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Noscapine: a study of its effects on the glioma vasculature

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Noscapine: a study of its effects on the glioma vasculature

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NOSCAPINE: A STUDY OF ITS EFFECTS ON THE GLIOMA VASCULATURE

by
Niyati Jhaveri

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

December 2009

Copyright 2009 Niyati Jhaveri
ii

ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my advisor Dr. Florence Hofman for
giving me the wonderful opportunity to work in her laboratory. She has been a constant source
of support and encouragement. I am very grateful for her patience and belief in me. Seeing her
dedication and passion for research has inspired me to work harder. This last year and a half, as
part of her team, I have grown considerably as a person and as a scientist. I have learnt how to
think and work in collaboration as well as developed the skills to have an independent approach
to research. I would also like to thank members of the Hofman and Chen laboratory especially
Jenny Virrey, Zhi Liu and Heeyeon Cho for teaching me different techniques and for providing
valuable insights in solving experimental problems.
I would also like to express my gratitude to my committee members Dr. Zoltan Tokes
and Dr. Young Hong for their support and guidance. I wish to acknowledge the Glioma Research
Group especially Dr. Chen, Dr. Schonthal, Dr. Louie and Dr. Tahara for participating in valuable
discussions about my project. I would also like to thank Weijun Wang for his help with
intracranial implantations. Lastly, I would like to thank my family and friends whose support and
faith in me have made this possible.

iii

TABLE OF CONTENTS
Acknowledgements ii
List of Figures iv
Abbreviations v
Abstract vii
Chapter I – Introduction 1
- Angiogenesis and the tumor vasculature 1
- Noscapine 9
- Glioblastoma multiforme 12
- Noscapine’s role in glioblastoma multiforme 17
- Purpose of study 18

Chapter II – Materials and Methods 21
- Part 1 – Noscapine’s effect on the tumor vasculature 21
- Part 2 – Noscapine’s effect on tumor progression and survival 25

Chapter III – Results and Conclusion 28
- Part 1 – Noscapine’s effect on the tumor vasculature 28
- Part 2 - Noscapine’s effect on tumor progression and survival 34
- Conclusion 38

Chapter IV – Discussion 39
Bibliography 42

iv

LIST OF FIGURES
Figure 1-1. The process of angiogenesis. 2
Figure 1-2. The phases of tumor-associated angiogenesis. 3
Figure 1-3. Vessel cooption by the tumor. 6
Figure 1-4. The normal and tumor vasculature. 7
Figure 1-5. Normal brain endothelial cells and tumor-associated endothelial cells. 8
Figure 1-6. Noscapine. 9
Figure 1-7. Glioblastoma multiforme. 13
Figure 1-8. Temozolomide. 15
Figure 3-1. Effect of Noscapine on cell viability. 29
Figure 3-2. Effect of Noscapine on migration of BEC. 30
Figure 3-3. Effect of Noscapine on migration of TuBEC. 31
Figure 3-4. Effect of Noscapine on secretion of IL-8 in BEC. 32
Figure 3-5. Effect of Noscapine on microvessel density. 34
Figure 3-6. Effect of Noscapine on tumor progression in a subcutaneous model
of human glioma. 35

Figure 3-7. Effect of Noscapine and TMZ on survival in an intracranial model of
human glioma. 36

Figure 3-8. Effect of Noscapine and TMZ in a subcutaneous TMZ-resistant model 37
v

ABBREVIATIONS
BEC Normal brain endothelial cell
bFGF/FGFR Basic fibroblast growth factor/receptor
COX-2 Cyclooxygenase 2
EGCG Epigallocatechin gallate
EGFR Epidermal growth factor receptor
ELISA Enzyme linked Immunosorbent Assay
ER Endoplasmic reticulum
ERK Extracellular signal regulated kinase
ET-1 Endothelin 1
FAK Focal adhesion kinase
FCS Fetal calf serum
GBM Glioblastoma multiforme
GRP78 Glucose regulated protein 78
HGF Hepatocyte growth factor
HIF1 α Hypoxia-inducible factor 1 α
IGF/IGFR Insulin-like growth factor/receptor
IL-8 Interleukin 8
vi

IMG Intussusceptive microvascular growth
JNK Jun-NH
2
terminal kinase
MDR Multi-drug resistance
MGMT O6- methylguanine-DNA methyltransferase
MTT Monotetrazolium
Nos Noscapine
PDGF/PDGFR Platelet derived growth factor/receptor
SEM Standard error of the mean
TGF- α Transforming growth factor α
TGF-β Transforming growth factor β
TMZ Temozolomide
TNF-α Tumor necrosis factor α
TuBEC Tumor (glioma) associated brain endothelial cell
VEGF/VEGFR Vascular endothelial growth factor/receptor

vii

ABSTRACT
Noscapine has been widely used as an oral antitussive agent. More recently, studies
have shown that noscapine significantly affects microtubule dynamics and has potent antitumor
and antiangiogenic activity. The aim of our study was to investigate noscapine’s effects in
glioblastoma multiforme (GBM) focusing on its effects on the glioma vasculature. We studied
the effects of noscapine on cytotoxicity, migration and cytokine secretion of normal (BEC) and
tumor-associated brain endothelial cells (TuBEC). We found that noscapine significantly
decreased migration of both BEC and TuBEC. In accordance with this, we found that noscapine
also decreased microvessel density in vivo.
Since noscapine has been shown to be effective against a wide variety of cancers, we
hypothesized that it may also be effective against glioma. Our study has indicated that
noscapine at 550 mg/kg/day significantly delayed tumor progression in a subcutaneous murine
model of human glioma without any observable side effects. Moreover, noscapine alone
significantly prolonged survival in mice implanted intracranially with human glioma cells but it
was not synergistic with temozolomide. However, in temozolomide- resistant tumors, noscapine
and TMZ are very synergistic.
Our studies showed that noscapine has potential as an antitumor and antiangiogenic
agent for GBM. To date, most studies on noscapine have been done on mouse or rat gliomas.
The novel aspect of our study is that we investigated noscapine’s effects on human glioma and
the associated vasculature. BEC and TuBEC used in our study were isolated from patients with
GBM and thus make for an excellent model. GBM, being a highly vascular tumor is dependent
on angiogenesis for tumor growth and progression. Noscapine attacks both the tumor cells and
the vasculature feeding the tumor simultaneously and this makes it an attractive drug for GBM
viii

treatment. Moreover, it chemosensitizes TMZ-resistant tumors to TMZ and may thus be
effective in treating tumors that are no longer responsive to temozolomide.
1

CHAPTER I – INTRODUCTION
1.1. ANGIOGENESIS AND THE TUMOR VASCULATURE
1.1.1. Physiological and Pathological angiogenesis
Blood vessels consist of endothelial cells that form the inner lining of the vessel wall and
pericytes that wrap around blood vessels for structural integrity. First used by British surgeon
John Hunter in 1787 (Figg and Folkman, 2008), the term ‘angiogenesis’ refers to the formation
of new blood vessels from pre-existing vessels. Physiological angiogenesis takes place during
the formation of placenta and involves expansion and remodeling of the primary vascular
network by endothelial cell sprouting and intussusceptive microvascular growth (IMG).
Sprouting occurs by endothelial cell proliferation, migration and tube formation. IMG involves
splitting of the vessel lumen (Kirsch and Black, 2004).
Angiogenesis is a complex, highly ordered process. The first step involves detachment of
pericytes and dilation of blood vessels followed by degradation of the basement membrane and
extracellular matrix. Tumor cells secrete a variety of proangiogenic factors which attract
endothelial cells. Endothelial cells then migrate, proliferate and form a lumen. A new basement
membrane is formed followed by attachment of pericytes. Fusion of blood vessel sprouts with
other sprouts results in a vascular network (Bergers, 2003). The last step, maturation, involves
the transition from an active growing phase to a quiescent mature phase. This requires
suppression of endothelial cell proliferation and sprouting and stabilization of the existing
vasculature (Adams and Alitalo, 2007). The normal adult vasculature is quiescent except in the
female reproductive system and in wound healing. After nerve cells, endothelial cells have the
longest lifespan with a turnover time of a few years (Hanahan and Folkman, 1996).
2

Figure 1-1. The process of angiogenesis. (a) New blood vessels arise from pre-existing vessels in
a tumor (b) Pericytes (green) detach, blood vessels dilate and basement membrane and ECM are
degraded (c) Endothelial cells (red) migrate towards tumor-secreted proangiogenic factors (d)
Endothelial cells proliferate and follow each other (e) Endothelial cells adhere to each other and
form a lumen, new basement membrane is formed and pericytes attach. Blood vessels fuse with
other sprouts and a new circulatory system is created (Figure credit: Bergers et al., 2003).

Normal physiological angiogenesis is tightly regulated. VEGF (Vascular endothelial
growth factor or vascular permeability factor) is the most important and most studied
angiogenic regulator. As its name suggests, it increases blood vessel permeability. It also
stimulates endothelial cell proliferation and migration. Hypoxia is another critical regulator
which induces HIF1α which in turn increases production of VEGF (Papetti and Herman, 2002).
Other important regulators of angiogenesis include positive factors like bFGF, PDGF, HGF, TGF-α,
IL-8, Angiopoeitin and negative factors like endostatin, tumstatin, angiostatin and
thrombospondin-1 (Kirsch and Black, 2004). An appropriate balance between proangiogenic and
antiangiogenic molecules regulates angiogenesis.
3

In tumors described as ‘wounds that never heal’, this balance is destroyed.
Consequently, there is uncontrolled, deregulated angiogenesis. The concept of tumor
angiogenesis – the requirement of angiogenesis for a tumor to grow beyond a certain size and
metastasise into distant sites was proposed by Judah Folkman in 1971. Tumor associated
angiogenesis has two phases (1) Avascular phase where the lesion is about 1-2 mm in diameter
and there is a balance between proliferation and apoptosis (2) Vascular phase which is brought
about by turning on the angiogenic switch after which the tumor starts to grow exponentially
(Bergers, 2003). The angiogenic switch is an active area of investigation. It is primarily turned on
by an excess of proangiogenic factors which may come from the tumor cells or from the
surrounding microenvironment.

Figure 1-2. The phases of tumor-associated angiogenesis. During the avascular phase, the
tumor is small (1-2 mm) and there is a balance between proliferation and apoptosis. The
switching ‘on’ of the angiogenic switch begins the vascular phase where there is exponential
growth of the tumor (Figure credit: www.researchvegf.com).

4

1.1.2. Angiogenesis as a target for cancer therapy
Angiogenesis is a rate limiting step in tumor progression and so drugs that interfere with
angiogenesis would inhibit tumor progression. Its potential as a target in cancer therapy was
first proposed in 1970 by Judah Folkman, but it was not until 2004 that the first antiangiogenic
drug Bevacizumab got clinical approval by the USFDA for use in metastatic colorectal cancer in
combination with 5-fluorouracil. Bevacizumab (Avastin) is a humanized monoclonal antibody
again VEGF (vascular endothelial growth factor), an important regulator of angiogenesis.
Thereafter several agents with antiangiogenic potential have been discovered and are currently
in clinical trials for efficacy and safety against a wide variety of cancers (Ferrara and Kerbel,
2005).
Antiangiogenic therapy targets the genetically stable endothelial cells and so there
would be no problems of conventional drug resistance (Carmeliet, 2005). Problems associated
with drug delivery are also circumvented because endothelial cells being in close contact with
blood are easily accessible to circulating antiangiogenic agents. But inhibition of angiogenesis is
only cytostatic and is not enough on its own. Combination of chemotherapy with antiangiogenic
drugs would attack both the tumor cells and the endothelial cells feeding the tumor and would
thus be a more effective strategy in combating cancer. Another important cell population to
target in tumors is the tumor stem cells which are responsible for sustained growth of cancer.
This population is not being targeted by conventional chemotherapeutics but the antiangiogenic
drugs might act on these and thus be beneficial for survival (Ferrara and Kerbel, 2005).
Since angiogenesis is virtually absent in adults, except in wound healing and the female
reproductive system, antiangiogenic therapy is tumor specific. Moreover, antiangiogenic agents
have low toxicity. Only 2 % of cases treated with Bevacizumab show serious side effects like
5

gastrointestinal perforations, hemorrhage, impaired wound healing and arterial
thromboembolic complications (Ferrara and Kerbel, 2005).
One of the criticisms against antiangiogenic therapy is that disrupting blood flow to the
tumor may interfere with delivery of drug and oxygen. Hypoxia, in turn would cause resistance
to radiotherapy. But appropriate dosing and scheduling of antiangiogenic agents could
‘normalize’ the abnormal tumor vasculature and this would allow for more efficient delivery of
drug and oxygen to the tumor site. Thus knowledge of the ‘vascular normalization time window’
is important to optimize the combination of chemotherapy, radiation and antiangiogenic
therapy (Lakka and Rao, 2008).
Moreover, antiangiogenic agents must be administered daily for a long period. The long
term side effects of these agents are not known. Targeting the VEGF pathway alone is not
sufficient. In gliomas, it has been observed that the tumor may switch to another angiogenic
pathway not dependent on VEGF. Thus a multidimensional antiangiogenic approach is
necessary. Gliomas are also known to coopt and grow along existing blood vessels. Antivascular
agents that target the existing vasculature may be effective in such cases. Moreover, bone
marrow derived angiogenic cells may be recruited to evade antiangiogenic agents. Recent
reports showing that antiangiogenic agents may in the long run select for tumors that are more
invasive and highly metastatic are discouraging (Ebos et al., 2009). The advantages and the
drawbacks of antiangiogenic therapy must be taken into consideration while designing new
drugs. But angiogenesis still remains an attractive target for cancer treatment.
6

Figure 1-3. Vessel cooption by the tumor. (a) Astrocytomas are non-angiogenic tumors which
coopt existing blood vessels by growing along them (b) When Grade III astrocytomas progress to
GBM, they become hypoxic and necrotic due to increased proliferation and regression of vessels
(c) New blood vessels are induced which promote tumor growth (Figure credit: Bergers et al.,
2003).

1.1.3. The tumor vasculature
Blood vessels are organized in a hierarchy of arterioles, capillaries and venules each
having its own characteristic structure and function. Blood vessels in different organs share
some common features and some organ specific characteristics. The normal brain vasculature is
highly organized having a thin monolayer of endothelial cells lining the wall of the blood vessels.
The endothelial cells are closely packed together via tight junctions. Tumor vessels, on the other
hand are structurally and functionally very different. They are highly disorganized, tortuous,
dilated, have uneven diameter and excess branches. The blood flow is chaotic which in turn
leads to hypoxic and acidic regions in tumors. These conditions lower the efficacy of
chemotherapeutics and also select for cancer cells that are more malignant and metastatic. The
tumor vessels are also leaky due to discontinuous basement membrane and loss of tight
junction proteins like CD144, ZO-1, occludin, claudin-1 and claudin-5. They are characterized by
high vascular permeability. In addition, the expression of surface markers on the tumor
endothelium is different from that on the normal vasculature. VEGF and TNF-α is known to be
7

upregulated while bFGF and TGF-β is downregulated. Targeting molecules that are specific to
tumor endothelium is an attractive therapeutic strategy (Carmeliet and Jain, 2000).

Figure 1-4. The normal and tumor vasculature. Normal blood vessels have a thin endothelial
cell lining and are highly organized. Blood vessels in a tumor are tortuous, dilated and have
many dead-end structures (Figure adapted from Jain et al., 2007).

In our laboratory, we have extensively characterized normal brain endothelial cells (BEC)
and glioma-associated endothelial cells (TuBEC) with the purpose of identifying key differences
between them which can then be translated to designing drugs that target the tumor
endothelium specifically. Our results indicate significant differences. Morphologically, TuBECs
(GBM-associated endothelial cells) have a large, flat, veil like appearance with large nuclei and
abundant cytoplasm whereas normal brain endothelial cells are small and plump. Another
important difference is that TUBECs proliferate much slower than BECs. This is important
because most drugs target rapidly proliferating cells. TuBECs undergo less apoptosis and are
resistant to a wide variety of cytotoxic drugs. This increased resistance has been shown to be
partly due to upregulation of protective factors like GRP78 and survivin, an inhibitor of apoptosis
(Virrey et al., 2008).
8

Figure 1-5. Normal brain endothelial cells and tumor-associated endothelial cells. (a) BECs are
small and plump whereas (b) TuBECs are large, flat and have a veil like appearance.

Boyden chamber migration assays show that TuBECs migrate faster than BECs which
may be partly due to expression of α smooth muscle actin, a contractile protein which is not
found in BECs (Charalambous et al., 2006). Flow cytometry studies have shown that both tumor
and normal brain endothelial cells express receptors for VEGF and ET-1 but only BECs are
sensitive to these angiogenic factors, TuBECs are unresponsive. This may be because
constitutive production of these factors by TuBECs saturates the receptors (Charalambous,
Hofman et al., 2005). ELISA and immunostaining studies confirm constitutive production of pro-
angiogenic growth factors like ET-1, IL-8 and VEGF by TuBECs which also explains their enhanced
migration and survival. Even though they have constitutive production of IL-8, they can be
stimulated by TNF-α or ET-1 to produce more (Charalambous, Pen et al., 2005).
TuBECs show signs characteristic of senescent cells. They express SA-β-gal, a marker for
senescent cells. Cell cycle analysis shows an increased number of cells in G0/G1 indicating G1
arrest. Also, TuBECs have lower levels of cyclin A and higher levels of cyclin D1 relative to BECs.
p53 is highly expressed in TuBECs. Cyclin dependent kinase inhibitors p21 and p27 are also
elevated (Charalambous, Virrey et al., 2007).
a b
9

Further characterization of the differences between these cells will help in designing
drugs to selectively target the tumor endothelium while minimally affecting normal endothelial
cells of the brain.

1.2. NOSCAPINE
1.2.1. Introduction
Noscapine [L-a-methyl-8-meth-oxy-6,7-methylenedioxy-1-1(6, 7-dimethoxy-3-phthal-
idyl)-1,2,3,4-tetrahydroisoquinoline] is a pthalideisoquinoline alkaloid derived from opium which
is structurally similar to the microtubule targeting drug colchicine. It was first isolated in 1817 by
Roboquet (Schuler et al., 1999). Noscapine has been widely used as an oral antitussive agent
with 94 % efficacy (Segal et al., 1957). Being a noncompetitive inhibitor of bradykinin, it also
plays a role in treatment of stroke and stroke-associated edema (Mahmoudian et al., 2003).

Figure 1-6. Noscapine. Noscapine is a microtubule targeting agent that has great potential as an
antitumor and antiangiogenic drug for GBM (Figure credit: www.answers.com).

1.2.2. Noscapine and microtubules
Microtubules are highly dynamic polymers of tubulin that are an integral part of the cell
cytoskeleton. They are involved in maintaining cell shape and polarity, intracellular transport of
10

vesicles and organelles and ciliary and flagellar motility. They play a critical role in cell cycle by
formation of the mitotic spindle which is involved in chromosome segregation during the M
phase of the cell cycle. The spindle assembly checkpoint ensures that chromosomes are properly
attached to the spindle microtubules. If not, it arrests the cells in M phase eventually causing
apoptosis. Microtubules are thus an important target in cancer therapy.
Noscapine has a prominent effect on microtubule dynamics. It significantly increases the
time microtubules spend in an idle or paused state without causing gross abnormalities. It has a
differential effect on normal and transformed cells. Normal cells undergo mitotic arrest due to
activation of the mitotic check point but this is reversible. Because of the short half life of
noscapine, when levels fall below threshold, cells resume the cell cycle. Transformed cells, on
the other hand are more susceptible to noscapine because of loss of mitotic checkpoints. These
cells undergo polyploidy ultimately resulting in cell death (Landen et al., 2002, Ye et al., 1998
and Schuler et al., 1999). Although noscapine interacts only weakly with tubulin compared to
colchicines, it is enough to induce arrest.

1.2.3. Noscapine as an antitumor agent
Noscapine has been shown to be effective against a wide variety of human cancers. It
has been shown to have potent antitumor activity against human non-small cell lung cancer, the
most common type of lung cancer. In vivo, noscapine reduced tumor growth by 49, 65 and 86 %
respectively at 300, 450 and 550 mg/kg/day. It caused mitochondrial mediated apoptosis as was
reflected by upregulation of PARP, Bax, caspase-3 and downregulation of Bcl2. Mitochondrial
mediated apoptosis of noscapine has also been demonstrated in human myelogenous leukemic
and colon cancer cells (Heidari et al., 2007 and Jackson et al., 2008).
11

Noscapine also reduced tumor growth in a subcutaneous murine model of E.G7-OVA T
cell lymphoma (Ke et al., 2000). Moreover, noscapine showed significant inhibition of melanoma
progression alone and in combination with paclitaxel. Paclitaxel is associated with peripheral
neuropathy which is not seen with noscapine (Landen et al., 2002). In vitro studies have shown
that noscapine can inhibit proliferation of both paclitaxel sensitive and resistant human ovarian
cancer cells at clinically applicable doses via JNK mediated apoptosis. Since it has an effect on
paclitaxel resistant cells it can be used in treating ovarian cancer that has become refractory to
paclitaxel. In vivo studies need to be carried out to confirm noscapine’s effect on paclitaxel
resistant tumors (Zhou et al., 2002).
Noscapine has also been shown to reduce growth and metastasis of PC3 human
prostate tumor when administered orally at 300 mg/kg (Barken et al., 2008). Morever,
noscapine administered intraperitoneally for 21 days at 120 mg/kg showed 80 % tumor
regression of human MCF-7 breast cancer in a murine model (Ye et al., 1998).
Using four HCT116 colorectal cancer cell lines with different p53, p21 and Bax
expression status, it was shown that p53 is necessary but not sufficient for noscapine induced
apoptosis. p21 plays a proapoptotic role and is necessary for p53 mediated apoptosis (Aneja,
Ghaleb et al., 2007). Thus the genotype of the tumor may be important in its response to
noscapine treatment.
Noscapine does not have any analgesic, sedative, euphoric or respiratory depressant
properties. Moreover, it is not addictive. Histological studies have shown that noscapine is not
toxic to normal tissues. Moreover, it does not interfere with immune responses. No
hematological toxicity has been observed. Existing microtubule targeting drugs are associated
with severe side effects like myelosuppression, peripheral neuropathy, alopecia and
gastrointestinal toxicity. Moreover, there are problems of poor solubility, bioavailability, and
12

drug resistance. Noscapine has an excellent pharmacological profile. Since it can be
administered orally, complications associated with intravenous injections like hypersensitivity,
infection, etc can be avoided. Noscapine is also water soluble so there is no need to dissolve it in
a vehicle solution that may have toxic side effects. One of the mechanisms of tumor resistance is
through upregulation of drug efflux proteins like p-glycoprotein. Noscapine is a poor substrate
for these transporters and this makes it an attractive treatment strategy for drug resistant
tumors (Aneja, Dhiman et al., 2007).
Currently, noscapine is in Phase I/II clinical trials for the treatment of low grade non-
Hodgkin’s lymphoma or chronic lymphocytic leukemia unresponsive to chemotherapy and
hematological malignancies (Jackson et al., 2008).

1.3. GLIOBLASTOMA MULTIFORME
1.3.1. Introduction
Gliomas are tumors of the glial cells of the brain and constitute about 60 % of human
central nervous system malignancies. Histologically, there are two classes of gliomas:
astrocytomas and oligodendrogliomas (Markert et al., 2005). Astrocytomas are the most
common in humans and they are further classified into different grades by the World Health
Organization. These are: (a) Grade I – Non-invasive pilocytic astrocytoma (b) Grade II –
Astrocytoma (c) Grade III – Anaplastic (malignant) astroctyoma and (d) Grade IV – Glioblastoma
(http://www.sd-neurosurgeon.com/diseases/astrocytomas.html). Glioblastoma multiforme is
the most common astrocytoma representating 82 % of diagnosed cases in the US. They are also
the most aggressive. Astrocytomas are non-angiogenic tumors which coopt existing blood
vessels for their growth. But progression to Grade IV GBM involves switching ‘on’ of the
13

angiogenic switch which causes highly invasive and metastatic growth of the tumor (Bergers,
2003). GBMs as their name suggests are multiforme. They exhibit regions of pseudopalisading
necrosis, pleomorphic nuclei and cells and high endothelial cell proliferation (Holland, 2000).

Figure 1-7. Glioblastoma multiforme. (a) MRI scan of a glioblastoma patient. (b)
Pseudopalisading necrosis and (c) endothelial cell proliferation are hallmarks of glioblastoma
multiforme (Figures adapted from www.mir.wustl.edu, www.neuropathology.neoucom.edu and
Pathweb).

Several environmental and genetic factors have been shown to play a role in GBM.
Genetic profiling studies have shown a number of modifications in the signal transduction
pathways and the cell cycle machinery. There are reports of amplifications or activating
mutations in EGFR, FGF/FGFR, PDGF/PDGFR, IGF/IGFR, CDK4, CDK6, cyclin D1 and MDM2 and
deletions in PTEN, INK4A-ARF, RB and p53. Moreover, there are losses in chromosomes 6, 9, 10,
13, 17, 19 and 22 and gains or amplifications of chromosomes 1, 5, 7, 8, 11, 12 (Markert et al.,
2005). These mutations may play an important role in the tumor’s response to
chemotherapeutic drugs.
a
b
c
14

In addition, GBMs have elevated expression of VEGF, which is the major mediator of
angiogenesis in gliomas. COX-2 expression is also elevated and is associated with tumor grade
and poor patient prognosis. Cytokines like IL-8, IL-12 and CXCL-12 (SDF-1) are also important in
GBM. SDF-1 and its receptor CXCR-4 are important in endothelial cell migration and
angiogenesis (Lakka and Rao, 2008). Knowledge of the molecular signature of the tumors will
be helpful in designing new treatment strategies as well as understanding the pathogenesis of
the disease.
A formidable challenge in glioma therapy is the blood brain barrier which consists of
endothelial cells, pericytes, astrocytes and neurons in close association with each other to
prevent the entry of toxic compounds into the brain. Drugs to treat GBM must be able to cross
the blood brain barrier.

1.3.2. Temozolomide – the standard of care for GBM treatment
The first step in treating GBM is usually surgery to resect out the tumor but this is highly
risky owing to the location of the tumor and its diffuse nature. Surgery is followed by radiation
and chemotherapy. Even with surgery, the tumor is known to recur almost always and the
survival time is generally 12-15 months. The standard treatment for glioblastoma is
temozolomide combined with radiation. This has been shown to increase survival from 12.1 to
14.6 months (Norden et al., 2008).
Temozolomide is an imidazotetrazine derivative which gets metabolized in vivo to an
active compound that methylates DNA at the O6 position of guanine. This induces the mismatch
repair system which causes DNA damage and kills the tumor cells. Temozolomide also kills cells
by inducing ER stress and autophagy. Temozolomide delays tumor progression and improves
15

patient survival but patients ultimately develop drug resistance and the tumor recurs. One of
the mechanisms of resistance is through overexpression of MGMT. MGMT (O6-methylguanine-
DNA methyltransferase) is a DNA repair enzyme that confers resistance to DNA alkylating agents
like TMZ by removing the alkyl adducts. Another mechanism of drug resistance is due to
multidrug resistance defined as simultaneous resistance of tumor cells to a broad spectrum of
structurally and functionally diverse drugs. Several MDR proteins have been identified to be
important in glioma chemoresistance like those belonging to the ATP-binding cassette (ABC)
superfamily of efflux pumps - P-glycoprotein (P-gp) and multidrug resistance associated protein
1 (MRP1) (Lu and Shervington, 2008).

Figure 1-8. Temozolomide – the standard of care for GBM treatment. Temozolomide is a DNA
alkylating agent that significantly delays tumor progression and improves survival of GBM
patients (Figure credit: Wikipedia).

In our laboratory we have shown that GRP78, an important component of the unfolded
protein response also confers resistance to temozolomide. Knocking down GRP78 using siRNA or
chemically with EGCG makes glioma cells more sensitive to TMZ (Pyrko et al., 2007). GRP78 also
increases tumor proliferation and promotes angiogenesis and is thus an attractive target in
glioma therapy (Dong et al., 2008).
16

Tumor recurrence may be because temozolomide has no effect on the tumor
vasculature. Studies in our laboratory have shown that glioma associated endothelial cells are
resistant to temozolomide. TMZ has no effect on viability, proliferation or migration of glioma
associated brain endothelial cells (TuBEC). Although TMZ greatly increased survival of mice
bearing intracranial tumors, it did not have any effect on microvessel density. Migration of
TuBEC was not affected even at high concentrations of TMZ (300 µM) (Virrey et al., 2009). Thus
TMZ does not have any antiangiogenic activity. This has also been confirmed by two other
groups (Kim et al., 2006). Moreover, TMZ does not have any effect on cancer stem cells.
TMZ has also been shown to be effective against melanomas, ovarian and colon cancers.
TMZ is lipophilic, can be delivered orally, has wide distribution in tissues and can cross the blood
brain barrier (Kim et al., 2006). But due to increasing TMZ-resistance to gliomas, there is an
urgent need to develop more effective therapies for treating GBM.

1.3.3. Antiangiogenic treatment for GBM
Gliomas are highly vascular tumors that depend on angiogenesis for growth and
progression. Thus antiangiogenic therapy may be an effective therapeutic strategy for GBM.
Since the VEGF pathway is the major proangiogenic pathway in gliomas, VEGF and its receptor
VEGFR have been the prime targets for antiangiogenic therapies for GBM. Bevacizumab, a
humanized monoclonal antibody against VEGF is a popular antiangiogenic drug and phase II
trials with bevacizumab and the topoisomerase inhibitor irinotecan have shown response rates
of 50-66% for recurrent and anaplastic glioma. Other drugs that target VEGF or its receptor like
cediranib, vatalanib, sorafenib and sunitinib are also been investigated for their role in
malignant glioma therapy (Norden et al., 2008).
17

1.4. NOSCAPINE’S ROLE IN GLIOBLASTOMA MULTIFORME
Noscapine’s potent antitumor activity in a variety of human tumors along with its easy
solubility, oral bioavailablity and low toxicity profile make it an attractive therapy for use in
treating gliomas. An important consideration in GBM therapy is the blood brain barrier. In vitro
and in vivo assays have confirmed that noscapine crosses the blood brain barrier. (Landen et al.,
2004).
Moreover, treatment of U87MG, U118MG, LN229 and T98G human glioma cell lines for
72 hours with noscapine (20-150 µmol/L) resulted in a dose dependent inhibition of cell
proliferation. Noscapine induced M phase arrest in all cell lines within 24 hours. The mechanism
of noscapine mediated apoptosis was found to be via activation of the JNK signaling pathway
together with the inactivation of the ERK signaling pathway (Newcomb, Lukyanov et al., 2008).
In vivo studies showed that noscapine inhibited the growth of both subcutaneous and
intracranially implanted rat C6 glioma tumors in a murine model. Noscapine administered daily
by gavage at 300 mg/kg for 15 days reduced tumor volume by 60 % (Landen et al., 2004).
In a subcutaneous GL261 model, noscapine in combination with radiation significantly
delayed tumor progression. Moreover, an in vitro tubule formation assay with murine
endothelial 2H11 cells showed that noscapine and radiation together significantly inhibited
tubule formation. At a concentration of 150µM, it caused 97 % inhibition of tubule formation. It
also decreased proliferation, increased apoptosis and reduced microvessel density. Radiation
reduced microvessel density 1.5 fold whereas the combination reduced it 2.3 fold. Thus
noscapine sensitizes GL261 tumors to radiation therapy in vivo. In vitro this effect was not
observed which implies an important role of tumor microenvironment in enhancing the
radioresponse (Newcomb et al., 2008).
18

Noscapine has also been shown to have potent antiangiogenic effects in human glioma
cells. This was shown in U87 and T98 glioma cells exposed to hypoxia. Treatment with 150 µM
noscapine in these cell lines inhibited HIF-1α expression by 60-90 %. The inhibition was found to
be at the posttranslational level by preventing accumulation in the nucleus and targeting it for
proteasomal degradation. It also decreased secretion of VEGF whose expression is under the
control of HIF-1α (Newcomb et al., 2006).
Noscapine’s ability to cross the blood brain barrier together with its antitumor and
antiangiogenic effects makes it a promising drug for use in treatment of GBM.

1.5. PURPOSE OF STUDY
Firstly we hypothesized that since noscapine has a significant effect on microtubule
dynamics causing them to spend an increased amount of time in the paused state, it would
significantly decrease migration of endothelial cells. Migration is a critical step in angiogenesis
and inhibiting migration would be an important antiangiogenic strategy. If noscapine decreases
migration of endothelial cells, it would be a promising antiangiogenic drug for cancer.
Glioblastoma multiforme, being a highly vascular tumor, noscapine would be an excellent drug
which can be used in combination with the current standard of care – temozolomide. In order to
test this hypothesis, we carried out Boyden chamber migration assays with normal as well as
glioma-associated brain endothelial cells. Noscapine’s antimigratory role has been studied in
murine endothelial cells (Newcomb et al., 2008) but no information is available on its effects on
human endothelial cells. The normal brain endothelial cells (BEC) and glioma-associated
endothelial cells (TuBEC) that we used in our study are isolated from patients with glioblastoma
multiforme. They are more representative of the normal and GBM brain endothelium
19

respectively and thus are better model systems. We studied noscapine’s effects on cytotoxicity,
migration and cytokine secretion of BEC and TuBEC.
Several studies have shown that noscapine has potent antitumor activity in a wide
variety of human cancers. In the second part of this study, we hypothesized that noscapine may
also show antitumor activity for glioma. We tested noscapine’s effect on tumor progression in a
subcutaneous murine model of human glioma. Since noscapine decreases endothelial cell
migration, we hypothesized that we would see a decrease in microvessel density in tumors from
noscapine-treated mice. We carried out CD31 immunostaining and microvessel density analysis
to test this theory. Moreover, since noscapine decreases migration and shows promise as an
antiangiogenic agent, we believed it would act synergistically in combination with
temozolomide. Athymic mice implanted with human glioma cells intracranially were treated
with noscapine, temozolomide and the combination. The intracranial model was chosen
because this is more representative of the orthotopic location of the tumor in humans. Since
noscapine is a weak substrate for drug efflux pumps, we also hypothesized that it may be
effective against tumors that have become resistant to chemotherapy due to overexpression of
drug efflux pumps. We tested noscapine and TMZ in a TMZ-resistant subcutaneous model for
this purpose.
In summary, noscapine is a widely used antitussive agent that has shown antitumor and
antiangiogenic activities in a variety of human cancers. Noscapine is able to cross the blood
brain barrier. Moreover, it is orally bioavailable, water soluble and has an excellent
pharmacological profile. Because of noscapine’s effects on both tumor cells as well as the
vasculature, it is a promising drug for highly vascular tumors like GBM which are dependent on
angiogenesis for tumor growth and progression. Most of the work with noscapine has been
done using models of mouse or rat glioma. No studies have been carried out using a model of
20

human glioma. In addition, there is no information about noscapine’s effects on human brain
endothelial cells and tumor associated brain endothelial cells. Our studies focus on these novel
aspects. These studies will be helpful in designing strategies for treating GBM that are more
effective because they target both the tumor and the tumor vasculature simultaneously.

21

CHAPTER II – MATERIALS AND METHODS

2.1. PART 1 – NOSCAPINE’S EFFECT ON THE TUMOR VASCULATURE
2.1.1. Endothelial Cell isolation and culture
Primary cultures of BEC (non-malignant brain endothelial cells) and TuBEC (glioma-
associated brain endothelial cells) were used for all experiments. Tissues were obtained and
handled in accordance with USC Institutional Review Board guidelines. BEC were isolated from
normal brain tissues of trauma or epilepsy patients whereas TuBEC were isolated from brains of
patients suffering from stage IV glioblastoma multiforme (GBM). Isolation is described briefly.
Tissues were rinsed thrice in PBS and then cut into small pieces. These were resuspended in
RPMI (USC Cell Culture Core Facility) containing 2 % fetal calf serum (FCS) (Omega Scientific) and
homogenized using a Douncer. Equal volume of 30 % dextran (Sigma Aldrich) was added and the
mixture was centrifuged for 10 minutes at 10,000 rpm at 4°C. The red pellet containing brain
microvessels was recovered and fresh RPMI 2 % FCS medium containing 1 mg/ml of collagenase-
dispase was added. After 1 hour incubation in a shaking water bath at 37°C, 10 ml RPMI 2 % FCS
was added and the cells were centrifuged at 1200 rpm for 5 minutes. This process was repeated
thrice to get rid of excess collagenase-dispase. The final cell pellet was resuspended in RPMI
medium supplemented with 100 ng/ml Endogro – endothelial cell growth supplement (Upstate
Biotechnologies), 2 mmol/L L-glutamine (Life Technologies, Inc.), 10 mmol/L HEPES (Life
Technologies, Inc.), 24 mmol/L sodium bicarbonate (Life Technologies, Inc.), 300 U heparin USP
(Sigma Aldrich) and 1 % penicillin-streptomycin and plated onto precoated gelatin flasks.
Endothelial cells were further purified by selecting cells that bind diacetylated low density
lipoprotein (di-LDL) using fluorescence activated cell sorting analysis. Endothelial cell status was
22

confirmed by immunostaining for endothelial cell specific markers: CD31/PECAM-1 (Santa Cruz
Biotechnology), von Willebrand Factor (DAKO), VE-cadherin (R&D Systems) and CD105/endoglin
(Santa Cruz Biotechnology). Cells were negative for astrocyte cell marker glial fibrillary acidic
protein (DAKO), progenitor endothelial cell marker CD34 (DAKO) and macrophage/microglia
marker CD11b (DAKO). Cells were cultured on gelatin coated flasks in Endogro medium. For all
experimental purposes, Endogro medium was replaced with RPMI 10 % FCS medium. Cells were
used upto passage 6.
2.1.2. Cell lines
U251 cell lines were a gift from Frank B. Furnari and Webster K. Cavenee (Ludwig
Institute of Cancer Research). Cells were propagated in DMEM medium (USC Cell Culture Core
Facility) supplemented with 10 % FCS (Omega Scientific).
2.1.3. MTT Assay for Cell Viability
Cells were seeded in triplicates onto a 96 well plate in RPMI 10 % FCS medium at a
density of 5 x 10
3
cells/50 µL/well. For endothelial cells, wells were coated with 1 % gelatin
before seeding. After 24 hours, cells were treated with the drug noscapine hydrochloride (Sigma
Aldrich) at increasing concentrations (50 µL). After 72 hours of drug treatment, 10 µL of 5 mg/ml
stock of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye (Sigma Aldrich)
was added. The tetrazolium salt is cleaved by mitochondrial enzymes in viable cells forming a
colored formazan dye. Following 4 hours of incubation with MTT, 100 µL lysis buffer containing
10 % SDS and 0.01 % HCl was added. Absorbance of the colored product was measured the next
day at 490 nm using a microtiter plate reader (Dynatech MR4000). Percentage cell viability was
calculated relative to untreated control and a plot of percent cell viability versus drug
23

concentration was constructed. IC50 – the concentration of drug required to inhibit cell
proliferation by 50 % was evaluated. Experiments were repeated thrice to confirm results.
2.1.4. Endothelial cell Migration Assay
Migration assays were done using transwell chambers with 8 µm pore, 6.5 mm diameter
polyethylene terephthalate filters (BD Biocoat). The filters were coated on both sides with 0.5 %
gelatin by immersion for 2 hours followed by overnight airdrying. Subconfluent cultures of BEC
and TuBEC were harvested with trypsin/EDTA (USC Cell Culture Core Facility) and seeded at a
density of 5 x 10
4
cells/100 µL/well in RPMI 10 % FCS medium in the upper chamber. The next
day, chemoattractant and noscapine were added to the lower chamber (600 µL) at the
appropriate concentrations. Recombinant human IL-8 (R&D Systems) was used as a
chemoattractant. For control, only RPMI 10 % FCS medium was added to the lower chamber.
The transwell plate was then incubated at 37°C in 5 % CO
2
for 6 hours. At the end of the
incubation period, all medium was removed. Cells in the upper chamber were removed using a
wet Q-tip and the filter was then stained with Diff Quick staining solutions (aqua, orange and
purple for 2 minutes each) (EMD Chemicals). Stained cells that had migrated through the filter
were counted under high power magnification (40 X). Ten fields were counted per chamber.
Groups were plated in duplicate. Experiments were repeated atleast three times to confirm
results.
2.1.5. Interleukin-8 ELISA
To determine the effect of noscapine on cytokine production in BEC, endothelial cells
were seeded in duplicate in equal number onto gelatin coated 6-well plates in Endogro medium.
After cells had reached 80 % confluency, medium was changed to RPMI 10 % FCS. Noscapine
and tumor necrosis factor-α (30 ng/ml) (R&D Systems) which serves as an activator of IL-8
24

production were added simultaneously. After 48 hour incubation, supernatants were collected
and analyzed for IL-8 levels using commercially available ELISA kits (Biosource Invitrogen). Cell
counts were measured from each well atleast twice and the amount of IL-8 per 10
4
cells was
calculated.
2.1.6. Microvessel density analysis
Frozen mouse tumor tissues were sectioned at 8 µM, fixed with acetone and blocked
with 5 % goat serum followed by overnight staining with rat anti-mouse CD31 antibody (1:100,
BD Pharmingen). The following day, sections were washed with PBS and incubated with
biotinylated anti-rat antibody (1:200, Vector Laboratories) for 45 minutes. Sections were then
treated with avidin-biotin peroxidase complex (Vector Laboratories) for 30 minutes and
aminoethyl carbazol substrate kit (Vector Laboratories) for 10 minutes followed by
counterstaining with Mayer’s hematoxylin for 2 minutes. Images were taken using a Fluorescent
Leica microscope (USC Imaging Core Facility) at 200X magnification. Positive staining indicated
by a red precipitate was quantified using ImageJ software (NIH). 2 % goat serum was used
instead of the primary antibody as a control. Tumor microvessel density was analysed in three
mice and atleast six to ten fields were evaluated for each tumor.
2.1.7. Statistical analysis
Values are presented as the mean +/- SEM. Statistical significance was evaluated using
the Student’s two-tailed t-test. A p-value < 0.05 was considered significant.

25

2.2. PART 2 – NOSCAPINE’S EFFECT ON TUMOR PROGRESSION AND SURVIVAL
2.2.1. Cell lines
U87 cell lines were obtained from the American Tissue Culture Collection. U251 cell
lines were a gift from Frank B. Furnari and Webster K. Cavenee (Ludwig Institute of Cancer
Research). Cells were propagated in DMEM medium (USC Cell Culture Core Facility)
supplemented with 10 % FCS (Omega Scientific). U87 TMZ-resistant and U251 TMZ-resistant cell
lines were developed by Adel Kardosh in the Schonthal laboratory by subjecting the cells to high
concentrations of temozolomide (Schering Plough) and selecting for cells that survived. These
were maintained in DMEM medium containing TMZ at a high concentration (100 μM) to
maintain TMZ resistance in these cell lines.
All animal protocols were approved by the Institutional Animal Care and Use Committee
(IACUC) of the University of Southern California (USC) and all guidelines were strictly adhered to.
2.2.2. In vivo study with noscapine in a subcutaneous U251 model
Four to six week old female athymic nu/nu mice (average weight 20-30gms) were
obtained from Harlan and housed in a pathogen free environment in the USC Animal Care
Facility. Animals were anesthesised with a 9:1 ketamine: xylazine solution (20 µL administered
intraperitoneally) and subsequently 1 x 10
6
sterile U251 cells in 100 µL PBS were injected
subcutaneously into the right flank. Animals were followed closely and treatment was initiated
when tumors became palpable approximately after two weeks.
To determine the effective dose of noscapine that slows down tumor progression, mice
were randomly divided into four groups (5 mice/group): three groups received 300, 550 and
1000 mg/kg noscapine hydrochloride in distilled water respectively; the fourth group served as
26

control and received vehicle solution (distilled water) only. Noscapine was administered daily by
gavage using a rounded-tip animal feeding needle.
Tumor dimensions (length, width and height) were measured every three days using a
vernier caliper and the tumor volume was calculated as length x width x height x 0.5 mm
3
. No
significant changes in body weight or apparent signs of clinical toxicity were observed in the
treated groups. Mice were euthanized at the end of treatment or when tumors became
ulcerated. Tumors were harvested and frozen in OCT at -125° for future analysis. Since there
was variability in tumor volumes amongst the different groups before start of treatment,
percent tumor volume with respect to day 0 of treatment was calculated and a graph of percent
tumor volume versus time was plotted.
2.2.3. In vivo study with noscapine and TMZ in an intracranial U87 model
4-6 week old female athymic nu/nu mice (Harlan) were anesthesised with a 9:1 ketamine:
xylazine solution and held in a stereotactic headframe. A paramedian incision was made and a
1.5 mm bur hole was drilled 1 mm anterior to the coronal suture on the right hemisphere and 2
mm lateral from the midline. A Hamilton syringe was used to slowly inject 2 x 10
5
sterile U87
cells in a total volume of 5 µL into the right frontal lobe of the brain. The incision was sutured
with a silk thread. Seven days after implantation, mice were randomly divided into four groups
(n=4): 1) Control (distilled water), 2) Noscapine (550 mg/kg), 3) TMZ (5 mg/kg) and 4) Noscapine
and TMZ combination. Drugs were administered by gavage. Mice were treated with noscapine
daily whereas they were treated with TMZ for 7 days and then left untreated for the next 7 days.
This cycle was repeated until the time of sacrifice as determined by preset criteria like
immotility, loss in body weight, hunched back, loss of balance and general morbidity.

27

2.2.4. In vivo study with noscapine and TMZ in a TMZ-resistant U251 subcutaneous model
Four to six week old female athymic nu/nu mice were anesthesised with a 9:1 ketamine:
xylazine solution and subsequently 2 x 10
6
sterile TMZ-resistant U251 cells in 100 µL PBS were
injected subcutaneously into the right flank. After tumors became palpable, mice were
randomly divided into four groups (n=3-5): 1) Control (distilled water), 2) Noscapine (550
mg/kg), 3) TMZ (5 mg/kg) and 4) Noscapine and TMZ combination. Drugs were administered by
gavage.
Tumor dimensions were measured every three days using a vernier caliper and the
tumor volume was calculated as length x width x height x 0.5 mm
3
. No significant changes in
body weight or apparent signs of clinical toxicity were observed in the treated groups. Mice
were euthanized at the end of treatment or when tumors became ulcerated. Percent change in
tumor volume with respect to day 0 of treatment was calculated and a graph of percent tumor
volume change versus time was plotted.
2.2.5. Statistical analysis
Values are presented as the mean +/- SEM. Statistical significance was evaluated using
the Student’s two-tailed t-test. A p-value < 0.05 was considered significant. For the in vivo study
in the TMZ-resistant model, statistical significance was evaluated using the linear mixed effects
model because the experiment was unbalanced.

28

CHAPTER III – RESULTS AND CONCLUSION
3.1. PART 1 – NOSCAPINE’S EFFECT ON THE TUMOR VASCULATURE

3.1.1. BEC and TuBEC are resistant to Noscapine
Since noscapine has been shown to have cytotoxic effects on human glioma cell lines
(Newcomb et al., 2008), we wanted to test the effect of noscapine on the glioma vasculature.
Normal brain endothelial cells and glioma associated endothelial cells were treated with
noscapine at different concentrations between 0-100 µM for 72 hours. MTT cell viability assay
was used to quantitate percentage cell survival. BEC and TuBEC were resistant to noscapine
even at higher concentrations. U251 human glioma cell line was used as a positive control. IC50
– the concentration at which there is 50 % cell death was between 50-60 µM for U251 (Figure 3-
1) whereas BEC and TuBEC showed less than 30 % cell death between this ranges. These results
show that noscapine shows differential cytotoxic effects depending on the cell type. It is
cytotoxic to glioma cells but has less cytotoxic effect on normal and tumor associated
endothelial cells of the brain.
29

Figure 3-1. Effect of Noscapine on cell viability. BEC, TuBEC and U251 were plated (5 x 10
3

cells/well) and treated with increasing concentrations of noscapine as shown in the graph. After
72 hours, MTT assay was performed and percent cell viability was calculated. BEC and TuBEC
were resistant to noscapine whereas U251 which serves as positive control shows dose
dependent cell death.

3.1.2. Noscapine decreases migration of activated brain endothelial cells
Noscapine is a microtubule targeting drug that significantly increases the time
microtubules spend in an idle state (Landen et al., 2002). So we hypothesized that noscapine
would have an inhibitory effect on migration. To test this, we seeded normal brain endothelial
cells on the upper chamber of transwell plates and added chemokine IL-8 (200ng/ml) and
noscapine (20µM) to the lower chamber. The noscapine concentration was chosen based on
studies in our laboratory that show that noscapine significantly decreases migration of human
glioma cells at 20 µM (unpublished data). After allowing cells to migrate for 6 hours, the number
of cells that migrate through the filter are counted using a high magnification.
30

Our results show that noscapine alone slightly increases migration (Figure 3-2). IL-8, an
important proangiogenic factor has been shown to increase migration of BEC (Charalambous,
Pen et al., 2005). In accordance with that, we see a 2-fold increase in migration with IL-8. This is
characteristic of activated BEC. Noscapine at 20 µM significantly decreases migration of
activated BEC (p < 0.0001). Migration is lowered even below the baseline. Thus noscapine has a
potent inhibitory effect on migration of activated BEC and this makes it a promising
antiangiogenic drug for treatment of GBM.

Figure 3-2. Effect of Noscapine on migration of BEC. BEC were plated (5 x 10
4
/well) in the upper
chamber of transwell plates. Medium, noscapine (20 μM), IL-8 (200 ng/ml) and noscapine + IL-8
were added to the lower chamber. After 6 hours, migrated cells were stained and counted. Data
are expressed as number of cells in ten high power fields using 400 X magnification. ** signifies
p < 0.0001 in the IL-8 treated versus noscapine + IL-8 treated BEC.

31

3.1.3. Noscapine decreases migration of untreated and IL-8-treated tumor-associated brain
endothelial cells
Since noscapine decreases migration of activated BEC, next we wanted to test if
noscapine has a similar effect on tumor-associated endothelial cells. TuBEC were plated in the
upper chamber of transwell plates and IL-8 (200ng/ml) and noscapine (1, 5 and 20 µM) were
added to the lower chamber. Cells were allowed to migrate for 6 hours. The results indicate
that noscapine decreases migration of TuBEC (Figure 3-3a) in a dose dependent manner with
more than 50 % inhibition seen at 20 µM (p < 0.0001). We found that TuBEC have a higher
baseline migration rate than BEC consistent with previous findings (Charalambous, Pen et al.,
2005). Moreover, TuBEC is unresponsive to IL-8 stimulation because of constitutive production
of IL-8 by TuBEC. IL-8 does not increase migration in TuBEC. Moreover, noscapine also
significantly decreases migration of IL-8-treated TuBEC (Figure 3-3b).

Figure 3-3. Effect of Noscapine on migration of TuBEC. TuBEC were plated (4-5 x 10
4
/well) in
the upper chamber of transwell plates. Medium, noscapine and IL-8 were added to the lower
chamber as indicated. After 6 hours, migrated cells were stained and counted. Data are
expressed as number of cells in ten high power fields using 400 X magnification. ** signifies p <
0.0001 in the medium (untreated) versus noscapine/noscapine + IL-8 treated BEC.
32

3.1.4. Noscapine has no effect on IL-8 secretion in normal and TNF-α activated BEC
Noscapine significantly decreases migration of activated BEC. To test whether its
mechanism of action is through lowering of cytokine levels, we carried out ELISAs for IL-8 with
supernatants from BEC treated with different concentrations of noscapine and TNF-α for 48
hours. TNF-α is known to stimulate IL-8 production in BEC and this is characteristic of activated
BEC. Our results show no effect of noscapine on IL-8 secretion in both normal (Figure 3-4a) and
TNF-α activated BEC (Figure 3-4b). Though there is a statistically significant difference with 10
µM noscapine in activated BEC, it is not clinically relevant. Thus noscapine’s effect on migration
is not due to decreasing levels of cytokines important in migration.

Figure 3-4. Effect of Noscapine on secretion of IL-8 in BEC. BEC were treated with (a) noscapine
(1, 10 and 30 μM) and (b) noscapine (1, 10 and 30 μM) and TNF-α (30ng/ml). After 48 hours,
supernatants were collected and analyzed for IL-8 levels. TNF-α stimulated IL-8 secretion.
Noscapine had no significant effect on IL-8 secretion in normal and activated BEC.

33

3.1.5. Noscapine significantly reduces microvessel density in vivo
Noscapine has been shown to have an antiangiogenic effect in human glioma cell lines
by decreasing HIF1α and VEGF expression (Newcomb et al., 2006). Moreover it also inhibits
tubule formation in vitro (Newcomb et al., 2008). We wanted to study noscapine’s
antiangiogenic effects in vivo. Tumor sections from athymic nude mice treated with different
doses of noscapine for 19 days were analyzed for CD31 expression by immunostaining. Intensity
of staining was quantitated using Image J software from NIH and microvessel density was
calculated.
Noscapine significantly reduced microvessel density compared to untreated control
(Figure 3-5a) mice (Figure 3-5d). Microvessel density was decreased by 36.8 % (p < 0.05) and 39
% (p <0.05) respectively at 300 mg/kg/day (Figure 3-5b) and 550 mg/kg/day noscapine (Figure 3-
5c). The morphology of blood vessels was different amongst control and treated mice. Control
showed elongated tubulated structures whereas treated mice had short, truncated vessels.
These results indicate that noscapine may have potent antiangiogenic effects in vivo.

34

Figure 3-5. Effect of Noscapine on microvessel density. Frozen sections of tumors from
noscapine treated (300 mg/kg/day and 550 mg/kg/day) and untreated mice were stained for
CD31. Red staining indicates positive staining for blood vessels. A representative image is shown
for untreated (a), noscapine 300 (b) and noscapine 550 (c) treated groups. Quantitation of
microvessel density (d) was done using ImageJ software (NIH). Data is presented as mean +/-
SEM. * signifies p <0.05 in the control (untreated) versus noscapine treated groups.

3.2. PART 2 – NOSCAPINE’S EFFECT ON TUMOR PROGRESSION AND SURVIVAL
3.2.1. Noscapine delays tumor progression in a subcutaneous U251 model
Noscapine has been shown to significantly delay tumor progression in lung cancer,
prostate cancer and T-cell lymphoma (Jackson et al., 2008; Ke et al., 2000 and Barken et al.,
2008). We wanted to study the effect of noscapine on tumor progression in a murine model of
human glioma. Human glioma U251 cells were implanted subcutaneously into nude mice and
treatment with increasing doses of noscapine was initiated once tumors became palpable.
35

In previous studies, noscapine has been shown to be effective in delaying tumor growth
at 300 mg/kg/day (Jackson et al., 2008) but in our study we did not find that dose to be effective
against glioma. However, noscapine at 550 mg/kg/day significantly decreased tumor
progression (p < 0.05) from day 15 of treatment without any toxic side effects (Figure 3-6).
Noscapine at 1000 mg/kg/day is also effective but this dose was toxic and was associated with
loss in body weight and death. Thus, noscapine at 550 mg/kg/day seems to be an effective dose
in delaying tumor progression of human glioma. However, the results are not dramatic and
noscapine alone may not be clinically significant.

Figure 3-6. Effect of Noscapine on tumor progression in a subcutaneous model of human
glioma. U251 glioma cells were implanted subcutaneously into the right flank of nude mice.
Once the tumors became palpable, the mice were treated with either vehicle control (distilled
water) or increasing concentrations of noscapine (300, 550 and 1000 mg/kg/day) daily by
gavage. Tumor size was measured every 3-4 days and tumor volumes were calculated. The
percentage change in tumor volume from day 0 versus the number of days is plotted. * signifies
statistical significance (p < 0.05).

36

3.2.2. Noscapine alone significantly prolongs survival in an intracranial U87 model but does not
synergize with temozolomide
Since noscapine alone does not dramatically reduce tumor progression in our
subcutaneous model, we next studied the combination of noscapine and temozolomide. We
used an intracranial model because it represents the orthotopic location of the tumor in
humans. U87 human glioma cells were implanted intracranially and treatment was initiated
seven days after implantation. We found that noscapine alone slightly prolonged survival in
these mice (Figure 3-7). However, contrary to our hypothesis, noscapine did not synergize with
temozolomide. Temozolomide alone was the most effective in prolonging survival. Thus
noscapine does not chemosensitize the tumors to temozolomide.

Figure 3-7. Effect of Noscapine and TMZ on survival in an intracranial model of human glioma.
U87 cells were implanted intracranially into nude mice and after seven days, mice were
randomly divided into four groups and treatment was initiated as indicated. Noscapine was
administered daily while TMZ was administered in a 7day on/off cycle. Mice were monitored
closely for loss in body weight, changes in behavior, loss of balance or general morbidity. A plot
of survival versus time in days is plotted. ** signifies statistical significance (p< 0.0001).

37

3.2.3. Noscapine chemosensitizes TMZ-resistant human gliomas to temozolomide
Tumors develop chemoresistance by overexpression of drug efflux pumps like p-
glycoprotein. Noscapine is a weak substrate for these drug efflux pumps. Thus it may be
effective in resistant tumors. We tested noscapine and temozolomide in a TMZ-resistant
subcutaneous model. TMZ-resistant U251 glioma cells were implanted subcutaneously into
nude mice and after tumors became palpable, treatment was initiated with noscapine,
temozolomide and the combination.
We found that tumors in the control, noscapine only and temozolomide only groups
progressed rapidly (Figure 3-8). Thus the tumors cells are truly resistant even in vivo. However,
tumors in mice treated with noscapine and temozolomide showed significant growth delay (p <
0.001). Thus, Noscapine chemosensitizes the tumor to temozolomide.

Figure 3-8. Effect of Noscapine and TMZ in a subcutaneous TMZ-resistant model. TMZ-resistant
U251 glioma cells were implanted subcutaneously into the right flank of nude mice. After
palpable tumors develop, treatment was initiated as indicated. Tumor size was measured every
3-4 days and tumor volumes were calculated. The percentage change in tumor volume from day
0 versus the number of days is plotted. ** signifies statistical significance (p < 0.001).
**
38

3.3. CONCLUSION
In summary, our studies indicate that noscapine has less cytotoxic effect on normal and
tumor associated endothelial cells compared to human glioma cells. However, it significantly
decreases cell migration independent of proliferation and cytotoxicity. Inhibition of migration is
not due to decrease in cytokine secretion as demonstrated by no change in IL-8 levels after
noscapine treatment. The mechanism of decreased migration remains to be investigated.
In addition, noscapine at 550 mg/kg/day significantly delays tumor progression in a
subcutaneous murine model of human glioma without any toxic side effects. Noscapine also
prolongs survival in mice bearing intracranial tumors. However, it does not show any synergistic
effects with temozolomide as expected. Temozolomide alone is still the most effective in
prolonging survival in these mice. Interestingly, in temozolomide-resistant tumors, noscapine
and TMZ show significant synergism. Microvessel density analysis of noscapine treated tumors
show that it significantly decreases microvessel density. Thus noscapine shows antiangiogenic
activity in vivo as well.
Overall, our studies indicate that noscapine is a promising drug for GBM treatment and
needs to be further investigated.

39

CHAPTER IV - DISCUSSION
Glioblastoma multiforme is a hypervascular tumor dependent on angiogenesis for
growth and progression. Noscapine is a widely used antitussive agent that has shown
antiangiogenic and antitumor activity against a wide variety of cancers. However, most studies
with noscapine have been done on rat and mouse gliomas. A few in vitro studies have been
done with human glioma cell lines and show promising results. In our study, we focused on
noscapine’s role in glioblastoma with respect to the glioma vasculature. Normal and tumor
associated brain endothelial cells were isolated from GBM patients and used for our study.
These are representative of the normal and GBM brain endothelium respectively and thus are
an excellent model.
Our studies showed that noscapine significantly decreased migration of both BEC and
TuBEC. We wanted to investigate the mechanism of decreased migration so we studied the
levels of cytokine IL-8 which is critical for migration after treatment with noscapine. Our results
indicated that noscapine had no effect on IL-8 secretion in normal and activated BEC.
Noscapine’s effect on IL-8 secretion in TuBEC remains to be investigated. Moreover, we will also
be studying other cytokines like VEGF and ET-1 which are important in migration and
angiogenesis.
Another potential target of noscapine could be the protein focal adhesion kinase (FAK).
FAK is a non-receptor tyrosine kinase that plays an important role in migration. It is activated by
autophosphorylation of tyrosine 397 followed by phosphorylation of several other tyrosine and
serine/threonine residues. Phosphorylation at tyrosine 407 is critical for endothelial cell
migration. It is required to recruit other proteins like paxillin and vinculin and ensure focal
adhesion formation (Lin et al., 2009). We hypothesize that since noscapine decreases migration,
40

it would decrease phosphorylation of FAK at tyrosine 407. To test this hypothesis, we will be
carrying out Western blots for pY407-FAK and total FAK on noscapine treated BEC and TuBEC
lysates. Another pathway of endothelial cell migration is mediated by p38MAPK. It would be
interesting to see if noscapine has any effect on phosphorylation of p38MAPK. Noscapine’s
primary action is on migration and by studying the effects of noscapine on several pathways of
cell migration we would be able to enhance its antimigratory effect.
Our in vivo studies showed that noscapine delayed tumor progression significantly in
mice bearing subcutaneous human glioma tumors. This is without any toxic side effects.
However, noscapine on its own does not dramatically decrease tumor progression and it may be
more effective in combination with other drugs. To test if noscapine works synergistically with
temozolomide – the current standard of care for GBM we used an intracranial model. This is a
more accurate model because it reflects the orthotopic location of human glioma. Noscapine on
its own only slightly prolonged survival in these mice but in combination with TMZ, we did not
see any synergism. Noscapine does not chemosensitize the tumors to temozolomide.
Microvessel density of noscapine treated tumors showed a decrease in microvessel density.
Thus noscapine also shows antiangiogenic potential in vivo.
Noscapine’s primary activity is on microtubules and migration. Thus it may not have a
direct effect on tumor growth and progression. But it may be more effective in combination
with other drugs. Studies need to be carried out with noscapine and other drugs used for cancer
like DMC and celecoxib.
Moreover, the major problem encountered in GBM therapy is chemoresistance. Tumors
become resistant to temozolomide over time. Resistance may be due to overexpression of
MGMT, P-gp, GRP78 or survivin. Noscapine is not a good substrate for P-glycoprotein and so it
41

may be helpful in tackling tumors that have become resistant because of increased P-gp. Results
from our in vivo studies with noscapine and temozolomide in a TMZ-resistant subcutaneous
model show that noscapine chemosensitizes these tumors to TMZ. These results are very
promising and further studies on the mechanism by which noscapine acts as a chemosensitizing
agent would be useful. Studies of noscapine’s effect on MGMT, GRP78 and survivin levels would
also be an interesting future step.
In conclusion, noscapine shows promise as an antitumor and antiangiogenic
drug for GBM. Noscapine is orally bioavailable, water soluble, can cross the blood brain barrier
easily and does not show any toxic side effects. Since it attacks the tumor cells and the
vasculature simultaneously it may be an attractive drug for GBM treatment.

42

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Abstract (if available)

Abstract Noscapine has been widely used as an oral antitussive agent. More recently, studies have shown that noscapine significantly affects microtubule dynamics and has potent antitumor and antiangiogenic activity. The aim of our study was to investigate noscapine's effects in glioblastoma multiforme (GBM) focusing on its effects on the glioma vasculature. We studied the effects of noscapine on cytotoxicity, migration and cytokine secretion of normal (BEC) and tumor-associated brain endothelial cells (TuBEC). We found that noscapine significantly decreased migration of both BEC and TuBEC. In accordance with this, we found that noscapine also decreased microvessel density in vivo.

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Asset Metadata

Creator Jhaveri, Niyati (author)

Core Title Noscapine: a study of its effects on the glioma vasculature

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2009-12

Publication Date 10/30/2009

Defense Date 10/20/2009

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

Tag angiogenesis,endothelial cell migration,glioma,noscapine,OAI-PMH Harvest,TMZ-resistant,vasculature

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Hofman, Florence M. (committee member), Hong, Young (committee member)

Creator Email jhaveri_niyati@yahoo.com,nsjhaver@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2701

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Document Type Thesis

Rights Jhaveri, Niyati

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

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