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Examination of the effect of oleandrin on head and neck cancer cells

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Examination of the effect of oleandrin on head and neck cancer cells

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Examination of the effect of oleandrin on head and neck cancer cells

by

Yuru Wu

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)

August 2021

Copyright 2021

Yuru Wu

ii
Acknowledgments
I would like to express my most profound gratitude to my research mentor and thesis committee
chair, Dr. Amy Lee, for her patient mentoring, helpful criticism and constant supervision of this
research over the past two years. It has been a great opportunity for me to have a mentor with such
rigorous demeanor and professionalism: she has constantly and convincingly conveyed her passion
and dedication for science, and this will have a profound impact on me in my future career.
Despite being trained as a scientist, she also provided me with the invaluable suggestion to train me
as a multi-faceted talent that can adapt to a variety of work environments from academic to
industrial. I am grateful for her patience, vast knowledge, and constant guidance and supervision.
In addition, I would like to express my sincere gratitude to the other members of my dissertation
committee, Dr. Ite. A Offringa, Dr. Pragna Patel, and Dr. Reginald Hill, for their generous help in my
application process to PhD program and their invaluable guidance in this project.
I would also like to thank my academic advisor, Dr. Vicky Yamamoto, who has been guiding me
through my master's program and advising me on my presentations in student seminars. She is such
a great mentor and a friend who gives me warm encouragements and professional help any time I
need it.
In addition, I would like to give sincere thanks to other members in Lee's lab. I appreciate all their
help and the great time we had in the lab. I would like to thank Dr. Ze Liu for his extremely valuable

iii
help and advice on my career development. I would also like to thank Dat Ha for his help and patient
guidance. He taught me the experimental techniques, advised on data analysis and interpretation,
and provided invaluable experience for the experiments. I would like to thank Anthony Carlos and
John Johnson for all the advice and encouragement they gave me and for their kind help and great
experience.

iv
Table of Contents
Acknowledgments .............................................................................................................................................. ii
List of Figures ..................................................................................................................................................... v
Abstract ............................................................................................................................................................. vi
Chapter I: Introduction ..................................................................................................................................... 1
Chapter II: Materials and Methods ................................................................................................................... 9
2.1 Cell Culture .............................................................................................................................................. 9
2.2 Cell viability assay ................................................................................................................................... 9
2.3 Immunoblot analysis ............................................................................................................................. 10
Chapter III: Results .......................................................................................................................................... 12
3.1 Oleandrin suppresses the cell viability of Head & Neck cancer cell line SCC15 ........................ 12
3.2 Oleandrin inhibits the expression of GRP78 in Head & Neck cancer cell line SCC15 ................ 13
3.3 Oleandrin-cetuximab combination treatment exhibit better efficacy in suppressing viability
and inducing apoptosis of Head & Neck cancer cell line SCC15 ........................................................ 14
Chapter IV: Discussion .................................................................................................................................... 19
Chapter V: Conclusion ..................................................................................................................................... 22
References ..............................................................................................................................................................................24

v
List of Figures
Figure. 1-1 Schematic representation of mechanism of action of oleandrin against cancer ............ 6
Figure. 1-2 Role of GRP78 in the unfolded protein response and the stress response ......................... 7
Figure. 3-1 The dose-dependent effects of oleandrin on SCC15 cell viability ....................................... 12
Figure. 3-2 The dose-dependent inhibitory effects of oleandrin on GRP78 level in SCC15 cells ....... 13
Figure. 3-3-1 Oleandrin combination treatment with cetuximab 50μg/ml and 100μg/ml causes
greater cytotoxicity in SCC15 cells than oleandrin and cetuximab mono-treatment .......................... 16
Figure. 3-3-2 Oleandrin combination treatment with cetuximab causes greater apoptosis induction
in SCC15 cells than oleandrin and cetuximab mono-treatment ............................................................... 17
Figure. 3-3-3 GRP78 expression is inhibited by oleandrin mono-treatment and combo-treatment 18

vi
Abstract
Oleandrin is a small molecule that belongs to a family of cardiac glycosides, which are naturally
derived compounds that inhibit Na
+
/K
+
ATPase. Traditionally, members from this family have been
used to treat heart failure. Our lab screened compounds that can suppress GRP78 gene expression,
an important protein that regulates the balance between cancer cell viability and apoptosis. We
further identified oleandrin, a drug that is currently in clinic trial phase two, as a GRP78 inhibitor.
We tested the therapeutic potential of oleandrin in head and neck cancer. We found that oleandrin
can successfully reduce GRP78 expression level and increase apoptosis in head and neck cancer. We
also found that, when combined with cetuximab, a monoclonal antibody against EGFR which is
currently used to treat head and neck cancer, oleandrin enhanced the efficacy of cetuximab-induced
cytotoxicity. In conclusion, although the mechanism of enhanced cytotoxicity warrants further
studies, oleandrin could be a promising drug for head and neck cancer treatment.

1
Introduction
Cardiac glycosides (CGs) are a diverse family of natural compounds with a steroid-like structure (Zhu
et al., 2017). Since first described by William Withering in the 18th century, members of CGs have
been used in clinics for the treatment of heart failure for over 200 years (Elbaz et al., 2012). The
mechanism of CGs was later identified as the ability to bind to and inhibit the Na
+
/K
+
ATPase.
Na
+
/K
+
ATPase is a ubiquitous membrane protein that actively transports potassium ions into cells
and sodium ions out of cells. Through inhibiting the inotropic function of Na
+
/K
+
ATPase, CGs raise
the level of sodium ions in cardiac myocytes, which induces a calcium ion level increase and leads
to an increase in cardiac contractile force (Prassas & Diamandis, 2008).
Besides producing inotropic effects and regulating myocyte contraction, the Na
+
/K
+
ATPase also acts
as an important regulator for many cellular events, including those associated with tumor cell
growth (Wang & O’Doherty, 2012). Moreover, certain Na
+
/K
+
ATPase isoforms have higher
expression in malignant cells than normal cells, which provides an opportunity for the development
of precision-targeted therapy (Mijatovic et al., 2012). This has aroused new interest in the
development of cardiac glycosides as anticancer agents. Different cardiac glycosides present
different affinities to different isoforms of ATPase and lead to unique sensitivity and toxicity. Thus,
for instance, oleandrin (an active principal component of Nerium oleander), whose sensitivity
positively correlates with the ratio of Na
+
/K
+
ATPase 3 subunit over 1 subunit, has less toxicity
and cardiotoxicity than other CGs (Yang et al., 2009; Wang & O’Doherty, 2012; Mekhail et al.,
2006).The plant extract of Nerium oleander has long been used for the treatment of dermatitis and

2
congestive heart failure in China, and oleandrin is now under phase II clinical trial for pancreatic
cancer in the United States (Roth et al., 2020). Recent studies of oleandrin have extensively
described its anticancer activities, such as inducing apoptosis, inhibiting cancer progression,
metastasis, stemness, angiogenesis, and radio-resistance (Sreenivasan et al., 2003; Nasu et al., 2002;
Manna et al., 2006; Colapietro et al., 2020). The mechanisms of its role in cancer treatment have
also been widely reported (Figure 1-1), including 1 ）the inactivation of the nuclear transcription
factor-κB (NF-κB) and activator protein-1 (AP-1) ， 2 ） the activation of caspase-3 dependent apoptosis
response, 3) the inhibition of Akt/mTOR signaling pathway, and 4) the downregulation of cancerous
stemness markers SOX2, CD44, and CXCR4 (Sreenivasan et al., 2003; Nasu et al., 2002; Colapietro et
al., 2020).In addition, oleandrin's role in inhibiting ER stress pathway PERK/eIF2a/ATF4/CHOP and
inducing immunogenic cell death in breast cancer has aroused our attention and interest (Li et al.,
2021). However, the interaction between oleandrin and glucose-related protein 78kDa (GRP78), a
crucial ER stress regulatory chaperone, was not investigated.
GRP78, also referred to as the immunoglobulin heavy chain binding protein (BiP), is a member of
the HSP70 family of proteins that are mostly expressed in the endoplasmic reticulum (ER) (Li & Lee,
2006). Since first discovered in 1977 by Ira Pastan through glucose starvation, GRP78’s traditional
role as an ER chaperone that facilitates protein folding and assembly, has been extensively studied
(Lee A., 2014). GRP78 is ubiquitously expressed in the ER to maintain the integrity and
homeostasis of the ER and mitochondria. It can be induced by a variety of environmental and
physiological stress conditions such as cell metabolism alteration, hyperproliferation, hypoglycemia,

3
hypoxia, acidosis, viral infection, and genetic lesion. These adverse conditions lead to the production
of mutant proteins and consequently, the accumulation of misfolded or unfolded proteins. In
response to the surging demand on ER protein-folding capacity caused by misfolded protein
aggregation, GRP78 is induced and assists in processes including protein folding and assembly,
misfolded protein targeting for degradation, ER Ca2+- binding, and the induction of unfolded protein
response (UPR). UPR is an adaptive survival mechanism through which cells can maintain a balance
between cell viability and apoptosis when the protein load exceeds the folding capacity of the ER
(Li & Lee, 2006).
GRP78 regulates the UPR by releasing and activating the UPR sensors: activating transcription factor
6 (ATF6), PERK-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1). The activated UPR
sensors mediate a series of downstream cyto-protective measurements, including arresting
translation, increasing ER-associated protein degradation, generating more ER folding proteins, and
producing cell surface, secretory, cytosolic, and mitochondrial GRPs that inactive p53 and facilitate
cell survival (Lee A., 2014; Lee A., 2006) (Figure. 1-2). When these corrective efforts are insufficient,
UPR will activate the apoptotic response by inducing pro-apoptotic transcription factor C/EBP
Homologous Protein (CHOP) and releasing caspase 7 and caspase 12(Wang and Kaufman, 2014).
Thus, the UPR is a key component for the survival of malignant cells under pathophysiological and
pharmacological insults, such as nutrient deprivation, oxygen limitation, high metabolic demand,
and oxidative stress (Glimcher, 2016; Lee A., 2014).

4
Therefore, GRP78, as the major UPR regulator, plays an important role in tumor development and
progression. As a result, the tumor-associated function of GRP78 has been extensively studied
during the past two decades. The induction of GRP78 has been reported in a wide variety of
cancer cell line and biopsy tissue (Kim et al., 2006) and accounts for ample cancerous adaptive
advantages, such as stemness, drug resistance, invasions, metastasis, and immune evasion (Wu et
al., 2010; Kabakov et al., 2020; Li & Lee, 2006; Dong et al., 2011; Lee E. et al., 2006; Lee A., 2014).
Studies have shown that partial knockdown of GRP78 in a transgenic mouse model exhibits
positive phenotypes such as reduced tumor cell proliferation and angiogenesis, suppressed
tumorigenesis, and increased apoptosis (Ninkovic et al., 2020; Picon & Guddati, 2020). GRP78 level
elevation has also been extensively documented across various patient cases with cancer
aggressiveness and poor survival (Lee A., 2014). Given that GRP78 plays such an important role in
nearly all phases of tumor development, the value of GRP78 as a prognostic marker and
therapeutic target has been of great interest (Gifford & Hill, 2018). Specifically, GRP78 has shown
great therapeutic and diagnostic potential in head and neck squamous cell carcinoma (HNSCC). By
blocking total and surface GRP78 expression, the growth of head and neck tumor initiation cell can
be suppressed in a mouse xenograft model (Lee A., 2014). Also, the activation of GRP78 is
associated with HNC aggressiveness and stemness and can predict the worse survival prognosis of
HNSCC patients (Chen et al., 2018).
Apart from the above, head and neck cancer patients have shown the best response to the
treatment of cardiac glycosides. Retrospective report of clinical data has shown that head and

5
neck cancer patients who received cardiac glycosides during conventional carcinoma therapies
exhibited significantly improved overall survival, when compared to patients who only received
cancer treatments (Menger et al., 2012). Thus, this study intends to utilize the head and neck
cancer cell model to test the therapeutic effects of oleandrin and the suppressive effect of
oleandrin (OLN) on GRP78 expression. In addition, we want to explore the possibilities of
oleandrin in combination treatment with currently used therapies for head and neck cancer.
Head and neck squamous cell carcinoma (HNSCC) is a highly lethal cancer with cellular and
functional heterogeneity. The worldwide incidence of HNSCC is roughly 600,000 cases per year, and
approximately half of these cases result in death (Picon & Guddati, 2020). One reason for this
high mortality is a lack of effective therapies. Some of the current prevalent clinical treatments for
HNSCC are cetuximab, a recombinant monoclonal antibody against epidermal growth factor
receptor (EGFR), which have been used since 2006 (Cramer et al., 2019; Gonzalez-Gronow et al.,
2021). As the only molecular targeted therapy for head and neck cancer, cetuximab improves
survival in the curative and recurrent and/or metastatic settings; however, the response rate is
only 13% and the efficacy is strongly limited by acquired resistance and recurrence (Picon &
Guddati, 2020).
Therefore, there is an urgent need to find a new drug that has a superior effect and can enhance
the efficacy of the current therapies for head and neck cancer. Studies have shown that the
upregulation of GRP78 can lead to treatment resistance and development of cancer stemness (Wu

6
et al., 2010). The inhibition of GRP78 expression can enhance the drug-sensitivity of cancer cells
(Kabakov et al., 2020). Therefore, we will also study the effect of oleandrin in combination with
cetuximab (CTX) in this study.

Figure. 1-1 Schematic representation of the mechanism of action of oleandrin against cancer.

7

Figure. 1-2 The role of GRP78 in the unfolded protein response and the stress response.
Endoplasmic reticulum (ER) glucose-regulated protein 78 (GRP78) functions as an unfolded protein
response (UPR) regulator. When pathophysiological or pharmacological insults disrupt the cell
homeostasis and lead to the accumulation of mis-folded protein, GRP78 responds by binding the
unfolded protein and helping the protein folding and degradation. At the same time, GRP78 leaves
and activates the UPR sensors: PRKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)
and inositol-requiring enzyme 1 (IRE1). The activation of the UPR sensors activate downstream
signaling pathways of UPR, which leads to the arrested translation and the generation of ATF4,
active nuclear form of ATF6 (ATF6(N)), and spliced form of X box-binding protein 1 (XBP1s) to

8
activate ER stress response element (ERSE) and subsequently the translation of ER stress-
responsive genes. Three major categories of ER stress-responsive genes lead to different
responses: 1) The translation of ER-associated protein degradation components leads to the
protein degradation. 2) The translation of pro-apoptosis transcription factor CHOP , together with
caspase7 and caspase12 that released by GRP78, induce the apoptosis response. 3) The
expression of GRP78 in ER, cytosol, mitochondria, cell surface and secretory GRP78 will carry out
cyto-protective functions, such as apoptosis inhibition, cell survival promotion, and angiogenesis.

9
Materials and Methods
2.1 Cell culture
The human head and neck cancer cell line SCC15 (kindly provided by Dr. Vicky Yamamoto) was
cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Inc. Manassas, VA)
containing 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA) and 1%
penicillin/streptomycin antibiotics. Cells were maintained at 37°C in a humidified atmosphere of
5% CO2 and 95% relative humidity. For oleandrin treatment, SCC15 cells were treated with
oleandrin (Santa Cruz Biotechnology, Inc., Dallas, TX) at 30 nM for 48 hours; for cetuximab
treatment, SCC15 cells were treated with cetuximab (MedChem Express, Monmouth Junction,
New Jersey) at 50 μg/ml and 100 μg/ml for 48 hours; for oleandrin-cetuximab combination
treatment, SCC15 were treated with 30 nM oleandrin-50 μg/ml cetuximab or 30 nM oleandrin-100
μg/ml cetuximab; as a positive control, SCC15 were treated with thapsigargin (Tg) (MilliporeSigma,
Billerica, MA) at 300 nM for 24 hours.

2.2 Cell viability analysis
Cell viability was assessed with the WST-1 reagent (Roche, Indianapolis, IN). 1 x 10
4
cells per well
were seeded into 96-well culture plates with 100 μl culture medium per well. Forty-eight hours
after oleandrin/cetuximab/combination treatment, the cell viability was measured by incubating
each plate with 10 μl per well of WST-1 substrate for 1-2 hours, and then the plates were read at a
wavelength of 450 nm with a reference wavelength of 655 nm.

10
2.3 Immunoblot analysis
The cell lysates from treated or untreated cells were homogenized in lysis buffer (0.5% Nonidet P-
40, 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1 mm EDTA, 10% glycerol, 1 mm dithiothreitol, and
proteinase inhibitor mixture (1 tablet/10 ml of lysis buffer, Roche Applied Science). After
incubation on ice for 30 min, the homogenate was centrifuged at 15,000 revolutions/min for 15
min at 4 °C. Thirty micrograms of total protein of the clarified supernatants was separated by 10 or
12% SDS-PAGE followed by transfer to nitrocellulose membrane at 4°C at 30 V overnight.
Nitrocellulose membranes containing the transferred proteins were blocked in Tris-buffered saline
containing 5% non-fat dry milk for 1 h at room temperature and were probed by primary
antibodies as follow: anti-GRP78 mouse antibody (610979, BD Biosciences, San Jose, CA), 1:2000;
anti-GAPDH mouse antibody (sc-32233, Santa Cruz Biotechnology, Inc. Dallas, TX),1:2000; anti-
cleaved PARP rabbit antibody (5625S, Cell Signaling Technology, Danvers, MA), 1:1000; anti-
cleaved caspase 7 rabbit antibody (9492P , Cell Signaling Technology, Danvers, MA), 1:1000. After
overnight incubation with primary antibodies at 4°C overnight, secondary antibodies were
incubated in secondary antibody for 2h in room temperature (m-IgGκ BP-HRP (sc-516102, Santa
Cruz Biotechnology, Inc. Dallas, TX) and mouse anti-rabbit IgG-HRP (sc-2357 Santa Cruz
Biotechnology, Inc. Dallas, TX)). The HRP signal was detected using SuperSignal
West Pico Plus chemiluminescent substrate (34580, Thermo Scientific, Waltham, MA)
andvisualized in a ChemiDocTMXRS + Imager (Bio-Rad Lab, Hercules, CA). The band intensities
were quantified with Image Lab software. And to normalize signals, the intensities of the target
proteins (GRP78, cleaved PARP , cleaved caspase 7) were divided by the intensity of the loading

11
control protein (GAPDH). The biological replicates were statistically analyzed using Student’s t-test,
data are presented as mean ± standard deviation. P values of ≤ 0.05 designated *P values of ≤ 0.01
designated as ** and P values of ≤ 0.001 designated as *** were considered statistically
significant.

12
Results
3.1 Oleandrin suppresses the cell viability of head & neck cancer cell line SCC15.
Although several studies have described some positive antitumor effects of cardiac glycosides
(digoxin or αldiginoside) in head and neck cancer (Ninkovic et al., 2020; Weng et al., 2020), few
studies have reported the role of oleandrin in head and neck cancer. Therefore, to determine the
effect of oleandrin in head and neck cancer, we treated SCC15 cells with different concentrations of
oleandrin for 48 hours. As shown in the Figure. 3-1, oleandrin at 20 nM, which is a concentration
below the clinically tolerated dose, can effectively reduce the viability of SCC15 cells to
approximately 30%. We also determined the best working concentration of oleandrin in the
subsequent experiments from this experiment.

Figure. 3-1 The dose-dependent effects of oleandrin on SCC15 cell viability. SCC15 cells were
treated with oleandrin at a concentration of 0, 10, 15, 20, and 30 nM for 48 hours. The cells were
incubated with WST-1 substrate and the cell viability was measured at a wavelength of 450 nm with
a reference wavelength of 655 nm. Result in Figure. 3-1 were obtained from Dr. Vicky Yamamoto.

13
3.2 Oleandrin inhibits the expression of GRP78 in Head & Neck cancer cell line SCC15.
To test our hypothesis that oleandrin can inhibit GRP78 expression, we added different
concentrations of oleandrin to SCC15 cells. As shown in the Figure 3-2, oleandrin at 20 nM can
effectively reduce GRP78 expression, again at a concentration lower than the clinical tolerance dose.

Figure. 3-2 The dose-dependent inhibitory effects of oleandrin on GRP78 level in SCC15 cells. A.
SCC15 cells were treated with oleandrin at a concentration of 0, 10, 20, 30, 40, and 50 nM for 48
hours. Target proteins were analyzed by Western blotting with GAPDH serving as a loading control.
B. Densitometric quantitation and comparison of GRP78 levels after normalization to GAPDH levels
in each lane of Panel A.

14
3.3 Oleandrin-cetuximab combination treatment exhibits better efficacy in suppressing viability
and inducing apoptosis of Head & Neck cancer cell line SCC15.
Through the experiments above, we confirmed that oleandrin can inhibit cancer cell viability and
down-regulate GRP78 expression in head and neck cancer cells. Overexpressed GRP78 levels have
been reported in many studies to contribute to cancer stemness and drug resistance, which leads
to the development of recalcitrant, refractory cancers. In addition, downregulation of GRP78 or the
use of GRP78 inhibitors has been shown to enhance drug sensitivity in treatment-naïve or drug-
resistant tumors, resulting in better therapeutic outcomes (Cook et al., 2013; Farshbaf et al., 2020;
Ramirez et al., 2019; Sisinni et al., 2019). The GRP78 inhibition feature of oleandrin opens up the
possibility for using oleandrin in combination therapy. The main reason why the efficacy of
cetuximab, a widely used targeted therapy in the clinical treatment of cancer, has been limited in
head and neck cancer is low responsiveness and drug resistance.
Therefore, we wanted to test whether the combination of oleandrin with cetuximab could enhance
the efficacy of cetuximab. We performed oleandrin monotherapy, cetuximab monotherapy and
oleandrin-cetuximab combination therapy on SCC15 cells and compared the extent to which cell
viability was affected under these treatments. As shown in the Figure 3-3-1, we found that the
combination treatment showed superior efficacy in triggering cytotoxicity compared to
monotherapy. Our next step was to explore the reasons behind this phenomenon. So, we performed
immuno-blot analysis on cell lysates and examined the expression of apoptosis markers, cleaved
PARP and cleaved caspase 7. From the result shown in the Figure 3-3-2, we found that the

15
combination treatment resulted in stronger induction of apoptotic markers. These data lead us to n
conclude that the oleandrin-cetuximab combination treatment triggers a stronger apoptotic
response in SCC15 cells, resulting in a more potent deleterious effect in cancer cells.
Next, we examined whether the reduction of the cyto-protective effect of GRP78 by oleandrin
triggered a stronger apoptotic response. Surprisingly, the GRP78 levels of cells under combination
treatment did not show noticeable differences from those of cells treated with oleandrin
monotherapy, as shown in Figure. 3-3-3. Therefore, the better efficacy of the combination
treatment may not be, at least not solely, dependent on the downregulation of GRP78. Therefore,
we examined the GRP78 expression levels of cells under different treatments.

16

Figure. 3-3-1 Oleandrin combination treatment with cetuximab at 50 μg/ml and 100 μg/ml causes
greater cytotoxicity in SCC15 cells than oleandrin and cetuximab mono-treatment. SCC15 cells
were treated with DMSO, Tg, 30 nM oleandrin, 50 μg/ml cetuximab, combination treatment of 30
nM oleandrin & 50 μg/ml cetuximab, and combination treatment of 30 nM oleandrin & 100 μg/ml
cetuximab for 48 hours. The cell was incubated with WST-1 substrate and the cell viability was
measured under a wavelength of 450 nm with a reference wavelength of 655 nm. The results in
Figure. 3-3-1 were obtained from Dr. Vicky Yamamoto.

17

Figure. 3-3-2 oleandrin combination treatment with cetuximab causes greater apoptosis
induction in SCC15 cells than oleandrin and cetuximab mono-treatment. A. and B. SCC15 cells were
treated with DMSO, Tg, 30 nM oleandrin, 5 0μg/ml cetuximab, combination treatment of 3 0nM
oleandrin & 50 μg/ml cetuximab, and combination treatment of 30 nM oleandrin & 100 μg/ml
cetuximab for 48 hours. Target proteins were analyzed by Western blotting with GAPDH serving as
a loading control. C. Densitometric quantitation of normalized cleaved PARP levels from Panel A and
its biological replicates. D. Densitometric quantitation of normalized cleaved caspase 7 levels from
Panel B and its biological replicates. The error is the standard deviation from the mean. Data
represent an average of three independent biological replicates. Treated samples and DMSO
controls were compared via t -tests.*=p<0.05, **=p<0.001

18

Figure. 3-3-3 GRP78 expression is inhibited by oleandrin mono-treatment and combo-treatment.
A. SCC15 cells were treated with DMSO, Tg, 30nM oleandrin, 50μg/ml cetuximab, combination
treatment of 30 nM oleandrin & 50 μg/ml cetuximab, and combination treatment of 30 nM
oleandrin & 100 μg/ml cetuximab for 48 hours. Target proteins were analyzed by Western blotting
with GAPDH serving as a loading control. B. Densitometric quantitation of normalized GRP78
levels from Panel A and its biological replicates. The error is the standard deviation from the
mean. Treated samples and DMSO controls were compared via t -tests.*=p<0.05, **=p<0.001.

19
Discussion
Previous studies have highlighted the role and value of oleandrin for its anticancer activity.
Oleandrin can suppress malignant tumor that is unresponsive to existing drugs and radiation
therapy, which is undoubtedly of high value for many malignant tumors with no effective
therapeutic options. In addition, the high affinity of oleandrin for 3 subunits of Na
+
/K
+
ATPase
that are specifically expressed in some malignant cells, allows it to be a more precise lethal agent
against cancer cells. This provides the basis for its development as a treatment that can effectively
kill cancer cells while causing minimal or no side effects on normal cells. The analysis on
expression levels of different subunits of Na
+
/K
+
ATPase in various cancer types and cell line is still
lacking. Filling this gap can help predict the therapeutic utility of oleandrin in other types of
cancer.
In in vitro experiments, oleandrin can induce cancer cell apoptosis at non-toxic concentrations in
normal cells. In clinical trials, oleandrin was generally well tolerated, with common treatment-
related side effects that included vomiting, nausea, decreased appetite, and diarrhea (Gonzalez-
Gronow et al., 2021; Wang & O’Doherty, 2012). In short, oleandrin is a very promising anti-
cancer molecule. Further understanding of this anti-cancer molecule will contribute to the
discovery of new application possibilities.
In this study, we found that treating cells with a clinically tolerated dose of 30 nM of oleandrin
resulted in a distinct decrease in GRP78 levels in SCC15 cells. The inhibitory effect of oleandrin on

20
GRP78 expression and the promoting effect of GRP78 on recalcitrant cancer cells are consistent
with the previously reported repressive effect of oleandrin on treatment-resistant cancer cells.
This finding adds a very important piece to the understanding of the mechanism of action of
oleandrin. Given the important role GRP78 plays in a variety of cancers, oleandrin’s inhibitory
effect on GRP78 suggests that oleandrin may hold great therapeutic potential for some refractory
cancer types. GRP78 could also serve as a predictive marker of oleandrin sensitivity and offers the
possibility of further expansion of the therapeutic scope of oleandrin.
The detailed mechanism by which GRP78 expression levels are inhibited by oleandrin is still
unclear. Continuing investigation of the relationship between oleandrin and GRP78 will facilitate
the understanding of the pathway of GRP78 action and thus identify new therapeutic targets or
predictive markers of oleandrin sensitivity.
In this study, we also characterized the therapeutic effect of oleandrin in reducing the viability of
head and neck cancer cells by inducing apoptosis. We found that at clinically safe doses, oleandrin
significantly reduced the cell viability of SCC15 and induced expression of the apoptosis biomarker,
cleaved PARP . Although this study did not include in vivo experiments, previous clinical studies
have shown that oleandrin did not exhibit cardiotoxicity even at the maximum tolerated dose,
which undoubtedly further increases the potential and value of oleandrin as an anti-cancer drug
(Wang & O’Doherty, 2012).

21
Similarly, we found that in combination with cetuximab, oleandrin can lead to greater reduction in
cell viability and elevated apoptotic signaling than single agent treatment. Surprisingly, cells
treated by the combination of oleandrin and cetuximab did not display a noticeable decrease in
GRP78 levels relative to monotherapy-treated cells. This suggests that the enhanced cytotoxicity in
combination therapy may not be solely dependent on the downregulation of GRP78 expression,
and that possible mechanisms require further elucidation.

22
Conclusion
Nowadays, oleandrin has received an increasing attention owing to its anti-cancer properties
characterized by its high specificity, low side-effects, and high potency against refractory cancers.
By investigating the mechanism of oleandrin and its efficacy in head and neck cancer, we have
successfully expanded our understanding of the mechanism of action of oleandrin and extended
the applicability of oleandrin. We found that oleandrin can reduce the viability of head and neck
cancer cells by triggering apoptosis, which suggests its value as a new therapeutic agent for HNC.
We further confirmed the enhancing effect of oleandrin on existing therapies when used in
combination with cetuximab, which strengthens the value of oleandrin in the continuing clinical
development process. However, the enhanced anti-tumor property of combination therapy is still
unclear and warrants further investigation. Taken together, these findings highlight the potential of
oleandrin as a new cancer therapeutic agent and raise the need for further understanding of the
mechanism of oleandrin.
However, this study still has some deficiencies that merit further investigation. Further suggestions
for future directions are listed below. Firstly, it is true that GRP78 levels in combination therapy did
not show significant differences from oleandrin monotherapy. However, this does not mean that
the oleandrin-induced downregulation of GRP78 levels is not related to the higher cytotoxicity in
the combination treatment. In fact, the SCC15 cells used in this study are not cetuximab-resistant
cells, so the upregulation of GRP78 levels in the cells themselves may not be sufficient to show the

23
stronger inhibitory properties for GRP78 in combination therapy. Therefore, the establishment of a
therapy-resistant cell line allows a better assessment of the effect of oleandrin and its ability to
downregulate GRP78 on drug-resistant tumors that overexpress GRP78.
Secondly, oleandrin has been widely reported as a radiosensitizer. And it was also found that GRP78
downregulation could enhance the sensitivity of radiotherapy (Lee E. et al., 2006). Therefore, the
efficacy of oleandrin in combination with radiotherapy for head and neck cancer is worthy of further
investigation.
Finally, this experiment did not include an in vivo model, and thus, further in vivo experiments would
allow a more accurate simulation of the patient's response to the drug and a better detection of the
toxicity of both the oleandrin as well as the combination therapy.

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Abstract (if available)

Abstract Oleandrin is a small molecule that belongs to a family of cardiac glycosides, which are naturally derived compounds that inhibit Na⁺/K⁺ ATPase. Traditionally, members from this family have been used to treat heart failure. Our lab screened compounds that can suppress GRP78 gene expression, an important protein that regulates the balance between cancer cell viability and apoptosis. We further identified oleandrin, a drug that is currently in clinical trial phase two, as a GRP78 inhibitor. We tested the therapeutic potential of oleandrin in head and neck cancer. We found that oleandrin can successfully reduce GRP78 expression level and increase apoptosis in head and neck cancer. We also found that, when combined with cetuximab, a monoclonal antibody against EGFR which is currently used to treat head and neck cancer, oleandrin enhanced the efficacy of cetuximab-induced cytotoxicity. In conclusion, although the mechanism of enhanced cytotoxicity warrants further studies, oleandrin could be a promising drug for head and neck cancer treatment.

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Creator Wu, Yuru (author)

Core Title Examination of the effect of oleandrin on head and neck cancer cells

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2021-08

Publication Date 07/28/2021

Defense Date 06/02/2021

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

Tag cardiac glycoside,cetuximab,GRP78,head and neck cancer,OAI-PMH Harvest,oleandrin

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Advisor Lee, Amy (committee chair), Hill, Reginald (committee member), Offringa, Ite (committee member), Patel, Pragna (committee member)

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