
MCF-10A cells (dark gray) as assessed by
the sulforhodamine B assay. Results are
expressed as growth inhibition and were obtained
from three independent experiments performed
in triplicate and presented as mean ± SD.
Significant increases between vehicle (MeOH) and
treatments are indicated by *** p < 0.001; ** p < 0.01;
*p < 0.05. (B) Genistein-induced G2-M cell cycle
arrest in T47D ( ) and MCF10A ( z ) cells.
Exponentially growing cells at 70-75 % confluency
were exposed to genistein (0.3-30 µM). Cells
were trypsinized, pelleted and collected prior to
fixing/digestion with RNase. Cellular DNA was stained
with PI, and analyzed by flow cytometry at 24 h
after genistein exposure. Results are expressed as % of
cells in G2-M phase of the cell cycle 24 h post genistein
exposure and were obtained from three independent
experiments performed in duplicate and presented as
mean ± SD. Significant increases between vehicle
(MeOH) and treatments are indicated by
*** p < 0.001; *p < 0.05. 26

vii
Figure 5 Cellular association of genistein with T47D cells and
MCF10A cells. Typical HPLC traces shown
with UV-visable spectral data of cell lysates from
cells exposed to genistein (10 µM) for 24h.
Panel A: T47D cells; Panel B: MCF10A cells.
Genistein elutes at a retention time of 60.2 min.
Three major metabolites are detected at 42.9, 53.7
and 54.8 minutes. C) Quantitative association
(ng/mg protein) of genistein with T47D and MCF10A
cells. Cells were exposed to genistein (0.3, 3, 30 µM
for 2 or 24 h after which cells were lysed, de-protonated
and analyzed by HPLC-PDA. T47D cells (light gray);
MCF10A cells: (dark gray). Resulted are represented
as mean + SD of 4 separate experiments performed
in duplicate. Significant decrease in genistein association
between 2 h and 24 h in T47D cell are indicated
by *** p < 0.001; **p < 0.01. 28

Figure 6 Determination of genistein metabolites. (B): Mass
spectrum of 5,7,3,4-tetrahydroxyisoflavone (THIF).
Spectrum shows the molecular ion ([M + H+)+ of
THIF (molecular weight: 286 Da). The structure
of the conjugate is shown after further confirmation
by 1HNMR. (B): Co-elution of synthesized glutathionyl
conjugate of genistein with T47D intracellular genistein
metabolites. Panel C: MS of 5-S-glutathonyl-THIF.
(C): LC-MS/MS spectrum of the peak at 53.7 min
relating to a 5-S-glutathionyl THIF conjugate.
The fragment ion spectrum of the conjugate is
recorded at collision energy of 35%. The inserted
spectrum shows the molecular ion ([M+H+]+)
of the conjugate (molecular weight 575 Da).
The structure of the conjugate is shown after
further confirmation by 1HNMR. 32

viii
Figure 7 (A) Quantitative association (ng/mg protein) of
the genistein, THIF and glutathionyl-THIF conjugates
with T47D and MCF10A cells. Cells were exposed
to genistein (3 µM) for 2, or 24 h after which cells
were lysed, de-protonated and analyzed by HPLC-PDA.
Quantification of the THIF was achieved using a
synthesized standard and all glutathionyl conjugates
were quantified using 5-S-glutathonyl-THIF as
a standard (see Materials and Methods). Genistein
(white bars); Glutathionyl conjugates (dark gray bars);
THIF (light gray bars). (B) Medium levels of
glutathionyl-THIF conjugates (µM) following
genistein administration to T47D and MCF10A cells.
Cells were exposed to genistein (0.3 to 30 µM) and
medium levels of conjugates were assessed at 24 h
by HPLC-PDA analysis. Black columns: T47D cells,
White columns: MCF10A cells. 36

ix

Figure 8 Effects of the CYP450 inhibitor cimetidine on G2-M
cell cycle arrest and genistein metabolite formation in
T47D cells. (A) The influence of cimetidine on
genistein-induced G2-M cell cycle arrest in
T47D cells. Exponentially growing cells at
70-75% confluency were exposed to genistein
(0.3-30 µM). Cells were trypsinized, pelleted and
collected prior to fixing/digestion with RNase.
Cellular DNA was stained with PI, and analyzed by
flow cytometry at 24 h after genistein exposure.
Results are expressed as % of cells in G2-M phase
of the cell cycle 24 h post genistein exposure and
were obtained from three independent experiments
performed in duplicate and presented as mean ± SD.
Significant increases between vehicle (MeOH)
and treatments are indicated by *** p < 0.001;
*p < 0.05. (B) Effect of cimetidine on the
intracellular formation of THIF and glutathionyl-THIF
conjugates in T47D cells. Cells were exposed to
genistein (3 µM) for 24 h after which cells were lysed,
de-protonated and analyzed by HPLC-PDA.
Quantification of the THIF was achieved using a
synthesized standard and all glutathionyl conjugates
were quantified using 5-S-glutathonyl-THIF as a
standard (see Materials and Methods). Glutathionyl
conjugates (dark gray bars); THIF (light gray bars).
Results are expressed as cell associated levels
(ng/mg protein) 24h were obtained from
three independent experiments performed in
duplicate and presented as mean ± SD. Significant
decreases between genistein and genistein and
cimetidine treatment are indicated by *** p < 0.001; 38

x
Figure 9 Anti-proliferative effects of 5,7,3,4-
tetrahydroxyisoflavone (THIF). (A) % Growth
inhibition induced by THIF (0.1-30 µM) in T47D
cells (dark gray), and MCF-10A cells (light gray) as
assessed by the sulforhodamine B assay. Results
are expressed as growth inhibition and were
obtained from three independent experiments
performed in triplicate and presented as mean ± SD.
Significant increases between vehicle (MeOH) and
treatments are indicated by *** p < 0.001; *p < 0.05.
(B) Inhibition of cdc2 phosphorylation in T47D
cells exposed to THIF. Crude lysates (30 µg)
prepared from T47D cells exposed to vehicle (MeOH),
or THIF (0.3-30 µM) for 1h were immunoblotted
with an antibody that specifically recognizes
phosphorylated cdc2 (Thr 161). The same crude
lysates (30 µg) were immunoblotted with an
antibody that recognizes total levels of cdc2.
Blots are representative blots of three independent
experiments on different cultures that yielded
similar results. Data obtained from immunoblot
experiments in (B) were analyzed using BioRad
Quantity One 1-D Analysis software ( and solid line).
Also plotted is % of cells in G2-M phase of the cell
cycle analysis ( z and dashed line). Results are
expressed as % of cells in G2-M phase of the
cell cycle 24h post THIF exposure and were
obtained from three independent experiments
performed in duplicate and presented as
± SD. Significant increases between vehicle
(MeOH) and treatments are indicated by
*** p < 0.001; *p < 0.05. 42

xi

Figure 10 Phosphorylation of JNK1/2, p38 and cyclin B1 in
T47D cells exposed to 5,7,3,4-tetrahydroxyisoflavone
(THIF). (A) Crude lysates (30 µg) prepared from
T47D cells exposed to vehicle (MeOH), or THIF
(0.3-30 µM) for 0.5 h were immunoblotted with
an antibody that specifically recognizes the
dually phosphorylated region of the active form
of JNK1 and JNK2 (pJNK1/2), the dually
phosphorylated motif Thr
180
-Pro-Tyr
182
within
activated p38 or cyclin B1 when phosphorylated at
Ser 147. Lysates were also immunoblotted with
antibodies for total p38 and total cyclin B1 levels.
(B) Data obtained from phospho-p38 and
phospho-cyclin B1 immunoblot experiments
represented in (A) were analyzed using BioRad
Quantity One 1-D Analysis software. Each point
represents the mean ± SD of four independent
experiments (*** p < 0.001; **p < 0.05). 46

Figure 11 Inhibition of THIF-induced signaling and growth
arrest by the p38 inhibitor SB203580. (A) Crude
homogenates (30 µg) prepared from T47D cells
exposed to vehicle (MeOH), SB203580 (2.5 µM),
THIF (3 µM) or THIF (3 µM) + SB203580 (2.5 µM;
0.5 h pre-treatment) were immunoblotted with an
antibody that recognizes phospho-38, total p38,
phospho-cyclin B1 or phospho-cdc2. Data
obtained from immunoblots were analyzed using
BioRad Quantity One 1-D Analysis software and
are presented with significant differences between
THIF treated and THIF + inhibitor are indicated
by *** p < 0.001. Significant differences between
control and inhibitor are indicated by ^ p < 0.05.
(B) Reversal of genistein (3 µM) and THIF (3 µM)-
Induced growth inhibition and cell cycle arrest by
SB203580. Results are expressed as growth
inhibition and were obtained from three
independent experiments performed
in duplicate and presented as mean ± SD.
Significant increases between vehicle (MeOH) and
treatments are indicated by *** p < 0.001; ** p < 0.01. 50
xii
Figure 12 Proposed mechanism for the anti-proliferative
effects of genistein in T47D cells. Genistein enters
cells where it is subject the CYP450-induced
intracellular metabolism yielding THIF. THIF
may undergo oxidation to form an o-quinone
that reacts with GSH and is exported from cells
(this may also be catalyzed by glutathione-
S-transferase). Rises in intracellular oxidative
stress, which is accompanied by depletion
of GSH, trigger the activation of the MAP
kinase p38 and two downstream targets of
ATM/ATR, p53 and Chk1. Active p38 prevents
the phosphorylation of cyclin B1 and hence
its transport to the nucleus, an event essential for
correct functioning of the cdc2-cyclin B1
complex. In addition, active p38 may undergo
translocation to the nucleus where it directly
inhibits the phosphorylation/activation of
cdc2, thereby blocking entry of cells into
mitosis (G2-M block). 52

Figure 13 The effects of genistein on cell cycle
progression in (A) T47D tumorigenic breast
epithelial cells and (B) MCF10A nontumorigenic
breast epithelial cells. Cells were treated with
varying concentrations of genistein for 96 h,
fixed in ethanol, stained with propidium iodide,
and analyzed using flow cytometry. Each data
point is representative of three independent
experiments, where results are expressed
as Mean + SEM. 66

Figure 14 The effects of genistein on the catalytic kinase
activities in T47D tumorigenic breast epithelial cells
of α-cdk2 (A.2) a-cyclin B kinase, and the catalytic
kinase activity of (B) a-cdk2 in MCF10A
nontumorigenic breast epithelial cells. Aliquots of
100 µg protein were immuoprecipitated with anti-
α-cdk2 or a-cyclin B kinase, and histone H1-
associated activity of immunopreciptates was
evaluated as described. 69

xiii
Figure 15. The effects of genistein on cyclin A expression
in (A) T47D tumorigenic breast epithelial cells,
and (B) MCF10A nontumorigenic breast
epithelial cells. Results shown are representative
of three separate western blots. 30 µg of total
protein was separated on a 12% SDS-PAGE gel,
transferred to a nitrocellulose membrane, and
incubated with cyclin A antibody. 71

Figure 16. The effects of genistein on p27 expression in
(A) T47D tumorigenic breast epithelial cells,
and (B) MCF10A nontumorigenic breast epithelial
cells. Experiments shown are representative of three
separate western blots. 30 µg of total protein was
separated on a 12% SDS-PAGE gel, transferred
to a nitrocellulose membrane, and incubated
with p27 antibody. 73

Figure 17. Links between c-myc, selected putative target genes,
cellular functions, and cell growth. This diagram shows
the complex relationship between c-Myc and its putative
target genes, which are grouped according to their
functions. The various cellular functions cooperate to
promote cell growth. 84

Figure 18 The effects of genistein on colony formation in
c-myc RAT1A fibroblast. Cells were grown
in 0.7 % soft agar, and treated with genistein
for 3 weeks. Fresh genistein and media were
added every fifth day. At three weeks, pictures
were taken using an electron photocapture microscope. 88
xiv
Figure 19 (A) The effects of genistein on cell viability
measured by MTT reduction in RAT1A and
(B) c-myc RAT1A fibroblast cells. Cells
were plated in 96 well culture dishes at a density
of 1000 cells/well. Cells were treated with
Control- EtOH (open circle), 5 µM (solid square),
25 µM (solid triangle), 50 µM (Star) of genistein for
6-7 days. The ability to reduce MTT was measured
following 4 h of incubation with MTT, followed
by addition of solubilization buffer. Samples were
measured at 590 nm. In c-myc RAT1A cells,
genistein exposure caused in a dose-dependent
reduction in cell viability with an approximate
IC50 of 50 µM. 90

Figure 20 The effects of genistein on annexin V binding in
RAT1A (Black square)and c-myc RAT1A
(Black diamond)fibroblasts. Cells were seeded at
100 000 cells and treated with genistein for 4 days.
Following that, cells were washed with PBS, and
stained with annexin/propidium iodide and analyzed
using flow cytometry. Results are expressed at
mean + SEM, where n=3. Experiments are
representative of three separate experiments. 93

Figure 21 Effects of genistein on BRDU incorporation on
(A) RAT1A and (B) RAT1A c-myc fibroblasts cells.
Cells were seeded at a density of 100 000, and exposed
to genistein [5-50 µM] for 4 days. On the fourth day,
cells were washed, and fresh media with BRDU was
added for 1 h. Following this, cells were washed, and
stained with an FITC-anti-BRDU dye, and analyzed
using flow cytometry. Data are expressed as mean,
where n=2. Experiment was performed once. 96
xv
Abbreviations

Akt/PKB protein kinase B

ATM Ataxia telangiectasia–mutated protein kinase
ATR ATM- and Rad3 related
BRDU 5-Bromo-2′-deoxyuridine
CDK cyclin dependent kinase
CDKI cyclin dependent kinase inhibitor
c-myc c-myelocytomatosis
CYP450 cytochrome P450
DCDHF-DA dichlorodihydrofluorescin diacetate
DNA deoxyribonucleic acid
ECL electrocheminiluminescence
EDTA ethylene diamine tetraacetic acid.
EGTA ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra
acetic acid

ERK1/2 extracellular signal-regulated kinase
FBS fetal bovine serum
FSH follicle stimulating hormone
LH lutenizing hormone
GSH glutathione
HDL high density lipid
xvi
HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
HME human mammary epithelial cells
HPLC-PDA high performance liquid chromatography photodiode array
HRP horse radish peroxidase
LC-MS liquid chromatography-mass spectrometry
LDL low density lipid
JNK c-Jun N-terminal kinase
MAPK mitogen-activated protein kinase
MTT 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazolium
bromide

NMR nuclear magnetic resonance
PI propidium iodide
PS phosphatidylserine
RNAse Ribonucleate 3′-pyrimidinooligonucleotidohydrolase
THIF 5,7,3,4-tetrahydroxyisoflavone

xvii
Abstract

Epidemiological studies suggest that dietary soy intake is associated with a
decrease risk of breast cancer. A soy isoflavone, genistein (4,5,7-
trihydroxyisoflavone), has been shown to prevent tumor formation in several in in
vivo models of cancer, and can exert antiproliferative effects in different cancer cell
types, including breast cancer cells. However, the mechanisms for the
chemopreventive and antiproliferative effects of soy remain unclear. Two aims
outlined in this study included: (1) To investigate the intracellular metabolism of
genistein and effect on antiproliferative-related signaling pathways in T47D
tumorigenic and MCF-10A nontumorigenic cells, (2) The investigate the
chemopreventive effects of genistein in terms of its effects on c-myc-mediated
oncogenic transformation by establishing the effects of genistein on apoptosis, cell
proliferation, cell viability, and anchorage-independent growth in RAT1A fibroblast
cells, and RAT1A fibroblast stably transfected with c-myc. The significant findings
that were established included: (1) selective genistein uptake into T47D cells but not
MCF10A cells (2) identification of CYP-mediated formation of bioactive
metabolites of genistein (5,7,3,4-tetrahydroxyisoflavone (THIF) and two glutathionyl
conjugates of THIF) (3) THIF regulated cell cycle via activation of p38 with
subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation.
These two events are critical for the correct functioning of the cdc2-cyclin B1
complex. (4) Genistein affected cell viability, induced apoptosis, and inhibited cell
xviii
proliferation to a greater extent in RAT1A fibroblasts that overexpressed c-myc, and
inhibition of anchorage-independent growth (a property of transformed cells), thus
suggesting that genistein’s possible selectivity and/or synergism for the c-myc
oncogene may represent an underlying explanation for its reported chemopreventive
abilities. Collectively, these results suggest that genistein is selectively metabolized
to a bioactive metabolite in cancer cells alone leading to inhibition of cell
proliferation and cell cycle progression, and that it may possess in vitro
chemopreventive effects via inhibition of c-myc-mediated oncogenic transformation
in RAT1A fibroblasts.
1
Chapter 1- Introduction

1.1- Phytoestrogens: isoflavones and lignans
Phytoestrogens or plant estrogens are natural plant substances (Setchell,
1989). The three main classes are isoflavones, coumestans, and lignans. Isoflavones,
the most frequently studied, exhibit structural similarity to mammalian estrogens and
are present in large amounts in soybeans, and soy products. Isoflavones contain a
number of phenolic hydroxyl groups attached to ring structures. The main
isoflavones with estrogenic activity are genistein, daidzein, their glucosides (genistin
and daidzin), and their methyl esters, biochanin A and formonometin (Fig 1). The
major molecular and cellular mechanisms of phytoestrogens that may underlie their
health benefits can be summarized as follows (Fig 2)

2

Genistein Daidzein
Genistin Biochanin A Daidzin Formononetin
Fig 1. Structure of major isoflavones
3

Fig 2. The molecular and cellular actions of phytoestrogens and their contributions
to beneficial health effects
- Inhibition of key enzymes involved in signal transduction, cell proliferation, and
DNA replication. Agents that induce terminal differentiation inhibit cancer cell
proliferation. For example, two important enzymes that can promote cell
proliferation are protein tyrosine kinesis and DNA topoisomerases. Tyrosine kinases
catalyze the phosphorylation of tyrosine residues in factors involved in tumor cell
signaling and proliferation, and are involved in control of mitogenesis, cell cycle
regulation, cell survival, and cellular transformation via growth factor binding. DNA
topoisomerases are required for DNA replication, and catalyzes topological changes
Phytoestrogen Molecular Actions Cellular Effects Health Effects
Isoflavone
or
Lignans

Inhibition of
protein tyrosine
kinases

Inhibition of DNA
topoisomerases
Inhibition of tumor
cell proliferation

Inhibition of
angiogenesis
Cancer
Free radical quencher

Inducers of
antioxidant enzymes
Antioxidant
Cardiovascular
disease
Binding to ERr-α and
ER-β

Inhibition of aromatase
Hormonal
Osteoporosis
4
in DNA. Genistein inhibits both protein tyrosine kinase, and DNA topoisomerase.
Accumulating evidence also suggests that genistein can inhibit angiogenesis, the
formation of new vessels in vivo and vitro, and that this effect is due to modulation
of the above-mentioned enzymes. This effect is important inasmuch as angiogenesis
is an initial factor in tumor growth and establishment of a blood supply.
-Free radical quenching and induction of antioxidant enzymes. Isoflavones have
been reported to have direct antioxidant ability, albeit weak. This function may
involve direct interactions with oxygen free radicals (Ruiz-Larrea et al., 1997, Guo et
al., 2002, Arora et al., 2000). Genistein ingestion has also been shown to increase
levels of antioxidant enzymes, including superoxide dismutase, glutathione
reductase, superoxide dismutase, glutathione peroxidase, and catalase (Wei et al.,
1995, Cai and Wei, 1996).
-Binding to estrogen receptors and inhibition of aromatase: estrogenic and
antiestrogenic effects. Isoflavones have attracted intense interest due to their
estrogenic potential. The postulated anticarcinogenic mechanism involves a weak
estrogen-like activity (Maggiolini et al., 2001). Isoflavones bind preferentially to the
estrogen receptor beta. It has been suggested that binding to the estrogen receptors
leads to a modest response while simultaneously inhibiting the binding of more
potent estrogens. Although binding to estrogen receptor alpha results in proliferation,
binding to estrogen receptor beta mediates a cytotoxic effects on human breast
cancer cells (Maggiolini et al., 2001). Evidence from in vitro studies demonstrates
that genistein inhibits the growth of both estrogen receptor positive and negative
Interestingly, isoflavones also share structural similarity to the potent synthetic
5
antiestrogen, tamoxifen, a drug that is effective in breast cancer treatment and
prevention (Cersosimo, 2003, Pappas and Jordan, 2002).

1.2- Health benefits associated with dietary phytoestrogen
Epidemiological evidence suggest that dietary phytoestrogens may protect
against cancer, osteoporosis, and cardiovascular disease: (1) Cancer:
Epidemiological studies (Ingram et al., 1997, Mangtani and Silva Idos, 1998) and
studies in cancer models (Cotroneo et al., 2002, Lamartiniere, 2002) suggest that
phytoestrogens reduce the risk of breast, colon, and prostate cancer risk. Although
the direct mechanism of action whereby isoflavones mediate a chemopreventive
effect is unknown, isoflavones have been shown to possess antiestrogenic effects,
reduce the expression of stress response genes (Zhou and Lee, 1998), and act as
antioxidants (Ruiz-Larrea et al., 1997). (2) Osteoporosis: The estrogenic effects of
phytoestrogens have been proposed to prevent bone resorption and promote
increased bone density, thereby having beneficial effects on osteoporosis (Potter et
al., 1998). Other possible direct effects on osteoblast or other estrogenic-
independent processes, such as favorable calcium intake and retention associated
with dietary patterns rich in phytoestrogens cannot be excluded. (3) Cardiovascular
disease: Phytoestrogens intake is associated with a decrease risk of heart disease, by
both estrogenic-dependent and -independent effects. The former mechanism
accounts for cholesterol contents in LDL and HDL similar to those observed in
estrogen replacement therapy (Potter, 1998). The estrogenic-independent effects
may be related to antioxidant functions of isoflavones, which allow protection
6
against lipid peroxidation (Wiseman et al., 1996, Wiseman, 1996), an important step
in the sequence of events that lead to formation of atherosclerotic plaques.

1.3- In vivo chemopreventive effects
In animal studies, soybean products have generally reduced tumors induced
by chemical carcinogens, while diets supplemented with soybean revealed lower
number of tumors in rats (Constantinou et al., 2001). Furthermore,

it has

also been
suggested that exposure to genistein during the prepubertal period in rats protected
against chemically-induced mammary cancer in rats, possibly by inducing terminal
differentation of mammary ducts, a process that is believed to protect against future
hormonal and/or xenobiotic exposure (Lamartiniere, 2002, Cotroneo et al., 2002,
Sarkar and Li, 2002). In contrast, Helferich and colleagues have reported that
genistein can promote tumor formation in nude mice implanted with breast cancer
cells (Allred et al., 2001a, Allred et al., 2001b, Ju et al., 2001). These conflicting
results may be in part due to the different models used for induction of breast cancer.
Intervention studies in humans have documented the effects of soy
consumption on hormone levels. Specifically, these studies showed that soy intake in
premenopausal women was correlated with either a decrease in serum 17 beta
estradiol (Nagata et al., 1998) and of plasma FSH and LH (Duncan et al., 1999) or an
increase in the urinary 2-hydroxyestrone/16α-hydroxyestrone ratio (Lu et al., 2000).
As genotoxic metabolites of estrogen are associated with an increased risk of
developing breast cancer, it is possible that isoflavones may exert chemoprotective
effects in premenopausal women by modulation of estrogen metabolism towards
7
nongenotoxic metabolites metabolites. The same effect remains consistent in
postmenopausal women, although less pronounced (Xu et al., 2000).
However, it is noteworthy that lifetime exposure to estrogens is associated
with an increased risk of developing breast cancer (Thomas et al., 1997a, Thomas et
al., 1997b, Verkasalo et al., 2001). Therefore, it remains unclear whether or not the
estrogenecity or antiestrogenecity of phytoestrogens would result in the promotion or
prevention of cancer.

1.4- Cell cycle control and phytoestrogens
Genistein can inhibit growth of both estrogen-dependent and estrogen-
independent breast tumor cells with an IC50 of 10-50 µM (Chen et al., 2003, Chinni
et al., 2003, Nomoto et al., 2002). However, in the nanamolar range, genistein can
stimulate the growth of estrogen-dependent MCF7 breast cancer cells (An et al.,
2001, Miodini et al., 1999), but not of estrogen-independent breast cancer cells
(Santell et al., 2000). This biphasic effect may be attributed to the estrogenic effects
of genistein associated with lower concentrations of genistein, whereas the effects
observed at higher concentrations are not related to estrogenic actions. The relevance
of these studies remains questionable because the high micromolar concentrations
used in these experiments generally exceeded the level of free genistein in serum
(Messina, 1999). Because its effective concentration in vitro is in the high
micromolar range, it is plausible that some metabolites of genistein are the
biologically active compound that exerts effects at lower concentrations
approximating levels found in vivo.

8
1.5- Metabolism of genistein
Isoflavones from soy protein are biotransformed by intestinal microflora,
absorbed, undergo enterohepatic recycling, and reach circulating concentrations that
exceed by several orders of magnitude the amounts of endogenous estrogens
(Rowland et al., 2003). These phytoestrogens and their metabolites have many potent
hormonal and nonhormonal activities that may explain some of the biological effects
of diets rich in phytoestrogens.
Genistein is metabolized in vitro by the cytochrome P450 family of enzymes
(Breinholt et al., 2002, Breinholt et al., 2003, Kulling et al., 2001, Kulling et al.,
2000, Kulling et al., 2002). The cytochrome P450 (CYP450) monooxgenase family
of genes encodes a variety of proteins that are involved in the metabolism of
endogenous compounds and xenobiotics (Guengerich et al., 1998). Although
CYP450 mediated oxidation can result in detoxification of xenobiotics (Guengerich
et al., 1998), it has also been linked to activation of carcinogens and various drugs
(Hall et al., 1989). Incubation of genistein with recombinant cytochrome P450
(CYP) 1A2, 3A4, 2C9 or 2D6 enzymes resulted in the detection of five different
metabolites. Incubation of genistein with CYP1A2 resulted in the production of
orobol alone, a 3’-hydroxylated metabolite. The other metabolites detected were
demethylated at the 4',7-, 4',6-positions or hydroxylated at the 3'- and demethylated
at the 4'-positions respectively. Overall, these observations suggest a major
involvement of CYP1A2 in the hepatic metabolism of genistein (Breinholt et al.,
2003). Roberts-Kirchoff and colleagues have also identified 5-6 similar hydroxylated
metabolites using recombinant human microsomes that contained various CYP450
9
isoforms (Roberts-Kirchhoff et al., 1999). These studies also suggested that the
formation of different genistein metabolites if is mediated by specific CYP450
isoforms. In biological systems, CYP-mediated metabolism of other flavonoids may
result in the production of bioactive metabolites that have different biological
properties to those of the parent compound (Rice-Evans et al., 1996, Edenharder et
al., 1997). Other studies have also shown that genistein is oxidatively metabolized,
and then sulfated, glucuronidated (Doerge et al., 2000), or hydroylated, and
methylated. However, the bioactivity of these metabolites remains unknown.
Although much is known about the oxidative metabolism of genistein in
vitro, little is known about the metabolism of genistein in tumorigenic and
nontumorigenic breast epithelial cells.

1.6- Cell cycle effects of genistein in cancer cells
Therapeutic agents that inhibit cancer cell proliferation, such as genistein, can
target the activity of key proteins controlling the cell cycle progression (Sartorelli et
al., 1986). Cell cycle regulatory proteins include cyclin-dependent kinases (CDK),
cyclin-dependent kinase inhibitors (p17, p21, p27), p53, signal transducer and
activator of transcription (STATs), and tyrosine kinases. As mentioned above,
genistein inhibits enzymes that promote cell proliferation, such as protein tyrosine
kinases (Boutin, 1994, Hunter et al., 1984) and DNA topoisomerases (Hartwell and
Kastan, 1994, Kiguchi et al., 1990). Tyrosine kinases catalyze the phosphorylation of
tyrosine residues in proteins involved in tumor cell signal transduction and
proliferation (Boutin, 1994, Hunter et al., 1984) and are involved in control of
10
mitogenesis, cell cycle regulation, cell survival, and cellular transformation via
growth factor binding.
Progression through the cell cycle is regulated by cyclin-dependent protein
kinases (CDKs), the activity of which is positively controlled by cyclins and
negatively controlled by CDK inhibitory proteins (CKIs), such as p21, p27, and p57.
One of the roles of CDKs in G1 is to phosphorylate the retinoblastoma gene product
(pRb) and to release the transactivator E2F, an essential factor for cell cycle
progression. Hence, compounds that inhibit CDK activity by upregulating CKI
expression may prove to be effective in arresting cell cycle, and, thus may have
utility as anticancer agents (fig 3) (Hartwell and Kastan, 1994). Indeed, genistein
has been shown to upregulate p21 protein levels in several cancer cell models (Shao
et al., 1998a).
As an inhibitor of CDK, p21 is a critical effector that integrates diverse
signals that impact on cell division, and causes cell cycle arrest. Its expression can
occur by p53-dependent and p53-independent pathways. Furthermore, p21
expression can be modulated in cancer cells by intracellular redox changes (Qiu et
al., 1996). The tumor suppressor p53 is also a cell cycle regulatory protein, and
leads to cell cycle arrest in G1 and the induction of apoptosis in response to DNA
damage (Constantinou et al., 1990). Expression of p21 in cancer cells may take
place in a p53-independent fashion resulting in inhibition of cell proliferation and
eventually apoptosis that is prevented by pRb (Qiu et al., 1998). Consequently,
induction of p21 by several proteins, including growth factors and p53, inhibits cell
cycle progression (Wu and El-Diery, 1996)). Thus, the modulation of p21 levels
11
appears to be an effective strategy to inhibit cancer cell proliferation, independent of
the presence of functional p53.

1.7- Study Objectives
Although there is an abundance of evidence on the effects of genistein in
many breast cancer cell types (Shao et al., 1998b, Shao et al., 1998c, Shao et al.,
1998a, Santell et al., 2000, Maggiolini et al., 2001, Fioravanti et al., 1998, Dampier
et al., 2001, Constantinou et al., 1998a), little is known about its effects in
nontumorigenic breast epithelial cells (Frey and Singletary, 2003, Frey et al., 2001,
Peterson et al., 1996, Upadhyay et al., 2001). It should also be recognized that these
Fig 3. Cell cycle phases and regulation. Progression through the cell cycle is
tightly controlled by phase-specific cyclin-dependent kinases (CDK), cyclin
dependent kinase inhibitors, and cyclins. (Taken from http://www-
rcf.usc.edu/~forsburg/cclecture.html)
12
studies were conducted with high micromolar concentrations, which exceeded the
level of free genistein in serum (Messina, 1999). Furthermore, the bioactivity of
genistein metabolite(s) in nontumorigenic compared with tumorigenic breast
epithelial cells have not been determined. Thus, this dissertation work describes
how differences in metabolism underlie the differential cellular effects of the
isoflavone genistein on tumorigenic and nontumorigenic breast epithelial cells. This
dissertation work documents a selective uptake and metabolism of genistein in
tumorigenic breast epithelial cells as compared with nontumorigenic breast epithelial
cells. In tumorigenic breast epithelial cells, a link between CYP450-mediated
oxidation of genistein to a bioactive metabolite of genistein that is involved in cell
cycle arrest was discovered, providing a direct link between a genistein metabolite
and its bioactivity that directly impacts cell cycle progression.
Due to conflicting evidence on the in vivo chemopreventive effects of
genistein, we used an in vitro model of oncogenic transformation to examine the
hypothesis that genistein can inhibit c-myc-mediated oncogenic transformation.
Specifically, the effects of genistein on anchorage-independent growth, a property of
transformed cells, were examined in pilot studies. Our preliminary data suggest that
genistein may effectively prevent anchorage-independent growth in soft agar, via
mechanisms that may involve modulation of cell cycle machinery and/or apoptosis.

13

1.8- Hypothesis
The overall hypothesis is that genistein exerts both antiproliferative and
chemopreventive actions.
1.8.1- Hypothesis I (Antiproliferative)
The hypothesis tested was that differences in the cellular responses between
tumorigenic and nontumorigenic breast epithelial cells to genistein are due to
differences in its metabolism within these respective cell lines. This idea was
examined in two steps: (1) by determining differences in uptake, metabolism, and
bioactivity, and (2) by comparing the effects on cell cycle progression and key
proteins that regulate cell cycle progression.

1.8.2- Hypothesis II (Chemopreventive)
The second idea that was explored was that genistein has chemopreventive
potential. The chemopreventive effects of genistein were explored with respect to its
ability to impact on anchorage-independent growth, a property of transformed cells.

14
Chapter 2- The intracellular genistein metabolite 5,7,3,4-
tetrahydroxyisoflavone mediates G2-M cell cycle arrest in breast cancer cells
via two distinct signaling pathways

2.0- Abstract
Epidemiological studies suggest that consumption of isoflavones is
associated with a decrease risk of breast cancer. However, intracellular actions of
genistein will be dependent upon cellular uptake and metabolism. We have
investigated the intracellular metabolism of genistein and effect on signaling
pathways in T47D tumorigenic and MCF10A nontumorigenic cells. Genistein
selectively induced growth arrest and G2-M phase cell cycle block in T47D
tumorigenic but not MCF10A non-tumorigenic breast epithelial cells. These anti-
proliferative effects were paralleled by significant differences in the association of
genistein and in particular its intracellular metabolism. Genistein was selectively
taken up into T47D cells and was subject to significant metabolism by CYP450
enzymes leading to the formation of both 5,7,3,4-tetrahydroxyisoflavone (THIF) and
two glutathionyl conjugates of THIF. THIF inhibited cdc2 activation via two distinct
routes suggesting that this species may mediate the cellular actions of genistein.
THIF led to the activation of p38 and subsequent inhibition of cyclin B1 (Ser 147)
and cdc2 (Thr 161) phosphorylation, two events critical for the correct functioning of
the cdc2-cyclin B1 complex. We suggest that the formation of THIF mediates the
cellular actions of genistein in tumorigenic breast epithelial cells by the activation of
p38 signaling pathway.
15
2.1- Introduction
Epidemiological studies suggest that high dietary soy intake is associated
with a decrease risk of breast cancer (Bradlow, 2005; Barnes, 1997; Lamartiniere,
2000). Specifically isoflavones such as genistein are believed to be the key
components that exhibit anti-carcinogenic effects in breast, colon and prostate cells
(Barnes, 1995; Lambert et al., 2005; Magee & Rowland, 2004). Genistein has been
shown to inhibit tyrosine kinase (Akiyama et al., 1987), DNA topoisomerase
(Constantinou et al 1995), and angiogeneis (Fotsis et al., 1995), bind to peroxisome
proliferator-activated receptor gamma receptors (Dang et al, 2003) and induce stress
response genes (Zhou et al., 1998). However, the precise cellular mechanism of
action of genistein in vivo is likely to be dependent on the cellular uptake and
metabolism of this isoflavone. Although some studies suggest that isoflavones such
as genistein accumulate in breast tissue (Chang et al., 2000), the relative uptake and
metabolism of genistein in tumorigenic and non-tumorigenic breast epithelial cells is
not known. Thus, many reported effects based on in vitro experiments are difficult
to extrapolate to in vivo situations. Moreover, the influence of metabolism on the
bioactivity of genistein is also not well understood. Although studies have
demonstrated that genistein may be oxidatively metabolized (Kulling et al. 2000;
Kulling et al. 2001; Kulling et al. 2002; Roberts-Kirchhoff, et al., 1999), sulfated and
glucuronidated (Doerge et al., 2000), or hydroxylated and methylated (Peterson, et
al., 1996), possible biologically active genistein metabolites remain unidentified.

16
Genistein has been shown to evoke G2-M cell cycle arrest in cancer cell lines
(Cappelletti et al., 2000; Constantinou et al. 1998). Mechanisms for genistein-
mediated modulation of cell cycle may involve inhibition of cdc2 activity
(Casagrande & Darbon, 2000,), induction of the cell cycle inhibitor p21cip1/waf1
(Kuzumaki et al., 1998) and inhibition of cdc25C protein expression (Frey et al.,
2000). More recently, genistein has also been linked with the activation of p38 and
inactivation of ERK1/2 in human mammary epithelial cells (Frey & Singletary,
2003), indicating that genistein may induce its cellular effects via modulation of
MAP kinase signaling cascade. Recent investigations have indicated that the
modulation of MAP kinase signaling is important in mediating the effects of another
group of polyphenols, the flavonoids (Schroeter et al., 2002). For example,
epicatechin and its 3′-O-methylated metabolite protect primary striatal neurons
against oxidized LDL-induced death by potently inhibiting oxidized LDL-induced
activation of JNK1/2, c-jun and caspase-3 (Schroeter et al., 2001) and quercetin
induces neuronal death via inhibition of neuronal survival signaling, that includes
both Akt/PKB and ERK pathways (Spencer et al., 2003). These data suggest that
flavonoids and isoflavones might exert many cellular effects via direct interactions
with signaling proteins, independent of their antioxidant ability.
There has been much attention regarding the effects of genistein on the
proliferation of malignant breast cells (Fotsis et al, 1995; Constantinou et al., 1998).
However, less is known regarding its effects on breast epithelial cells at the earlier
stages of the oncogenic process and if there is a differential effect of genistein on
these two types of cells. In the study described here, we have investigated the
17
mechanisms underlying the growth inhibition effects of genistein in breast epithelial
cells and human breast carcinoma cells. We investigated the effects of genistein in
terms of its differential intracellular metabolism in these two cell types and its ability
to modulate signaling pathways pivotal in cell cycle control. We identified a novel
intracellular genistein metabolite that is generated specifically in tumorigenic breast
epithelial cells and provide evidence that this metabolite may mediate the anti-
carcinogenic effects of genistein in breast cancer cells.

2.2- Materials and Methods
Reagents- Specialized chemicals used were obtained from Sigma Chemical
Company (Poole, Dorset, UK): sulforhodamine B, cimetidine, caffeine, mammalian
protease inhibitor cocktail. Genistein was purchased from Extrasynthese (Genay
Cedex, France). Primary antibodies used: anti-ACTIVE JNK pAb (Promega,
Madison, MI, USA); anti-ACTIVE-p38 pAb, anti-phospho-cyclin B1 (Ser 147) pAb,
anti-phospho-cdc2 (Thr 161), anti-phospho p53 (Ser 15) pAb, anti-phospho Chk1
pAb, anti p38, pAb, or anti cyclin B1 pAb (Cell Signaling Technology, Beverley,
MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody
(Sigma) and enhanced chemiluminescence reagent (ECL) and Hyperfilm-ECL were
purchased from Amersham (Little Chalfont, UK). SB203580 was obtained from
Tocris Bioscience (Avonmouth, UK). Specialized HPLC solvents were purchased
from Rathburn (Walkerburn, UK) and HPLC columns were from Waters (Watford,
UK). All other reagents used were of the analytical grade and obtained from Sigma.
18
Cell culture- T47D breast cancer cells were cultured in RPMI (Gibco BRL,
MD, USA) with 10% fetal bovine serum (Gemini) and 1 % penicillin and
streptomycin (Hyclone Laboratories, UT, USA) in 5% carbon dioxide-air at 37
0
C.
Cells were plated onto 10 cm dishes. Non-tumorigenic breast epithelial MCF10A
cells were cultured in DMEM/F12; 5% (v/v) Horse Serum (Gemini, USA, 2.5 mM
HEPES (Sigma, USA), 2 mM L-glutamine (Sigma, USA), 100 U/ml penicillin
(Gibco), 100 µg/ml streptomycin (Gibco), 20 ng/ml EGF (Sigma), 100 ng/ml cholera
toxin (Sigma), 10 µg/ml bovine insulin (Sigma), 500 ng/ml hydrocortisone (Sigma).
All cells were grown to 70-75 % confluence, and were treated with genistein (Sigma,
USA) [0.3, 1.0, 3.0, 10, and 30 µM] for 0.5, 1, 2 or 24 h depending on the
experiment. In cell cycle progression studies, cells were also challenged with
genistein with or without 250 µM of a general CYP450 inhibitor cimetidine (Sigma).
Cell-association studies- The cell-associated levels of genistein and possible
genistein metabolites from T47D tumorigenic and MCF10A nontumorigenic breast
epithelial cells was assessed following 2 and 24 h incubation. After exposure, cells
were washed 3 times in 30 mL ice cold PBS before lysing with aqueous acidified
methanol. The lysed cells were scraped from the plates, collected, vortexed, and
centrifuges (5000 rpm for 10 min at 4
o
C). The supernatant was recovered and
analyzed by HPLC with photodiode array detection as previously described (Spencer
et al., 2001). HPLC analysis was performed on a Agilent 1100 system with a Nova-
Pak C18 column (250 mm x 4.6 mm i.d., 4 µm) and guard column (15 x 4.6 mm i.d.,
4 µm). Mobile phase A consisted of methanol/water/5N HCl (5/94.9/0.1 v/v/v) and
mobile phase B of acetonitrile/water/5N HCl (50/49.9/0.1 v/v/v). The following
19
gradient system was used (min/% acetonitrile): 0/0, 5/0, 40/50, 60/100, 65/100, and
65.1/0 with a flow rate of 0.7 ml/min. The eluent was monitored by photodiode array
detection at 220 nm for NDMA detection and 280 nm for 3-NT measurements with
spectra of products obtained over the 220-600 nm range. Protein concentration of
lysate was determined using the Bradford method. Quantification of cell-associated
genistein was achieved by use of an authentic standard (retention time and unique
spectral characteristics) and expressed as ng/mg protein.
Synthesis of 5,7,3,4-tetrahydroxyisoflavone and glutathionyl conjugates of
genistein-Synthesis of glutathionyl adducts was achieved using a procedure similar
to that previously reported (Spencer et al., 2003). GSH (0.4 g) and 20 mg of
mushroom tyrosinase (2000 units/ml) were incubated in ammonium acetate buffer
(0.1 M), pH 5.8, 25 °C (total volume of 50 ml) before a drop-wise addition of
genistein (0.1 g) under constant stirring for 2 hours. Resulting products were
analyzed by analytical HPLC with diode array detection. 5,7,3,4-
Tetrahydroxyisoflavone was synthesized in a similar manner to glutathione adducts,
although here no GSH was added to the reaction mixture. Sample mixtures were
filtered through a 0.22 µm Centriprep

particle separator (Amicon, UK), and de-
protonated by addition of an equal volume of ice-cold methanol. The de-protonated
preparation was rotary-evaporated to remove the methanol and then freeze dried.
The dried residue was dissolved in 10 ml of aqueous methanol (50% v/v) and 2 ml
fractions were purified by preparative RP-HPLC, using a Zorbax ODS (21.2 × 250
mm) (HPLC Technology, Macclesfield, Cheshire, UK), eluted isocratically at a flow
rate of 4 ml/min with 6 % (
v
/v) methanol in 10 mM ammonium acetate buffer pH
20
4.7. Fractions containing 5,7,3,4-tetrahydroxyisoflavone were lyophilized, and
dissolved in methanol (50 %) to obtain a 10 mM stock solution for use in cell
experiments.
Characterization of 5,7,3,4-tetrahydroxyisoflavone (THIF) and GSH
conjugates was performed by LC-MS and
1
H NMR. Electrospray mass spectroscopy
was performed using a LCQ Deca XP quadrupole ion-trap mass spectrometer
(ThermoFinnigan, San Jose, CA). The identity was confirmed by the molecular
mass of the [M+H
+
]
+
ion and the specific fragments generated by low energy
collision induced dissociation.
1
H NMR

spectra were recorded using a Bruker DRX
500 spectrometer with an operating frequency of 500.13 MHz (Bruker Biospin,
Rheinstetten, Germany). The samples were dissolved in deuterated DMSO
(Cambridge Isotopes, Cambridge, UK).
Assessment of cellular proliferation- Exponentially growing T47D and
MCF10A cells at 70-75 % confluence were exposed to genistein or synthesized
5,7,3,4-tetrahydroxyisoflavone (0-30 µM) for 48h. Growth inhibition elicited by
THIF was evaluated by analysis of cell biomass determined by the sulforhodamine B
assay. Cells were fixed by addition of 25 µl ice-cold acetic acid at 4°C for 1h and
the total biomass was determined by staining with sulforhodamine. An increase or
decrease in the number of cells (total biomass) results in a concomitant change in the
amount of dye incorporated by the cells in the culture, which was measured at 490
nm, and subtracted from the background absorbance at 690 nm, using a Tecan
GENios micro-plate photometer (Tecan, Reading, UK). Results are plotted as
21
percentage of growth inhibition, which was calculated as follows: ODcells exposed
to vehicle - ODcells exposed to flavanols/ ODcells exposed to vehicle x 100.
Cell cycle analysis- Exponentially growing cells at 70-75 % confluence were
treated with genistein or synthesized 5,7,3,4-tetrahydroxyisoflavone (0.3-30 µM).
Cells were harvested by typsinization at 24 h, pelleted by low-speed centrifugation,
re-suspended in 200 µl of citrate buffer: 250 mM sucrose (250 mM), trisodium
citrate (40 mM) DMSO (5%); pH 7.6. Before staining, cells were treated with
RNase A (0.1 mg/ml). Following fixing, nuclei were stained with propidium iodide.
All solutions were prepared in buffer containing: trisodium citrate (3.4 mM), NP-40
(0.1%), spermine hydrochloride (1.5 mM), Tris-base (0.5 mM); pH 7.6. DNA
content in each cell nucleus was determined in a FACScan flow cytometer (Becton-
Dickinson, San Jose, CA) and populations of G0/G1, S, and G2/M were quantified
using Cellquest software as previously described (Qiu et al., 1998).
Immunoblotting- Following exposures, cells were washed with ice-cold PBS
(+ EGTA 200 µM) and lysed on ice using Tris (50 mM), Triton X-100 (0.1%), NaCl
(150 mM) and EGTA/EDTA (2 mM), containing mammalian protease inhibitor
cocktail (1:100 dilution), sodium pyrophosphate (1 mM), PMSF (10 µg/ml), sodium
vanadate (1 mM) and sodium fluoride (50 mM). Lysed cells were scraped and left
on ice to solubilize for 45 min. Lysates were centrifuged at 1,000 xg for 5 min at
4
o
C to remove unbroken cell debris and nuclei. Protein concentration in the
supernatants was determined by the Bio-Rad Bradford protein assay (Bio-Rad).
Samples were incubated for 5 min at 95°C in boiling buffer (final conc. 62.5 mM
Tris, pH 6.8, 2 % SDS, 5 % 2-mercaptoethanol, 10 % glycerol and 0.0025 %
22
bromophenol blue). Boiled samples (20-30 µg/lane) were run on 9 -12 % SDS-
polyacrylamide gels and proteins were transferred to nitrocellulose membranes
(Hybond-ECL; Amersham) by semi-dry electroblotting (1.5 mA/cm2). The
nitrocellulose membrane was then incubated in a blocking buffer (20mM Tris, pH
7.5, 150 mM NaCl; TBS) containing 4% (w/v) skimmed milk powder for 30 min at
room temperature followed by 2 x 5 min washes in TBS supplemented with 0.05 %
(v/v) Tween 20 (TTBS). Blots were then incubated with either anti-ACTIVE JNK
pAb (1:5000), anti-ACTIVE-p38 pAb (1:2000), anti-phospho-cyclin B1 (Ser 147)
pAb (1:1000), anti-phospho-cdc2 (Thr 161) (1:500), anti p38, pAb (1:5000), or anti
cyclin B1 pAb (1:1000) in TTBS containing 1% (w/v) skimmed milk powder
(antibody buffer) overnight at room temperature on a three dimensional rocking
table. The blots were washed 2 x 10 min in TTBS and then incubated with goat anti-
rabbit IgG conjugated to HRP (1:1000 dilution), rabbit anti-sheep IgG conjugated to
HRP (1:2000 dilution), or goat anti-mouse IgG conjugated to HRP (1:3000 dilution,
Upstate), in antibody buffer for 60 min. Finally the blots were washed 2 x 10 min in
TTBS rinsed in TBS and exposed to ECL-reagent for 1-2 min (Amersham, UK).
Blots were exposed to Hyperfilm-ECL

(Amersham, UK) for 2-5 min in an auto-
radiographic cassette and developed. Bands were analyzed using BioRad Quantity
One 1-D Analysis software. Molecular weights of the bands were calculated from
comparison with pre-stained molecular weight markers (MW 27,000 – 180,000 and
MW 6,500 - 45,000, Sigma) that were run in parallel with the samples. The equal
loading and efficient transfer of proteins was confirmed by staining the nitrocellulose
with Ponceau Red (Sigma, UK).
23
Analysis of intracellular oxidative stress and cellular GSH- The effects of the
THIF on the intracellular redox status was performed using 2′,7′-
dichlorodihydrofluorescin diacetate (DCDHF-DA) (Wang & Joseph, 1999). THIF
(0.3-30 µM; 2 h) exposed T47D cells and control cells (MeOH) were washed twice
with HBM and loaded with DCDHF-DA (100 µM) for 30 minutes at 37°C. Cultures
were then washed thoroughly with PBS and cellular fluorescence (ex. 500 nm; em.
530 nm) was monitored using a CytoFluor

fluorescent multi-well plate reader
(PerSeptive Biosystems, USA). Changes in intracellular ROS levels were expressed
as fold increase over control cells. GSH was detected according to the method
described by Reed et al. (Reed et al;, 1980) with modifications. This method is
based on the derivatisation reaction between glutathione and iodoacetic acid to form
5-carboxymethyl glutathione which complexes with dinitrofluorobenzene and can
therefore be detected by HPLC with ultraviolet detection (Sian et al., 1997). Cells
were washed twice and scraped on ice with 1ml PBS and then centrifuged (1500 rpm
for 10 min at 4°C). Trichloroacetic acid (5 %) containing 0.2 mM desferal was
added to each sample. After vortexing and centrifuging at 12000 rpm for 5 min at
4°C, 180 µl of supernatant was transferred to an eppendorf that contained 50 mg of
NaHCO
3
. Following addition of 45 µl of 108 µM iodoacetic acid, the mixture was
vortexed and incubated in the dark for one hour after which 180 µl of Sanger reagent
was added. The samples were then vortexed, incubated for at least 12 h and
centrifuged at 12000 rpm for 10 min at 4°C prior to analysis by reverse phase HPLC
using a mobile phase consisting of: (A) aqueous methanol (75 %) and (B) 2 M
24
sodium acetate pH 4.6 - methanol 36:34 v/v. Separation was achieved using the
following gradient system: Flow rate: 1.2 ml/min. Starting conditions: 25 % phase B
followed by a 30 min linear gradient to 95 % phase B followed by 5 min linear
gradient to 25 % phase B.
Statistical Analysis- Data were expressed as mean + SD. Statistical
comparisons were made using an unpaired, two-tailed Student’s t-test with a
confidence level of 95 %. Significance level was set at p<0.05.

2.3- Results
Inhibition of growth and cell-association of genistein -The ability of
genistein to influence the growth of tumorigenic T47D and non-tumorigenic
MCF10A breast epithelial cells was investigated using two distinct assay systems.
The growth of T47D cells, derived from a human ductal carcinoma, was markedly
inhibited by genistein (0.3-30 µM), as indicated by reductions in cell biomass
determined by the sulforhodamine B assay. In contrast, the proliferation of human
mammary epithelial

cells, MCF10A, was only inhibited at higher concentrations (3-
30 µM) and to a much lesser degree than that observed in T47D cells (Fig. 4A). In
agreement with these observations, genistein (0.3-30 µM) induced significant G2/M
cell cycle arrest in T47D cells but not in MCF10A cells (Fig. 4B). The percent of
cells in the G2-M phase increased from 40.6 % in control cells to 60.4 % in those
exposed to genistein (30 µM). In contrast, no G2/M block was observed in MCF10A
cells treated with genistein at any concentration. There was no significant change in
the % of cells in the S phase following any of the treatments.
25
In order to understand the differential effect of genistein on these two cell
lines we investigated the cellular association and metabolism of genistein. Typical
HPLC chromatograms obtained from cell lysates of genistein-treated T47D cells
showed the presence of genistein (RT: 60.3 min) and three major metabolites; the
latter were more polar than genistein and had similar spectral characteristics (Fig.
5A). In MCF10A cells, genistein association was also observed, although only one
of the metabolites that were present in T47D cell was observed (53.6 min) (Fig. 5B).
β-Glucuronidase and sulphatase treatment of cellular lysates did not result in the
cleavage and disappearance of any of the metabolites observed, thus indicating that
neither genistein glucuronide nor genistein sulphate was present in the cells.
Genistein association with T47D cells was greater than that observed with MCF10A
cells following 2h of exposure (Fig. 5C). However, at 24h of exposure the levels of
genistein were significantly reduced in T47D cells suggesting that genistein was
subject to intracellular metabolism as is observed in Fig. 5A. Indeed, levels of
genistein associated with T47D cells at 24 h were not significantly different to that
measured in MCF10A cells.

26
Fig. 4: Anti-proliferative effects of genistein on the proliferation of T47D and
MCF10A cells. (A) % Growth inhibition induced by genistein (0.1-30 µM) in T47D
cells (white), and MCF-10A cells (black) as assessed by the sulforhodamine B assay.
Results are expressed as growth inhibition and were obtained from three independent
experiments performed in triplicate and presented as mean ± SD. Significant
increases between vehicle (MeOH) and treatments are indicated by *** p < 0.001; **
p < 0.01; *p < 0.05. (B) Genistein-induced G2-M cell cycle arrest in T47D ( ) and
MCF10A ( z ) cells. Exponentially growing cells at 70-75 % confluency were
exposed to genistein (0.3-30 µM). Cells were trypsinized, pelleted and collected
prior to fixing/digestion with RNase. Cellular DNA was stained with PI, and
analyzed by flow cytometry at 24 h after genistein exposure. Results are expressed
as % of cells in G2-M phase of the cell cycle 24 h post genistein exposure and were
obtained from three independent experiments performed in duplicate and presented
as mean ± SD. Significant increases between vehicle (MeOH) and treatments are
indicated by *** p < 0.001; *p < 0.01

27

Genistein (µM)
0.1 0.3 1 3 10 30
% Growth
Inhibition
0
20
40
60

T47D
MCF10A
**
***
***
***
***
*
**
***
A
% Cells
in
G2/M
*
**
**
Genistein (µM)
0. 3 3
B
3
4
5
5
7
Fig 4: Continued
28
Fig. 5: Cellular association of genistein with T47D cells and MCF10A cells. Typical
HPLC traces shown with UV-visable spectral data of cell lysates from cells exposed
to genistein (10 µM) for 24h. Panel A: T47D cells; Panel B: MCF10A cells.
Genistein elutes at a retention time of 60.2 min. Three major metabolites are
detected at 42.9, 53.7 and 54.8 minutes. C) Quantitative association (ng/mg protein)
of genistein with T47D and MCF10A cells. Cells were exposed to genistein (0.3, 3,
30 µM) for 2 or 24h after which cells were lysed, de-protonated and analyzed by
HPLC-PDA. T47D cells (white); MCF10A cells: (black). Resulted are represented
as mean + SD of 4 separate experiments performed in duplicate. Significant
decrease in genistein association between 2h and 24h in T47D cell are indicated by
*** p < 0.001; **p < 0.01.

29

C
(ng/mg protein)
2h 24h
0.3 3 30 0.3 3 30
0
500
1000
1500
2000
2500
3000
3500
T47D
MCF10A
***
**
A
4 4 4 52 5 60 6
0.0
0.0
0.0
A
60.
53.
54.
42.
n
30
40
50
42. 53. 60. 54.
Retention Time
Retention Time
B
A
0.0
0.0
0.0
60.
53.
n
30
40
50
53. 60.
4 4452 5 60 6
Fig 5: Continued
30
Identification of intracellular genistein metabolites- In order to obtain
structural information regarding the genistein metabolites, lysates initially underwent
LC-MS/MS analysis. The most polar product at 42.9 minutes, which was unique to
T47D cells, had an m/z ratio of 287.0 (positive ion mode) suggesting that the
metabolite was a hydroxylated form of genistein (MW: 270.2) (Fig. 6A). To further
characterize this metabolite and to determine the position of hydroxylation, the peak
at 42.9 minutes was collected from analytical HPLC runs and subjected to NMR
analysis. 1H-NMR provided further evidence of hydroxylation and indicated that the
addition of an –OH group took place at the C3 position of the B-ring: Genistein:
1
H-
NMR (DMSO- K 6) K (ppm) 5.91 (s, H6), 5.94 (s, H8), 6.61 (d, H3), 6.63 (d, H5),
7.11 (d, H2), 7.15 (d H6), 7.71 (s, H2); Hydroxylated-genistein:
1
H-NMR (DMSO-
K 6) K (ppm) 5.90 (s, H6), 5.96 (s, H8), 6.48 (d, H5), 6.69 (d, H2), 6.74 (d H6), 7.67
(s, H2). MS and
1
H-NMR data led to this metabolite being characterized as 5,7,3,4-
tetrahydroxyisoflavone (THIF). This hydroxylated form of genistein was not
observed in MCF10A cells following genistein treatment and was proposed to result
from its oxidative metabolism by CYP450 enzymes present in T47D cells as
discussed later.
The T47D cell metabolites at 53.7 min and 54.8 min (Fig. 5A) had identical
retention times and spectral characteristics to two synthesized glutathionyl
conjugates formed during the reaction of genistein with tyrosinase in the presence of
GSH (Fig. 6B; solid line). This reaction led to the formation of three new peaks
(50.9, 58.8 and 54.9 min), two of which were present in T47D cell lysates (Fig. 6B;
31
dashed line) and one in MCF10A cells (Fig. 6B). Such conjugates may result from
the nucleophilic addition of the thiolate for of GSH (GS
-
) on 5,7,3,4-
tetrahydroxyisoflavone following its intracellular oxidation to an o-quinone. Similar
reactions have been reported following the intracellular accumulation and oxidative
metabolism of the flavonol quercetin where a 2-glutathionyl quercetin adduct was
formed (Spencer et al., 2003). HPLC data suggested that the intracellular formation
of glutathionyl-THIF adducts may occur in both cell types (Fig. 5A and B), although
to a greater extent in T47D cells. The LC retention time, spectral characteristics and
the MS fragmentation spectrum (Fig. 6C) of the peak at 53.7 min (m/z of [M+H
+
]
+
:
592.1) allowed characterization of the peak as a predicted glutathionyl-genistein
adduct. Fragment ion scans of this compound confirmed the presence of a
glutathionyl moiety, although they were unable to provide information regarding the
position of conjugation.
1
H-NMR provided confirmation that the major metabolite at
53.7 min was 5-S-glutathionyl-THIF:
1
H-NMR (DMSO- K 6) K (ppm) 5.97 (s, H
6
),
5.99 (s, H
8
), 6.39 (d, H
2
| ), 6.60 (d H
6
| ), 7.54 (s, H
2
) (information regarding GSH
proton shifts have been removed to aid viewing). It is likely that the other conjugate
in T47D cells (Figure 5A; 54.8 min) is a THIF conjugates in the C2 or C6 positions
of the B-ring, although we have not determined the precise structure of this
component.

32
Fig. 6: Determination of genistein metabolites. (A): Mass spectrum of 5,7,3,4-
tetrahydroxyisoflavone (THIF). Spectrum shows the molecular ion ([M + H+)+ of
THIF (molecular weight: 286 Da). The structure of the conjugate is shown after
further confirmation by 1HNMR. (B): Co-elution of synthesized glutathionyl
conjugate of genistein with T47D intracellular genistein metabolites. Panel C: MS of
5-S-glutathonyl-THIF. (C): LC-MS/MS spectrum of the peak at 53.7 min relating to
a 5-S-glutathionyl THIF conjugate. The fragment ion spectrum of the conjugate is
recorded at collision energy of 35%. The inserted spectrum shows the molecular ion
([M+H+]+) of the conjugate (molecular weight 575 Da). The structure of the
conjugate is shown after further confirmation by 1HNMR.
33
Retention Time (min)
20
40
60
80
60
53
54
50
B

40 44 48 52 56 60 64
50.9
53.8
54.9
nm
300 400 500
A
0 100 200 300 400
0
20
80

60
100
m/z
153.1
287
288.1
240.9
Peak at 42.9
min
MW: 286
O
O
OH
HO
OH
OH
C
O
O
OH
HO
OH
OH
NH
HN
NH 2
O
S
O
O
OH
OOH
K
z2
b2
y2
317
462
517.1
446.1
k
z2
y2
b2
Peak at 53.7
min
MW: 575
10
592.
593.
594.
200 300 400 500 600
0
20
40
60
80
100
Relative
Density
mAU
Relative
Density

40
Fig 6: Continued
34
In order to quantify these products the synthesis and purification of THIF and
the 5-S-glutathuionyl-THIF were carried out. Both THIF and glutathionyl
conjugates of THIF (quantified using 5-S-GSH-THIF as standard) were present in
higher amounts in T47D cells than in MCF10A cells (Fig. 7A). In particular, THIF
was only detected in T47D cells and was observed to maintain its concentration
within cells, being at a higher concentration than genistein at 24h (Fig. 7A). The
greater accumulation of metabolites in T47D cells is likely to be linked to the greater
amount of genistein uptake in these cells and/or due to a greater extent of genistein
metabolism. It does not appear to be due to impaired export of metabolites from
T47D cells as we also measure high amounts of glutathionyl conjugates in the
medium of T47D cells (Fig. 7B). Therefore, it seems that T47D cells accumulate and
metabolize genistein to a greater extent than MCF10A cells, yielding in particular the
unique metabolite THIF.

Effects of cimetidine on genistein-induced G2-M phase cell cycle arrest in
T47D cells- To determine if the formation of THIF and glutathionyl conjugates is
dependent on the action of CYP450 in cells (Breinholt et al., 2003), we utilized the
general CYP450 inhibitor cimetidine. In T47D cells pre-treated with cimetidine
(250 µM), large reductions in the formation of both metabolites were observed (Fig.
8A), suggesting that CYP450 enzymes mediate the formation of THIF and
subsequent glutathione conjugates. Cimetidine was also effective at reversing G2-M
cell cycle arrest induced by genistein (Fig. 8B), thus supporting the notion that the
growth arrest induced by genistein is dependent on the formation of THIF in T47D
35
cells. Consistent with this, in MCF10A cells, no G2-M cell cycle arrest was
observed in response to genistein (Fig. 4B) and no THIF was detected (Fig. 7A).
These data suggest that the selective uptake and metabolism of genistein by CYP450
in T47D tumorigenic breast epithelial cells leads to the formation of a possible
bioactive form of genistein, identified as THIF.
36

Fig. 7: (A) Quantitative association (ng/mg protein) of genistein, THIF and
glutathionyl-THIF conjugates with T47D and MCF10A cells. Cells were exposed to
genistein (3 µM) for 2, or 24 h after which cells were lysed, de-protonated and
analyzed by HPLC-PDA. Quantification of the THIF was achieved using a
synthesized standard and all glutathionyl conjugates were quantified using 5-S-
glutathonyl-THIF as a standard (see Materials and Methods). Genistein (white bars);
Glutathionyl conjugates (black bars); THIF (white and black striped bars). (B)
Medium levels of glutathionyl-THIF conjugates (µM) following genistein
administration to T47D and MCF10A cells. Cells were exposed to genistein (0.3 to
30 µM) and medium levels of conjugates were assessed at 24 h by HPLC-PDA
analysis. Black columns: T47D cells, White columns: MCF10A cells.
37
Fig 7: Continued
B
0.3 1 3 10 30
Medium
concentration
[mM]

Genistein (µM)
0
1
2
4
5
T47D
MCF10A
A
Genistein
GSH conjugates
THIF
2h 24h
T47D T47D MCF10A MCF10A
0
100
200
300
400
500
600
(ng/mg
protein)
Cell
Association
38
Fig. 8: Effects of the CYP450 inhibitor cimetidine on G2-M cell cycle arrest and
genistein metabolite formation in T47D cells. (A) Effect of cimetidine on the
intracellular formation of THIF and glutathionyl-THIF conjugates in T47D cells.
Cells were exposed to genistein (3 µM) for 24 h after which cells were lysed, de-
protonated and analyzed by HPLC-PDA. Quantification of the THIF was achieved
using a synthesized standard and all glutathionyl conjugates were quantified using 5-
S-glutathonyl-THIF as a standard (see Materials and Methods). Glutathionyl
conjugates (white bars); THIF (black bars). Results are expressed as cell associated
levels (ng/mg protein) 24h were obtained from three independent experiments
performed in duplicate and presented as mean ± SD. Significant decreases between
genistein and genistein and cimetidine treatment are indicated by *** p < 0.001; (B)
The influence of cimetidine on genistein-induced G2-M cell cycle arrest in T47D
cells. Exponentially growing cells at 70-75% confluency were exposed to genistein
(0.3-30 µM). Cells were trypsinized, pelleted and collected prior to fixing/digestion
with RNase. Cellular DNA was stained with PI, and analyzed by flow cytometry at
24 h after genistein exposure. Results are expressed as % of cells in G2-M phase of
the cell cycle 24 h post genistein exposure and were obtained from three independent
experiments performed in duplicate and presented as mean ± SD. Significant
increases between vehicle (MeOH) and treatments are indicated by *** p < 0.001; *p
< 0.05.

39
Fig 8: Continued
Control Gen Cim Gen (30)
+ Cim
G
S
G2-M
B
**
**
0
10
20
30
40
50
60
70
0
50
100
150
200
250
300
A
Cell
Association
(ng/mg
protein)
Genistein Genistein +
Cimetidine
**
**
GSH conjugates

THIF
Cell
population
(%)
40
Effects THIF on T47D cell growth and phosphorylation state of cdc-2 - To
further investigate whether THIF, may mediate the anti-proliferative effects of
genistein we studied its direct cytotoxic properties in both T47D and MCF10A cells.
We obtained pure THIF following synthesis using tyrosinase and semi-preparative
HPLC of the reaction mixtures (see Materials and Methods section). Growth of both
T47D and MCF10A cells was markedly inhibited by THIF (0.3-30 µM) (Fig. 9A).
Significant inhibitory effects were observed at 0.3 µM and above and these effects
were more potent than those seen with similar concentrations of genistein (Fig. 4A).
Genistein (3 µM) inhibited growth of T47D cells by 29.9% + 3.8%, whilst THIF (3
µM) inhibited growth by 45.6% + 3.4%. Interestingly, THIF induced strong growth
inhibition in MCF10A cells, whilst genistein was able to induce only mild growth
arrest in the same cell line (which, apparently failed to metabolize genistein to THIF:
genistein (3 µM): 3.6% + 1.2%; THIF (3 µM): 38.7% + 3.2%. This strengthens the
notion that the intracellular formation of THIF from genistein is necessary for
growth arrest to occur.
THIF (0.1-30 µM) was also observed to induce potent G2-M cell cycle arrest
in T47D cells (Fig. 9B; dashed line). Again these inhibitory effects were more
potent than those seen with genistein at similar concentrations (Fig. 4B). To begin
exploring a potential mechanism by which THIF exerts its anti-proliferative effects,
we initially examined its effect on the activation state of cdc2 kinase, a major kinase
involved in G2-M cell cycle control and the entry of all eukaryotic cells into mitosis
(Norbury & Nurse, 1992; Galaktionov et al., 1995). The influence of THIF on cdc2
41
phosphorylation in T47D cells was investigated by immunoblotting of cellular
homogenates with an anti-phospho cdc2 polyclonal antibody that detects cdc2 when
it is phosphorylated at Thr161, an event known to be important for full activation of
the kinase (Norbury & Nurse, 1992). The strong inhibition of proliferation and G2-
M block observed in T47D cells correlated with de-phosphorylation of cdc2 (Thr
161) at 2h post THIF exposure (Fig. 9B).
42
Fig. 9: Anti-proliferative effects of 5,7,3,4-tetrahydroxyisoflavone (THIF). (A) %
Growth inhibition induced by THIF (0.1-30 µM) in T47D cells (dark gray), and
MCF-10A cells (light gray) as assessed by the sulforhodamine B assay. Results are
expressed as growth inhibition and were obtained from three independent
experiments performed in triplicate and presented as mean ± SD. Significant
increases between vehicle (MeOH) and treatments are indicated by *** p < 0.001; *p
< 0.05. (B) Inhibition of cdc2 phosphorylation in T47D cells exposed to THIF.
Crude lysates (30 µg) prepared from T47D cells exposed to vehicle (MeOH), or
THIF (0.3-30 µM) for 1h were immunoblotted with an antibody that specifically
recognizes phosphorylated cdc2 (Thr 161). The same crude lysates (30 µg) were
immunoblotted with an antibody that recognizes total levels of cdc2. Blots are
representative blots of three independent experiments on different cultures that
yielded similar results. Data obtained from immunoblot experiments in (B) were
analyzed using BioRad Quantity One 1-D Analysis software ( and solid line). Also
plotted is % of cells in G2-M phase of the cell cycle analysis ( z and dashed line).
Results are expressed as % of cells in G2-M phase of the cell cycle 24h post THIF
exposure and were obtained from three independent experiments performed in
duplicate and presented as mean ± SD. Significant increases between vehicle
(MeOH) and treatments are indicated by *** p < 0.001; *p < 0.05.
43
A
5,7,3,4-tetrahydroxyisoflavone (µM)
0
25
50
75
100
T47D
MCF10A
0.1 0.3 1.0 3.0 10.0 30.0
*
***
***
***
***
B
(Thr 161)
C10 0.3 1 3 30 THIF (µM)
C10 0.3 1 3 30
% Cells in
G2-M at 24 h
.
***
***
***
0.2
0.4
0.6
0.8
1.0
1.2
1.4
40
50
60
70
80
*
*
C10 13 30
***
***
***
0.2
0.4
0.6
0.8
1.0
1.2
1.4
40
50
60
70
80
*
*
Total cdc2
THIF [µM]
% Growth
Inhibition
Phospho-cdc2
Phospho
-cdc2
Relative
Band
Intensity
at 0.5 h
(n)
Fig 9: Continued
44
Effects of THIF on activation of MAP kinase signaling and cyclin B1 in T47D
cells- As there was a clear inhibition of cdc2 activation in response to THIF
exposure, we were interested in determining upstream events that may lead to the
deactivation of cdc2. JNK has been implicated in pro-apoptotic signaling in various
cell types and consequently we investigated the ability of THIF to modulate the
phosphorylation of this kinase. The phosphorylation state of JNK was investigated
by immunoblotting of neuronal homogenates with anti-active JNK1/2 antibody. This
antibody detects JNK when it is dually phosphorylated within the Thr
138
-Pro-Tyr-
185

motif (pTPpY) in the catalytic core of active JNK. Treatment of T47D cells with
THIF (0.3-30 µM) for 0.5 h resulted in no significant measurable alteration in the
relative intensities of the two immuno-detectable bands, corresponding to the
activated JNK isoforms, JNK (54 kDa and 46 kDa) as compared with basal levels
(Fig. 10A). In contrast, the phosphorylation state of p38, which was probed using a
phospho specific antibody, which recognizes the dually phosphorylated motif Thr
180
-
Pro-Tyr
182
within activated p38, was observed to increase in a dose-dependent
manner in response to THIF (0.3-30 µM; 0.5h) treatment (Fig. 10A). Parallel
immunoblots with an antibody that detects total levels (non-phosphorylated and
phosphorylated p38) indicated that there were no changes in total p38.
As p38 is known to be able to affect another protein important in cell cycle
control, (Bulavin & Fornace, 2004; Garner et al, 2002; Chao & Yang, 2001) cyclin
B1, we also probed the ability of THIF to regulate the activation of this important
cyclin. Four cyclin B1 phosphorylation sites (Ser126, 128, 133 and 147) are located
in the cytoplasmic retention signal (CRS) domain and are thought to regulate the
45
translocation of cyclin B1 to the nucleus at the G2/M checkpoint (Li et al., 1997).
Exposure of T47D cells to THIF (0.3-30 µM; 0.5h) resulted in a strong dose-
dependent decrease in the phosphorylation below basal levels in bands
corresponding to cyclin B1 phosphorylated at serine 147 (Fig. 10A and B). There
were no alterations in the total levels of cyclin B1 at any concentration (Fig. 10A).
These data suggest that THIF may suppress the activation of cyclin B1 and prevent
its translocation to the nucleus where it may regulate entry of cells into mitosis.
46
Fig. 10: Phosphorylation of JNK1/2, p38 and cyclin B1 in T47D cells exposed to
5,7,3,4-tetrahydroxyisoflavone (THIF). (A) Crude lysates (30 µg) prepared from
T47D cells exposed to vehicle (MeOH), or THIF (0.3-30 µM) for 0.5 h were
immunoblotted with an antibody that specifically recognizes the dually
phosphorylated region of the active form of JNK1 and JNK2 (pJNK1/2), the dually
phosphorylated motif Thr
180
-Pro-Tyr
182
within activated p38 or cyclin B1 when
phosphorylated at Ser 147. Lysates were also immunoblotted with antibodies for
total p38 and total cyclin B1 levels. (B) Data obtained from phospho-p38 and
phospho-cyclin B1 immunoblot experiments represented in (A) were analyzed using
BioRad Quantity One 1-D Analysis software. Each point represents the mean ± SD
of four independent experiments (*** p < 0.001; **p < 0.05).
47
Fig 10: Continued
B
Relative
Band
Intensity
C 10 0.3 1 3 30
THIF (µM)
0.0
0.5
1.0
1.5
2.0
phospho-p38
phospho-cyclin B1 (ser 147)
A
phospho-
pJNK
p3
C 1 0. 1 3
pJNK
3 THIF
phospho-Cyclin
B1 (ser 147)
Cyclin
48
Effects of THIF on cyclin B1 and cdc2 are mediated by p38 activation- To
investigate a possible link between the increases in p38 activity in T47D cells and
the observed de-phosphorylation of cdc2 and cyclin B1 in cells exposed to THIF, a
specific p38 inhibitor, SB203580, was employed (Patterson & Murray, 2002). While
SB203580 inhibits p38 activity, it does not significantly affect the activation of p38
itself and does not inhibit other kinases such as PKA, PKC or ERK and JNK MAP
kinases. In agreement with this, SB203580 (2.5 µM) had no significant effect on the
phosphorylation state of p38 and did not restore p38 phosphorylation to baseline
levels following THIF exposure (Figure 11A). However, pre-treatment of cells with
SB203580 (2.5 µM) for 0.5 h completely reversed THIF-induced de-phosphorylation
of cyclin B1 (Fig. 11A), thus suggesting that p38 activation in cells may mediate de-
phosphorylation of cyclin B1, an event which causes it sequestration in the cytosol
and thus cell cycle arrest at the G2-M checkpoint (Fig 12). Exposure to SB203580
(2.5 µM; 0.5 h) led to a similar reversal of THIF-induced cdc2 deactivation (Fig.
11A). In addition, a small but significant increase in cdc2 phosphorylation was
observed in cells treated with SB203580 (2.5 µM; 0.5 h) alone (Figure 11A). This
may reflect the inhibition of active p38 following its translocation to the nucleus, an
event that would reduce its inhibitory effect on cdc2 (Fig 12).
Pre-treatment of T47D cells with SB203580 (2.5 µM; 0.5 h) prior to exposure
with THIF or genistein led to significant reductions in both growth inhibition and the
percentage of cells in the G2-M phase (Fig. 11B). Both genistein and THIF (3 µM)
induced growth inhibition, which was significantly inhibited by SB203580.
49
Furthermore, the percentage of cells in the G2-M phase was almost
completely restored to basal levels by pre-incubation of cells with SB203580. These
observations support the mechanism by which both genistein and THIF induce their
anti-proliferative effects in T47D cells via p38 activation.
50

Fig. 11: Inhibition of THIF-induced signaling and growth arrest by the p38 inhibitor
SB203580 (or SB). (A) Crude homogenates (30 µg) prepared from T47D cells
exposed to vehicle (MeOH), SB203580 (2.5 µM), THIF (3 µM) or THIF (3 µM) +
SB203580 (2.5 µM; 0.5 h pre-treatment) were immunoblotted with an antibody that
recognizes phospho-38, total p38, phospho-cyclin B1 or phospho-cdc2. Data
obtained from immunoblots were analyzed using BioRad Quantity One 1-D Analysis
software and are presented with significant differences between THIF treated and
THIF + inhibitor are indicated by *** p < 0.001. Significant differences between
control and inhibitor are indicated by ^ p < 0.05. (B) Reversal of genistein (3 µM)
and THIF (3 µM)-induced growth inhibition and cell cycle arrest by SB203580.
Results are expressed as growth inhibition and were obtained from three independent
experiments performed in duplicate and presented as mean ± SD. Significant
increases between vehicle (MeOH) and treatments are indicated by *** p < 0.001; **
p < 0.01.

51
B
A
Phospho-p38 -
Phospho-cyclinB1 -
(ser 147)
Phospho-cdc2
(Thr 161)
C
Gen(3) Gen(3)THIF(3) THIF (3)
+ SB203580 + SB203580

% Inhibition
of growth
(bars)
% Cells in
G2/M phase
( l )
***
***
0
20
40
60
80
0
20
40
60
80
**
***
Total p38
C SB THIF THIF
C
SB203580 (2.5 µM)
THIF (3 µM)
THIF (3 µM)
+SB203580

Relative
Band
Intensity
0.0
0.5
1.0
1.5
p - cyclin B1
(ser 147)
p -
(Thr 161)
0.0
0.5
1.0
1.5
p38
p p cdc2
***
**
^
+SB
Fig 11: Continued
52
Fig. 12: Proposed mechanism for the anti-proliferative effects of genistein in T47D
cells. Genistein enters cells where it is subject the CYP450-induced intracellular
metabolism yielding THIF. THIF may undergo oxidation to form an o-quinone that
reacts with GSH and is exported from cells (this may also be catalyzed by
glutathione-S-transferase). Rises in intracellular oxidative stress, which is
accompanied by depletion of GSH, trigger the activation of the MAP kinase p38 and
two downstream targets of ATM/ATR, p53 and Chk1. Active p38 prevents the
phosphorylation of cyclin B1 and hence its transport to the nucleus, an event
essential for correct functioning of the cdc2-cyclin B1 complex. In addition, active
p38 may undergo translocation to the nucleus where it directly inhibits the
phosphorylation/activation of cdc2, thereby blocking entry of cells into mitosis (G2-
M block).
53
Fig 12: Continued

54
2.4- Discussion
Although genistein has been reported to exert anti-proliferative actions in
tumor cell models, including breast cancer cells (Salti et al., 2000; Constantinou et
al., 1990, Constantinou et al., 1996) the relevance of these studies remain
questionable due the high micromolar concentrations used and the fact that they may
exceed the level of free genistein in serum (Messina at al, 1999). However,
accumulating evidence suggests that serum levels may not reflect tissue or cellular
uptake of genistein in endocrine-responsive tissues including brain, liver, mammary
gland, ovary, prostate, testis, thyroid and uterus; in these tissues genistein
concentration was reported to be greater than in serum (Chang et al., 2000).
Furthermore, dietary genistein has been shown to modulate levels of epidermal
growth factor and estrogen receptor in rat prostate tissue following feeding studies
suggesting that tissue-selective accumulation of genistein may lead to biological
responses at the cellular level (Dalu et al., 1998, Dalu et al., 2002, Fritz et al., 2002).
We demonstrated that the exposure of breast epithelial cells to nana-molar or low
micromolar concentrations of genistein selectively induces growth arrest and G2-M
phase cell cycle block in T47D tumorigenic but not MCF10A non-tumorigenic breast
epithelial cells. These anti-proliferative effects were paralleled by significant
differences in the association of genistein with cells and in particular its intracellular
metabolism. Genistein is selectively taken up into T47D tumorigenic breast
epithelial cells and was subjected to significant intracellular metabolism by CYP450
enzymes leading to the formation of both 5,7,3,4-tetrahydroxyisoflavone (THIF) and
two glutathionyl conjugates of THIF (Fig. 9). In contrast, there was minimal cell-
55
association of genistein with MCF10A cells and no subsequent formation of free
THIF (although small levels of one glutathionyl conjugate of THIF was detected).
Cytochrome P450 proteins are located in most tissues where they metabolize both
endogenous compounds and xenobiotics by epoxidation, N-dealkylation, O-
dealkylation, S-oxidation and hydroxylation (Guengerich et al., 1998). Furthermore,
the expression of several CYP450 isoforms has been linked to many types of human
cancers. For example, CYP1B1 is over-expressed in breast, lung, liver,
gastrointestinal tract, prostate, and bladder tumors (Patterson et al, 2002) and it has
been suggested that anticancer agents may be designed to exploit this feature
(Jounaidi, 2002; Waxman et al., 1999).
Our data suggest that that the formation of THIF is pivotal in genistein-
induced G2-M cell cycle arrest and inhibition of cellular proliferation. Cimetidine,
the general CYP450 inhibitor blocked THIF formation in T47D cells and prevented
genistein-induced G2-M cell cycle arrest. In addition, it was demonstrated that
genistein selectively induced G2-M phase cell cycle block and growth inhibition in
T47D tumorigenic but not in MCF10A non-tumorigenic breast epithelial cells that
did not metabolize genistein to THIF. Previously, it has been suggested that
isoflavone metabolism in transformed but not non-transformed breast epithelial cells
may modulate the growth inhibitory effects of genistein (Peterson et al., 1996).
Furthermore, the metabolism of genistein has been investigated using recombinant
human CYP450 where the isoforms 1A1, 1A2, 1B1, 2B6, 2C8, 2E1, or 3A4 were
observed to metabolize genistein to form one main hydroxylated product, while
CYPP450 3A4 produces two different hydroxylated products.
56

The formation of glutathionyl conjugates of THIF in cells may result either
from the action of glutathione-S-transferase or form the oxidation THIF and
subsequent reaction of THIF o-quinine with cellular thiols including GSH. Because
of the inherent nucleophilicity of the sulphydryl group, protein and non-protein
sulphydryls represent a major target for quinones and the detoxification of quinones
by GSH is generally considered to be cytoprotective (Monks & Lau, 1997; Monks &
Lau, 1998). Glutathionyl conjugates from a variety of polyphenol quinones have
been observed in cellular systems and display a wide array of biological activities
(Monks & Lau, 1998). Indeed, the redox activity of polyphenols is frequently
enhanced following conjugation with GSH (Monks & Lau, 1998) and thus does not
necessarily result in detoxification. Only when GSH conjugation is coupled to the
subsequent export of the adduct form cells, as we observe in our studies, will
detoxification be the predominant pathway (Fig. 12). In the studies described here,
export of 5-S-glutathionyl- and 2-S-glutathionyl-THIF is observed, and did not
induce cytotoxicity when added to the extracellular milieu. However, the export of
glutathionyl-THIF from cells may result in the loss of GSH from cells.
Much recent interest has concentrated on the potential of genistein to interact
with intracellular signaling pathways such as the MAP kinase cascade (Shimizu et
al., 2005) and its ability to modulate cell cycle regulatory apparatus (Frey et al.,
2001). When cells are exposed to stressful or toxic stimuli that cause DNA damage,
they undergo cell cycle arrest at G1 or G2 to prevent the replication or segregation of
mutated DNA. Mammalian cells possess at least two stress-activated protein
57
kinases, p38 and JNK, which are activated by common protein kinase cascades.
Activation of p38 may cause cells to arrest at G1, by increasing the expression of the
cell cycle inhibitor protein p21/CIP1, or at G2, by inhibiting the expression of cyclin
B1 (Bulavin & Fornace, 2004). We show that the genistein metabolite THIF induces
a strong activation of p38 but not JNK in T47D cells. The activation of p38 by THIF
was accompanied by potent de-activation of cdc2, a major kinase involved in G2-M
cell cycle control and the entry of all eukaryotic cells into mitosis (Norbury & Nurse,
1992; Watanabe et al., 1995) and deactivation of cyclin B1. The latter requires
activation by phosphorylation to facilitate its translocation to the nucleus (Fig. 9).
Consequently, THIF may prevent entry of cells into mitosis via inhibition of cdc2
activation and cyclin B1 nuclear localization to the G2-M checkpoint. In addition,
the inability of THIF to activate JNK agrees with previous studies (Frey &
Singletary, 2003) and indicates that THIF does not induce an apoptotic mode of
death in T47D cells. As expected the p38 inhibitor, SB203580 (2.5 µM) had no
effect on THIF-induced p38 activation. However, it significantly inhibited THIF-
induced cyclin B1 phosphorylation indicating that activation of p38 in the cytosol by
THIF may lead to the de-phosphorylation of cyclin B1 thus preventing its
translocation to the nucleus where it may control the G2-M transition (Fig 12).
Furthermore, the p38 inhibitor restored cdc2 phosphorylation following THIF
treatment indicating that p38 phosphorylation may also mediate the loss of cdc2
phosphorylation in T47D cells. Treatment with SB203580 provided significant
protection against THIF-induced growth inhibition and G2-M cell cycle arrest
(Figure 11B) and prevented genistein-induced effects on cellular proliferation.
58
These observations indicate that p38 activation is likely to mediate the effects of both
compounds in T47D cells and also strengthens our proposal that THIF mediates the
effects of genistein in these cells.

2.5- Summary
Our study proposes a mechanism by which genistein inhibits cancer cell
growth that is mediated by the formation of the metabolite, 5,7,3,4-
tetrahydroxyisoflavone (THIF) (Summarized in Fig 9). Genistein enters cells where
it is subject the CYP450-induced intracellular metabolism yielding THIF. THIF may
undergo oxidation to form an o-quinone that reacts with GSH and is exported from
cells (this may also be catalyzed by glutathione-S-transferase). THIF-induced
activation of the p38 MAP kinase leads to the inhibition of cyclin B1
phosphorylation and hence its transport to the nucleus, an event essential for correct
functioning of the cdc2-cyclin B1 complex. In addition, active p38 may undergo
translocation to the nucleus where it directly inhibits the phosphorylation/activation
of cdc2, thereby blocking entry of cells into mitosis (G2-M block). We suggest that
one mechanism by which genistein may exert its cellular activity are related to the
interactions of its cellular metabolite with p38 signaling pathway.

59
CHAPTER 3 Contrasting the effects of genistein on cell proliferation and cell
cycle arrest in nontumorigenic human breast epithelial cells and human breast
cancer cells: Involvement of α-cdk2 kinase, cyclin B1 kinase and cyclin
dependent kinase inhibitor, p27

3.0- Abstract
Few studies have compared the cellular effects of genistein in nonmalignant
versus malignant breast epithelial cells. Therefore, we investigated the effects of
genistein on key cell cycle proteins in nonmalignant MCF10A and malignant T47D
breast epithelial cells. In agreement with our previous study, genistein caused G2-M
arrest in malignant T47D cells but not in nonmalignant MCF10A breast epithelial
cells. We demonstrated that there was upregulation of protein expression of negative
cell cycle regulator, p27/kip1 in response to genistein in malignant T47D breast cells
and not in nonmalignant MCF10A breast epithelial cells. In general, genistein
inhibited cyclin dependent kinase (CDK) activity in T47D malignant cells while
increasing CDK activity in MCF10A nonmalignant cells (48 h). In summary,
genistein upregulated p27/kip1, leading to inhibition of CDK that blocks cell cycle
progression in T47D malignant but not in MCF10A nonmalignant breast epithelial
cells. However, the upregulation of cyclin A by genistein in T47D cells is
paradoxical. Speculatively, this may represent either an accumulation of cyclin A in
the G2/M phase prior to its degradation or its ability to interact with

the negative cell
cycle regulators p27/kip1 does not exclusively rely on its ability

as a positive
regulator of G
2
progression. Furthermore, cyclin A may possess other functions,
independent of Cdk2 activation

and p27/kip1 binding that contributes significantly to

60
its ability to prevent cell cycle progression in malignant T47D breast epithelial cells.
In these two distinct breast cell lines genistein may regulate cell cycle machinery by
differentially modulating key cell cycle proteins.
3.1- Introduction
Phytoestrogens present in soybean have been the subject of intense
investigation due to their reported benefits in many diseases, including breast cancer.
Genistein is one the principle phytoestrogens found in soybeans. Although its exact
mechanism of action remains unclear, it has been found to be an inhibitor of tyrosine
kinases (Akiyama et al., 1987), DNA topoisomerase (Bertrand et al., 1993, Salti et
al., 2000) and angiogenesis (Fotsis et al., 1995, Fotsis et al., 1998). It also exerts
antioxidant activity (Ruiz-Larrea et al., 1997), targets stress response elements (Zhou
and Lee, 1998) as well as the peroxisome proliferating-activated receptor (Dang et
al., 2003). The ability of genistein to inhibit tyrosine kinases and DNA
topoisomerases may contribute to its antiproliferative effects (Cappelletti et al., 2000,
Alhasan et al., 2001, Constantinou et al., 1998b).
Indeed, genistein has been shown to block cell proliferation and cell cycle in
various cancer cell types by modulating various proteins that are essential in cell
cycle progression (Shao et al., 1998c). Tight regulation of cell cycle progression is
controlled by many different proteins and enzymes. For example, the accumulation
of cyclins D, E, and A bind to and activate different CDK catalytic subunits to
regulate progression from G1 to S phase of the cell cycle while the binding and

61
activation of cdk4–cyclin D and/or cdk6–cyclin D complex controls transition from
early to mid G1 phase. In addition, the activation of the Cdk2–cyclin E complex is
necessary for transition through mid G1 to S. Progression through late G1 to S phase
also requires the presence of cdk2–cyclin A complex (Sherr, 1996). CDK regulation
is also negatively controlled by a family of proteins called the Cyclin-dependent
kinase inhibitors (CDKI) by binding and subsequent inactivation of the cdk–cyclin
complexes. Two classes of CDKIs have been described: (i) p21
(Cip1/Waf1/Cap20/Sdi1/Pic1), p27 (Kip1), and p57 (Kip2) are related proteins with
a preference for cdk2– and cdk4–cyclin complexes; (ii) p16INK4A, p15INK4B,
p18INK4C and p19INK4D are closely related CKIs specific for cdk4– and cdk6–
cyclin complexes (Sherr & Roberts, 1995).
Previous studies have shown that the growth inhibitory effects of genistein in
various cancer cell types were accompanied by G2 or G1 cell cycle arrest (Davis et
al., 1998, Shen et al., 2000, Lian et al., 1998). Genistein has also been shown to
cause a dose-dependent growth inhibition of different breast cancer cells with
accumulation of cells in G2 phase of cell cycle without deregulation of the p34(cdc-
2)/cyclin B1 complex (Cappelletti et al., 2000). Conversely, several other studies
have shown that genistein-induced cell cycle arrest was related to an inhibition of
cyclin B levels, which contributed to the deregulation of the cyclin B1/p34 complex
(Choi et al., 2000). In addition, genistein can also exert pronounced antiproliferative
effects on both estrogen receptor-positive and -negative human breast carcinoma

62
cells through G2-M arrest, induction of p21/WAF1 expression, and apoptosis (Shao
et al., 1998c).
Although genistein has been reported to inhibit the multiplication of
numerous neoplastic cells, including those in the breast, there is limited information
on the effects of genistein on nontumorigenic human breast epithelial cells. It has
been reported that genistein inhibited proliferation of MCF10F human
nontumorigenic breast epithelial cell line with an IC50 of 19-22 µM, and, caused a
reversible G2/M block in cell cycle progression. This particular study suggested that
genistein also inhibited the growth of nontumorigenic MCF10F human breast cells
by preventing the G2/M phase transition, induced the expression of the cell cycle
inhibitor p21(waf/cip1) as well as its interaction with Cdc2, and inhibited the activity
of Cdc2 in a phosphorylation-dependent manner (Frey et al., 2001). Inhibition of cell
proliferation of this nontumorigenic breast epithelial cell line is believed to involve
MAP kinase signaling (Frey and Singletary, 2003). However, Upadhyay and workers
suggested that genistein causes a greater degree of G2-M arrest and induces
apoptosis in malignant cell lines compared with normal breast epithelial cells.
Interestingly, in these experiments, genistein treatment resulted in a hyperdiploid
population in tumorigenic but not nontumorigenic breast epithelial cells (Upadhyay
et al., 2001).

63
3.2- Materials and methods
Cell Culture- MCF10A nontumorigenic human breast epithelial cells were
incubated in DMEM/F12 media supplemented with 5% (v/v) Horse Serum (Gemini
Bioproducts), 2.5 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml
streptomycin (Gibco), 20 ng/ml EGF, 100 ng/ml cholera toxin, 10 µg/ml bovine
insulin, 500 ng/ml hydrocortisone. T47D tumorigenic human breast epithelial cells
were cultured in RPMI media supplemented with 10% Fetal Bovine Serum (Gemini
Bioproducts) and 1 % penicillin/streptomycin (Gibco). Cells were placed in an
incubator with 5 % carbon dioxide-air at 370C. Cells were continuously exposed to
varying concentrations (0, 5, 10, 25, 50, 100 µM) of genistein for 24, 48, 72, and 96
hours depending upon the assay.
Cell Cycle Analysis- Cells were grown to 60 % confluence and then DNA
content per duplicate was analyzed using FACStar flow cytometer as previously
described (Qiu et al., 1998). Cells (stained with propidium iodide containing RNase)
will be analyzed by flow cytometry. Populations of G0/G1, S, and G2/M will be
quantitated using Cellquest software.
Immunoblot and immunoprecipitation analysis for histone H1 kinase- Cells
were harvested and lysed with RIPA buffer following treatments. Protein
concentration is determined by the Bradford method (Biorad). 30 µg of total protein
from each sample is separated on a 12 % SDS-polyacrylamide gel, and then
transferred to a nitrocellulose membrane (Amersham). The membrane is then

64
blocked using 5 % casein/tris buffered saline (TBS) (Pierce) for 30 minutes prior to
overnight incubation with the appropriate primary antibody at 4
o
C. Following that,
the membrane is washed for 10 minutes for 4 times in TBS/tween and then incubated
with secondary antibody (anti-mouse or anti-rabbit). Detection is achieved using
electrochemiluminescence (ECL) (Amersham) at 1 and 5 minutes of exposure of
radiographic film (Kodak). For histone H1 kinase assay, the cell lysate will be
subjected to immunoprecipitation with cdk2 or cyclin B1 kinase antibody (Santa
Cruz Antibodies), as previously described (Qiu et al., 1998). Autoradiographs were
obtained after 24 h of exposure of the gel to the film. The equal loading and efficient
transfer of proteins was confirmed by staining the nitrocellulose with Ponceau Red
(Sigma, USA)
3.3 Results
Genistein induces G2 Cell Cycle Arrest in T47D tumorigenic breast
epithelial cells- Previous studies have shown that genistein potently inhibits
proliferation of cancer cells (Cappelletti et al., 2000; Choi et al., 2000). Our earlier
study demonstrated that selective uptake and metabolism of genistein underlies its
antiproliferative effects on T47D tumorigenic breast epithelials cells and not in
nontumorigenic MCF10A cells. In this study, we demonstrated that genistein
selectively induced G2 cell cycle arrest in T47D cells. In order to determine whether
genistein affects cell cycle progression, both T47D and MCF10A cells were treated
with genistein for 96 h prior to being subjected to cell cycle analyses. Consistent
with its inhibitory effects on cell proliferation as demonstrated in Chapter 2, T47D

65
cancer cells did not progress through the cell cycle as did MCF10A cells in response
to genistein exposure (Fig 13). These results are consistent with results obtained in
Chapter 2 for the shorter exposure duration of 24 h, demonstrating a longer lasting
effect of genistein.

66

Fig 13. The effects of genistein on cell cycle progression in (A) T47D tumorigenic
breast epithelial cells and (B) MCF10A nontumorigenic breast epithelial cells. Cells
were treated with varying concentrations of genistein for 96 h, fixed in ethanol,
stained with propidium iodide, and analyzed using flow cytometry. Legend:

0
10
20
30
40
50
60
70
80
0 5 10 25 50 100
0
10
20
30
40
50
60
70
80
90
100
0 5 10 25 50 100
Genistein [µM]
A. T47D
B. MCF10A
Cell population (%)
Cell population (%)
G S G
Genistein [µM]

67
Effects of genistein on cyclin-dependent kinase activity α−cdk2 and α-
cyclinB1 in T47D tumorigenic and MCF10A nontumorigenic breast epithelial cells-
Existing evidence supports a role for α-cdk2-cyclin A and/or α-cyclin B1 kinase-
cyclin B complexes in regulating the transition of cells from G2 to M phase.
Therefore, we determined whether genistein modulation of α-cdk2 or cyclin B1
kinase activity may be involved in the cell cycle arrest in T47D tumorigenic cells. In
T47D cells, an inhibition of α-cdk2 histone-associated kinase activity was observed
with concentrations at 24 and 48 h in a dose-dependent manner but not at 96 h. A
similar pattern was also observed with α-cyclin B1 kinase activity in T47D cells. In
contrast, in MCF10A cells, genistein treatment was associated with an initial
inhibition of α-cyclin B1 histone-associated kinase activity at 24 h, followed by an
increase in activity at 48 h [5-25 µM] (Fig 14), that was paralleled by progression
through the cell cycle. These results suggest that an overall inhibition of cyclin-
dependent kinases is associated with cell cycle blockage in T47D cells whilst an
inhibition is associated with cell cycle progression in MCF10A cells.

68
Fig 14. The effects of genistein (Gen) on the catalytic kinase activities in T47D
tumorigenic breast epithelial cells of (A.1) α-cdk2 (A.2) α-cyclin B kinase, and the
catalytic kinase activity of (B) a-cdk2 in MCF10A nontumorigenic breast epithelial
cells. Aliquots of 100 µg protein were immuoprecipitated with anti-α-cdk2 or α-
cyclin B kinase, and histone H1-associated activity of immunopreciptates was
evaluated and samples were analyzed by SDS-PAGE, followed by autoradiography.
In T47D cells, an inhibition of α-cdk2 histone-associated kinase activity was
observed with concentrations at 24 and 48 h in a dose-dependent manner but not at
96 h. A similar pattern was also observed with α-cyclin B1 kinase activity in T47D
cells. In MCF10A cells, genistein treatment was associated with an initial inhibition
of α-cyclin B1 histone-associated kinase activity at 24 h, followed by an increase in
activity at 48 h [5-25 µM]

69
Fig 14: Continued

Gen [µM] 0 5 10 25 50 100 0 5 10 25 50 100
24 h
48 h
96 h
Coom.
Blue stain
α-cdk2 α-cyclin B kinase
32
-P-HI
% cpm
32
-P-HI
% cpm
A.1
B.
32
-P-HI
α-cdk2
A.2
24 h

48 h

96 h
Gen [µM] 0 5 10 25 50 100

70
Effects of genistein on cyclin A/p60 expression in T47D tumorigenic and
MCF10A nontumorigenic breast epithelial cells- The mechanism of genistein-
induced accumulation of cells in T47D cells in the G2/M phase of the cell cycle
mediated through alterations in the catalytic subunit of p60-cdc-2 activity and the
expression of cyclin A was investigated. Treatment with genistein resulted in a time-
and dose-dependent increase in cyclin A (p60) expression in T47D cells. At 24 h,
concentrations at 25 µM or greater effectively increased cyclin A expression
between 24 and 48 h. (Fig 15). Furthermore, the concentrations of genistein that
upregulate cyclin A protein expression corresponds with those that result in
inhibitory effects on cell cycle kinases (Fig 15). Interestingly, cell cycle arrest is
usually accompanied by a decrease in cyclin levels rather than an increase as
observed in these studies. Similarly, in MCF10A cells, there was an also an
upregulation of cyclin A (5-100 µM) at 24 h, which decreases at 48 h (Fig 15). At
48 and 96 h, cyclin A upregulation in MCF10A cells is associated with an
upregulation of α-cdk2 activity while at 24 h, cyclin A upregulation is associated
with upregulation of α-cdk2.

71

Fig 15. The effects of genistein on cyclin A expression in (A) T47D tumorigenic
breast epithelial cells, and (B) MCF10A nontumorigenic breast epithelial cells. Blots
are representative blots of three independent experiments on different cultures that
yielded similar results. 30 µg of total protein was separated on a 12% SDS-PAGE
gel, transferred to a nitrocellulose membrane, and incubated with cyclin A antibody.
Genistein caused a time- and dose-dependent increase in cyclin A (p60) expression
in T47D cells. In MCF10A cells, there was an also an upregulation of cyclin A (5-
100 µM) at 24 h, which decreases at 48 h.
24 h 48 h 96 h
Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50 100
24 h 48 h 96 h
Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50 100
A.
B.
55kD
55kD
24 h 48 h 96 h
Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50 100
24 h 48 h 96 h
Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50 100
24 h 48 h 96 h
Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50 100
A.
B.
55kD
55kD

72
Effects of genistein on protein expression of cdk inhibitors p27/kip 1
expression in T47D tumorigenic and MCF10A nontumorigenic breast epithelial
cells- Current studies demonstrate that p21/waf1 protein was a regulatory factor of
cell cycle in G1 phase but is also involved in G2/M phase arrest. By binding to a
variety of CDK and cyclins such as cyclin A-cdk1 and cyclin B1-cdk1, p21/waf1
exerts its inhibitory effects on cyclin/CDK complexes. The CDK inhibitor, p27, is a
universal inhibitor that is active during both the G1 and G2 phases of the cell cycle
by binding to the cdk2-cyclin E/A complex to inhibit cdc-2 activity. Hence, we
further examined whether genistein could modulate expression of one of members of
the CDK family, namely p27/kip1. In T47D cells, levels of p27/kip1 expression
increased in a time- and dose-dependent manner (Fig 16). In contrast to cyclin A
expression, which was only affected at concentrations greater than 25 µM at 24-48 h,
p27/kip1 expression was modulated by genistein with 5 µM exposure at 24 h in
T47D cells. This suggests that p27/kip1 is more sensitive to genistein-induced
modulation than cyclin A. In MCF10A cells, p27/kip1 expression was not altered,
suggesting that CDK inhibitors are not as sensitive to genistein challenge as in T47D
cells. Collectively, this is demonstrative that the inhibitory effect of genistein on
human tumorigenic breast epithelial cells is associated with genistein-induced
expression of p27/kip1 and genistein arrests tumor cells in G2/M phase. Conversely,
genistein did not alter the expression of p27/kip1 expression in MCF10A
nontumorigenic breast epithelial cells (Fig. 16). A similar pattern of results for CDK
inhibitor, p21/waf1 was also observed using fluorescent labeling and FACS analyses
(unpublished data).

73

Fig 16. The effects of genistein on p27 expression in (A) T47D tumorigenic breast
epithelial cells, and (B) MCF10A nontumorigenic breast epithelial cells.
Experiments shown are representative of three separate western blots. 30 µg of total
protein was separated on a 12% SDS-PAGE gel, transferred to a nitrocellulose
membrane, and incubated with p27 antibody.

27 kD
Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50 100
24 h 48 h 96 h

Genistein [µM] 0 5 10 25 50 100 0 5 10 25 50 100 0 5 10 25 50
24 h 48 h 96 h
27 kD

74
3.4- Discussion
Cyclins are a group of proteins with cell cycle specificity. Cyclin A and
cyclin-dependent kinase 1 (cdk2) are two proteins required for cells to traverse from
S into G2. We investigated the expression of cyclin A in T47D and MCF10A cells
treated with various concentrations of genistein for 24, 48, and 96 h. Although cell
cycle arrest is usually associated with an inhibition of cyclin levels, these results
showed that the expression of cyclin A increased with exposure to genistein in T47D
cancer cells. Similar studies have shown that a sustained increase of cyclin B1
resulted in cell cycle arrest in cell cleavage phase. In particular, another study has
also shown that cell cycle arrest is associated with a parallel upregulation and an
accumulation of cyclin B1 (Cappelletti et al., 2001). By and large, genistein blocks
many breast cancer cells in the G2/M phase of the cell cycle. However, G2/M
blockage does not always follow the decrease of cyclin B1 expression. Similarly,
another study demonstrated that genistein blocked SGC-7901 gastric cancer cell
proliferation and increased the number of cells in G2/M phase more than three-fold,
as well as the expression of cyclin B1 while decreasing cyclin D expression (Cui et
al., 2005). It has been suggested that cyclin B1 protein accumulates during
interphase, while cell cycle progression is arrested at G2/M phase. It is plausible that
a similar mechanism is in place in these two cell lines.
To find out the effect of genistein on cell proliferation cycle, we detected the
expression of CKI-p27/kip1protein by FACS labeling. The negative cell cycle
regulator, p27/Kip1, is a protein that binds to cyclin and cdk complexes blocking

75
entry into S phase. When T47D cells are incubated with genistein, the expression of
p27/kip1 is increased in a dose- dependent manner, demonstrating that the inhibitory
effect of genistein on tumorigenic human breast epithelial cells is related to
genistein-induced expression of p27/kip1 and genistein arrests tumor cells in G2/M
phase.
Cell cycle regulation is a complicated process involving many factors. This
study was designed to compare the differences in the effects of genistein on the cell
cycle components in T47D tumorigenic and MCF10A nontumorigenic human breast
epithelial cells. The data from our studies indicate that genistein could arrest cell
cycle progression of T47D cells at G2/M phase. The possible underlying mechanism
may be that genistein promotes the expression of p27/kip1, consequently inhibiting
CDK activity, and rendering tumorigenic breast epithelial cells incapable of
traversing the checkpoint pathway of G2/M and procession to mitosis. Contrary to
this, genistein did not inhibit either cell proliferation (Chapter 2) or cell cycle
blockage in MCF10A nontumorigenic cells. In support of these findings, genistein
generally increased CDK activity (48-96 h) while it did not alter expression of
p27/kip1 or cyclin A.
In agreement with existing studies, genistein inhibited cell cycle progression
of T47D cells (Dampier et al., 2001) but not MCF10A cells. Similarly, Upadhyay
and colleagues (Upadhyay et al., 2001) suggested that there is differential sensitivity
between nontumorigenic and tumorigenic breast epithelial cells to genistein. Their
investigation showed that genistein induced a greater G2/M phase arrest as well as

76
apoptosis in tumorigenic compared with nontumorigenic breast epithelial cells. In
addition, Peterson et al. reported that normal human mammary epithelial (HME)
cells were less sensitive to growth inhibition than MCF-7 tumorigenic cells. The
proposed basis for the differential sensitivity of cultured HME cells and a
transformed human breast cancer MCF-7 cell line to growth inhibition by genistein
was due to a lack of metabolism of genistein by HME cells (Peterson et al., 1996).
In contrast, Frey et al., suggested that genistein also exerted antiproliferative effects,
induced cell cycle arrest, but did not induce apoptosis in MCF10F nontumorigenic
breast epithelial cells (Frey et al., 2001). These effects were correlated with
Mitogen-Activated-Protein-Kinase signaling (Frey and Singletary, 2003). These
studies were conducted for shorter durations (8 and 24 h) compared with longer
time-courses for the present studies. Indeed, previous studies conducted in our
laboratory (Chapter 2) also demonstrated that the difference in the cellular responses
elicited by genistein is associated with its selective uptake and metabolism in
tumorigenic and not nontumorigenic breast epithelial cell line.
In T47D cells, cell cycle arrest corresponded with a decrease in cyclin
dependent kinase, namely α-cdk2 at 24 and 48 h. In contrast, MCF10A cells had
extremely low and almost undetectable levels of α-cdk2. There was an initial
inhibition of α-cdk2 activity after 24 h treatment in MCF10A cells treated with
genistein. However, by 48 h, there was an increase in α-cdk2 activity in response to
genistein treatment [5-25 µM]; there was an increase in α-cdk2 activity, whereas 50-
100 µM inhibited α-cdk2 activity. At 48 h, genistein increased α-cdk2 activity.

77
However, in T47D cells, genistein inhibits CDK activity at concentrations greater
than 25 µM at 24 and 48 h. At 96 h, despite a large accumulation of cells in the
G2/M phase in T47D cells with 50 µM genistein, there is no inhibition of both α-
cdk2 and cyclin B1 kinase activities.
In eukaryotic cells, entry into mitosis involves the formation of α-cdk2
and/or cyclin A α-cdk2 complexes, which activates protein kinase activity. As a cell
enters the G1 and G2 phase of the cell cycle, cyclin A levels increase, and cyclin A
bind to α-cdk2 to form a complex (REF). Thus, the effects of genistein on cyclin A
protein expression were ascertained. In T47D cells, there was an increase in cyclin
A expression in a time- and dose-dependent manner. Similarly, the cyclin B1
response of various tumorigenic cells to genistein has been variable. Some existing
data suggest that a decrease in cyclin B1 expression paralleled with a decrease in
CDK activity in response to genistein underlies cell cycle arrest induced by genistein
(Alhasan et al., 2000, Choi et al., 2000). In addition, another report shows that
genistein treatment can result in a biphasic response on cyclin B1: 70% increase in
cyclin B1 level at 25 µM, and 50 and 70% decrease at 50 and 100 µM, respectively
(Balabhadrapathruni et al., 2000). However, our results are consistent with the work
of Capelletti and colleagues (Cappelletti et al., 2000) who demonstrated that
genistein caused an increase in cyclin B1 expression in various breast tumorigenic
cells, which they hypothesized to be attributed to an accumulation of cyclin B1 in the
G2/M phase prior to its degradation. In MCF10A cells, there was also upregulation
of cyclin A at 24 h in a dose-dependent manner, albeit less pronounced than in T47D

78
cells. However, this upregulation of cyclin A was generally associated with an
inhibition of CDK activity and lack of cell cycle arrest. It may also be possible that
ability of cyclin A to interact with

the negative cell cycle regulators p27/kip1 or
p21/waf1 does not exclusively rely on its ability

as a positive regulator of G
2
progression. Rather, we speculate

that cyclin A may possess other functions,
independent of Cdk2 activation

and p27/kip1 binding that contribute significantly to
its ability to prevent cell cycle progression in malignant T47D breast epithelial cells.
In Chapter 1, we show that a genistein metabolite, THIF, induces cell cycle arrest via
inhibition of cyclin B1. Therefore, it is highly likely that the interaction between
cyclin B1 and negative cell cycle regulators is responsible for the overall cell cycle
block in T47D cells.
The activity of CDK can be regulated by CDKIs. Both p21/waf1 and
p27/kip1 are CDKIs that enters in the G1 phase of the cell cycle and binds to cdk2-
cyclin A/E complex to inhibit cdc-2 activity. Genistein selectively induced a time-
and dose-dependent on upregulation of p21/kip1 and p27/kip1 expressions in T47D
cells but not in MCF10A cells. Our results suggest that p27/kip1 and p21/waf 1 may
be key regulatory factors that contribute to genistein-induced G2 arrest of T47D
cells.

79
3.5-Summary
This study demonstrates that there is a differential sensitivity of T47D
tumorigenic and MCF10A nontumorigenic breast epithelial cells to genistein-
induced cell cycle arrest. This effect appears to be related to the different effects of
genistein on CDK activity. With an increase in soy consumption in health conscious
individuals, it is important to conduct further studies to understand and compare the
effects of soy phytoestrogen genistein on nontumorigenic as well as tumorigenic
breast epithelial cells.

80
Chapter 4- The effects of genistein on cellular transformation in RAT1A and c-
myc RAT1A fibroblast cells

4.0- Abstract
In an effort to understand the chemopreventive effects of genistein, it was
assumed that genistein might target specific oncogenes, and, as a consequence, be a
more effective chemopreventive agent in the presence of certain oncogenes. The
effects of genistein on apoptosis, cell proliferation, cell viability, and anchorage-
independent growth in RAT1A fibroblast cells, and RAT1A fibroblast stably
transfected with c-myc were compared. Genistein affected cell viability, as
measured by MTT reduction, to a greater extent in RAT1A fibroblasts that
overexpress c-myc, thus suggesting that genistein may have selectivity for the c-myc
oncogene. This may be due to either a differential effect on apoptosis and/or cell
proliferation. Genistein induced apoptosis and inhibited cell proliferation to a greater
extent in RAT1A fibroblasts that overexpress c-myc, indicating that apoptosis and/or
inhibition of cell proliferation may account for difference in cell viability between
these two cell lines. The effects of genistein on cellular transformation, using soft
agar, was also examined. Approximately 30-70% of breast tumors are c-myc
positive. Cells that overexpress c-myc are readily transformable. Thus, it was
examined whether or not genistein could prevent cellular/oncogenic transformation
in vitro. At a concentration of 25 µM genistein reduced the number of visible
colonies formed by c-myc-mediated transformation of RAT1A fibroblasts. Future
experiments should include a comparison of the effects of genistein on other
oncogenes (i.e. her2/neu, ras) and attempt to elucidate the effects of genistein on

81
neoplastic transformation and its cellular mechanisms in more relevant human
neoplastic models.

4.1- Introduction
Although epidemiological studies have linked dietary soy intake with a
decreased risk of breast cancer, the present experimental evidence is conflicting. In
particular, genistein and/or soy products have been shown to reduce chemically-
induced tumors. In addition, a timely exposure to genistein during the prepubertal
period in rats is associated with protection against chemically-induced mammary
cancer. The proposed underlying mechanism is the induction of terminal
differentation of mammary ducts by genistein, which is believed to protect against
hormonal or xenobiotic exposure (Lamartiniere, 2002, Cotroneo et al., 2002, Sarkar
and Li, 2002, Gallo et al., 2001). Conversely, genistein has also been reported to
promote tumor formation in nude mice implanted with estrogen-dependent breast
cancer cells (Allred et al., 2001b, Allred et al., 2001a). However, Constantinou and
colleagues showed that genistein can protect against tumor formation using the nude
mouse model. Thus, the idea that genistein is chemopreventive warrants further
investigation.
Protoocogenes are genes involved in the regulation of cell proliferation; their
abnormal expression can lead to the development of cancer. Oncogenes are mutated
versions of protooncogenes. The c-myc gene was originally identified as the
oncogene v-myc (viral myc) of the MC29 avian myelocytomatosis virus, which
induced carcinomas, endotheliomas, sarcomas, and myelocytomatosis in birds

82
(Vennstrom et al., 1982. Subsequently, human, rat, and mouse c-myc genes have
also been cloned and characterized {Dalla-Favera, 1983 #256, Yang et al., 1984,
Hayashi et al., 1987). C-myc can also transform fibroblasts and macrophages in
vitro (Stone et al., 1987). Regulated c-myc gene expression is crucial for controlled
cell proliferation, whereas deregulated c-myc expression is associated with tumor
cells. A central role for c-myc in the activation of the cell

cycle machinery is
supported by the ability of c-myc to increase the activities

of enzymes that are
required for DNA metabolism and other metabolic

pathways. Collectively, this
suggests that c-myc may be a critical component

of cell cycle machinery and
metabolism (Dang, 1999) [Fig 18]. Interestingly, altered c-myc expression has been
associated with a variety of cancers, including breast cancer (Marcu et al., 1992,
Spencer and Groudine, 1991). Amplification of the c-myc gene is associated with
lung, colon, and breast

carcinomas (Dang, 1999).

Elevated expression of the c-myc
gene is found in

almost one-third of breast and colon carcinomas (Escot et al., 1986,
Erisman et al., 1985).
Because the chemopreventive properties of genistein using in vivo animal
models are conflicting, the objective of this study was to determine the effects of
genistein on in vitro c-myc-mediated oncogenic transformation in rat fibroblasts. In
this system, rat fibroblasts were transformed using fibroblasts that stably
overexpressed the protooncogene, c-myc. Preliminary studies in soft agar assays
show that genistein may prevent oncogenic transformation by c-myc in rat
fibroblasts at 25 µM. This effect may be a result of the selective induction of
apoptosis and inhibition of cell proliferation, as shown by annexin V binding and

83
BRDU incorporation (DNA synthesis indicator) respectively. These data suggest
that genistein may act synergistically with c-myc by impacting pathways that
regulate apoptotic or cell cycle progression, thus, resulting in the inhibition of
oncogenic transformation. However, further studies are required to dissect the
mechanisms by which genistein can inhibit transformation of overexpressing c-myc
fibroblasts.

84

Fig 17. Links between c-myc, selected putative target genes, cellular functions, and
cell growth. This diagram shows the complex relationship between c-Myc and its
putative target genes, which are grouped according to their functions. The various
cellular functions cooperate to promote cell growth. Taken from (Dang, 1999)

85
4.2- Methods
Cell exposures- RAT1A fibroblast and c-myc RAT1A fibroblasts were grown
in DMEM with low glucose with 5 % FBS and 1 % penicillin-streptomycin. RAT1A
fibroblasts overexpressing c-myc were also grown in the presence of G418 (300
ng/ml) to select for stable transfects. All cells were grown in a humidified condition
at 37
o
C. Cells were exposed to genistein [5, 25, 50 µM] for 24, 48, 72, 96, 144 h,
depending upon the actual parameter measured.
Cell growth by MTT reduction- Assessment of cell growth was determined
using the Cell Titer 967 Non-Radioactive Cell Proliferation Assay kit (Promega):
Cells were seeded at 1000 per well into 96 well plates and cell growth was MTT
reduction. Briefly, MTT dye was added to the wells for 4 hours prior to the addition
of solubilization buffer. The microtiter plate was then read at 590 nm.
BRDU incorporation- BRDU incorporation is a widely accepted non-
radioactive method of measuring of DNA synthesis. Cells were plated at a density of
10
5
in 10 cm culture dishes, and exposed to varying concentration of genistein for
24, 48, 72, and 96 h. Following this, media were removed from plates, and 10 µM
BRDU was added with fresh media for 60 min at 37
0
C. Cells were fixed with 70 %
ice-cold ethanol, denatured with 2 N HCl with 0.5 % Triton-100 for 30 min, and
resuspended in 1 ml of 0.1 M Borax/ sodium tetraborate, pH 8.5 and 10 ml water to
neutralize the acid. Cells were then incubated with 10 ml anti-BRDU [Promega] for
30 min, washed twice with 1 ml 0.5 % Tween 20/1.0 % BSA in PBS, and then
resuspended in 1 ml of PBS with 5 µg propidium iodide. Samples were analyzed
using flow cytometry at laser excitation of 488 nm.

86
Annexin V/propidium iodide stain- Early apoptosis is characterized by the
translocation of the membrane phosphatidylserine (PS) from the inner to the outer
plasma membrane. Annexin V, Ca 2+-dependent, phospholipid binding protein with
a high affinity for PS. Following treatment, cells were washed 2X with ice-cold
PBS, and trypinized. Cells were then resuspended in calcium binding buffer (10 mM
HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl
2
) containing 0.5 µg annexin V
and 5 µg/ml propidium iodide. After 10 minutes of incubation in the dark, samples
were analyzed using flow cytometry. Results are expressed as a total percentage of
annexin V positive cells (BD Biosciences).
Soft Agar Assay- Cells were transferred to six-well plates layered with 1 ml
of 0.7 % basal agar dissolved in DMEM with low glucose, 5 % FBS, 1% penicillin-
streptomycin, and G418 (300 ng/ml). Prior to transferring, 0.1 ml of trypsinized cell
suspension (10
5
cells/ml) was added to 0.9 ml of 0.35 % agar dissolved in DMEM
with low glucose, 5 % FBS, 1% penicillin-streptomycin, and G418 (300 ng/ml) at
39
o
C, with varying concentrations of genistein (5, 10, 25 µM). After gently mixing,
the cells were poured into each well. Colony formation was monitored at 14 days.
This assay exploits the anchorage-independent growth of transformed cells.

87
4.3- Results
Effects of genistein on colony formation in RAT1A and c-myc RAT1A
fibroblasts- Cell anchorage-dependent growth was measured by using the soft agar
assay. Anchorage independence can be described in terms of fibroblastic cell lines
(e.g. BALB/c3T3, NIH-3T3, etc.) that must attach to a solid surface for cell division
to occur. Therefore, these cells do not grow in a gel such agarose. Once transformed,
these cells grow in a gel and are anchorage-independent. Cell growth ability in soft
agar is believed to be an in vitro indicator of malignant phenotype (Dang et al.,
2003). These results demonstrated that genistein could inhibit c-myc-mediated
oncogenic transformation of RAT1A fibroblasts at a concentration 25 µM (Fig 17).
Because this model is generally regarded as a reasonably good predictor of in vivo
activity, it is possible that this positive result here could be viewed as potential
indications that genistein may have the ability to prevent oncogenic transformation.
Future experiments should be conducted to include a positive carcinogenic agent
such as N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 12-O-
Tetradecanoylphorbol 13-acetate (TPA).

88

Fig.18 The effects of genistein on colony formation in c-myc RAT1A fibroblast.
Cells were grown in 0.7 % soft agar, and treated with genistein for 3 weeks. Fresh
genistein and media were added every fifth day. At three weeks, pictures were taken
using an electron photocapture microscope. Genistein inhibited c-myc-mediated
oncogenic transformation of RAT1A fibroblasts at a concentration of 25 µM

Untreated
40X
Genistein 5 µM
40X
40X
Untreated 100X
Genistein 25 µM

89
Effects of genistein on MTT reduction in RAT1A and c-myc RAT1A
fibroblasts- The inhibitory effects of genistein on c-myc-dependent transformation
may be an indirect effect of toxicity. MTT cytotoxocity studies in both RAT1A
fibroblasts and RAT1A cells that overexpressed c-myc revealed that the latter are
more susceptible to genistein than the former cell line. This effect is most evident
with exposure to genistein at days 3 and 4, where genistein resulted in a dose-
dependent reduction in cell viability with an approximate IC50 of 50 µM.
Interestingly, genistein treatment of RAT1A parental fibroblasts did not exhibit the
same degree of toxicity. Since, RAT1A fibroblasts transected with c-myc are more
susceptible to genistein-induced cell death than nontransfected cells; it is possible
that genistein and c-myc may be acting synergistically to cause differential cytoxicity
in c-myc transfected cells (Fig 19).

90
Fig 19. (A) The effects of genistein on cell viability measured by MTT reduction in
RAT1A and (B) c-myc RAT1A fibroblast cells. Cells were plated in 96 well culture
dishes at a density of 1000 cells/well. Cells were treated with Control- EtOH (open
circle), 5 µM (solid square), 25 µM (solid triangle), 50 µM (Star) of genistein for 6-7
days. The ability to reduce MTT was measured following 4 h of incubation with
MTT, followed by addition of solubilization buffer. Samples were measured at 590
nm. In c-myc RAT1A cells, genistein exposure caused in a dose-dependent
reduction in cell viability with an approximate IC50 of 50 µM.

:

91

A. RAT1A
0
0.2
0.4
0.6
0.8
1.0
1 234 56
Day
ABS
(590 nm)
0
0.2
0.4
0.6
0.8
1.1
1.2
1 2 3 4 5 6
Day
B. c-myc RAT1A
ABS
(590 nm)
Fig 19: Continued

92
Effects of genistein on BDRU incorporation in RAT1A and c-myc RAT1A
fibroblasts- One possible explanation through which genistein prevents anchorage-
independent growth is via inhibition of cellular proliferation. Therefore, the effect of
genistein on BRDU incorporation was determined. Incorporation of BRDU is a
measure of DNA synthesis, and, therefore it is also an indicator of cell proliferation.
In RAT1A cells that overexpressed c-myc, genistein preferentially inhibited cell
proliferation, as shown by BRDU incorporation, in a time- and dose-dependent
manner. Genistein did not inhibit cell proliferation to the same extent in RAT1A
parental fibroblasts (Fig 20). These results may suggest that the effect of genistein
on colony formation in soft agar may be in part due to the inhibition of cell
proliferation in c-myc overexpressing RAT1A fibroblasts.

93
Fig 20. .Effects of genistein on BRDU incorporation on (A) RAT1A and (B) RAT1A
c-myc fibroblasts cells. Cells were seeded at a density of 100 000, and exposed to
genistein [5-50 µM] for 4 days. On the fourth day, cells were washed, and fresh
media with BRDU was added for 1 h. Following this, cells were washed, and
stained with an FITC-anti-BRDU dye, and analyzed using flow cytometry. Data are
expressed as mean, where n=2. Legend: Gen (0 µM) white bars, Gen (5 µM) black
bars, Gen (25 µM) black and white striped bar, Gen (50 µM) white with black dots.
Experiment was performed once. Genistein preferentially inhibited cell proliferation
in RAT1A cells that overexpressed c-myc but did not inhibit cell proliferation to the
same extent in RAT1A parental fibroblasts.

94
0
100
200
300
400
500
600
12 3 4
(%) BRDU incorporation
A.
B.
0
100
200
300
400
500
600
12 34
(%) BRDU incorporation
Days
Days
Fig 20: Continued

95

Effects of genistein on annexin V staining in RAT1A and c-myc RAT1A
fibroblasts- Inhibition of c-myc-mediated cellular transformation of RAT1A
fibroblasts may be attributed to the ability of genistein to induce apoptosis. Early
detection of apoptosis revealed that RAT1A fibroblasts that overexpressed c-myc
had a relatively greater percentage of annexin V binding when treated with 50 µM of
genistein. However, there were no significant differences at the lower concentrations
of genistein that were used in this study (Fig 21).

96

Fig 21. The effects of genistein on annexin V binding in RAT1A (Black square) and
c-myc RAT1A (Black diamond) fibroblasts. Cells were seeded at 100 000 cells and
treated with genistein for 4 days. Following that, cells were washed with PBS, and
stained with annexin/propidium iodide and analyzed using flow cytometry. Results
are expressed at mean + SEM, where n=3. Experiments are representative of three
separate experiments.
0
20
40
60
80
100
120
0 10 25 50
Genistein [uM]
Annexin V positive cells (%)

97
4.4- Discussion
The results of this study are in agreement with two existing studies, which
documented that genistein exhibited the reverting effect on the transformed
phenotypes of H ras-3T3 fibroblasts. Specifically, treatment with 25 µM of genistein
could effectively reverse the transformed morphology of ras-3T3 cells into flatter
cells with contact inhibition. Colony formation in soft agar was also decreased by 25
µM of genistein. This effect was accompanied by a dose-dependent manner
inhibition of cell proliferation of ras-3T3 cells. The possible mechanism of this
reversion was suggested to involve inhibition of tyrosine kinases, which can
subsequently lead to the inhibition of the p21ras-mediated signal transduction
pathway (Kuo et al., 1993, Kuo et al., 1994).
While regulated c-myc gene expression is essential for controlled cell
proliferation, deregulated c-myc expression is associated with tumor cells. Firstly,
altered c-myc expression has been associated with a variety of cancers, including
breast cancer (Marcu et al., 1992, Spencer and Groudine, 1991). Secondly, genistein
has also been shown to induce apoptosis in a variety of cancer cell lines (Lian et al.,
1998, Katdare et al., 2002, Constantinou et al., 1998a). Taken together, it may be
hypothesized that genistein may target specific oncogenes, such as c-myc, and that
its effectiveness is potentiated by particular oncogenes.
Further studies are warranted to examine the mechanisms whereby genistein
can prevent c-myc-mediated transformation of RAT1A fibroblasts. Based upon
these preliminary findings (genistein can inhibit both apoptosis and cell proliferation
of RAT1A cells that over express c-myc), the mechanisms that should be explored

98
may involve the regulation of cell cycle progression and apoptosis signaling cascade.
Future studies must also include the effects of genistein on c-myc mediated
transformation of human mammary epithelial cells, as this would better mimic a
human model of breast cancer development. The effects of genistein on other
important oncogenes, including Her-2/neu and ras should also be considered.

99
Chapter 5- Conclusions
The studies within this dissertation demonstrated that a differential uptake
and metabolism of the isoflavone genistein in tumorigenic but nontumorigenic breast
epithelial cells accounts for the ability of genistein to cause G2-M phase cell cycle
arrest. We identified THIF as a CYP450-derived bioactive genistein metabolite
associated with cell cycle arrest. Indeed, existing evidence suggests that isoforms of
CYP450 can oxidize genistein in vitro. Existing chemotherapeutic drugs are
associated with adverse effects because they nonspecifically target both tumorigenic
and nontumorigenic cells. The specific uptake and metabolism of genistein cancer
cells suggests its potential as a selective chemotherapeutic agent. As cancer cells are
associated with increased CYP450 levels, anticancer agents can be designed to
exploit this feature (Waxman et al., 1999, Jounaidi, 2002). The findings of this
dissertation suggest that genistein may be an effective prodrug that requires specific
CYP450 activation in tumorigenic cells only.
The different effects on cell cycle progression and cell cycle components that
include CDK and cyclins may be a result of differential uptake and metabolism of
genistein in tumorigenic but not nontumorigenic breast epithelial cells. Future
studies would involve a more in-depth characterization of the signal transduction
pathways involved in cell cycle arrest. In addition, the effects of the genistein
metabolite on cell cycle progression and cellular signaling should be investigated,
and compared with the effects of genistein itself.
Present studies on the chemopreventive effects of genistein are conflicting.
The preliminary studies using c-myc transformed rat fibroblast suggests that

100
genistein may be able to prevent oncogenic transformation through mechanisms that
involve control of apoptosis and cell proliferation. Importantly, the effects of
genistein on foci formation, colony formation in soft agar, and tumor formation in
the nude mouse model need to be determined. Further studies are also required to
investigate the molecular mechanisms through which genistein may inhibit
oncogenic transformation. From a practical standpoint, fibroblasts are an excellent
model for studying cellular transformation. However, the relevance of these findings
would be increased by using human mammary epithelial cells, and also by
transforming cells with other oncogenes that are more commonly associated with
human breast cancers, such as Her-2/neu.

101
References
Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya,
and Fukami, Y. (1987) J Biol Chem, 262, 5592-5.

Alhasan, S. A., Aranha, O. and Sarkar, F. H. (2001) Clin Cancer Res, 7, 4174-81.

Alhasan, S. A., Ensley, J. F. and Sarkar, F. H. (2000) Int J Oncol, 16, 333-8.

Allred, C. D., Allred, K. F., Ju, Y. H., Virant, S. M. and Helferich, W. G. (2001a)
Cancer Res, 61, 5045-50.

Allred, C. D., Ju, Y. H., Allred, K. F., Chang, J. and Helferich, W. G. (2001b)
Carcinogenesis, 22, 1667-73.

An, J., Tzagarakis-Foster, C., Scharschmidt, T. C., Lomri, N. and Leitman, D. C.
(2001) J Biol Chem, 276, 17808-14.

Arora, A., Valcic, S., Cornejo, S., Nair, M. G., Timmermann, B. N. and Liebler, D.
C. (2000) Chem Res Toxicol, 13, 638-45.

Balabhadrapathruni, S., Thomas, T. J., Yurkow, E. J., Amenta, P. S. and Thomas, T.
(2000) Oncol Rep, 7, 3-12.

Barnes, S. (1995) J. Nutr. 125, 777S-783S

Barnes, S. (1997) Breast Cancer Res Treat. 46, 169-179

Bertrand, R., Solary, E., Jenkins, J. and Pommier, Y. (1993) Exp Cell Res, 207, 388-
97.

,J. A. (1994) Int J Biochem, 26, 1203-26.

Bradlow, H. L. and Sepkovic, D. W. (2002) Ann N Y Acad Sci, 963, 247-67.

Breinholt, V. M., Offord, E. A., Brouwer, C., Nielsen, S. E., Brosen, K. and

Friedberg, T. (2002) Food Chem Toxicol, 40, 609-16.

Breinholt, V. M., Rasmussen, S. E., Brosen, K. and Friedberg, T. H. (2003)
Pharmacol Toxicol, 93, 14-22.

Bulavin, D. V. and Fornace, A. J., Jr. (2004) Adv. Cancer Res 92, 95-118

Cai, Q. and Wei, H. (1996) Nutr Cancer, 25, 1-7.

102

Cappelletti, V., Fioravanti, L., Miodini, P. and Di Fronzo, G. (2000) J Cell Biochem,
79, 594-600.

Casagrande, F. and Darbon, J. M. (2000) Exp Cell Res, 258, 101-8.

Cersosimo, R. J. (2003) Ann Pharmacother, 37, 268-73.

Chang, H. C., Churchwell, M. I., Delclos, K. B., Newbold, R. R. and Doerge, D. R.
(2000) J Nutr, 130, 1963-70.

Chen, W. F., Huang, M. H., Tzang, C. H., Yang, M. and Wong, M. S. (2003)
Biochim Biophys Acta, 1638, 187-196.

Chinni, S. R., Alhasan, S. A., Multani, A. S., Pathak, S. and Sarkar, F. H. (2003) Int
J Mol Med, 12, 29-34.

Choi, Y. H., Lee, W. H., Park, K. Y. and Zhang, L. (2000) Jpn J Cancer Res, 91,
164-73.

Constantinou, A., Kiguchi, K. and Huberman, E. (1990) Cancer Res, 50, 2618-24.

Constantinou, A., Mehta, R., Runyan, C., Rao, K., Vaughan, A. and Moon, R. (1995)
J Nat Prod, 58, 217-25.
Constantinou, A. I., Kamath, N. and Murley, J. S. (1998a) Eur J Cancer, 34, 1927-
34.

Constantinou, A. I., Krygier, A. E. and Mehta, R. R. (1998b) Am J Clin Nutr, 68,
1426S-1430S.

Constantinou, A. I., Lantvit, D., Hawthorne, M., Xu, X., van Breemen, R. B. and
Pezzuto, J. M. (2001) Nutr Cancer, 41, 75-81.

Constantinou, A. I., Mehta, R. G. and Vaughan, A. (1996) Anticancer Res, 16, 3293-
8.
Cotroneo, M. S., Wang, J., Fritz, W. A., Eltoum, I. E. and Lamartiniere, C. A. (2002)
Carcinogenesis, 23, 1467-74.

Cui H.B., Na X.L, Song D.F, and Liu Y. (2005) World J. Gastroenterology11(1):69-
72

Dalu, A., Blaydes, B. S., Bryant, C. W., Latendresse, J. R., Weis, C. C. and Barry

Delclos, K. (2002) J Chromatogr B Analyt Technol Biomed Life Sci, 777, 249-60.

103
Dalu, A., Haskell, J. F., Coward, L. and Lamartiniere, C. A. (1998) Prostate, 37, 36-
43.
Dampier, K., Hudson, E. A., Howells, L. M., Manson, M. M., Walker, R. A. and

Gescher, A. (2001) Br J Cancer, 85, 618-24.

Dang, C. V. (1999) Mol Cell Biol, 19, 1-11.

Dang, Z. C., Audinot, V., Papapoulos, S. E., Boutin, J. A. and Lowik, C. W. (2003) J
Biol Chem, 278, 962-7.

Davis, J. N., Singh, B., Bhuiyan, M. and Sarkar, F. H. (1998) Nutr Cancer, 32, 123-
31.

Doerge, D. R., Chang, H. C., Churchwell, M. I. and Holder, C. L. (2000) Drug
Metab Dispos, 28, 298-307.

Duncan, A. M., Underhill, K. E., Xu, X., Lavalleur, J., Phipps, W. R. and Kurzer, M.
S. (1999) J Clin Endocrinol Metab, 84, 3479-84.

Edenharder, R., Rauscher, R. and Platt, K. L. (1997) Mutat Res, 379, 21-32.

Erisman, M. D., Rothberg, P. G., Diehl, R. E., Morse, C. C., Spandorfer, J. M. and
Astrin, S. M. (1985) Mol Cell Biol, 5, 1969-76.

Escot, C., Theillet, C., Lidereau, R., Spyratos, F., Champeme, M. H., Gest, J. and
Callahan, R. (1986) Proc Natl Acad Sci U S A, 83, 4834-8.

Fioravanti, L., Cappelletti, V., Miodini, P., Ronchi, E., Brivio, M. and Di Fronzo, G.
(1998) Cancer Lett, 130, 143-52.

Fotsis, T., Pepper, M., Adlercreutz, H., Hase, T., Montesano, R. and Schweigerer, L.
(1995) J Nutr, 125, 790S-797S.

Fotsis, T., Pepper, M. S., Montesano, R., Aktas, E., Breit, S., Schweigerer, L., Rasku,
S., Wahala, K. and Adlercreutz, H. (1998) Baillieres Clin Endocrinol Metab, 12,
649-66.

Frey, R. S., Li, J. and Singletary, K. W. (2001) Biochem Pharmacol, 61, 979-89.

Frey, R. S. and Singletary, K. W. (2003) J Nutr, 133, 226-31.

Fritz, W. A., Wang, J., Eltoum, I. E. and Lamartiniere, C. A. (2002) Mol Cell
Endocrinol, 186, 89-99.

104

Gallo, D., Giacomelli, S., Cantelmo, F., Zannoni, G. F., Ferrandina, G., Fruscella, E.,
Riva, A., Morazzoni, P., Bombardelli, E., Mancuso, S. and Scambia, G. (2001)
Breast Cancer Res Treat, 69, 153-64.

Guengerich, F. P., Hosea, N. A., Parikh, A., Bell-Parikh, L. C., Johnson, W. W.,
Gillam, E. M. and Shimada, T. (1998) Drug Metab Dispos, 26, 1175-8.

Guo, Q., Rimbach, G., Moini, H., Weber, S. and Packer, L. (2002) Toxicology, 179,
171-80.

Hall, M., Forrester, L. M., Parker, D. K., Grover, P. L. and Wolf, C. R. (1989)
Carcinogenesis, 10, 1815-21.

Hartwell, L. H. and Kastan, M. B. (1994) Science, 266, 1821-8.

Hayashi, K., Makino, R., Kawamura, H., Arisawa, A. and Yoneda, K. (1987) Nucleic
Acids Res, 15, 6419-36.

Hunter, T., Gould, K. L. and Cooper, J. A. (1984) Biochem Soc Trans, 12, 757-9.

Ingram, D., Sanders, K., Kolybaba, M. and Lopez, D. (1997) Lancet, 350, 990-4.

Jounaidi, Y. (2002) Curr Drug Metab, 3, 609-22.
Ju, Y. H., Allred, C. D., Allred, K. F., Karko, K. L., Doerge, D. R. and Helferich, W.
G. (2001) J Nutr, 131, 2957-62.

Kastan, M. B. and Lim, D. S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186

Katdare, M., Osborne, M. and Telang, N. T. (2002) Int J Oncol, 21, 809-15.

Kiguchi, K., Constantinou, A. I. and Huberman, E. (1990) Cancer Commun, 2, 271-
7.

Kulling, S. E., Honig, D. M. and Metzler, M. (2001) J Agric Food Chem, 49, 3024-
33.

Kulling, S. E., Honig, D. M., Simat, T. J. and Metzler, M. (2000) J Agric Food
Chem, 48, 4963-72.

Kulling, S. E., Lehmann, L. and Metzler, M. (2002) J Chromatogr B Analyt Technol
Biomed Life Sci, 777, 211-8.

Kuo, M. L., Kang, J. J. and Yang, N. C. (1993) Cancer Lett, 74, 197-202.

105
Kuo, M. L., Lin, J. K., Huang, T. S. and Yang, N. C. (1994) Cancer Lett, 87, 91-7.

Kuzumaki, T., Kobayashi, T., and Ishikawa, K. (1998) Biochem Biophys Res
Commun 251, 291-295

Lambert, J. D., Hong, J., Yang, G. Y., Liao, J., and Yang, C. S. (2005) Am. J. Clin.
Nutr. 81, 284S-291S

Lamartiniere, C. A. (2000) Am. J. Clin. Nutr. 71, 1705S-1707S

Lamartiniere, C. A. (2002) J Mammary Gland Biol Neoplasia, 7, 67-76.

Lee, J. C., Kassis, S., Kumar, S., Badger, A., and Adams, J. L. (1999) Pharmacol.
Ther. 82, 389-397

Levine, A. J. (1997) Cell 88, 323-331

Li, J., Meyer, A. N., and Donoghue, D. J. (1997) Proc. Natl. Acad. Sci. U. S. A 94,
502-507

Lian, F., Bhuiyan, M., Li, Y. W., Wall, N., Kraut, M. and Sarkar, F. H. (1998) Nutr
Cancer, 31, 184-91.

Lu, L. J., Cree, M., Josyula, S., Nagamani, M., Grady, J. J. and Anderson, K. E.
(2000) Cancer Res, 60, 1299-305.
Magee, P. J. and Rowland, I. R. (2004) Br. J. Nutr. 91, 513-531

Maggiolini, M., Bonofiglio, D., Marsico, S., Panno, M. L., Cenni, B., Picard, D. and
Ando, S. (2001) Mol Pharmacol, 60, 595-602.

Mangtani, P. and Silva Idos, S. (1998) Lancet, 351, 137; author reply 138-9.

Marcu, K. B., Bossone, S. A. and Patel, A. J. (1992) Annu Rev Biochem, 61, 809-60.

Messina, M. J. (1999) Am J Clin Nutr, 70, 439S-450S.

Miodini, P., Fioravanti, L., Di Fronzo, G. and Cappelletti, V. (1999) Br J Cancer, 80,
1150-5.

Monks, T. J. and Lau, S. S. (1998) Annu. Rev. Pharmacol. Toxicol. 38, 229-255

Monks, T. J. and Lau, S. S. (1997) Chem. Res Toxicol. 10, 1296-1313

Nagata, C., Takatsuka, N., Inaba, S., Kawakami, N. and Shimizu, H. (1998) J Natl
Cancer Inst, 90, 1830-5.

106

Nomoto, S., Arao, Y., Horiguchi, H., Ikeda, K. and Kayama, F. (2002) Oncol Rep, 9,
773-6.
Norbury, C. and Nurse, P. (1992) Annu. Rev. Biochem 61, 441-470

Pappas, S. G. and Jordan, V. C. (2002) Cancer Metastasis Rev, 21, 311-21.

Patterson, L. H. and Murray, G. I. (2002) Curr Pharm Des, 8, 1335-47.

Peterson, T. G., Coward, L., Kirk, M., Falany, C. N. and Barnes, S. (1996)
Carcinogenesis, 17, 1861-9.

Potter, S. M. (1998) Nutr Rev, 56, 231-5.

Potter, S. M., Baum, J. A., Teng, H., Stillman, R. J., Shay, N. F. and Erdman, J. W.,
Jr. (1998) Am J Clin Nutr, 68, 1375S-1379S.

Qiu, X., Forman, H. J., Schonthal, A. H. and Cadenas, E. (1996) J Biol Chem, 271,
31915-21.

Qiu, X. B., Schonthal, A. H. and Cadenas, E. (1998) Free Radic Biol Med, 24, 848-
54.

Reed, D. J., Babson, J. R., Beatty, P. W., Brodie, A. E., Ellis, W. W., and Potter, D.
W. (1980) Anal. Biochem 106, 55-62

Rice-Evans, C. A., Miller, N. J. and Paganga, G. (1996) Free Radic Biol Med, 20,
933-56.

Roberts-Kirchhoff, E. S., Crowley, J. R., Hollenberg, P. F. and Kim, H. (1999) Chem
Res Toxicol, 12, 610-6.

Rowland, I., Faughnan, M., Hoey, L., Wahala, K., Williamson, G. and Cassidy, A.
(2003) Br J Nutr, 89 Suppl 1, S45-58.

Ruiz-Larrea, M. B., Mohan, A. R., Paganga, G., Miller, N. J., Bolwell, G. P. and
Rice-Evans, C. A. (1997) Free Radic Res, 26, 63-70.

Salti, G. I., Grewal, S., Mehta, R. R., Das Gupta, T. K., Boddie, A. W., Jr. and
Constantinou, A. I. (2000) Eur J Cancer, 36, 796-802.

Santell, R. C., Kieu, N. and Helferich, W. G. (2000) J Nutr, 130, 1665-9.

Sarkar, F. H. and Li, Y. (2002) Cancer Metastasis Rev, 21, 265-80.

107
Sartorelli, A. C., Ishiguro, K., King, C. L., Morin, M. J. and Reiss, M. (1986) Adv
Enzyme Regul, 25, 507-29.

Schroeter, H., Boyd, C., Spencer, J. P., Williams, R. J., Cadenas, E., and Rice-Evans,
C. (2002) Neurobiol. Aging 23, 861-880

Schroeter, H., Spencer, J. P., Rice-Evans, C., and Williams, R. J. (2001) Biochem J.
358, 547-557

Shao, Z. M., Alpaugh, M. L., Fontana, J. A. and Barsky, S. H. (1998a) J Cell
Biochem, 69, 44-54.

Shao, Z. M., Wu, J., Shen, Z. Z. and Barsky, S. H. (1998b) Cancer Res, 58, 4851-7.

Shao, Z. M., Wu, J., Shen, Z. Z. and Barsky, S. H. (1998c) Anticancer Res, 18, 1435-
9.

Shen, J. C., Klein, R. D., Wei, Q., Guan, Y., Contois, J. H., Wang, T. T., Chang, S.
and Hursting, S. D. (2000) Mol Carcinog, 29, 92-102.

Sherr C.J., and Roberts J.M. (1995) Genes Dev 9(10) 1149-63

Sherr C.J. (1996) Science 274(5293):1672-7

Sian, J., Dexter, D. T., Cohen, G., Jenner, P. G., and Marsden, C. D. (1997) J.
Pharm. Pharmacol. 49, 332-335
Shimizu, M. and Weinstein, I. B. (2005) Mutat. Res

Spencer, C. A. and Groudine, M. (1991) Adv Cancer Res, 56, 1-48.

Spencer, J. P. E., Rice-Evans, C., and Williams, R. J. (2003) J. Biol. Chem. 278,
34783-34793

Spencer, J. P. E., Kuhnle, G. G., Williams, R.

Stone, J., de Lange, T., Ramsay, G., Jakobovits, E., Bishop, J. M., Varmus, H. and
Lee, W. (1987) Mol Cell Biol, 7, 1697-709.

Strakowski, S. M., Keck, P. E., Jr., Wong, Y. W., Thyrum, P. T. and Yeh, C. (2002)
J Clin Psychopharmacol, 22, 201-5.

Thomas, H. V., Key, T. J., Allen, D. S., Moore, J. W., Dowsett, M., Fentiman, I. S.
and Wang, D. Y. (1997a) Br J Cancer, 76, 401-5.

Watanabe, N., Broome, M., and Hunter, T. (1995) EMBO J. 14, 1878-1891

108

Thomas, H. V., Reeves, G. K. and Key, T. J. (1997b) Cancer Causes Control, 8,
922-8.

Upadhyay, S., Neburi, M., Chinni, S. R., Alhasan, S., Miller, F. and Sarkar, F. H.
(2001) Clin Cancer Res, 7, 1782-9.

Vennstrom, B., Sheiness, D., Zabielski, J. and Bishop, J. M. (1982) J Virol, 42, 773-
9.

Verkasalo, P. K., Thomas, H. V., Appleby, P. N., Davey, G. K. and Key, T. J. (2001)
Cancer Causes Control, 12, 47-59.

Wang, H. and Joseph, J. A. (1999) Free Radic. Biol. Med. 27, 612-616

Waxman, D. J., Chen, L., Hecht, J. E. and Jounaidi, Y. (1999) Drug Metab Rev, 31,
503-22.

Wei, H., Bowen, R., Cai, Q., Barnes, S. and Wang, Y. (1995) Proc Soc Exp Biol
Med, 208, 124-30.

Wiseman, H. (1996) Biochem Soc Trans, 24, 795-800.

Wiseman, H., Lim, P. and O'Reilly, J. (1996) Biochem Soc Trans, 24, 392S.

Wu, G. S. and El-Diery, W. S. (1996) Nat Med, 2, 255-6.

Xu, X., Duncan, A. M., Wangen, K. E. and Kurzer, M. S. (2000) Cancer Epidemiol
Biomarkers Prev, 9, 781-6.

Yang, J. Q., Mushinski, J. F., Stanton, L. W., Fahrlander, P. D., Tesser, P. C. and
Marcu, K. B. (1984) Curr Top Microbiol Immunol, 113, 146-53.

Zhao, H. and Piwnica-Worms, H. (2001) Mol. Cell Biol. 21, 4129-4139

Zeng, Y., Forbes, K. C., Wu, Z., Moreno, S., Piwnica-Worms, H., and Enoch, T.
(1998) Nature 395, 507-510

Zhou, Y. and Lee, A. S. (1998) J Natl Cancer Inst, 90, 381-8.

Abstract (if available)

Abstract Epidemiological studies suggest that dietary soy intake is associated with a decrease risk of breast cancer. A soy isoflavone, genistein (4,5,7-trihydroxyisoflavone), has been shown to prevent tumor formation in several in in vivo models of cancer, and can exert antiproliferative effects in different cancer cell types, including breast cancer cells. However, the mechanisms for the chemopreventive and antiproliferative effects of soy remain unclear. Two aims outlined in this study included: (1) To investigate the intracellular metabolism of genistein and effect on antiproliferative-related signaling pathways in T47D tumorigenic and MCF-10A nontumorigenic cells, (2) The investigate the chemopreventive effects of genistein in terms of its effects on c-myc-mediated oncogenic transformation by establishing the effects of genistein on apoptosis, cell proliferation, cell viability, and anchorage-independent growth in RAT1A fibroblast cells, and RAT1A fibroblast stably transfected with c-myc. The significant findings that were established included: (1) selective genistein uptake into T47D cells but not MCF10A cells (2) identification of CYP-mediated formation of bioactive metabolites of genistein (5,7,3,4-tetrahydroxyisoflavone (THIF) and two glutathionyl conjugates of THIF) (3) THIF regulated cell cycle via activation of p38 with subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation. These two events are critical for the correct functioning of the cdc2-cyclin B1 complex. (4) Genistein affected cell viability, induced apoptosis, and inhibited cell proliferation to a greater extent in RAT1A fibroblasts that overexpressed c-myc, and inhibition of anchorage-independent growth (a property of transformed cells), thus suggesting that genistein's possible selectivity and/or synergism for the c-myc oncogene may represent an underlying explanation for its reported chemopreventive abilities.

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Creator Nguyen, Dominique Truong-Giang (author)

Core Title The differential effects of genistein on cellular effects in T47D tumorigenic and MCF10A nontumorigenic breast epithelial cells: role of metabolism

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Publication Date 05/04/2007

Defense Date 07/31/2003

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

Tag breast cancer,genistein,metabolism,OAI-PMH Harvest

Language English

Advisor Cadenas, Enrique (committee chair), Schonthal, Axel (committee member), Sevanian, Alex (committee member)

Creator Email dominiquenguy@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m485

Unique identifier UC1225566

Identifier etd-Nguyen-20070504 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-485495 (legacy record id),usctheses-m485 (legacy record id)

Legacy Identifier etd-Nguyen-20070504.pdf

Dmrecord 485495

Document Type Dissertation

Rights Nguyen, Dominique Truong-Giang

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