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Anti-cancer effects of novel glidobactin type proteasome inhibitors

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Anti-cancer effects of novel glidobactin type proteasome inhibitors

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ANTI-CANCER EFFECTS OF NOVEL GLIDOBACTIN TYPE PROTEASOME
INHIBITORS

by

FNU Ashish Anshu

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2011

Copyright 2011 FNU Ashish Anshu

ii
Acknowledgements

Exchange of ideas creates a new object working in a better way. Apart from the ability,
labor and time dedication, guidance and assistance are the two pillars for the success of a
project. Whenever others aid a person his heart is bound to pay gratitude to them. To
realize a project it involves a lot of effort and contribution from a number of people. My
most sincere gratitude is reserved for my guide Dr. Axel Schönthal for his permission to
commence this thesis in the first instance and to do the necessary research work. He by
virtue of his office constantly encouraged me and I tender my utmost gratitude and
appreciation for priceless guidance and suggestions extended by him. His inspiring
suggestions and encouragement helped me in all the time of research for and writing of
this thesis.
It is my privilege to express words of gratitude to Dr. Florence Hofman and Dr. Stanley
Tahara for being the most admirable and talented professor’s I have come across in my
lifespan. Their knowledge, interactive skills and innovative approach in teaching are in
itself a state of art.
I am immensely thankful and convey my sincere gratitude to Dr. Thomas Chen and Dr.
Stan Louie for their guidance and encouragement for all that period that I got to spend
during the completion of project.
I take this opportunity to say my sincere thanks to one of the most enduring figures that
taught the basics to a naïve person like me and made me able enough to successfully

iii
complete this project. From this platform I would like to thank Dr. Simmy Thomas for
her sincere effort and near perfect professional approach that is instrumental in this thesis
completion. I will be deeply obliged to her for the rest of my life.
Taking this moment into account I would also like to thank my lab members Puneet
Agarwal, Natasha Sharma, Dr. Heeyon Cho, Francisco Vasquez and Reyna Gonzalez for
their invaluable support and guidance all the way through my project.
Last but not the least I would like to thank my parents for all their support and
encouragement.

iv
Table of Contents

Acknowledgements

ii
List of Figures

v
Abbreviations

viii
Abstract

ix
1. Introduction

1.1. Hematological Malignancies
1.1.1 Multiple Myeloma
1.1.2 Waldenström Macroglobulinemia
1.1.3 Acute Lymphocytic Leukemia (All)

1.2. Apoptosis
1.3. Endoplasmic Reticulum (ER) Stress
1.4. 26 S Proteasome and its function
1.5. Potential of 26s Proteasome inhibition in cancer therapy
1.6. Novel Glidobactin Analogues

1
2
3
4

4
7
10
14
15

2. Materials and Methods

2.1. Materials
2.2. Cell Lines and Culture conditions
2.3. 26S Proteasome Assay
2.4. 20S Proteasome Assay
2.5. MTT Assay
2.6. Immunoblots and Antibodies

17
17
18
18
19
19
3. Results
Table 1: Showing Ic
50
Values of T-01, T-02, T-03 and T-04.

21
26
4. Discussion

44
Bibliography

49

v
List of Figures

Figure 1: Showing six different hallmarks of cancer, adapted from Hanahan
and Weinberg, 2011

5 5 5
Figure 2: Showing role of GRP-78 in three signaling pathways resulting
from unfolded protein response (adapted from Szegezdi et al.
2006).

8
Figure 3: Figure 3a shows the heptameric arrangement of α and β subunit of
20S proteasome (adapted from Creative Commons Attribution-
Share Alike 3.0 Unported); figure 3 b shows composition and
arrangement of 26S proteasome (McNaught et al. 2001).

13
Figure 4: Figure 4a,b, c and d depicts the structure of T-01, T-02, T-03 and
T-04 respectively

16
Figure 5: Effect of T-01 (B), T-02 (A, C), T-03 (B) and T-04 (A,C) on
proteasome activity of BCWM1. Figure 5a shows the
chymotrypsin like activity of 26S proteasome of BCWM1 cells
under different treatment conditions in a dose dependent manner
whereas; figure 5b depicts the similar activity of 20S proteasome
in BCWM1. Figure 5c shows the proteasome inhibition of
BCWM1 cells treated with T-04 and T-02 in a time dependent
manner (8h, 16h, and 24h).

22
Figure 6: Accumulation of ubiquitinated proteins as a direct consequence of
T-04 treatment of RPMI/8226 cells. RPMI/8226 multiple
myeloma cells were treated with increasing concentrations of T-04
for 24 hours. Total cell lysates were harvested and the analyzed by
Western blot with an antibody recognizing ubiquitinated proteins
(as an indirect readout of proteasome inhibition) and with an
antibody against PARP (as a direct readout of ongoing apoptosis).
Each blot was stripped and re-probed with an antibody detecting
actin levels (as a loading control; only one blot is shown).

23

vi
Figure 7: Reduced cell growth and survival by syrbactin analogues in
hematological cancer cell lines. RPMI/8226(7a), BCWM1 (7b)
and REH (7c) cells were treated with indicated concentrations of
glidobactin analogues. After incubation period of 72 hours, cell
viability & survival of the treated cells were measured by an MTT
assay. The experiment was repeated several times under same
conditions and produced similar outcomes.
25

Figure 8:

Induction of ER stress and apoptosis markers in cells treated with
T-04 and BZM. Hematological cancer cell lines (RPMI/8226 (c),
BCWM1 (a) and REH (d)) were treated with T-04 individually at
indicated concentrations for 20 h (controls are non-treated cells)
and cell lysates were subjected to western blot analysis with
specific antibodies to CHOP (pro-apoptotic protein and a specific
ER stress marker), Pro caspase-7 and active (cleaved) caspase-3
and 7(an ER stress and apoptosis related protein), Mcl-1 and
Survivin (a pro survival protein), PARP (cleavage of PARP
indicates ongoing apoptosis) and ATF-3 (a protein associated with
general stress). Figure 8b represent the time dependent western
blot (WB) analysis in T-04 treated BCWM1 cells.

28
Figure 9: Depiction of autophagy cycle and formation of autophagosome.
Figure also shows the localization of lipidated and non-lipidated
form of LC3.

30
Figure 10: Western blot analysis showing accumulation of lipidated form of
LC3 (LC3-II) and down-regulation of P-62 in BCWM1 cells when
treated with different concentrations of T-04 for 20 hour indicating
induction of autophagy. Actin was used as a loading control.

31
Figure 11: Reduced cell growth and survival of RPMI/8226 and BCWM1 in
combination drug treatment. Both cell lines were treated with T-
04 in combination with thapsigargin (Tg) in indicated
concentrations. Cell viability was determined by MTT assay after
72 hours of drug treatment. These experiments were repeated
several times with almost similar outcomes.

33
Figure 12: Showing effects of celecoxib and its analogues. DMC and UMC
individually (fig 12a) or in combination (12b) with T-04 in
BCWM1 cell through an MTT assay after treatment for 72 hours.
Fig 12c shows the basal level expression of COX-2 in MIA-PaCa,
U-87, REH and BCWM1 cells by a western blot analysis.

35

vii
Figure 13: Reduced cell growth and survival of RPMI/8226 and BCWM1 in
combination drug treatment. Both cell lines were treated with T-
04 in combination with dimethyl celecoxib (DMC) in indicated
concentrations. Viability of cells was determined by an MTT assay
after 72 hours of drug treatment. These experiments were repeated
several times with almost similar outcomes.

36

Figure 14:

Reduced cell growth and survival of RPMI/8226 and BCWM1 in
combination drug treatment. Both cell lines were treated with T-
04 in combination with bortezomib in indicated concentrations.
Viability of cells was determined by an MTT assay after 72 hours
of drug treatment. These experiments were repeated several times
with almost similar outcomes.

37
Figure 15: Reduced cell growth and survival of RPMI/8226 and BCWM1 in
combination drug treatment. Both cell lines were treated with T-
04 in combination with tunicamycin indicated concentrations.
Viability of cells was determined by an MTT assay after 72 hours
of drug treatment. These experiments were repeated several times
with almost similar outcomes.

39
Figure 16: Enhanced expression of markers for ER stress and apoptosis after
combination treatment BCWM1 cells were treated with 50 nM T-
04 in the presence or absence of 0.25 or 0.5 µM thapsigargin (Tg).
Parallel cell cultures remained untreated (control, C) or received
vehicle (Veh) only. After 20 hours, cell lysates were harvested and
analyzed by Western blot.

40
Figure 17: Enhanced expression of markers for ER stress and apoptosis after
combination treatment. BCWM1 cells were treated with 50 nM T-
04 in the presence or absence of 20 and 30 µM DMC. Parallel
control cell cultures were untreated (C) or received vehicle only
(Veh). After 20 hours, cell lysates were harvested and analyzed by
Western blot.

41
Figure 18: T-04 modulates growth signaling pathway in BCWM1 cells. Cells
were treated with T-04 in varying concentrations and the whole
lyasate were subjected to western blotting using anti-p-Akt, anti-
Akt, anti p-ERK and anti-ERK.

43

viii
Abbreviations

WM Waldenström Macroglobulinemia
ALL Acute Lymphocytic Leukemia
ERS Endoplasmic Reticulum Stress
UPR Unfolded Protein Response
ATF6 Activating Transcription Factor 6
CHOP CCAAT/enhancer binding protein homologous
transcription factor

GlbA Glidobactin A
SylA Syringolin A
DMSO Dimethyl Sulfoxide
PBS Phosphate Buffered Saline
PARP Poly ADP-Ribose Polymerase
LC3 Light Chain–3
Tg Thapsigargin
SERCA Sarcoplasmic/Endoplasmic Reticulum Calcium
ATPase
DMC Dimethyl Celecoxib
UMC Unmethylated Celecoxib
PDK1 3-Phosphoinositide-Dependent Protein Kinase-1
IKK IκB Kinase
Mcl-1 Myeloid Leukemia Cell Differentiation Protein
GRP-78 Glucose Related Protein-78
ATF-3 Activation Transcription Factor-3
NF-Κb Nuclear Factor Kappa-Light-Chain-Enhancer of
activated B Cells

ix
Abstract

Proteasome inhibitors are widely used today as an important tool for anti-cancer
treatment, which has led to a wide search of novel proteasome inhibitors. Proteasome
inhibitors tested in this study are analogues of glidobactin, which are a new class of
proteasome inhibitors. Similarly, these compounds effectively inhibit the 26S proteasome
activity. In this study, we investigated the cytotoxic effect of these compounds on human
multiple myeloma (MM), human Waldenstrom macroglobulinemia (BCWM1), and
human lymphocytic leukemia cells (REH). Of the four compounds we tested, T-02 and
T-04 were most potent ones and their proteasome inhibitory effect directly correlates with
their cell cytotoxicity. Induction of mild ER stress provides a survival benefit to cells,
however severe ER stress is known to inhibit proliferation of cells leading to apoptosis.
Since a number of proteasome inhibitors have been shown to trigger ER stress induced
apoptosis, we investigated whether these novel proteasome inhibitors have this anti-
cancer efficacy. Our results suggest that it is indeed the case. We further explored
whether the combination of these compounds with other known ER stress inducers leads
to aggravated ER stress and enhancement of their anti-cancer efficacy. T-02 or T-04 in
combination with thapsigargin (a known inhibitor of sarco/endoplasmic reticulum Ca(2+)
ATPase) and dimethyl celecoxib (DMC), an analogue of celecoxib demonstrate enhanced
cytotoxic effect with more severe ER stress induction and apoptosis mediated cell death.
Taken together, our findings suggest these novel compounds are very effective
proteasome inhibitors and trigger the ER stress response with enhanced apoptosis

x
mediated cell death. In addition, in combination with other ER inducer(s), cytotoxic
effects of these compounds are highly potentiated.

1
1. Introduction

1.1 . Hematological Malignancies
Hematological malignancies are malignant neoplasia of blood, bone marrow
and lymph nodes. It is widely believed that tumors arise from single cells, which
accumulate different mutations and genetic lesions inducing uncontrolled cell
proliferation, ultimately leading to malignant or non-malignant tumors. Similarly, all
neoplastic cells of hematological disorders originate from hematopoietic stem cells and
are a direct consequence of the hierarchical developmental lineages of the hematopoietic
system (Warr et al. 2011). Hematological malignant cells are derived from two major
blood lineages, lymphoid and myeloid precursors and are distinguished based on different
parameters such as clinical, morphological and genetic features as well as
immunophenotype of cells (Warr et al. 2011 and Ireland et al. 2011). Common lymphoid
precursors produce B cells, T cells and natural killer (NK) cells whereas common
myeloid precursors produce granulocytes (polymorphonuclear leukocyte), erythrocytes,
macrophages, dendritic cells and mast cells. In the context of hematological malignancies
to these precursors: myelomas, lymphomas and lymphocytic leukemia arise from
common lymphoid progenitors and acute myelogenous leukemia and myeloproliferative
disorders arise from common myeloid progenitors (Warr et al. 2011). In this study, we
mainly focused on three hematological malignancies, i.e. multiple myeloma,
Waldenström macroglobulinemia and acute lymphocytic leukemia.

2
1.1.1 . Multiple Myeloma
Multiple myeloma or Kahler’s disease is the cancer of plasma cells that are
responsible for producing antibodies. It is the second most predominant hematological
malignancy in the USA after non-Hodgkin’s lymphomas. Occurrence of multiple
myeloma is more common in men compared to women and the incidence is twice as high
in black compared to white American populations. This disparity in incidence of disease
in different sex and race is however unknown (Anderson et al., 2009).
In multiple myeloma, a group of abnormal plasma cells (myelomas) multiply extensively
leading to clonal expansion of plasma cells and their accumulation at multiple sites in
bone marrow. The disease is characterized by an increase in bone marrow plasma cells,
osteolytic lesion and monoclonal proteins. (Anderson et al., 2009). It is diagnosed by the
presence of paraprotein in serum or urine, which are detected by the electrophoresis of
serum and immunofixation. The presence of malignant plasma cells in bone marrow is
also detected by analyzing the bone marrow aspirate. Since multiple myelomas are
known to cause bone lesions, its presence can be screened by magnetic resonance
imaging (MRI) and skeletal survey (Anderson et al., 2009). Treatment of multiple
myeloma includes conventional treatment and high dose treatment followed by
autologous stem cell transplant. Bortezomib, which is a proteasome inhibition based
drug, is FDA approved for the treatment of relapsed multiple myeloma (Anderson et al.,
2009).

3
1.1.2. Waldenström Macroglobulinemia
Waldenström macroglobulinemia (WM) is a rare non-Hodgkin lymphoma
and is characterized by infiltration of lymphoplasmacytic cells in bone marrow. WM cells
have a high protein turnover since they secrete large amounts of monoclonal IgM
antibody. Symptoms of this disease include hepatosplenomegaly, i.e simultaneous
enlargement of liver and spleen, anemia, lymphadenopathy, nose bleeds, visual and
neurological problems (Gertz, 2005 and Ghobrial et al., 2008). One of the most intriguing
problems with the diagnosis of Waldenström macroglobulinemia is that some patients do
not exhibit any symptoms, thus making an early detection of the disease problematic.
Initial diagnosis of WM includes blood test and bone marrow biopsy. Blood is analyzed
to check the levels of IgM protein and some other markers known to be associated with
this disease. Bone marrow biopsy mainly from the pelvic region is used to identify
certain subtypes of lymphocytes indicative of WM cells. If any positive indications for
the occurrence of WM based on these test are found, the diagnosis is then confirmed by a
computed axial tomography (CAT) scan (National Cancer Institute, USA). As for now
there is no known cure of WM, however a number of treatment options are available such
as alkylating agent based therapy, purine nucleoside analogues like fludarabine or
cladribine along with autologous stem cell transplant. Nonetheless a range of new agents
are currently being tested which includes ritumaxib, which is an antibody of CD20,
thalidomide and proteasome inhibition based drugs, primarily bortezomib (Ghobrial et
al., 2008).

4

1.1.3. Acute Lymphocytic Leukemia (ALL)
Acute lymphocytic leukemia is a type of cancer of white blood cells
(lymphocytes). It mainly affects immature blood cells. It is also known as acute
childhood leukemia and is one of the more common types of cancer in children (Collins
et al. 2010, Kasner et al. 2010 and Rowe et al. 2010). Symptoms of ALL are very similar
to general flu symptoms such as frequent nosebleeds, fever and formation of lumps due
to swallowing of lymph nodes, pale skin and weakness. However, contrary to flu, ALL
symptoms persist (Collins et al. 2010, Kasner et al. 2010 and Rowe et al. 2010).
Diagnosis of this disease mainly involves determining white blood cell count, the
presence of blast cells in blood smears and a bone marrow biopsy. The diagnosis can be
confirmed by pathological analysis, cytogenetics and immunophenotyping (Collins et al.
2010, Kasner et al. 2010 and Rowe et al. 2010). Treatment of ALL includes
chemotherapy and radiation therapy along with autologous stem cell transplant. Several
new agents are currently investigated such as purine nucleoside analogues (nelarabine,
clofarabine, and forodesine) and bortezomib in combination with mitoxantrone and
etoposide relapsed/refractory acute leukemia (Collins et al. 2010, Kasner et al. 2010 and
Rowe et al. 2010).

1.2. Apoptosis
In the midst of growing notion that cancer cells evolve from single cells,
various arguments and hypothesis have been put forward to explain the diversity and

5
evolution of neoplastic diseases. Hanahan and Weinberg have proposed that normal cells
must acquire various distinct capabilities to evolve to a neoplastic state (Hanahan and
Weinberg, 2011 and Hanahan and Weinberg, 2000). These capabilities were listed as:
evasion of apoptosis, sustained cell proliferation, substantial increase in angiogenesis,
tissue evasion and metastasis, evasion from growth suppressors and enabling replicative
immortality [figure 1] (Hanahan and Weinberg, 2011 and Hanahan and Weinberg, 2000).

Figure 1: Showing six different hallmarks of cancer, adapted from Hanahan and Weinberg, 2011.

Since malignant hematological cells are known to have a high rate of proliferation, we
mainly focused on checking its proliferation rate by subjecting these cells to severe stress
conditions leading to apoptosis. Cell death is generally induced by two mechanisms, i.e.
necrosis and apoptosis. If cells are subjected to external injury or insult, they undergo

6
necrosis whereas if an internal mechanism is activated, cells commit to programmed cell
death i.e. apoptosis. Apoptosis is the programmed cell death that occurs in multicellular
organisms. Different physiological and biochemical events leads to morphological
changes in the cell such as nuclear fragmentation, chromatin condensation and bulge in
plasmamembrane leading to disruption of cell integrity (Alberts et al. 2008). Apoptosis
thus induces production of cellular fragments known as apoptotic bodies, which are
engulfed and cleared by neighboring cells. Different cancer cells are known to have
evolved different evasion mechanisms for sustained growth. For example, B-cell
leukemias increase expression of pro-survival genes such as Bcl-2 and melanoma cells
block the expression of Apaf-1. Withdrawing the growth signals as well as induction of
death signals induces apoptosis (Fulda, 2009). Apoptosis is known to be induced by two
main mechanisms, the intrinsic pathway or mitochondrial pathway or extrinsic or the
death receptor pathway. In the intrinsic pathway, internal injury to a cell induces BAX
protein to migrate to the outer membrane of the mitochondria which inhibits the anti-
apoptotic effect of Bcl-2 by permeabilizing the mitochondrial membrane through which
cytochrome C leaks out from the organelle (Irene et al. 2005 and Reed et al. 2005).
Cytochrome C binds to Apaf-1 (apoptotic protease activating factor-1) to form
apoptosomes and in turn binds pro-caspase-9 for activation (Irene et al. 2005 and Reed et
al. 2005). Caspase-9, an initiator caspase activates executioner caspase-3 leading to a
caspase cascade and ultimately inducing apoptosis (Fulda, 2009). In the extrinsic
pathway, death receptors and other receptors like fas, upon proper stimulation by ligands
send a signal to the cytoplasm to stimulate caspase -8, which is an initiator caspase.

7
Caspase-8 in turn activates a caspase cascade leading to apoptosis (Fulda, 2009).
Different groups worldwide are currently investigating different stress conditions to
induce apoptosis in different cancer cells of which ER stress pathway is one of the most
prominent one.

1.3. Endoplasmic Reticulum (ER) Stress
Endoplasmic reticulum is known to be the site of protein synthesis and
folding. Proper functioning of endoplasmic reticulum is vital for the cell survival as any
disruption may lead to accumulation of unfolded or misfolded protein causing cellular
stress. As a corrective mechanism, endoplasmic reticulum stress (ERS) response is
mounted by the cell in which the transmembrane receptors of ER upon detection of stress
initiates an unfolded protein response (UPR) to minimize the effects of the insult and
maintain homeostasis (Szegezdi et al. 2006). The UPR is a corrective mechanism to
maintain proper functioning of ER and is mediated by a set of signaling mechanisms
involving three different ER transmembrane receptors: PERK (double-stranded RNA-
activated protein kinase-like ER kinase), ATF6 (Activating transcription factor 6) and
IRE1 (endoribonuclease inositol-requiring enzyme 1). In normal cells these three
signaling pathways are inhibited by the action of a molecular chaperone, i.e. GRP-78,
which binds to the three transmembrane receptors, and maintain them in an inactive state
(Szegezdi et al. 2006). This opinion is also supported by the fact that normal cells have
no basal level expression of GRP-78. In conditions of mild stress, GRP-78 dissociates
from these transmembrane receptors and in the process activates pro-survival signaling

8
pathways, which mount an UPR response. However, when an unmitigated ER stress is
induced, the pro-survival effects of these pathways are overcomed by the pro-apoptotic
signals (Szegezdi et al. 2006).

Figure 2: Showing role of GRP-78 in three signaling pathways resulting from unfolded protein response
(Szegezdi et al. 2006).

PERK upon dissociation from GRP-78 is activated and prevents protein translation by
phosphorylating eIF2 (eukaryotic initiation translation factor). This proves beneficial for
cell survival in response to conditions of accumulated, unfolded proteins. However, this
inhibition is incomplete and certain genes that have regulatory sequences in 5’
untranslated region can bypass eIF2 dependent inhibition (Szegezdi et al. 2006). One
such example is expression of ATF4, which induces several pro-survival genes that are
involved in the stress response, amino acid synthesis and transport. However, not all

9
functions of ATF4 are anti-apoptotic, since it also induces the expression of CHOP
(Szegezdi et al. 2006). This disparity can be explained in the context of the level of
stress, in which it has been proven that mild stress conditions are beneficial for cell
survival, however sustained stress can prove detrimental to the cell. Similarly, ATF6 after
dissociating with GRP-78 migrates to golgi apparatus where it gets activated by the
action of proteases. It then migrates to the nucleus and increases the expression of pro-
survival genes such as GRP-78, GRP-94 along with CHOP and x-box binding protein1
(XBP1) expression (Szegezdi et al. 2006). Intriguingly, IRE1 is involved in feedback
inhibition of PERK mediated translation inhibition through P58
IPK
thereby signifying the
termination of UPR response
.
IRE1 modifies XBP1 by removing a 26 nucleotide long
intron generating sXPB1 that encodes HSP40 family member P58
IPK
(Szegezdi et al.
2006).
On the basis of these signaling pathways, the UPR response is first mediated by PERK
and ATF6 and later on by IRE1. However, if the stress is prolonged, pro-apoptotic
signals are induced. One of the important proteins involved in this switch is C/EBP
homologous protein (CHOP), which is also known as GADD153 (growth arrest and
DNA damage-inducible gene 153) (Schönthal H. Axel. 2008). Tumor cells are known to
express small amounts of CHOP since high GRP-78 levels check its expression. However
in the advent of severe ER stress, significant induction of CHOP has been reported which
gravely reduces the survival potential of the cell and ensures its death (Schönthal H.
Axel. 2008).

10
Keeping up with these developments, our main focus of this study was to induce ER
stress mediated apoptosis in hematological cells. There are many different ways to induce
severe insult to cells in order to mount an ERS response such as, inhibition of 26S
proteasome activity, disruption of calcium homeostasis, hypoxia, hypoglycemia and
inhibition of HDAC (Schönthal H. Axel. 2008). Since the study mainly focused on
secretory cells that have been shown to be more susceptible to proteasome inhibition, we
mainly focused on exploiting this pathway to induce ER stress related apoptosis.

1.2 26 S Proteasome and its function

Proteasomes are large non-lysosomal, multienzyme complexes (ca. 2.4
MDa) and are found in all eukaryotes, archaea and some bacteria. They are constitutively
present in the cytoplasm and nucleus of all eukaryotic cells and are the principal pathway
for degradation of intracellular proteins, misfolded and unfolded proteins. Recent studies
show that proteasomes can enter nuclei through nuclear pores. The 26S proteasome
complex contains a 20S core catalytic subunit and two 19S regulatory subunits (Adams.
J. 2004). The 20S catalytic subunit is a 28-subunit core and consists of two outer and
inner heptameric rings arranged axially to form a hollow central core (McNaught et al.
2001). Two outer rings are known as α subunit, which consists of seven polypeptides and
are part of structural makeup of the 20S proteasome. The inner two rings are known as β
subunit and are made up of seven polypeptides (figure 3a and 3b). β subunits form the
central core chamber consisting of three catalytic subunits: β1 which has chymotrypsin
like activity i.e. it is specific for peptides having tyrosine and phenylalanine at the

11
carboxyl position, β2 which has trypsin like activity as it has specificity for peptides
having carboxyl arginine or lysine residues and β5 which has post-glutamyl peptidyl
hydrolytic–like activity since it cleaves the peptides after acidic residues like aspartate or
glutamate (Adams. J. 2004). Seven different genes encode the seven subunits of α and β
subunits and the two subunits are assembled in a specific manner to impart selectivity in
function of the 20S proteasome (McNaught et al. 2001). A regulatory subunit, 19S is a
700 KDa complex consisting 20 different polypeptides. The 19S complex is present on
both ends of the 20S proteasome and serves two main functions: it acts as a lid that needs
to be opened to allow the entry of proteins into the catalytic core (20S) for subsequent
degradation and it also regulates the unfolding of polyubiquitinated protein (Adams. J.
2004). The 19S regulatory subunit consists of six different ATPases along with other
polypeptides. ATP hydrolysis is necessary for conformational change in the 19S subunit
thereby opening the lid that covers the 20S subunit and thus facilitates the entry of
intracellular proteins into the catalytic core (McNaught et al. 2001). However, the first
step in the degradation pathway of unwanted proteins is poly-ubiquitination, i.e. tagging
of proteins with a protein molecule known as ubiquitin. Ubiquitin is a small protein
consisting of 76 amino acids and is highly conserved in eukaryotes. Ubiquitin is
synthesized as a precursor form and requires modification at the C-terminal end by
deubiquitinating enzymes (DUBs) to expose its substrate conjugation site i.e. glycine
carboxylate (Kerscher et al. 2006). Ubiquitin has seven lysine residues that are involved
in poly-ubiquitination (Kerscher et al. 2006). Ubiquitin (Ub) is covalently conjugated to
protein substrates and the process involves three different ubiquitin associated enzymes,

12
E1, E2 and E3. E1 (ubiquitin activating enzyme) binds to ubiquitin and activates it in the
presence of ATP. E2 (ubiquitin carrier enzyme) transfers the Ub molecule from E1 to an
ubiquitin ligase enzyme, E3 (Kerscher et al. 2006). Many different subtypes of E3 are
present which binds to specific substrates targeted for degradation thus imparting
specificity to the whole process. E2 and E3 coordinate the transfer of the ubiquitin
molecule to the lysine residue of the substrate via an isopeptide bond. This process is
repeated several times by three ubiquitin-associated enzyme leading to poly-
ubiquitination of the target protein. These poly-ubiquitinated proteins are then recognized
by the 19S subunit and are then processed for degradation (Adams. J. 2004).
From the description above, it can be said that the 26S proteasome functions are
regulated by the energy and ubiquitination state of the proteins in the cell. However,
some exceptions to this rule have been noted in the mammalian cells in which a different
regulatory subunit, 11S binds to the 20S subunit to form a 26S complex (figure 3b). This
complex also has protein degradation activity though in an energy and ubiquitination
independent manner (McNaught et al. 2001).

13
3a

3b

Figure 3: figure 3a shows the heptameric arrangement of α and β subunit of 20S proteasome (adapted from
Creative Commons Attribution-Share Alike 3.0 Unported); figure 3b shows composition and
arrangement of 26S proteasome (McNaught et al. 2001).

14
1.5. Potential of 26S Proteasome inhibition in cancer therapy
The proteasomes are known to regulate cell cycle and endoplasmic
reticulum homeostasis and therefore are considered to be an effective target in cancer
therapy. Their importance is much more pronounced in secretory cells such as
immunoglobulin producing multiple myeloma or other highly secretory cells such as
Waldenström macroglobulinemia cells since any disruption in normal functioning of
proteasome leads to accumulation and aggregation of large amounts of misfolded or
unfolded proteins which can prove detrimental to the cell by causing proteotoxicity
(Drexler, 2009 and Lonial, 2011) Several groups have tested different proteasome
inhibitors, however the first to be tested for clinical use was the dipeptidyl boronic acid
small molecule bortezomib (Velcade
®
). Since then, bortezomib continues to be the only
FDA approved drug for the treatment of multiple myeloma in this class. Recently,
clinical trials have shown some therapeutic potential of bortezomib in other
hematological malignancies such as Waldenström macroglobulinemia.
Notwithstanding the clinical benefits, concerns have been raised regarding bortezomib
toxicity presenting as thrombocytopenia and peripheral neuropathy (incidence ≥30%),
inhibitor specificity, and drug resistance in case of prolonged treatment (Hideshima et al.
2001). The success and associated concerns of bortezomib use have led many researchers
to search for novel proteasome inhibitors.
Recently, an entirely new class of proteasome inhibitors was identified termed the
syrbactins. This class of proteasome inhibitors is structurally distinct from other known

15
proteasome inhibitors and was shown to bind and inhibit the eukaryotic proteasome in a
novel manner (Schönthal A. H. 2009, Schönthal A.H. 2010 and Treiman, 1998).

1.6. Novel Glidobactin Analogues
Syrbactins include two structurally related, but nonetheless different natural
compounds i.e. the syringolins and the glidobactins. Syringolins, such as syringolin A
(SylA) and syringolin B, are plant virulence factors produced by the plant pathogen
Pseudomonas syringae pv. Syringae whereas glidobactins, such as glidobactin A (GlbA),
are produced by an unknown species of the Burkholderiales order of proteobacteria
(Archera et al. 2010). This study showed that the syrbactin class of proteasome inhibitors
have a prominent anti-cancer effect and induce apoptosis and autophagy in
neuroblastoma cells (Archera et al. 2010). They also found GIbA to have a more
profound anticancer effect compared to SylA and concluded that the difference lies in the
lipophilic tail of GIbA, which enables it to enter cells in a rapid and effective manner
(Archera etal. 2010). Based on these studies and proven anti-cancer ability of glidobactin
A, four analogues of glidobactin A were prepared namely, T-01, T-02, T-03 and T-04 by
the laboratory of Dr. Michael C. Pirrung (Department of Chemistry, University of
California, Riverside) (figure 4a,b c&d). These analoges are structurally similar to SylA
with a 12 membered ring system but have a lipophilic tail similar to glidobactin A
(GIbA). The structure of these glidobactin analogues consists in addition to the lipophilic
tails several amide and ester groups embedded either in the closed ring or in the side
chains. There are minor changes among the structure of these four analogues, for

16
example T-01 (fig. 4a) have an iso-propyl side chain compared to an iso-amyl side chain
in T-02 (fig. 4b). Similarly T-04 (fig. 4c) has a methyl group replacing the isopropyl side
chain of T-01 and T-02. T-03 (fig. 4d) seems to differ from rest of analogues in being
dehydrogenated in ring structure and an ester group replacing an amide group in the
closed ring.

4a 4b

4c 4d

Figure 4: Figure 4a,b, c and d depicts the structure of T-01, T-02, T-03 and T-04 respectively.

H
N
O
O
O
N
H
O
H
N
H
N
C
12
H
25
O
H
N
H
N
O
O
N
H
O
H
N
H
N
C
12
H
25
O
H
N
H
N
O
O
N
H
O
H
N
H
N
C
12
H
25
O

17
2. Materials and Methods

2.1. Materials
Four glidobactin analogs (T-01, T-02, T-03, T-04) were de-novo
synthesized and kindly provided Dr. Michael C. Pirrung from University of California,
Riverside. Each compound was dissolved in dimethyl sulfoxide (DMSO) to make a stock
solution of 10 mM. Thapsigargin and Tunicamycin were obtained from Sigma-Aldrich
(St. Louis, MO) and dissolved in DMSO. Bortezomib was acquired from Millenium
Pharmaceutical, Cambridge, MA and was dissolved in DMSO. All agents were added to
the cell culture medium in a manner so that the final concentration of solvent (DMSO)
did not exceed 1%, most incubation were well below that value.

2.2. Cell lines and Culture conditions
Human RPMI/8226 multiple myeloma cells were obtained from the
American Type Culture Collection (ATCC; Manassas, VA). The human Waldenström’s
macroglobulinemia cell line BCWM1 was kindly provided by Steven P. Treon [17], and
human REH lymphocytic leukemia cells were a kind gift from Alan L. Epstein. All cells
were propagated in RPMI (Cellgro, Herndon, VA) supplemented with 5% fetal bovine
serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin, and kept in a humidified
incubator at 37˚ C and a 5% CO
2
atmosphere.

18

2.3. 26S Proteasome Assay
After treatment with the drugs for 24 hours, cells were washed with buffer 1
(50 mM Tris pH 7.4, 0.1 mM EDTA, 2 mM DTT, 5 mM MgCl
2
, 2 mM ATP) and syringe lysed
10x in 100µl buffer 1 containing 250 mM sucrose and 0.04% NP40. Samples were spun
at 10k for 10 min and supernatant was collected. After protein concentration assay, 20
µg protein was diluted to 200 µl in buffer 1 in a 96 well plate. 80µM (1.6 µl) of
fluorogenic proteasome substrate, SucLLVY-AMC procured from Sigma Aldrich (St.
Louis, MO) was added to the 96-well plate. Proteolytic activity was measured by
monitoring the release of the fluorescent group 7-amido-4-methylcoumarin (AMC)
(excitation 360 nm, emission 460 nm) at 37
o
C using Perkin-Elmer multilabel reader,
Envision 2103 (Waltham, Massachusetts) and quantified by a free AMC standard curve.
Readings were taken at 0, 15, 30, 45 and 60 min of incubation.

2.4. 20S Proteasome Assay
After treatment with the drugs for 24 hours, cells were lysed in 100 µl
buffer 2 (50 mM Tris pH 7.4, 0.1 mM EDTA, 20 mM KCl, 5 mM MgCl
2
, 1 mM DTT, 0.03%
SDS) containing 0.04% NP40. After protein assay, 20 µg protein was diluted to 200 µl in
buffer 1 in a 96–well plate. 80µM (1.6µl) LLVY-AMC from stock solution was added to
the plate. Release of the fluorescent AMC group was followed at ex. 360 nm and em.
460 nm at 37
o
C using Perkin-Elmer multilabel reader, Envision 2103 (Waltham,

19
Massachusetts) and quantified by a free AMC standard curve. Readings were taken at 0,
15, 30, 45 and 60 min of incubation.

2.5. MTT Assay
A 96 well plated was with 50,000 cells/well in a 00 mL volume and treated
with desired drug concentrations along with a control group. 10 µl of 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole, is added to
each well after 72 hours of incubation at 37°C. Yellow MTT dye is reduced to purple
formazan in the mitochondria of living cells. The plate is then covered with aluminum
foil to protect it from light exposure. 100 µl of solubilization solution (a solution of
the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the
insoluble purple formazan product after 4 hours incubation. The culture plate is then read
at a 490 nm wavelength in a spectrophotometer. All assays were repeated several times at
variable cell densities from 1.0 to 5.0 × 10
4
.

2.6. Immunoblots and Antibodies
Cells were passaged a day before treatment in RPMI media with 5% FBS
to ensure most of the cells were in exponential phase. The cells were then treated with
drugs at desired concentrations along with a control group for the desired time-period
prior to harvesting the cells for western blot analysis. Cells were washed twice with
phosphate buffered saline (PBS) and then centrifuged at 14000 rpm for 30 sec. The
supernatant fraction was discarded and the pellet was lysed with RIPA lysis buffer

20
containing 10 µl PMSF, Protease inhibitor (1 tablet) and 100 µl phosphatase inhibitor.
Cells were then subjected to sonication and centrifugation at 14000 rpm for 20 min. The
supernatant fraction was collected as protein lysate. Protein concentrations were
measured in duplicate by Bradford protein assay using a BCA standard. The loading
lysate was prepared with at final volume containing 50 µg protein/lane and ran on an
SDS-PAGE minigel at 100 V. After completion of electrophoresis, proteins were
transferred onto a nitrocellulose membrane by the wet transfer method (Schönthal et al.
2009). After the completion of transfer, the membrane was blocked for 1 hour in 5% non-
fat dry milk at room temperature. They were then incubated overnight with primary
antibody (1:500 dilution of antibody in 5% milk). Membranes were then washed in PBST
(phosphate buffered saline and 0.2% Tween 20) for 10 minutes three times. The
membranes were then incubated with horseradish peroxidase (HRP) conjugated
secondary antibodies for one hour. The membranes were then washed three times with
PBST and developed using Supersignal substrate from Thermo Scientific, Rockford, IL.
Primary and secondary antibodies were purchased from Cell Signaling Technologies
(Beverly, MA), or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used according
to the manufacturer’s recommendations.

21
3. Results

3.1. Novel glidobactin analogues exhibit the 26S and 20S proteasome inhibitory
activity in hematological cell lines.
Glidobactins were discovered around 22 years ago and were shown to have
some anti-cancer properties. However recent studies showed that these small molecules
are actually potent proteasome inhibitors (Groll et al. 2008). Keeping with this effort to
further better the effects of these compounds, we developed four analogues of glidobactin
A (GlbA), T-01, T-02, T-03 and T-04 and probed their efficacy with respect to anti-
cancer effects.
Figure 5a shows that the four analogues have variable inhibitory effect on proteasome
activity implying that small changes in the structure of glidobactin A resulted in marked
differences in its ability to inhibit the 26S proteasome activity. T-02 was found to be the
most potent drug, narrowly followed by T-04, both of which showed marked proteasome
inhibition in the nanomolar range. By comparison, T-03 was able to inhibit the
proteasome only in the micromolar range and T-01 was the least effective with an IC50 >
100 µM.
We further tested T-02 and T-04, the two most potent drugs for their ability to affect the
20S proteasome activity. As shown in figure 5b, these two drugs were found to have a
more pronounced effect on the 20S proteasome with almost complete inhibition achieved
at 100 nM. We further investigated the effect of T-02 and T-04 on 26S proteasome

22

Figure 5: Effect of T-01 (B), T-02 (A, C), T-03 (B) and T-04 (A,C) on proteasome activity of BCWM1.
Figure 5a shows the chymotrypsin like activity of 26S proteasome of BCWM1 cells under different
treatment conditions in a dose dependent manner whereas; figure 5b depicts the similar activity of 20S
proteasome in BCWM1. Figure 5c shows the proteasome inhibition of BCWM1 cells treated with T-04
and T-02 in a time dependent manner (8h, 16h, and 24h).

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23
activity at different times of incubation, in BCWM1 cells to see the time-point at which
the inhibition is achieved. Figure 5c shows that these drugs inhibit the proteasome
activity in a time dependent manner with significant inhibition seen within 8 hours of
treatment of T-02 or T-04 and almost complete inhibition at 24 hours.

Figure 2

Figure 6: Accumulation of ubiquitinated proteins as a direct consequence of T-04 treatment of RPMI/8226
cells. RPMI/8226 multiple myeloma cells were treated with increasing concentrations of T-04 for 24 hours.
Total cell lysates were harvested and the analyzed by Western blot with an antibody recognizing
ubiquitinated proteins (as an indirect readout of proteasome inhibition) and with an antibody against PARP
(as a direct readout of ongoing apoptosis). Each blot was stripped and re-probed with an antibody detecting
actin levels (as a loading control; only one blot is shown). C: untreated control cells.

24
In order to cross check these results, we employed another method to determine
proteasome inhibition by analyzing the accumulation of polyubiquitinated protein. Since
binding of one ubiquitin molecule triggers binding of other ubiquitin molecules to the
same protein leading to polyubiquitination. Inhibtion of proteasome leads to
accumulation of polyubiquitinated proteins. Multiple myeloma cells, RPMI/8226 were
treated with T-04 for 20 hours and the cell lysate was probed for ubiquitin accumulation
by a western blot analysis. As shown in the figure 6, accumulation of ubiquitin can be
observed in a dose dependent manner. This confirms that the treatment of cells with these
drugs exhibit expected consequences associated with inhibition of proteasome, i.e.
accumulation of ubiquitin. Also shown in figure 6 is the effect of T-04 on the cleavage of
PARP-1 (poly ADP-ribose polymerase-1). It can be observed that T-04 effectively
cleaves PARP indicating that cytotoxic effects of this compound induces the onset of
apoptosis providing vital indications of the efficacy of these drugs for cancer therapy.

3.2. Glidobactin analogues have cytotoxic effect on malignant hematological cell
lines and inhibit their cell proliferation.
To determine how glidobactin analogues (T-01, T-02, T-03 & T-04)
would affect the cell viability and proliferation, we further investigated the cytotoxic
potential of our four glidobactin analogues in three cell lines (RPMI/8226, BCWM1, and
REH) representing different hematological malignancies. Multiple myeloma
(RPMI/8226), Waldenström macroglobulinemia (BCWM1), and acute lymphocytic
leukemia (REH) cells were treated with increasing concentrations of the four analogues

25

7a 7b

7c

Figure 7: Reduced cell growth and survival by syrbactin analogues in hematological cancer cell lines.
RPMI/8226(7a), BCWM1 (7b) and REH (7c) cells were treated with indicated concentrations of
glidobactin analogues. After incubation period of 72 hours, cell viability & survival of the treated cells
were measured by an MTT assay. The experiment was repeated several times under same conditions and
produced similar outcomes.

RPMI/8226
10
0
10
1
10
2
10
3
10
4
10
5
10
6
1
10
100
50
T-01
T-02
T-03
T-04
[ nM ]
% Cell Survival
10
0
10
1
10
2
10
3
10
4
10
5
10
6
1
10
100
50
T-01
T-02
T-03
T-04
BCWM1
[ nM ]
% Cell Survival
10
0
10
1
10
2
10
3
10
4
10
5
10
6
1
10
100
50 T-01
T-02
T-03
T-04
REH
[ nM ]
% Cell Survival

26
for 72 hours and cell survival was determined by MTT assay. As shown in figure 7a, 7b
& 7c, T-02 and T-04 were most potent with almost similar IC
50
i.e. ≈ 180 nM and 195 nM
respectively (Table-1). T-03 was less potent with an IC
50
around 1µM in RPMI/8226 and
REH cells but showed considerable potency in BCWM1 cells with an IC
50
around 500
nM.T-01 was least potent in all the three cell lines tested with an IC
50
of above 100

Table 1: Showing IC50 values of T-01, T-02, T-03 and T-04 when treated for 72 hours in BCWM1,
RPMI/8226 AND REH cells.

µM.These results are in direct correlation with the proteasome inhibitory capacity of
these analogues as shown in figure 5 and 6. Based on these results, cytotoxic potential of
these four glidobactins analogues can be attributed to T-02≈T-04>T-03>T-01.

Drug IC
50

T-01 >100 µM
T-02 ≈ 180 nM
T-03
≈ 1µM in RPMI/8226 and REH cells
≈ 500 nM in BCWM1 cells
T-04 ≈ 195 nM

27
3.3. Glidobactin analogue T-04 triggers severe ER stress and apoptosis in malignant
hematological cell lines.
Since previous studies showed that proteasome inhibitors induce ER stress
and apoptosis (Schönthal A., 2009), we tested one of these analogues (i.e. T-04) on
malignant hematopoietic cells (BCWM1, RPMI/8226 and REH cells) in varying
concentrations and several key cellular targets were analyzed which are markers for ER
stress and apoptosis. BCWM1 cells were exposed to increasing concentrations of T-04
for 20 hours, and cellular lysates were analyzed by Western blot analysis. As shown in
Fig. 8a, drug treatment resulted in down-regulation of survivin and Mcl-1 protein levels,
and increased expression of CHOP and ATF-3. At the same time, there was pronounced
conversion of inactive pro-caspases 3 and 7 to their activated effector counterparts, as
well as proteolytic cleavage of PARP-1.The induction of ER stress and apoptosis markers
coincide with inhibition of 26S proteasome function at a comparable drug concentration
indicating that the proteasome inhibition correlated with the triggering of ER stress and
subsequent advent of apoptosis under these treatment conditions. To investigate the time
course of some of these effects, BCWM1 cells were treated with 100 nM T-04 for various
time periods and stress markers were analyzed by western blot analysis. Figure 8b shows
that ER stress is triggered around 16-hours of drug treatment with induction of CHOP,
which became more prominent with increasing time. Down regulation of Mcl-1 was also
seen at around 24 hour with a pronounced effect at 32-hours with maximum PARP
cleavage. Elevated levels of ATF-3 also confirmed induction of stress in these cells after
this treatment.

28
8a 8b

8c

Figure 8: Induction of ER stress and apoptosis markers in cells treated with T-04 and BZM.
Hematological cancer cell lines (RPMI/8226 (c), BCWM1 (a) and REH (d)) were treated with T-04
individually at indicated concentrations for 20 h (controls are non-treated cells) and cell lysates were
subjected to western blot analysis with specific antibodies to CHOP (pro-apoptotic protein and a specific
ER stress marker), Pro caspase-7 and active (cleaved) caspase-3 and 7(an ER stress and apoptosis related
protein), Mcl-1 and Survivin (a pro survival protein), PARP (cleavage of PARP indicates ongoing
apoptosis) and ATF-3 (a protein associated with general stress). Figure 8b represent the time dependent
western blot (WB) analysis in T-04 treated BCWM1 cells. In these WB, actin was used as a loading
control.

29
Comparable effects of T-04 were observed when investigated in RPMI/8226 cell line
with down-regulation of Mcl-1, induction of ATF-3 and CHOP and cleavage of pro
caspase-3 into its active form preceding cleavage of PARP (figure 8c).However in REH
cells, although strong induction of CHOP was observed along with cleavage of caspase-3
and PARP, no induction of ATF-3 was seen along with marginal down-regulation of
Mcl-1 (figure 8d).
Summarizing the above results, it can be stated that T-04 treatment triggered ER stress
and eventual apoptosis of BCWM1, RPMI/8226 and REH cell lines in a dose, time and
cell line specific manner.

3.4. Glidobactin analogues induced proteasome inhibition promotes autophagy in
hematological cells.
Autophagy and proteasome degradation are two pathways employed by
the cell to clear out the unneeded proteins. It has been widely reported that proteasome
inhibition generally induces autophagy (Archera et al. 2010). To check whether our novel
compounds, which we have shown to be a potent proteasome inhibitors, behave in similar
fashion, we specifically probed the effect of T-04 on light chain–3 (LC3) protein. LC-3 is
constitutively present in the cytoplasm of the cell in non-lipidated form known as LC3-I
(Tanida I. et al 2008 and Tanida I. et al., Dec 2008). During autophagosome formation,
LC3–I is conjugated to phosphatidylethanolamine (PE) by two enzymes Atg7 and Atg3
to form a lipidated form known as LC3-II that migrates to the autophagosome membrane.
When an autophagosome fuses to a lysosome, LC3-II is degraded making it an

30
important marker for autophagosome formation (Tanida I. et al 2008 and Tanida I. et al.,
Dec 2008) (Figure 9). Another protein known as P-62 which is an ubiquitin associated
protein interacts with LC-3 through a specific region known as LRS (LC-3 recognition
sequence) (Komatsu. et al. 2010). Previous studies have shown that P-62 guides the
ubiquitinated proteins into the autophagosomes, interacts with LC-3 and is subsequently
degraded by lysosomes. Therefore P-62 is considered to be a strong marker for autophagy
i.e. its accumulation indicates inhibition of autophagy and its depletion indicates
induction of autophagy. Based on this hypothesis, we tested the ability of our novel
glidobactin analogue, T-04 on LC3 protein modification in BCWM1 cells through
western blot analysis.

Figure 9: Depiction of autophagy cycle and formation of autophagosome. Figure also shows the
localization of lipidated and non-lipidated form of LC3.

31

Figure 10: Western blot analysis showing accumulation of lipidated form of LC3 (LC3-II) and
downregulation of P-62 in BCWM1 cells when treated with different concentrations of T-04
for 20 hour indicating induction of autophagy. Actin was used as a loading control.

Figure 10 shows that control cells contained non-lipidated LC3 (LC3-I), however with
increasing concentration, a strong accumulation of lipidated LC3 (LC3-II) was apparent
at 100 nM concentrations suggesting that treatment by T-04 promoted formation of
autophagosome in BCWM1 cells. To further probe the effect of T-04 on autophagy,
whether it induces or inhibits the formation of autophagosomes, we looked at the
expression levels of P62, a well-known autophagy marker (Moscat et al. 2009). It has
been reported that impaired autophagy promotes accumulation of P-62 that aids
tumorigenesis and its ablation leads to induction of autophagy (Moscat et al. 2009).
Based on these findings, we looked at the expression level of P-62. Figure 10 shows that
with increasing concentration of T-04, P-62 expression was down regulated suggesting
that drug treatment was responsible for induction of autophagy.

32
3.5. Combination treatments of T-04 with other ER stress inducers enhance its
cytotoxic effects in hematologic malignant cells.
Since T-04 in previous experiments proved to be an able inducer of ER
stress evident by strong induction of CHOP, we further examined the possibilities of
combining T-04 with other known ER stress inducers and investigate their effect on
malignant hematological cell lines. Based on previous studies of the Schönthal laboratory
as well as other groups, we identified several compounds for combination treatments
such as thapsigargin (Tg), a widely used inhibitor of SERCA (sarcoplasmic/endoplasmic
reticulum calcium ATPase), dimethyl celecoxib (DMC), an analog of celecoxib,
tunicamycin, a known inhibitor of protein glycosylation and bortezomib, the best known
proteasome inhibitor. We combined the glidobactin T-02 or T-04 with different
concentrations of the above-mentioned compounds and determined cytotoxic efficacy by
observing reduction of viable cells in BCWM1 and RPMI/8226 cells. Relatively low
concentrations of all these drugs were specifically selected for treatment so that potential
enhancing effects would emerge more prominently. As shown in figure 11, combination
treatment of T-02 and T-04 with thapsigargin was extremely potent in both tumor cell
lines. For example, when 50 nM T-02 was combined with 0.5 µM thapsigargin, around
80% reduction was seen in number of viable cells and strikingly almost complete
inhibition of cell survival was seen, when 100 nM T-02 was combined with 1 µM
thapsigargin. This strongly indicates an enhancing effect of thapsigargin on T-04
cytotoxicity. Though slight variability was observed at certain concentrations, it can be

33

Figure 11: Reduced cell growth and survival of RPMI/8226 and BCWM1 in combination drug
treatment. Both cell lines were treated with T-04 in combination with thapsigargin (Tg) in indicated
concentrations. Cell viability was determined by MTT assay after 72 hours of drug treatment. These
experiments were repeated several times with almost similar outcomes.

safely proposed that inhibition of SERCA enhanced the effect of T-02 and T-04 in
RPMI/8226 and BCWM1 cell lines.For further investigation of different combination
drug effects, we investigated the effect of celecoxib, which is a prominent drug for
arthritis and its analogues, dimethyl celecoxib (DMC) and unmethylated celecoxib
(UMC). DMC has a profound anti-cancer effect on Raji lymphoma cells and its action
was shown to be independent of cox-2 inhibition (Chen et al. 2010). On the similar line,
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34
we next investigated the effect of celecoxib, DMC and UMC on BCWM1 cells in
different concentrations. Figure 12a shows that DMC was most potent among the three
drugs with an IC50 around 30 µM compared to CXB or UMC which had IC50s around
45 and 70 µM, respectively. In further investigation we tried a combination treatment of
these drugs with our novel glidobactin analogue, T-04, and found that the combination of
T-04 and DMC was most effective among those tested. For example, 30 µM DMC and
50 nM T-04 reduced cell survival by > 90% (figure 12b). We also investigated whether
COX-2 is expressed in BCWM1 cells. Figure 12c shows a western blot analysis of basal
level expression of COX-2 in BCWM1. MIA-PaCa (human pancreatic carcinoma cells)
was used as a negative control and U-87 (human glioblastoma cells) along with REH
cells were used as positive controls.

35

12a

12b

12c

Figure 12: Showing effects of celecoxib and its analogues. DMC and UMC individually (fig 12a) or in
combination (12b) with T-04 in BCWM1 cells through an MTT assay after treatment for 72
hours. Fig 12c shows the basal level expression of COX-2 in MIA-PaCa, U-87, REH and
BCWM1 cells by a western blot analysis.

36
Based on the finding that DMC was more potent than celecoxib and UMC, we combined
T-02 and T-04 with DMC in RPMI/8226 and BCWM1 cell line and analyzed cell
viability by the MTT assay. As shown in figure 13, DMC considerably enhanced the
cytotoxic potential of these analogues with a dramatic reduction in cell viability. For
example, in BCWM1 cells, 50 nM T-02 in combination with 20 µM of DMC reduces the

Figure 13: Reduced cell growth and survival of RPMI/8226 and BCWM1 in combination drug
treatment. Both cell lines were treated with T-04 in combination with dimethyl celecoxib (DMC) in
indicated concentrations. Viability of cells was determined by an MTT assay after 72 hours of drug
treatment. These experiments were repeated several times with almost similar outcomes.

37
cell viability by around 95% indicating the prominent enhancing effect of DMC on T-02
and T-04 action (figure 13). Intriguingly, a cell line specific effect was observed when
BZM was combined with T-02 and T-04. As evident from fig. 14, RPMI/8226 cells were
more sensitive to the combination treatment than BCWM1 cells.

Figure 14: Reduced cell growth and survival of RPMI/8226 and BCWM1 in combination drug
treatment. Both cell lines were treated with T-04 in combination with bortezomib in indicated
concentrations. Viability of cells was determined by an MTT assay after 72 hours of drug treatment. These
experiments were repeated several times with almost similar outcomes.

38
Since the primary targets of these two drugs are same i.e. both are potent proteasome
inhibitors, the result suggested that effects of these two drugs might get limited after
maximum possible inhibition of proteasome is achieved i.e. since no further inhibition
can be achieved therefore no enhancing effect can be observed. Continuing our further
investigation of combination potential of glidobactin analogues, we tested the potency of
these two drugs in combination with tunicamycin. Tunicamycin prevents glycosylation of
newly synthesized peptides and has been shown to induce an unfolded protein response
(UPR) (Szegezdi et al. 2006). Figure 15 shows that unexpectedly, this combination
treatment exhibited variable results with some clear antagonizing effect seen at certain
drug concentrations. For example, 100 nM T-02 in combination with 1 µM tunicamycin
in RPMI/8226 cells showed ca. 80% cell survival, which is more than that observed with
individual drug treatments. This suggested that tunicamycin by some mechanism
negatively interfered with the action of glidobactin analogues.

39

Figure 15: Reduced cell growth and survival of RPMI/8226 and BCWM1 in combination drug
treatment. Both cell lines were treated with T-04 in combination with tunicamycin indicated
concentrations. Viability of cells was determined by an MTT assay after 72 hours of drug treatment. These
experiments were repeated several times with almost similar outcomes.

Since combination treatments showed DMC and Tg potentiated the effects of glidobactin
analogues, we proposed that the enhanced cytotoxic combination effect of these drugs is
associated with ER stress aggravation which induced a more pronounced apoptosis at

40
lower concentrations of either DMC or Tg. BCWM1 cells were treated with either drug
alone or in combination at different T-04 concentrations and the expression of ER stress

Figure. 16. Enhanced expression of markers for ER stress and apoptosis after combination treatment
BCWM1 cells were treated with 50 nM T-04 in the presence or absence of 0.25 or 0.5 µM thapsigargin
(Tg). Parallel cell cultures remained untreated (control, C) or received vehicle (Veh) only. After 20 hours,
cell lysates were harvested and analyzed by Western blot.

markers and PARP cleavage were analyzed. As figure 16 depicts, T-04 and Tg together
triggered more pronounced CHOP induction than the single drug treatments.Consistent
with this effect, a marked increase in apoptotic PARP cleavage was observed in
combination treatments compared to individual treatments. In the case of anti-apoptotic
proteins, a slight down-regulation of Mcl-1 was noted. The increase in ER stress was
confirmed by increased induction of pro-survival protein GRP-78. Similarly, ATF-3
induction was more pronounced in combination treatments.

41
Next we observed the effect of combination treatment of T-04 and DMC on different
markers (figure 17).

Figure. 17. Enhanced expression of markers for ER stress and apoptosis after combination treatment.
BCWM1 cells were treated with 50 nM T-04 in the presence or absence of 20 and 30 µM DMC. Parallel
control cell cultures were untreated (C) or received vehicle only (Veh). After 20 hours, cell lysates were
harvested and analyzed by Western blot.

T-04 at 50 nM and DMC at 20 & 30 µM alone showed only slight effects, however the
combination treatment of these two drugs showed a substantial aggravation of ER stress
with prominent increase in CHOP and ATF-3 expression. Additionally, complete PARP
cleavage and down-regulation of anti-apoptotic protein Mcl-1 was observed suggesting
extensive apoptosis in these combination treatments, which directly correlated with the
aggravation of ER stress and higher cytotoxicity observed in the MTT assay (figure
13).

42

3.6. Signaling pathways regulated by Glidobactin analogue, T-04.
It has been previously shown that phosphatidylinositol inositol-3-kinase
(PI3K)/Akt pathway plays a critical role in cell survival and proliferation of malignant
hematological cells (Uddin et al. 2006). PIP3 present on the inner surface of the plasma
membrane helps in the recruitment of AKT after PI3K activation through interaction with
its PH domain. Thr308 of AKT is phosphorylated by the action of PDK1 (3-
phosphoinositide-dependent protein kinase-1), which is a PH-domain-containing
serine/threonine kinase, followed by phosphorylation at Ser473 by PDK2 (Uddin et al.
2006). Phosphorylation at Thr308 is sufficient to activate AKT, however its maximum
utilization is achieved only after subsequent phosphorylation at Ser473. Thus it has been
proposed that inhibition of phosphorylation at either of the two sites of AKT will impart a
growth disadvantage for cancer cells (Uddin et al. 2006). Based on these findings, we
investigated whether inhibition of cell proliferation is due to inhibition of the AKT
pathway in response to our novel compounds in BCWM1 cells. Figure 18 shows that T-
04 inhibited phosphorylation of Akt at Thr308 as well as at Ser473, in a dose dependent
manner thus rendering the enzyme inactive. Since it is known that dual phosphorylation
of Akt is necessary for its maximal activity, inhibition of Akt phosphorylation should
greatly impair the growth advantage of cancer cells.
We next investigated the effect of T-04 on the MEK/ERK pathway, which is considered
to be vital for growth and survival of cells. Figure 18 shows that T-04 inhibited the

43
phosphorylation of ERK1/2 (Thr-202/ Tyr-204) in a dose dependent manner with
complete inhibition achieved at 100 nM concentrations.

Figure.18. T-04 modulates growth signaling pathway in BCWM1 cells. Cells were treated with T-04 in
varying concentrations and the whole lyasate were subjected to western blotting using anti-p-Akt, anti-Akt,
anti p-ERK and anti-Erk. Actin was used as a loading control.

44
4. Discussion
The fact that proteasomes are a critical regulator of cell cycle has led to its
exploitation in cancer therapy. Experimental findings have shown that actively
proliferating malignant cells are more susceptible to proteasome inhibition induced
apoptosis than normal cells (Adams et al. 2004). This has led to the approval of the use of
bortezomib in multiple myeloma therapy. Many models have been put forward to explain
differential pattern of response in normal and cancer cells; suggesting the role of a single
factor alone or working in conjunction with other factors is responsible for growth
inhibition (Adams et al. 2004). One explanation put forward is that, since cancer cells
have a high proliferation rate, the maintenance of growth requires a greater dependency
on proteasome function for continuous removal of misfolded or unfolded proteins
(Adams et al. 2004). Any disturbance of proteasome function would thus prove to be
detrimental to cancer cells. Another ingenious model put forward is that that inhibition of
proteasome activity might induce certain corrections that reverse the pro-growth
mutation(s) or signals that are predominantly accelerated in cancerous cells (Adams et al.
2004). Another proposed mechanism highlights the importance of NF-κB in growth and
survival of some hematological malignant cells since it is known to control the
expression of anti-apoptotic genes (Leleu et al. 2008). AKT is a positive regulator of NF-
κB by phosphorylation thereby activating IκB kinase (IKK), which induces degradation
of NF-κB inhibitor, IκB. NF-κB pathway is a proteasome dependent pathway suggesting
that proteasome inhibition based drug can also impart a growth disadvantage in cancer
cells compared to normal cells. Based on the remarkable potential of proteasome

45
targeting in cancer therapy and the success of bortezomib, several next-generation drug
candidates are being investigated, including peptide boronic acid analogs MLN9708 and
CEP-18770, peptide epoxyketones carfilzomib and PR-047, and NPI-0052, a beta-lactone
compound. The mechanism of binding to the proteasome as well as the pharmacological
properties of the above–mentioned drugs are distinctive, which imply different efficacy
for these compounds in cancer cells. The four synthetic glidobactin analogues described
in this thesis are homologus but differ slightly in the macrocyclic lactam central structure
and exocyclic side chains. In this study, we demonstrated that two of the four glidobactin
analogues showed marked inhibition of proteasome as well as growth and cell
proliferation of malignant hematological cells tested. Contrary to this effect, the other two
analogues showed vague or no cellular effects even at high micromolar concentrations.
The effect of these drugs on cell survivability was in directly correlated to their
proteasome inhibition effect. For example, the most effective drug, T-02, was ca. 500-
fold more potent than T-01 suggesting that small structural changes impart vast
differences in pharmacological effects of these compounds. Since these analogues have a
lipophilic tail, these drugs presumably can enter the cell rapidly as suggested from the
26S proteasome assay. A significant inhibition was observed as early as 8 hours after
drug addition.
Further investigation revealed that T-04 induced ER stress induced apoptosis mediated by
caspases as was evident in western blot analysis. T-04 was also shown to increase
accumulation of the lipidated form of LC3 (LC3 II) and down-regulation of P-62
indicating an induction of autophagy. In summary these two results show that

46
proteasome inhibition by these drugs activated both apoptosis and autophagy in the
cancer cell lines. Induction of autophagy has also been previous shown to be associated
with proteasome inhibition since it acts as a compensatory or survival mechanism for the
removal of unfolded or misfolded proteins.
Furthermore we also showed that glidobactin analogue, T-04, targets the PI3K/AKT and
MEK/ERK pathways, which are key regulatory pathways for apoptosis, cell cycle and
cancer cell proliferation in hematological and other disorders. Previous studies showed
that AKT expression is up regulated in WM cells and has a pivotal role in the
malignanant property of these cells (Roccaro et al. 2008). So, suppression of the
expression of these regulatory molecules must create a negative growth environment for
these cancer cells. However, whether the inhibition of AKT is one of the primary targets
of T-04 or is secondary to other effects still needs to be investigated.
Lastly, we showed that thapsigargin and DMC potentiated the effect of T-02 and T-04 as
the modes of action of these drugs are notably distinctive. As expected, no enhancing
effect was seen when these analogues were combined with bortezomib, but intriguingly
Tunicamycin seems to have had an antagonizing effect on the potency of these drugs.
In summary, our work and study has described the physiological effects of a new class of
proteasome inhibitors derived from a natural product. This work also provided evidence
for potential use as anti-proliferative agents in multiple myeloma, Waldenström
macroglobulinemia and acute lymphocytic leukemia based on its predominent effect in
inducing apoptosis in these cells. Although bortezomib is widely used against relapsed
multiple myeloma and is being currently investigated for a host of other disorders, its

47
toxicity which includes thrombocytopenia and neuropathy is significant. Furthermore its
low specificity and associated drug resistance in clinical use are contraindications for
widespread use; thus this has propelled many researchers to look for other natural product
based proteasome inhibitors. Since proteasome inhibition is also related to
chemosensitization of cancer cells (Archera et al. 2010), the effects of these drugs hold
great promise for future applications.
Taken together, hematological malignancies account for 9.5% of new cancer diagnoses in
the United States, which suggests that there is a large potential need for novel effective
compounds which check the proliferation of cancer cells (BCC survey). Drug cost is an
important consideration, especially for bortezomib, since one course of velcade injections
costs about $30,000 (BCC research, July 2010). Consequently velcade use is simply
unavailable to a large population of this world due to treatment cost; therefore there is a
grave need to find new, more affordable, safe and efficacious replacements for this class
of drugs.
Currently we are investigating the effect of knockdown of Chop and GRP78 on the
growth of T-04 treated cells by siRNA transfection experiments to provide a definite
proof for the role of ER stress in the cytotoxic effects of these drugs. We are also
analyzing cell growth in solid agar to confirm the inhibition of colony formation in drug-
treated cells as a measure of anti-tumor effect. As for additional proof to show that the
these drugs induce autophagy, we are also planning to transfect cells with an LC3- GFP
construct to confirm the localization of LC3 to autophagosomes in response to drug

48
treatment and effects on autophagy. Last but not least, we are in the process of comparing
the toxicity of these compounds on normal B cells, lymphocytes and leukocytes with
correlation of its effect on malignant ones.

49

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Abstract (if available)

Abstract Proteasome inhibitors are widely used today as an important tool for anti-cancer treatment, which has led to a wide search of novel proteasome inhibitors. Proteasome inhibitors tested in this study are analogues of glidobactin, which are a new class of proteasome inhibitors. Similarly, these compounds effectively inhibit the 26S proteasome activity. In this study, we investigated the cytotoxic effect of these compounds on human multiple myeloma (MM), human Waldenstrom macroglobulinemia (BCWM1), and human lymphocytic leukemia cells (REH). Of the four compounds we tested, T-02 and T-04 were most potent ones and their proteasome inhibitory effect directly correlates with their cell cytotoxicity. Induction of mild ER stress provides a survival benefit to cells, however severe ER stress is known to inhibit proliferation of cells leading to apoptosis. Since a number of proteasome inhibitors have been shown to trigger ER stress induced apoptosis, we investigated whether these novel proteasome inhibitors have this anti-cancer efficacy. Our results suggest that it is indeed the case. We further explored whether the combination of these compounds with other known ER stress inducers leads to aggravated ER stress and enhancement of their anti-cancer efficacy. T-02 or T-04 in combination with thapsigargin (a known inhibitor of sarco/endoplasmic reticulum Ca(2+) ATPase) and dimethyl celecoxib (DMC), an analogue of celecoxib demonstrate enhanced cytotoxic effect with more severe ER stress induction and apoptosis mediated cell death. Taken together, our findings suggest these novel compounds are very effective proteasome inhibitors and trigger the ER stress response with enhanced apoptosis mediated cell death. In addition, in combination with other ER inducer(s), cytotoxic effects of these compounds are highly potentiated.

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Asset Metadata

Creator Ashish Anshu, Fnu (author)

Core Title Anti-cancer effects of novel glidobactin type proteasome inhibitors

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology

Publication Date 05/03/2011

Defense Date 03/25/2011

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

Tag apoptosis,autophagy,endoplasmic reticulum stress,glidobactin A,hematological malignancy,OAI-PMH Harvest,proteasome

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Schönthal, Axel H. (committee chair), Hofman, Florence M. (committee member), Tahara, Stanley M. (committee member)

Creator Email anshu@usc.edu,ashish.microbio@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3860

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Document Type Thesis

Rights Ashish Anshu, Fnu

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