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Radiosensitization of nasopharyngal carcinoma cells by NEO212, a perillyl alcohol-temozolomide conjugate drug

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Radiosensitization of nasopharyngal carcinoma cells by NEO212, a perillyl alcohol-temozolomide conjugate drug

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Content i
RADIOSENSITIZATION OF NASOPHARYNGEAL CARCINOMA
CELLS BY NEO212, A PERILLY ALCOHOL-TEMOZOLOMIDE
CONJUGATE DRUG
by
Megan Anika Dsouza
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
MOLECULAR MICROBIOLOGY AND IMMUNOLOGY
August 2024
Copyright 2024 Megan Anika Dsouza

ii
Acknowledgments
First and foremost, I am deeply grateful to Dr. Axel Schönthal for his unwavering support and for
allowing me to work in his esteemed laboratory. His guidance and encouragement have been instrumental
in my academic journey, allowing me to delve into a field of research that truly captivates me. His
mentorship has expanded my knowledge and instilled in me the confidence to ask the right questions and
navigate the complexities of this study.
I would also like to extend my sincere thanks to my thesis committee members for their unwavering
support and guidance. Dr. Steve Swenson, for his encouragement and friendly demeanor, which allowed
me to grow my knowledge and create a comfortable laboratory environment whenever I met him. Dr.
Stanley Tahara, who always encouraged me to think outside the box and broadened my knowledge with a
broad perspective on the subject. Lastly, Dr. Radu O. Minea, whose unwavering knowledge of the subject
and ever-ready enthusiasm paved the way for my broad understanding of the subject. I am genuinely
grateful for their role in shaping my academic journey.
I want to thank Dr. Thomas Chen for allowing the development of this project and for helping me
with his suggestions and support. I would also like to extend my appreciation to my fellow lab members for
their help during my time in the laboratory, especially Thu Zan Thein, for creating a welcoming environment
in the laboratory and guiding me to learn and improve in any way that I can.
Furthermore, I would like to express my deep appreciation to my friends, near and far. My old
friends scattered around the globe for their unending encouragement and support across time zones. My
new friends for making me feel at home and providing a safe and relaxing environment whenever needed.
They were the light in the dark times and never failed to put a smile on my face. Especially Wenzel, Isha,
and Max, who have been pillars of support, personally and academically, throughout this time.
Finally, I would like to thank my parents, Marietta and Darryl Dsouza, for allowing me to chase my
dreams and providing the support and guidance to be a strong and independent woman. I am forever
grateful for their unwavering belief in me and for teaching me that no dream is too big to achieve. I also
thank my sister, Danielle, for always being my shoulder to cry on and lifting my spirits whenever I was low.
None of my accomplishments would have been possible without all of them. Thank you.

iii
Table of Contents
Acknowledgments............................................................................................................................ii
List of Tables.....................................................................................................................................v
List of Figures...................................................................................................................................vi
List of Abbreviations......................................................................................................................viii
Abstract...........................................................................................................................................ix
Chapter 1 – Introduction..................................................................................................................1
1.1 Nasopharyngeal Carcinoma………………………..................................................................1
1.1.1 Introduction to Nasopharyngeal Carcinoma...............................................................1
1.1.2 Epidemiology and Etiology.........................................................................................2
1.1.3 Clinical Manifestation and Histopathology………………………........................................3
1.1.4 EBV and Nasopharyngeal Carcinoma .........................................................................3
1.1.5 Diagnostics and Staging..............................................................................................5
1.1.6 Available Treatments.................................................................................................7
1.2 NEO212 ......................................................................................................................................9
1.3 Radiosensitization of NEO212.....................................................................................10
1.4 Hypothesis………………...............................................................................................................12
Chapter 2 – Materials and Methods...............................................................................................14

iv
2.1 Pharmacological agents...............................................................................................14
2.2 Cell lines......................................................................................................................14
2.3 MTT assay....................................................................................................................14
2.4 Colony Formation Assay/Clonogenic Assay (CFA)........................................................15
2.5 Immunoblots...............................................................................................................16
2.6 DNA Damage Analysis by FACS Analysis ……………………………........................................16
2.7 Statistical Analysis.......................................................................................................17
Chapter 3 – Results........................................................................................................................18
3.1 NEO212 and Ionizing Radiation inhibit growth in TW1 and C666.1 NPC cell lines........18
3.1.1 Effect of IR on cell viability in TW1 and C666.1 cell lines………………………...................18
3.1.2 Effect of NEO212 on cell viability in TW1 and C666.1 cell lines..................................22
3.2 Combination therapy decreases cell survival in TW1 and C666.1 cell lines..................25
3.3 NEO212 radio-sensitizes NPC TW1 & C666.1 cell lines at clinically
relevant concentrations…………..…………………………………………………………………………………..34
3.4 NEO212 + IR increased cell death via DNA damage......................................................38
Chapter 4 – Discussion...................................................................................................................44
Bibliography...................................................................................................................................51

v
List of Tables
Table 1.1: Staging for NPC according to the 8th edition of the UICC/ANCC staging system................6
Table 1.2: Staging Groups of NPC according to the 8th edition of the UICC/ANCC staging
System……………………………………………………………………………………………………………………………….………

vi
List of Figures
Figure 1.1: Anatomy of the nasopharynx……………………………….........................................................1
Figure 1.2: Chemical Structure of TMZ and TMZ-Analog NEO212………………………………………..........10
Figure 3.1: Effect of IR on TW1 NPC cell line………………………………...................................................18
Figure 3.2: Effect of IR on EBV-positive C666.1 NPC cell line...........................................................20
Figure 3.3: Comparative effect of IR on EBV-negative and positive TW1 &C666.1 NPC cell line.....21
Figure 3.4: Effect of NEO212 TW1 NPC cell lines.............................................................................22
Figure 3.5 Effect of NEO212 on EBV-positive C666.1 NPC cell lines................................................23
Figure 3.6: Comparative effect of NEO212 on EBV-negative and positive TW1 and C666.1 NPC
Cell lines………………………………………………………………………………………………………………......................24
Figure 3.7 Combination effect of 4 Gy IR and NEO212 on TW1 NPC cell lines...............................26
Figure 3.8: Combination effect of 6 Gy IR and NEO212 on TW1 NPC cell lines.............................27
Figure 3.9: Comparative effect of single and concurrent NPC TW1 cell line treatment with IR
And NEO212………………….……………………………………………………………………………...............................28
Figure 3.10: Combination effect of 6 Gy IR and NEO212 on C666.1 NPC cell lines...........................29
Figure 3.11: Combination effect of 8 Gy IR and NEO212 on C666.1 NPC cell lines...........................30
Figure 3.12: Comparative effect of single and concurrent NPC C666.1 cell line treatment with
IR & NEO212...................................................................................................................................30

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Figure 3.13: NEO212 as a robust radiosensitizer of MGMT-negative TW1 NPC cell line.................31
Figure 3.14: NEO212 as a radiosensitizer of MGMT-positive C666.1 NPC cell line..........................32
Figure 3.15: NEO212 as a radiosensitizer of TW1 and C666.1 NPC cell line.....................................34
Figure 3.16: NEO212 as a robust radiosensitizer of TW1 NPC cell line............................................33
Figure 3.17: NEO212 as a radiosensitizer of C666.1 NPC cell line....................................................36
Figure 3.18: Determination of the levels of MGMT in the two NPC cell lines cells..........................37
Figure 3.19: Determination of the levels of Ƴ-H2AX TW1 NPC cells with treatment of IR,
NEO212, & NEO212 + IR................................................................................................................38
Figure 3.20: Determination of the levels of Ƴ-H2AX C666.1 NPC cells with treatment of IR,
NEO212, & NEO212 + IR.................................................................................................................39
Figure 3.21: Effect of NEO212 on cell death and DNA damage in TW1 NPC cells............................40
Figure 3.22: Effect of NEO212 on cell death and DNA damage in C666.1 NPC cells.........................41

viii
List of Abbreviations
γ-Ha2x: phosphorylated Ha2x protein IMRT: Intensity-Modulated Radiotherapy
AC: Adjuvant Chemoradiotherapy IR: Ionizing Radiation
AML: Acute Myeloid Leukemia LMP: Latent membrane protein
ATCC: American Type Culture Collection MGMT: methylguanine methyltransferase
AJCC: American Joint Committee on Cancer MMR: DNA Mismatch Repair
BER: Base Excision Repair MRI: Magnetic Resonance Imaging
CCRT: Concurrent Chemoradiotherapy MTT: methylthiazoletetrazolium
CDDP: Cis-platin Chemoradiotherapy NCRT: Neoadjuvant Chemoradiotherapy
CD21: Complement Receptor – 2 NEO212: perillyl alcohol covalently linked
CT: Computed Tomography to temozolomide (TMZ-POH)
DMEM: Dulbecco's Modified Eagle Medium NPC: Nasopharyngeal Carcinoma
EA: Early Antigens NRP1: Neuropilin1
EBV: Epstein-Barr Virus PAGE: Polyacrylamide gel electrophoresis
EBNA: Epstein-Barr Virus nuclear Antigen PBS: Phosphate Buffered saline
FACS: Fluorescence-activated cell sorting PET: Positron Emitting Tomography
FBS: Fetal Bovine Serum POH: Perillyl Alcohol
gb: Glycoprotein B PVDF: Polyvinylidene difluoride
GBM: Glioblastoma RT: Radiotherapy
GLOCBOCAN: Global Cancer Statistics ROS: Reactive Oxygen Species
Gy: Grey TNM: Tumor, node, and metastasis
ID50: The dose of radiation that reduces TMZ: Temozolomide
cell proliferation by 50% UICC: Union for International Cancer
IC50: concentration of drug that reduces Control
cell proliferation by 50% VCA: Viral capsid antigens
IgA: Immunoglobulin A WHO: World Health Organization
IgG: Immunoglobulin G

ix
Abstract
Nasopharyngeal carcinoma (NPC) is a head and neck tumor located at the back of the throat. It is
a specifically beguiling cancer to treat owing to its unique position, predisposing it to non-surgical treatment
outcomes. Moreover, current therapeutic measures for NPC are effective for early-stage, non-metastatic
tumors. In the case of late-stage NPC and nodal metastasis, treatment outcomes have been bleak, with low
rates of disease-free progression and overall survival post-treatment. Furthermore, current
chemoradiotherapies have proven to show off-target toxicity effects, further diminishing patient outcomes.
These obstacles to the effective treatment of NPC are closely related to the radio-resistance of NPC
cell lines. In this study, we aim to evaluate the effect of NEO212, a novel perillyl alcohol-temozolomide
conjugate, as a robust radiosensitizer of NPC cell lines when administered concurrently with ionizing
radiation (IR). We hypothesize that NEO212 will synergize with ionizing radiation (IR) at clinically relevant
concentrations to produce an efficacious and potent treatment outcome. Additionally, we also wanted to
assess the difference in the effect of NEO212 and IR on Epstein-Barr Virus (EBV) and O6-methylguanine–
DNA methyltransferase (MGMT) negative and positive TW1 and C666.1. NPC cell lines, respectively.
To carry out this study, the effect of NEO212 and IR, alone and together, were determined via cell
proliferation and viability assays. The impact on cell death and apoptosis was also determined using
relevant cell death, DNA damage, and apoptotic factors. Treatment conditions included various drug and
IR doses with different NPC cell lines. To achieve these results, we implemented methylthiazoletetrazolium
assays, colony formation assays, Western blots, and fluorescence-activated cell sorting.
Our results established that both NPC cell lines were susceptible to NEO212. MGMT and EBVpositive cell line C666.1 were slightly more radioresistant and chemo-sensitive than TW1. We established
NEO212 as a robust radiosensitizer in the TW1 cell line and found that the synergism between the two

x
modalities was most efficient at lower doses of NEO212. However, the C666.1 cell line presented
inconclusive results concerning the radiosensitization ability of NEO212 and its ability to synergize with IR.
We suspect the presence of robust, DNA-damage repair protein MGMT to be the underlying feature for
the unpredictability of this synergistic mechanism in the C666.1 cell line.
Overall, NEO212 proved to be a promising radiosensitizer for nasopharyngeal carcinoma and a
potent alternative to current therapies. Further studies are required to determine the best conditions for
synergism between NEO212 and IR in MGMT and EBV-positive NPC cell lines and to characterize this
radiosensitization ability of NEO212 with IR in vivo studies and clinical settings.

1
Chapter 1: Introduction
1.1. Nasopharyngeal Carcinoma
1.1.1 Introduction to Nasopharyngeal Carcinomas
Nasopharyngeal carcinoma (NPC) is known to be an endemic head and neck tumor, prevalent in
North Africa and Southeast Asia, predominantly in China (1,2,3). The tumor is located at the back of the
throat and arises from the epithelial cells of the nasopharynx. Nasopharyngeal carcinomas are known to be
significantly different from other tumors due to their unique position, clinical manifestation, and patient
prognosis (2).
NPC is typically complicated to detect and treat owing to its location at the back of the nose. It arises
at the junction of the nasal passage and auditory tubes, where it joins the upper respiratory tract. This
positioning makes it an arduous subject for effective treatment (2).

2
Figure 1.1 Anatomy of the nasopharynx. The anatomical structure of the head and neck is shown.
NPC arises from the back of the nasal passage above the roof of the mouth. Abnormal cells form a tumor
that can lead to nodal and distant metastasis. (Figure made with BioRender.com)
1.1.2 Epidemiology and Etiology
Epidemiology: NPC is a rare malignancy observed worldwide, unlike most other cancers. The
incidence rate of NPC is as low as 1 in every 100,000 individuals per year. However, regions such as
Southeast Asia, mainly Southern China, North Africa, the Arctic, and specific areas of Middle East Asia show
a higher incidence rate. NPC cases are found to be independent of race and ethnicity; however, it has been
observed to be higher in males than females with a ratio of 2-3:1 (male: female). In 2022 GLOBOCAN
statistics, >86,000 detected NPC cases were in males compared to the >36,000 cases observed in females
(4).
The correlation of NPC with age shows a steady increase in incidence with age in low-risk
populations like most epithelial cancers. In contrast, high-risk populations show an increase until the peak
age of 45-55 years, after which NPC cases observe a decline with age. Moderate-risk populations such as
North American, Malaysian, and Indian present a peak in incidence in adolescents and young adults (5).
According to the Global Cancer Statistics (GLOBOCAN - 2022) Report, more than 120,000 cases of NPC were
identified worldwide (4).
Etiology: NPC cases are usually first noticed by the detection of the Epstein-Barr Virus nuclear
antigen (EBNA) that is known to be associated with NPC. EBV is known to infect the epithelial cells of the
affected individuals, thus leading them to be growth-transformed. In addition, other etiological factors of
NPC are presumed to be ethnic descent, exposure to environmental carcinogens, and genetic susceptibility.
One of the most predominant ecological etiological factors of NPC is observed to be the consumption of
food products containing carcinogens (1).

3
1.1.3 Clinical Manifestations and Histopathology
Clinical Manifestations: Nasopharyngeal carcinomas typically arise from the lateral walls of the
nasopharynx, namely the fossa of Rosenmuller (pharyngeal recess) and the Eustachian tube. The presence
of the closely situated lymph nodes at the tumor site often serves as the initial site of metastasis. Thus,
initial presentations of Cervical lymphadenopathy serve as the primary diagnostic marker for NPC via lymph
node biopsies. Symptoms are often associated with the primary tumor and are manifested by obstructed
nasal passage, blood-traced sputum, nasal discharge, tinnitus, hearing loss, and cranial nerve paralysis (1,
6).
Histopathology: According to the World Health Organization (WHO), NPC is classified into three
distinct subtypes. Like most other head and neck cancers, type I is keratinizing squamous cell carcinomas
(SCC). Type II is non-keratinizing carcinoma, and Type III is undifferentiated carcinoma (7).
Type II and III are associated with EBV infection, while Type I is not (1). Type I carcinomas are usually
related to low-risk populations in Western countries, while Type III accounts for almost 97% of all NPC
cases. Type III undifferentiated carcinomas are associated with lymphocyte infiltration and
“lymphoepithelioma” (6). More specifically, the TW1 cell lines are known to be EBV-negative carcinomas,
while NPC C666.1 is positive for EBV.
1.1.4 EBV Virus and Nasopharyngeal Carcinomas
Epstein-Barr Virus is an oncogenic herpes 4-associated virus that is latently present in >90% of the
worldwide population (6). It exists asymptomatically as a B-cell infection in its latent stage. In some cases,
transient infection of EBV can lead to clinically relevant mononucleosis (8). However, reactivation of EBV
infection is linked to several malignancies, more specifically in NPC. In contrast to other head and neck

4
cancers, NPC Type II and III, namely non-keratinizing squamous cell carcinomas and undifferentiated
carcinomas, are known to be positively associated with the Epstein-Barr Virus (EBV) (6, 34).
Although frequently termed a silent infection, the latent stage of EBV infection encapsulates
several tightly regulated viral genes, namely, latent membrane proteins, LMP1, 2A, and 2B, six viral nuclear
antigens (EBNA1, 2, 3A,3B, 3C, and EBNA-LP). EBV also encodes two small, non-coding nuclear RNAs known
as EBERs. According to various genetic studies, this set of genes is critical to B-cell transformation. The
oncogenic characteristics of EBV have been proven to be associated with the expression of LMP1, LMP2A,
and LMP2B latent membrane proteins, as well as EBNA1 and EBNA2 viral-nuclear antigens (8).
Although the initial penetrance of the EBV infection in the nasopharyngeal epithelial cells is still
undetermined, particular research suggests CD21 receptors play a role in viral entry (8). A recent study
indicates that a class of novel receptors, neuropilin1 (NRP1) and ephrinA2, might be responsible for viral
entry via EBV glycoprotein B (gb) fusion (9).
Secretory immunoglobulin IgA-mediated transport is also known to facilitate endocytoses and
further infection of the nasopharyngeal epithelial cells (8). This is further validated by the presence of
increased IgA titers to viral capsid antigens (VCA) and increased IgG antibody titers to early antigens (EA) in
EBV positive, Type II and III NPC patients as compared to Type I, EBV negative patients that present normal
IgG levels (6, 10). It has also been observed that these titers are closely related to increased tumor burden,
disease progression, and relapse rates. EBV viral antigens are observed before the development of NPC,
thus implying that EBV reactivation occurs prior to cellular transformation and disease progression.
Under normal circumstances, viral replication occursin epithelial cells. However, in the case of NPC
progression, viral replication is prevented, and instead, genes responsible for cellular transformation are
expressed, thus generating proliferative EBV-infected cells. The presence of premalignant lesions and
tumorigenic cells is a rarity, suggesting that expression of the EBV-transforming genes is a tightly regulated

5
phenomenon. This critical activation of a subset of genes is significant for cellular proliferation and justifies
the sudden malignancy and invasiveness of EBV-infected epithelial cells to NPC (10).
1.1.5 Diagnostics and Staging
Diagnostics: Early diagnostics for NPC show an extremely successful prognosis for early-stage NPC.
However, what remains an obstacle is the long-term patient prognosis and overall survival (5). The primary
method of diagnosis of NPC is via lymph node biopsies and primary tumor biopsies. However, primary
tumor biopsies are rarely considered due to the intrinsic position of the tumor. On the other hand, lymph
node biopsies are generally indicators of late-stage metastatic NPC, wherein the primary tumor has
migrated to the surrounding lymph nodes. This late-stage detection further decreases the overall prognosis
and survival of the patient (11).
Additionally, since biopsies can result in tissue swelling and hematoma, other viable diagnostics
such as Computed Tomography (CT) scans, Magnetic Resonance imaging (MRI), EBNA viral antigen testing,
and cranial nerve examinations are undertaken before biopsies. (1, 10) As mentioned in the paper by Teo
and Chan (2002), MRI is observed to be a more sensitive diagnostic tool since it allows the direct soft tissue
imagining of the primary tumor, nodal metastasis, and possible perineural invasion. However, in the case
of prevalent bone erosion, CT scans are known to be more sensitive (12).
A recent study defining the benefits of PET (Positron Emission Tomography) and CT (Computed
Tomography) diagnostic scans showed that PET/CT scans provided a highly accurate and sensitive diagnosis
for lymph node metastatic NPC. PET/CT-diagnosed patients showed higher survival rates than MRI-staged
patients. The study emphasizes the advantageous nature of PET/CT diagnostics in providing enhanced
survival benefits owing to accurate diagnosis of lymph node metastasis. Combined with MRI scans, this is
beneficial in diagnosing high-risk NPC patients and providing the best-personalized treatment (11).

6
Staging: As the outcome of new imaging techniques for NPC advanced, the need for better staging
arose. This led to the formulation of a new and improved staging system by the American Joint Committee
on Cancer (AJCC). The new system works based on the Union for International Cancer Control (UICC) and
the American Joint Committee on Cancer (AJCC) System – tumor, node, and metastasis (TNM) staging
system. The 8th and most recent (2016) edition of the UICC/ANCC staging system has been explained in a
tabular format— (Table 1) (Table 2) (13).
Table 1. Staging for NPC according to the 8th
edition of the UICC/ANCC staging system
Primary Tumor (T) Regional Lymph Node (N) Distant Metastasis
T0
EBV-positive lymph
node, no identifiable
tumor
T1
The tumor extends to
the
nasopharynx/oropharynx
T2
Pharyngeal extension
with adjacent soft tissue
extension (medial
pterygoid, lateral
pterygoid, prevertebral
muscles)
T3
Extension into bone
structures and/or
paranasal sinuses
T4
Intracranial extension
involving cranial nerves,
hypopharynx, orbit,
extensive soft tissue
N0
No regional metastasis
N1
Unilateral cervical or uni-/
bilateral lymph node
metastasis, 6 cm or smaller
N2
Bilateral metastasis in
lymph nodes, 6cm or
smaller
N3
Lymph node metastasis,
>6cm and/or extension to
the supraclavicular fossa
M0
No distant
metastasis
M1
Distant metastasis

7

Table 2. Staging Groups of NPC according to the 8th
edition of the UICC/ANCC staging system
Staging Groups Staging
Stage I T1 N0 M0
Stage II T2 N0–1 M0, T0–1 N1 M0
Stage III T3 N0–2 M0, T0–2 N2 M0
Stage IVA T4, N3 M0
Stage IVB Any T, any N, M1
1.1.6 Available Treatments
Available Treatments: Owing to the anatomically intricate position of NPC tumors and their highly
radiosensitive nature, surgical removal of the cancer is not the recommended primary treatment, thus
making radiotherapy the first line of treatment for non-metastatic cases. (11, 14)
Radiotherapy: Radiotherapy for NPC was first described by Ho during the early 1990’s. Ho proposed
2D-RT as the preferred method of treatment. Radiotherapy utilizes ionizing radiation to directly target
nuclear DNA, resulting in direct DNA damage or indirect damage due to ROS production (3). 2D
conventional RT had a 6-7-week treatment period and a 60 to 70 Gy tumoricidal dose separated into 2 Gy
per fraction. This method of conventional 2D-RT showed initial success rates only in early disease prognosis
and prevention cases; it had a local control rate of up to 80%. With advances in the staging systems, and
therefore NPC diagnosis and progression, 2D-RT demonstrated decreased survival rates and increased local
and regional treatment failure, especially in the case of older patients (12, 15).
With the failure of 2D-RT, radiotherapy for NPC slowly transitioned into 3D-RT, more specifically
Intensity-Modulated-Radiotherapy (IMRT), which showed better progression-free-survival rates and

8
distant-metastasis-free survival—overall allowing better control of patients diagnosed with NPC (16). The
rise of IMRT benefited NPC treatment from various fronts; it allowed for better tumor-targeted therapy
when combined with MRI or CT diagnosis, thus preventing off-target effects on vital organs and reducing
the likelihood of tumor load misses. Overall, it increased local control rates for stage I patients from 80% to
90%. However, stage II patients showed a 30-50% survival rate, deeming IMRT less favorable for latterstage NPC treatment. (12, 14) Additionally, the outstanding shortcoming of RT alone was the lack of overall
survival in stage III/IV cases and an inability to combat distant metastasis as well as metastatic recurrence,
leading to the development of concurrent radiation and chemotherapeutic therapy (CCRT) (14).
Additionally, the persistence of radioresistance in NPC has been demonstrated; therefore, calling for better
treatment options is required to decrease NPC radioresistance and favor radiosensitization (17).
CCRT – Concurrent Chemoradiotherapy & Neoadjuvant Therapy: Owing to the limitations of RT,
multiple clinical trials emerged to assess the efficacy of concurrent, adjuvant chemotherapy (AC) and
neoadjuvant chemotherapy in combination with RT (NCRT). Several clinical trials and meta-analyses of such
trials demonstrated the ambiguity of overall survival for AC in comparison to CCRT/NCRT. Furthermore,
several trials state the increased effectiveness of CCRT over AC (18). These studies helped shape and focus
on CCRT as the primary treatment of NPC.
CDDP – Cis-platin-based CCRT: NPC, in addition to being highly radiosensitive, was also found to be
increasingly sensitive to platinum-based drugs. This led to the current standard treatment of locoregionally
advanced NPC cases. Cis-platin-based CCRT was first demonstrated in the US Intergroup 0099 trial. The trial
demonstrated the effectiveness of relatively high doses of cisplatin in combination with adjuvant cisplatin
and fluorouracil. Results showed a 78% 3-year progression-free survival rate for CCRT compared to the 24%
PFS rate for RT alone (19). However, there were adverse effects; cis-platin-based CCRT showed grade 3 and
4 gastrointestinal reactivity, hematological toxicities, and radiation-induced adversities (20, 21).
Additionally, a reduction in cisplatin doses was required to minimize toxicity responses. Concerning CCDP

9
dosing, studies also demonstrated that changes in dosage showed no differential outcome in toxicity
profiles and overall outcomes (22).
Although there have been significant generations of platinum-based drugs for the treatment of
NPC, it is a well-known fact that platinum-based chemotherapeutic drugs are not well tolerated and result
in several biological toxicities. The non-specificity of cis-platin causes cellular apoptosis to all dividing cells,
giving rise to extensive organ toxicities and thus showing limited dose tolerance for up to two or three
cycles at max (2,23,24). This leads to a new and efficacious molecule to replace Cis-platin-based CCRT, with
minimal toxicities, increased overall and progression-free survival, and decreased reoccurrence rates.
1.2. NEO212
NEO212 is a novel conjugate molecule generated by linking two anti-cancer agents, temozolomide
(TMZ) and POH (Perillyl Alcohol). Recent studies demonstrate NEO212 as a potent chemotherapeutic for
pre-clinical cancer models of glioblastoma (GBM), breast cancer, brain-metastatic breast cancer, and Acute
Myeloid Leukemia (AML) (25, 26, 27, 28).
Figure 1.2. Chemical Structure of TMZ and TMZ-Analog NEO212. TMZ was conjugated with perillyl
alcohol via a carbamate bridge to generate NEO212. NEO212 is a new chemical entity with new

10
physicochemical properties but retains the same alkylating properties of TMZ. This is because NEO212
breaks down to release intact TMZ. Therefore, the same methyl group (in red) in NEO212 as in TMZ will
eventually be donated by NEO212 to DNA nucleobases via the release of the same highly reactive methane
diazonium chemical species (29).
TMZ is a clinically relevant chemotherapeutic, primarily for glioblastoma. It is administered as the
primary treatment for GBM alone or with radiotherapy (30) and acts as a DNA alkylating agent, more
explicitly methylating the O6-guanine base moiety in the DNA. This represents the primary DNA lesions,
mediating its cytotoxic impact (25). The presence of multiple DNA repair mechanisms, such as Base Excision
Repair (BER) and DNA Mismatch Repair (MMR), are interpolated with the efficacy of TMZ. Since TMZ
generates O6-methyl guanine lesions, a prevalent enzyme, O6-methylguanine–DNA methyltransferase
(MGMT), repairs alky adducts in the DNA. MGMT is, therefore, demonstrated to be a large contributor to
drug resistance and is also assumed to play a significant role in cis-platin-based resistance (31). POH is a
naturally occurring monoterpene from lavender, lilac oil, cherries, spearmint, celery seeds, and other
plants. It has shown promising anti-tumor properties in various tumor models. (2, 32, 33)
NEO212 demonstrated increased potency and tumor uptake in NPC, generating increased tumor
cell death compared to TMZ and POH. NEO212 has proven to overcome the chemoprotective nature of the
DNA repair enzyme MGMT (O6-methylguanine methyltransferase), thereby chemo-sensitizing the cells to
secondary rounds of drug treatment. However, it has also been shown that cells without MGMT and
MGMT-inhibited cells showed a more enhanced response to NEO212. Additionally, xenotransplanted NPC
tumors in mice models showed decreased tumor growth with a 10-day treatment of NEO212 (2).
1.3. Radiosensitization of NEO212
RT and CCRT have been the primary treatments for NPC. However, 20% of treated patients show
local and/or nodal reoccurrence after primary treatment (34). Despite the clinical validity of RT and

11
combined CCRT, radioresistance in NPC remains a therapeutic obstacle. Various studies have implemented
radio-sensitizing compounds to overcome radioresistance and increase the potency of radiation-induced
DNA damage while having nominal toxicity and side effects (35,36). Radiosensitization of cancer cells is
achieved by small molecules or any chemical substance that enhances tumor inactivation. Compounds that
have these properties are called radiosensitizers, which increase the effect of radiation on the tumor load
when combined with the expected additive effect of the two treatment modalities (37).
As described by Minea et al., combatting radioresistance and increasing RT potency can be done
by generating a TMZ-analog such as NEO212. NEO212 exhibits more potent radiosensitization effects when
administered concurrently with RT; NEO212 gives rise to an overburdened repair system, allowing for
successful synergism of drug and radiation as observed in glioblastoma models. Concurrent treatment with
clinically relevant doses of NEO212 and radiation achieved increased cytotoxic and synergistic effects that
proved to be dependent on the DNA alkylating property of the drug (29).

12
1.4. Hypothesis
The anatomically unique position of nasopharyngeal carcinomas makes treating them extremely
difficult. The primary tumor forms at the back of the nose, above the throat, making surgical removal
unlikely. Moreover, this prevents primary tumor biopsies and leaves metastatic lymph node biopsies as the
only alternative (2, 38).
For decades, RT has been the standard treatment for primary NPC; however, clinical obstacles were
encountered with locoregional and metastatic disease progression. Conventional chemo-radiotherapy for
NPC is administered to treat advanced stages of the disease using platinum-based drugs; these studies
showed initial success rates; however, cases of patient relapses were prevalent, and 20% of treated
patients showed local and/or
nodal reoccurrence after primary treatment (34). The overall survival post-reoccurrence was as
low as 7-22 months. (2, 39). Additionally, the standard CCRT therapy for NPC with cis-platin was found to
be tolerated for only a few cycles owing to the high toxicities associated with treatment (2,24). The
increasing reoccurrence and distant metastasis of NPC seem to be associated with radioresistance in
patients post-RT (17). Owing to these clinical obstacles faced with the treatment of NPC, specifically the
development of radioresistance, new therapeutic strategies must be sought to induce radio-sensitization
of NPC tumors and combat disease progression.
NEO212, a novel analog of TMZ, has proven to be a potent and highly successful drug for the
treatment of NPC (2) as well as other cancer models such as glioblastoma, AML, and breast cancer
(25,26,28). NEO212 showed increased tumor uptake in mice models and was efficacious at low doses in
TMZ-resistant GBM. NEO212 also showed increasingly practical radiosensitization effects in these GBM cell
lines at clinically relevant concentrations by synergizing with concurrently administered IR, enabling a more
significant cytotoxic impact on tumor cells (26,29).

13
This radio-sensitizing power of NEO212 in GBM cell lines is proposed due to the drug's strong
potency and tumor uptake. Intracellular NEO212 possibly overburdens the DNA repair mechanism that
generally protects the cells from IR-induced DNA lesions. Manipulation of these repair pathways by
treatment with NEO212 results in replicative stress due to methylated DNA lesions during the cell cycle's
S-phase. This leaves the cells vulnerable to further IR-induced DNA breaks, thus allowing alkylating agents
such as NEO212 to radio-sensitize the tumor cells at the S-phase and bring about successful tumor cell
inactivation when treated with concurrent IR (29).
Based on these promising results of NEO212 as a potent anticancer drug in NPC and a strong
radiosensitizer in MGMT-positive and negative GBM cell lines, we hypothesize that NEO212 might yield
similar radio-sensitizing results in MGMT and EBV-positive and negative NPC cell lines. In this study, we aim
to evaluate the cytotoxic impact of concurrently administered NEO212 and IR to determine the radiosensitizing power of NEO212 in NPC cell lines and the mechanisms of action.

14
Chapter 2: Material and Methods
2.1. Pharmacological Agents
NEO212, in its crystalline powder form, was synthesized by Axon (Reston, VA, USA) under good
manufacturing practice (cGMP) conditions and graciously provided by NeOnc Technologies (Los Angeles,
CA, USA). 100 mM DMSO (Santa Cruz Biotechnology, Dallas, TX, USA) was used to dissolve NEO212 for all
in vitro experiments. The stock solution of the dissolved drug was stored at -80 °C. All in vitro experiments
were performed using DMSO concentrations equal to or less than 0.1%.
2.2. Cell Lines
Two different human nasopharyngeal carcinoma cell lines were used. TW1 and C666.1 were
obtained from the American Tissue Culture Collection (ATCC; Manassas, VA, USA). The cell lines were grown
in DMEM (HyClone Laboratories, Logan, UT, USC) supplemented with 10 % fetal bovine serum (FBS), 100
U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were kept in a humidified incubator at 37 °C and a 5 %
CO2 atmosphere.
2.3. MTT Assay
Methylthiazoletetrazolium (MTT) assays were carried out per the following protocol: both cell lines
were seeded in 96-well plates in volumes of 50 µL per well at 1.0-3.0 x 105 cells/ml. To this, an additional
50 µL of the medium was added with varying drug (or DMSO) concentrations (0 µM – 100 µM of NEO212);
the cells were then incubated for 1 hour after which they were irradiated at a range of doses ( 2 Gy – 10
Gy) using external beam radiotherapy in an X-RAD320 irradiator (Precision X-Ray, North Branford, CT). The
cells were then incubated for ~ 5 days (120 hours). For NEO212 to trigger cell death, two cycles of cell
division are necessary; thus, more extended incubation periods are required (40). After incubation, the cells

15
were subjected to 10 µL of thiazolyl blue tetrazolium bromide (methylthiazoletetrazolium, MTT; Sigma–
Aldrich, St. Louis, MO) for 4 hours. The stock solution of MTT is 5 mg/mL in PBS. After 4 hours, the MTT
reaction was stopped, and the cells were lysed with 100 µL of solubilization solution (10% sodium dodecyl
sulfate, SDS, in 0.01 N hydrochloric acid, HCl). To allow complete solubilization of the MTT crystals, the
plates were incubated in an incubator at 37 °C and a 5 % CO2 atmosphere overnight. The optical density
(OD) for each well was determined the following day using the Varioskan Lux Reader by Thermo Scientific
(Waltham, MA) at an absorbance of 570 nm. The background value (OD of control well-containing medium
without cells + MTT + solubilization solution) was subtracted from all measured values. Each treatment
condition was set up in duplicates or triplicates for individual experiments, and each experiment was
repeated several times independently.
2.4. Colony Formation Assay / Clonogenic Assay (CFA)
To determine the effect of clinically relevant concentrations of NEO212, the Clonogenic assay was
performed with either drug only, irradiation only, or a combination of both to test the long-term survival
of nasopharyngeal carcinoma. Depending on the respective cell line, 250 – 1000 cells were seeded in each
well. As per the experimental setup, 48, 24, and 12-well plates were used. Once fully attached to the
surface, the cells were treated after 24h with varying drug concentrations (10 µM – 100 µM or DMSO)
either on their own or concurrently with a range of doses or irradiation (2 Gy – 10 Gy). The drug was added
before for all experiments and treatment conditions, and cells were incubated for an hour before being
irradiated. External beam radiotherapy was administered in an X-RAD320 irradiator (Precision X-Ray, North
Branford, CT). Post-treatment, the cells were incubated and allowed to grow for 2-3 weeks, depending on
the formation of observed colonies. The colonies were stained with 1% methylene blue (in methanol). The
area percentage of the colonies was then determined via the ImageJ software (National Institutes of Health,
Bethesda, MD, USA) (41) using the colony area plugins (42).

16
2.5. Immunoblots
Cells were treated and harvested by centrifugation. Total cell lysates were prepared by lysing the
cell pellets with radio-immunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) supplemented
with 1 mm PMSF (phenylmethylsulfonyl fluoride, Sigma Aldrich) and Halt Protease & Phosphatase inhibitor
single use cocktail (1 micro tube = 100 µL; Thermo Fisher Scientific). Protein concentrations were
determined using the Pierce BCA protein assay reagent (Thermo Scientific), and 25-30 µg of total cell lysate
from each sample was added to each lane. The samples were separated by denaturing polyacrylamide gel
electrophoresis (PAGE) using the 12% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA). Trans-blot (BioRad, Hercules, CA) was used for the semi-dry transfer to Immuno-Blot Polyvinylidene Fluoride (PVDF)
membrane (Bio-Rad, Hercules, CA, USA) that was activated in methanol for 30 seconds. The following
antibodies were used: to detect γ-H2AX, a monoclonal antibody (sc-517348) from Santa Cruz Biotechnology
Inc. (Dallas, TX) was used. A monoclonal antibody (ab39253) from Abcam (Cambridge, UK) was used for
MGMT detection. Horseradish peroxidase-antibody conjugates (i.e., secondary antibodies) were obtained
from Cell Signaling Technology (Danvers, MA). All antibodies were used according to suppliers’
recommendations. For detection, SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher
Scientific, Waltham, MA, USA) was used, and all Immunoblots were repeated to confirm the results.
2.6. DNA Damage Analysis by FACS Analysis
For persistent DNA damage quantification by γH2AX staining, NPC cells were seeded in 10 cm plates
at 2 × 105
cells/mL, after which they were treated with either chemotherapy alone (NEO212) or IR alone (4
Gy) or concurrent chemotherapy and IR. Clinically relevant drug concentrations of the drug were used (1
µM – 20 µM). The cells were incubated for 72-120 hours (3-5 days), and the media was replenished if
required. Post incubation, the cells were washed with PBS, harvested, and transferred into centrifuge
tubes. TONBO Ghost Dye Violet 450 viability dye was added to the harvested cells and incubated for ~10

17
minutes. The cell pellets were then fixed in 4% paraformaldehyde in PBS, permeabilized in 0.05% Triton X100 in OBS, and incubated γH2AX antibody, clone JBW201 (EMD Millipore, Darmstadt, Germany) for an
additional 30 minutes. The cells were further analyzed using violet and blue lasers with a BD FACSAria II
instrument (BD Biosciences, Franklin Lakes, NJ). Cells incubated with an isotype control antibody (i.e.,
negative control) were used for setting cytometer voltages.
2.7. Statistical Analysis
All parametric data were analyzed using PRISM Software (GraphPad Software, San Diego, CA, USA).
Student t-tests were applied to calculate the significance values. The cell viability was assessed by analysis
of variance (ANOVA) followed by Tukey post-hoc multiple comparison tests of treatment groups relative to
control. A probability value (p) <0.05 was considered statistically significant.

18
Chapter 3: Results
3.1. NEO212 & Ionizing Radiation inhibit growth in TW1 & C666.1 NPC cell lines
MTT Assays were performed to determine the cytotoxic effects of NEO212 in TW1 and C666.1 cell
lines. The MTT assay is a colorimetric assay that measures the metabolic rate of the cells by analyzing the
breakdown of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow tetrazolium
salt to purple formazan crystals in metabolically active cells. Thus, viable cells induced the reduction of this
compound to form purple formazan, which is then solubilized to a colored solution and analyzed
calorimetrically. The higher the OD measurement, the more cell viability is (43).
3.1.1 Effect of IR on cell viability in TW1 and C666.1 Cell lines
First, we tested the response of NPC TW1 and C666.1 to ionizing radiation (IR) at a range of doses.
Control
2 Gy
4 Gy
6 Gy
8 Gy
0
20
40
60
80
100
120
Dose (Gy)
% Viability
ns
✱
✱✱
✱✱
0 2 4 6 8 10
0
20
40
60
80
100
120
TW1 ID50 - 2.4 Gy
IR Dose (Gy)
% Viability
A. B.

19
Figure 3.1 Effect of IR on TW1 NPC cell line. (A) MTT dose-dependent effect of increasing irradiation
doses. The cells were treated once and incubated for a period of 5 days. Percentage survival was determined
using untreated control cells. ns p-value > 0.05, *p-value <0.05, **p-value <0.0001 (B) Dose-dependent
survival of TW1 cell line with increasing IR doses. All values were normalized to the untreated control at the
same time point. The dotted line indicates the ID50 (dose of IR that reduces cell proliferation by 50%). For
TW1, the ID50 was 2.4 Gy.
The effect of single IR treatments on TW1 cells was significantly relevant at a dose of 4 Gy and
above (Fig.3.1). The calculated p-values show statistically significant values at 4, 6, and 8 Gy doses. In
addition, the ID50 (dose of IR that reduces cell proliferation by 50%) for TW1 cells was 2.4 Gy with a profile
likelihood range between 1.6 Gy and 3.4 Gy when CI - 95% (Fig 3.1). The effect of IR on TW1 was observed
to be dose-dependent and highly sensitive. This effect was consistently observed in repeated MTTs
performed to confirm these results.
With these results, we wanted to test the effect of IR on a different NPC cell line. We treated NPC
C666.1 cells with increasing doses of IR. The difference between these two cell lines is the EBV-positive
feature of C666.1 cells. EBV infection in NPC is reported to cause increasing radio-resistance in positive cell
lines. We wanted to test the difference in radio sensitivity between the two cell lines.

20
Figure 3.2 Effect of IR on EBV-positive C666.1 NPC cell line. (A) MTT dose-dependent effect of
increasing irradiation doses. The cells were treated once and incubated for 5 days. Percentage survival was
determined using untreated control cells. ****p-value <0.0001 (B) Dose-dependent survival of C666.1 cell
line with increasing IR doses. All values were normalized to vehicle control at the same time point. The dotted
line indicates the ID50 (dose of IR that reduces cell proliferation by 50%). For C666.1, the ID50 was 3.7 Gy.
The effect of IR on C666.1 was observed to be statistically significant at doses of 4 Gy and above
when compared with the untreated control cells. Increasing doses of IR reported a substantial decrease in
cell survival. C666.1 cells achieved their ID50 at around 3.7 Gy with a profile likelihood range between 3.117
and 4.432 Gy when CI – 95%, which was observed to be slightly higher than the TW1 cell lines. (Fig 3.2)
Control
4 GY
6 Gy
8 Gy
10 Gy
0
20
40
60
80
100
120
140
IR Dose (Gy)
% Viability
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
0 2 4 6 8 10
0
20
40
60
80
100
120
IR Dose (Gy)
% Viability
C666.1 ID50 - 3.7 Gy
A.
B.

21
Figure 3.3 Comparative effect of IR on EBV-negative and positive TW1 and C666.1 NPC cell lines.
The cells were treated once and incubated for 5 days. Percentage survival was determined using untreated
control cells. All values were normalized to vehicle control at the same time point. The dotted line indicates
the ID50 (dose of IR that reduces cell proliferation by 50%). The ID50 for TW1 was 2.4 Gy, while the ID50 for
C666.1 was 3.7 Gy.
We observed that by implementing the same dose-dependent IR treatments on both the cell lines,
the EBV-positive C666.1 cell line was observed to have a similar pattern of cell survival that was dosedependent with increasing doses of IR. The C666.1 are observed to achieve their Id50 at around 4 Gy, as
compared to 2 Gy in TW1 cell lines (Fig 3.3). Moreover, TW1 cell lines showed a significant decrease in cell
survival, with the highest-dose margin of IR being 8 Gy, and C666.1 cell lines, although showing decreased
cell survival, required a higher dose margin of IR of 10 Gy. Thus, C666.1 might be slightly more resistant to
radiotherapy, requiring twice as much IR to reach its ID50 than EBV-negative TW1 cells.
0 2 4 6 8 10
0
20
40
60
80
100
120
IR Dose (Gy)
% Viability
C666.1 TW1

22
3.1.2 Effect of NEO212 on cell viability in TW1 and C666.1 cell lines
In previously demonstrated studies, NEO212 was observed to suppress cell proliferation in the TW1
cell lines at an IC50 of around 32 µM (2). Considering these results, we now tested the response of EBVnegative and positive NPC TW1 and C666.1 cell lines to NEO212 at increasing concentrations from 0 to 100
µM via CFA (Colony formation Assay) analysis.
Figure 3.4 Effect of NEO212 TW1 NPC cell lines. (A.) Effect of NEO212 on NPC TW1 cell lines' cell
survival with increasing drug treatment concentrations. Ns p-value >0.05, */** p-value <0.05, ***pvalue<0.001. (B.) Dose-dependent survival curve of TW1 cell line with increasing concentrations of NEO212.
A colony formation Assay was performed. Cells were treated once and incubated for 2-3 weeks. All values
were normalized to untreated control at the same time point. The IC50 (concentration of drug that reduces
cell proliferation by 50%) for these cells was observed to be at 31 µM.
0 20 30 40 60 70 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
ns
✱ ✱✱
✱✱
✱✱✱
✱✱✱
✱✱✱
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
TW1 - IC50 - 31 uM
A.
B.

23
NEO212 inhibited the proliferation of TW1 cells at low concentrations. The p-values calculated
were statistically significant at concentrations of 30 µM and above. (Fig.3.4. A) The IC50 (concentration of
drug that reduces cell proliferation by 50%) for these cells was observed to be at 31 µM with a profile
likelihood range between 21 µM and 45 µM when CI – 95%. (Fig.3.4. B) The IC50 values were found to be
directly proportionate to the cell density in repeated experiments. The effect of NEO212 on TW1 was
observed to be drug-dependent and highly sensitive. The effect was consistent with repeated MTT and
CFAs, which confirmed these results.
After obtaining promising results for the effect of NEO212 on TW1 cell lines, we wanted to test its
impact on the EBV-positive NPC cell line. Despite C666.1's EBV-positive feature, there has been no evidence
of its sensitivity to NEO212. We treated C666.1 cells with similar doses of NEO212 in an increasing
concentration. The effect on cellular survival was observed by the cells' ability to form colonies posttreatment.
Control
20
30
40
60
70
80
10
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
✱✱✱
✱✱✱✱
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
C666.1 IC50 - 8 uM
A. B.

24
Figure 3.5 Effect of NEO212 on EBV-positive C666.1 NPC cell lines. (A) Effect of NEO212 on cell
survival of C666.1 cell lines with increasing concentrations of the drug. ns p-value >0.05, *** p-value 0.001,
****p-value<0.001. (B.) Dose-dependent survival of C666.1 cell line with increasing concentrations of
NEO212. A colony formation Assay was performed. Cells were treated once and incubated for 2-3 weeks.
All values were normalized to the untreated control at the same time point. The IC50 (concentration of drug
that reduces cell proliferation by 50%) for C666.1, the IC50 was 8 µM.
As a result, we observed similar outcomes in C666.1 cell lines. However, increased doses of NEO212
were observed to cause a more significant reduction in cell survival for C666.1 cells. Concentrations as low
as 20 µM showed statistically significant effects, and further dose increases produced more significant pvalues for cellular survival. The sensitivity NEO212 on the cells was striking, with almost no cells surviving
at doses above 40 µM. (FIG.3.5. A) Similar to the TW1 cell line, the response was dose-dependent. In
addition, the IC50 for C666.1 was observed to be 8 µM with a profile likelihood between 4 µM and 13 µM
of NEO212 when CI – 95%.
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
TW1 C666.1

25
Figure 3.6 Comparative effect of NEO212 on EBV-negative and positive TW1 and C666.1 NPC cell
lines. C666.1 cell line exhibited a lower IC50 when compared to TW1, thus further decreasing cell survival at
lower concentrations. A colony formation Assay was performed. Cells were treated once and incubated for
2-3 weeks. All values were normalized to the control at the same time point. The IC50 for TW1 was 31 µM
while the IC50 for C666.1 was 8 µM.
Thus, the effect of NEO212 on EBV-positive and negative cell lines TW1 and C666.1 was observed
to be statistically significant. Despite both cells being part NPC cells, the C666.1 was observed to be more
sensitive to NEO212 with an IC50 of 8 µM with a profile likelihood range between 4 to 13 µM, while the
TW1 cell line exhibited its IC50 at around 31 µM. (Fig 3.6) This observation of a much lower IC50 for C666.1
is substantiated by the presence of EBV in this cell line. Previously demonstrated results show that NEO212
induces the lytic cycle activation in the EBV-positive C666.1 cell line (44). Thus, the C666.1 cell line is further
sensitized to drug treatment compared to EBV-negative TW1 cell lines owing to the activation of the EBV
lytic cycle.
3.2. Combination therapy decreases cell survival in NPC TW1 and C666.1 cells
Both IR and NEO212 so far have been demonstrated to exert a significant effect on cell survival in
NPC TW1 and C666.1 cell lines. After establishing the robust effect of IR and NEO212 on NPC TW1 and
C666.1 cell viability and proliferation as independent entities, we decided to study the combined effect of
the two therapies synergistically.
To prove this hypothesis, we wanted to establish the difference in cell survival based on the
independent treatment outcomes, the theoretical additive effect of IR and NEO212, and the observed
combination effect of (IR + NEO212). To establish these results, we conducted a CFA analysis, wherein each
plate served as a specific treatment modality, including concurrent (IR + NEO212). The conditions for these

26
setups were based on the previously recorded effect of the individual treatment with the respective cell
lines.
For the TW1 cell line, we used increasing concentrations of NEO212 with two treatment groups of
constant IR (4 Gy and 6 Gy) to determine the point of synergism between the two treatment modalities.
Figure 3.7 Combination effect of 4 Gy IR and NEO212 on TW1 NPC cell lines. A dose-dependent
decrease in cell viability of TW1. A colony formation Assay was performed. Cells were treated once and
incubated for 2-3 weeks. All values were normalized to the control at the same time point, and the p-value
was statistically significant. (p <0.05).
This experiment showed that the combined effect of NEO212 and IR (4 Gy) further decreased
cellular viability. Based on the dose-dependent treatment of the TW1 cell line, the IC50 for the combination
treatment of NEO212 with IR of 4 Gy decreased from 31 µM to 30 µM. This decrease was insignificant and
could be due to the lack of synergism between the two modalities at the given concentrations.
From this, we carried out the same treatment conditions, this time using a higher dose of IR to
observe the effect of the combined therapy on the TW1 NPC cell line.
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
4 Gy
NEO212
NEO212 + 4 Gy

27
Figure 3.8 Combination effect of 6 Gy IR and NEO212 on TW1 NPC cell lines. A dose-dependent
decrease in cell viability of TW1. Cells were treated once and incubated for 2-3 weeks. All values were
normalized to the control at the same time point, and the p-value was statistically significant. (p <0.05)
As observed in (Fig 3.8), increasing the dose of IR with concurrent NEO212 treatment significantly
reduced cell survival. In this case, the IC50 of NEO212 with the combination treatment was reduced from
31 µM to 12 µM. This significant result was attributed to the increased cell death for combination therapy.
We further compared the effect of single and combined treatments on the TW1 cell line and
observed a statistically significant decrease in cell survival with both combined treatments.
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
6 Gy
NEO212
NEO212 + 6 Gy

28
Figure 3.9 Comparative effect of single and concurrent NPC TW1 cell line treatment with IR and
NEO212. A colony formation Assay was performed. Cells were treated once and incubated for 2-3 weeks.
All values were normalized to the control at the same time point. ** p-value <0.05, *** p-value<0.001.
The comparative graph denotes that NEO212 + 6 Gy had a more significant effect on cell survival
than NEO212 + 4 Gy (Fig 3.9). Nevertheless, both conditions showed a feasible decrease in cell survival with
concurrent treatment compared to singular treatment strategies. This further substantiates that
combination therapy of IR and NEO212 is more beneficial for cell death in the NPC TW1 cell line.
Observing these promising results with the TW1 cell line, we wanted to implement the same
treatment strategy in the EBV-positive C666.1 cell line. To observe the effect of a combination of NEO212
and IR on the C666.1 cell line, we carried out a similar CFA analysis with the same range of NEO212
4 Gy
6 Gy
NEO212
NEO212 + 4 Gy
NEO212 + 6 Gy
0
20
40
60
80
100
% Survival
6 Gy
NEO212
4 Gy
NEO212 + 4 Gy
NEO212 + 6 Gy
✱✱✱
✱✱

29
concentrations. From our previous results, we observed C666.1 to be slightly more radio-resistant than
TW1 and hence used a higher dosage of constant IR (6 Gy and 8 Gy).
Figure 3.10 Combination effect of 6 Gy IR and NEO212 on C666.1 NPC cell lines. A dose-dependent
decrease in cell viability of TW1. A colony formation Assay was performed. Cells were treated once and
incubated for 2-3 weeks. All values were normalized to the control at the same time point, and the p-value
was statistically significant. (p <0.05)
As observed (Fig 3.10), the concurrent treatment of C666.1 with NEO212 and 6 Gy showed an
increasingly effective decrease in cell survival. Concurrently treated cells were observed to have been
inhibited entirely, with almost no surviving cells—moreover, the IC50 for the cells reduced from 8 µM with
only NEO212 to nearly 0 µM.
We also wanted to test the similar effect of concurrent treatments when using a higher dose of IR
(8 Gy) with NEO212 in C666.1 cells owing to their radio-resistant property.
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
6 Gy
NEO212
NEO212 + 6 Gy

30
Figure 3.11 Combination effect of 8 Gy IR and NEO212 on C666.1 NPC cell lines. A dose-dependent
decrease in cell viability of C666.1 cells. A colony formation Assay was performed. Cells were treated once
and incubated for 2-3 weeks. All values were normalized to the control at the same time point, and the pvalue was statistically significant. (p <0.05)
We observed that increasing doses of NE0212 and a constant IR dose of 8 Gy exhibited robust cell
death in the C666.1 NPC cell line. (Fig 3.11) Once again, the IC50 was reduced from 8 µM to 0 µM with
almost no cell surviving with the concurrent treatment as compared to NEO212 alone.
0 20 40 60 80 100
0
20
40
60
80
100
120
NEO212 (uM)
% Survival
8 Gy
NEO212
NEO212 + 8 Gy
6 Gy
8 Gy
NEO212
NEO212 + 6 Gy
NEO212 + 8 Gy
0
20
40
60
80
% Survival
6 Gy
8 Gy
NEO212
NEO212 + 6 Gy
NEO212 + 8 Gy
✱

31
Figure 3.12 Comparative effect of single and concurrent NPC C666.1 cell line treatment with IR and
NEO212. Cells were treated once and incubated for 2-3 weeks. All values were normalized to the control at
the same time point. * p-value <0.05
In a comparative study, the IR dose significantly affects cell survival rates. The highest dose of IR
produced the most statistically significant effect on cell survival (Fig 3.12). The impact of concurrent
NEO212 + IR on the C666.1 was observed to decrease cell survival robustly, and the IC50s of the treatment
conditions were lowered from 8 µM to almost 0 µM. However, we suspect this extreme effect can be
attributed mainly to the high doses of radiation.
With this data, we went on to calculate the fold radiosensitization values for both NEO212 using
the same formula for all datasets (i.e., the ratio between the total cytotoxic effect observed with
combinatorial treatments divided by the calculated additive effects of treatments as monotherapies)

32
Figure 3.13 NEO212 as a robust radiosensitizer of MGMT-negative TW1 NPC cell line. A colony
formation Assay was performed. Cells were treated once and incubated for 2-3 weeks. Fold sensitization
values were determined from CFA datasets. Values >1 indicate synergistic effects (i.e., radiosensitization).
Values equal to 1 indicate additive effects, and values <1 indicate antagonistic effects.
The fold sensitization values of NEO212 in the TW1 cell line showed increasingly synergistic effects
at lower concentrations of NEO212. However, it is noteworthy to observe that there is a decrease in fold
sensitization (i.e., values <1) with increased drug dose (Fig 3.13). Thus, we can suggest that NEO212
combines with IR to synergize at low concentrations and provides a better cytotoxic effect.
These promising results led us to test the fold sensitization for the EBV and MGMT-positive cell line
C666.1. O6-methylguanine methyltransferase (MGMT) is known to provide drug resistance owing to its
robust DNA repair mechanism. This is especially true for TMZ-associated analogs like NEO212. This feature
of C666.1 led us to explore the effect of NEO212 as a radiosensitizer in the MGMT-positive cell line.
0 20 40 60 80 100
0.0
0.5
1.0
1.5
2.0
C666.1
NEO212 (uM)
Fold sensitization
NEO212 + 6 Gy NEO212 + 8 Gy
Synergistic Effects Antagonistic Effects

33
Figure 3.14 NEO212 as a radiosensitizer of MGMT-positive C666.1 NPC cell line. A colony formation
Assay was performed. Cells were treated once and incubated for 2-3 weeks. Fold sensitization values were
determined from CFA datasets. Values >1 indicate synergistic effects (i.e., radiosensitization). Values equal
to 1 indicate additive effects, and values <1 indicate antagonistic effects.
As we anticipated, the synergistic effects of NEO212 did not prevail in the C666.1 cell line(Fig 3.14).
This reaffirms our initial hypothesis that the drug synergizes with IR when the dose is much lower in both
treatment conditions, as observed at the 20 µM+ 6 Gy IR data point in Fig 3.14. Moreover, an increase in
dosage resulted in an increased antagonistic effect of the two treatments. This key finding underscores the
importance of maintaining the right drug and radiation dose balance for optimal treatment outcomes.
NEO212 + 6 Gy
NEO212 + 8 Gy
NEO212 + 4 Gy
NEO212 + 6 Gy
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Fold sensitization
TW1 C666.1
Synergistic Effects Antagonistic Effects

34
Figure 3.15 NEO212 as a radiosensitizer of TW1 and C666.1 NPC cell line. Cells were treated once
and incubated for 2-3 weeks. Fold sensitization values were determined from CFA datasets. Values >1
indicate synergistic effects (i.e., radiosensitization). Values equal to 1 indicate additive effects, and values
<1 indicate antagonistic effects.
Comparative analysis of concurrent NEO212 and IR therapy for NPC TW1 and C666.1 cell lines
proved highly effective in decreasing cell viability. However, when analyzing the synergistic effect of the
two treatment modalities for the respective cell lines, we observed that higher doses of IR and NEO212
prevented the synergism between the two treatments. In contrast, it leads to an antagonistic effect. (Fig
3.15) This could be attributed to synergism in drug therapy, wherein lower doses are required to observe
the synergistic effect between the two treatments. Higher doses can cause one treatment to overpower
the other, hence preventing the combinatory synergistic effects.
3.3. NEO212 radio-sensitizes NPC TW1 and C666.1 cell lines at clinically relevant
concentrations
Based on our current results, we are optimistic about the potential of our findings. We decided to
implement similar concurrent conditions. However, these experiments were set up using lower doses of
NEO212 to observe the desired synergistic effect of the two treatments. As observed previously, NEO212
can synergize with IR at lower drug doses. This suggests a potential strategy for enhancing the effectiveness
of the cancer treatment. Thus, we implemented the same mode of treatment but using doses between 1
µM and 30 µM only and a constant IR dose of 4 Gy. These findings could pave the way for more targeted
and effective cancer therapies in the future.
A
.

35
Figure 3.16 NEO212 as a robust radiosensitizer of TW1 NPC cell line. Panel A. Colony Formation
results of TW1 NPC with NEO212, IR, and NEO212 + IR. Panel B. Colony survival comparison of the
treatments. Cells were treated once and incubated for 2-3 weeks. All values were normalized to the control
at the same time point. The colony survival data show that NEO212 can synergize with ionizing radiation in
the clinically relevant concentration range (i.e., one μM – 30 μM)
As anticipated, lower concentrations of NEO212 demonstrated a powerful synergistic effect when
combined with an ionizing radiation dose of 4 Gy in the TW1 NPC cell line. The percent of cell survival data
clearly shows that the combination effect of NEO212 + IR was more efficient in reducing cell survival than
the additive effect of the individual treatments. This underscores the superior effectiveness of the
0 10 20 30
0
20
40
60
80
100
NEO212 (uM)
% Survival
Only IR
Only NEO212
Additive Effect
Combination Effect
B
.
A.
B.

36
combinatory effect of NEO212 + IR, which outperformed the sum of their effects when administered
individually.
Notably, the IC50 value for NEO212 alone was approximately 30 µM. In the case of an additive
effect, the IC50 dropped to 16 µM. However, the IC50 was further reduced to an impressive 11 µM with
combination therapy. This significant reduction in the IC50 value underscores the potential of the
combination of NEO212 + IR to facilitate more efficient and potent cell death in TW1 NPC cells, owing to
NEO212’s ability to radio-sensitize the cells and generate a synergism between the drug and IR.
We observed that despite the effective reduction in cell survival, the total reduction was still only
around 60%. This can be due to the low IR dose. An increased IR dose combined with NEO212 can increase
effective killing from 60% to less than 10%. Thus showing better results for combination therapy.
Following these results, we also wanted to observe if the decrease in NEO212 concentration
affected the synergism in C666.1 cells. Thus, we implemented the same treatment protocol; however,
considering the known sensitivity of C666.1 to NEO212, we used a concentration range (i.e., one μM—30
μM).
Figure 3.17 NEO212 as a radiosensitizer of C666.1 NPC cell line. Colony Formation results of C666.1
NPC with NEO212, IR, and NEO212 + IR. Cells were treated once and incubated for 2-3 weeks.

37
However, in line with our previous results, NEO212 could not synergize with IR in the C666.1, even
at low doses. Moreover, the results from the CFA were inconclusive since there was almost 100% cell
survival and a negligible decrease in cell growth between the three treatment modalities, implying no
statistical significance was observed. (Fig 3.17)
According to previous research, we assume that NEO212's inability to synergize with IR in the
C666.1 cells is due to the cell line's MGMT-positive feature of this cell line. The synergism between the two
treatments is known to overwhelm the BER repair system, thus producing efficient DNA damage effects on
the tumor cell. However, in the case of MGMT-positive cell lines, the ability of this DNA repair protein to
reverse O6-methylguanine lesions gives rise to treatment resistance.
Fig. 3.18 Determination of the levels of MGMT in the two NPC cell lines cells. C666.1 cells are MGMT
positive, while TW1 NPC cell lines are MGMT negative. Cell lysates were prepared and subjected to Western
blot analysis with an antibody for MGMT. Cells were treated once and harvested after 24 hours.
We showed that TW1 cells are MGMT-negative and C666.1 cells are MGMT-positive; this feature
of C666.1 cells might be the inherent obstacle to radiosensitization with NEO212 in the C666.1 cell line. It
is also noteworthy to explain that the drug has no observed effect at the expression level; further incubation
of treated cells for 1-2 days would be required to determine the effect of NEO212 on MGMT expression.

38
3.4. NEO212 + IR increases cell death via DNA damage
With these insightful results, we wanted to study the effect of NEO212 and IR at the molecular
level. For this purpose, we carried out the same treatment conditions for NPC TW1 and C666.1 cell lines
and isolated whole cell lysates. These were then used to analyze the DNA damage by Western blot analysis
using anti-Ƴ-H2AX as the primary antibody.
Fig. 3.19 Determination of the levels of Ƴ-H2AX TW1 NPC cells with treatment of IR, NEO212, and
NEO212 + IR. Ƴ-H2AX expression was observed at low concentrations (10 µM) in both NEO212 and NEO212
+ IR samples, indicating DNA damage. Cell lysates were prepared and subjected to Western blot analysis
with an antibody for Ƴ-H2AX. Cells were treated once and harvested after 24 hours. Actin was used as the
loading control.
We observed that NEO212 increased the levels of DNA damage marker phosphorylated-H2AX
protein (H2AX) at concentrations as low as 10 μM (Fig 3.18). This indicates that NEO212 can decrease cell
survival due to potent DNA damage. It is unclear why there was no DNA damage in the case of 20 μM and
30 μM alone; potential loss of dead cells could be attributed to the lack of expression; further
experimentation is required to understand the mechanism of actions. However, we can observe expression

39
of ƳH2AX at 10 μM + IR alone with a faint expression of ƳH2AX at 20 μM + IR and 30 μM + IR compared to
their standalone treatments. This could indicate that combination treatment could generate more DNA
damage at low concentrations.
Fig. 3.20 Determination of the levels of Ƴ-H2AX C666.1 NPC cells with treatment of IR, NEO212, and
NEO212 + IR. Ƴ-H2AX expression was observed at low concentrations (10 µM) in both NEO212 and NEO212
+ IR samples, indicating DNA damage. Expression was more in NEO212 alone than in NEO212 + IR. Cell
lysates were prepared and subjected to Western blot analysis with an antibody for Ƴ-H2AX. Cells were
treated once and harvested after 24 hours. Actin was used as the loading control.
We observed that C666.1 cells showed increased levels with NEO212 alone rather than NEO212 +
IR. (Fig 3.20) This directly translates to our previous findings that show that the C666.1 cell line is more
sensitive to NEO212 alone, thus showing more DNA damage as a standalone treatment. Moreover, with
these results, we could not characterize the synergistic effect of the two therapies in the C666.1, further
demonstrating the radiosensitization effect NEO212 on the C666.1 cell lines to be inconclusive.

40
To further test the level of DNA damage and correlate it to cell death, we subjected the two cell
lines to a FACS analysis. The treatment conditions were the same as in the Western Blot, with low doses of
NEO212 (1 µM to 30 µM) and a constant IR of 4 Gy.
Figure 3.21 Effect of NEO212 + IR on cell death and DNA damage in TW1 NPC cells. Panel A. FACS
cell death data in TW1 NPC with NEO212, IR, and NEO212 + IR. Panel B FACS data DNA damage in TW1 NPC
with NEO212, IR, and NEO212 + IR. Cells were treated once and incubated for 5 days. All values were
normalized to the control at the same time point. The data show that NEO212 can synergize with ionizing
Untreated
10 μΜ
20 μΜ
30 μM
RT
10 μΜ + IR
20 μΜ + IR
30 μΜ + IR
0
2
4
6
8
10
% Cell Death
Untreated 10 μΜ 20 μΜ 30 μM
RT 10 μΜ + IR 20 μΜ + IR 30 μΜ + IR
✱
✱
✱✱
Untreated
10 μΜ
20 μΜ
30 μM
RT
10 μΜ + IR
20 μΜ + IR
30 μΜ + IR
0
2
4
6
8
10
% YH2AX RT
Untreated 10 μΜ 20 μΜ 30 μM
10 μΜ + IR 20 μΜ + IR 30 μΜ + IR
✱ ✱
A
.
B
.
A.
B.

41
radiation and produce more cell death by DNA damage at 30 µM and 4 Gy IR concentrations. *p-value
<0.05, **p-value <0.001
Concurrent with our Western blot data, NEO212 synergized with IR at low concentrations,
providing more significant cell death with the simultaneous treatment than standalone NEO212-treated
cells. The 20 µM + 4 Gy treatment group exhibited the most significant increase in cell death. (Fig 3.20)
Thus, it can be said that the cell death in the TW1 NPC cells caused by NEO212 is related to DNA
damage; we observed a significant increase in DNA damage marker ƳH2AX in treatment groups 10 µM and
20 µM + 4 Gy IR. Thus, we can say that NEO212 synergizes with IR at low concentrations to cause increased
DNA damage that ultimately results in cell death.
In addition, we wanted to test the same for C666.1 cell lines. We subjected these cells to the same
treatment conditions as the western blot sample groups, with lower NEO212 concentrations and a constant
IR dose of 4 Gy.

42
Figure 3.22 Effect of NEO212 on cell death and DNA damage in C666.1 NPC cells. Panel A. FACS cell
death data in C666.1 NPC with NEO212, IR, and NEO212 + IR. Panel B FACS data DNA damage in C666.1
NPC with NEO212, IR, and NEO212 + IR. Cells were treated once and incubated for 3 days. All values were
normalized to the control at the same time point. The data shows no significant difference between
treatment conditions. ns p-value >0.05.
In line with our western blot results, we observed no significant increase in cell death with the
concurrent treatment of NEO212 + IR. Moreover, we observed approximately the same percentage of cell
Untreated
1 μΜ
5 μΜ
10 μM
RT
1 μΜ + IR
5 μΜ + IR
10 μΜ + IR
0
10
20
30
40
% Cell Death
Untreated 1 μΜ 5 μΜ 10 μM
RT 1 μΜ + IR 5 μΜ + IR 10 μΜ + IR
ns
Untreated
1 μΜ
5 μΜ
10 μM
RT
1 μΜ + IR
5 μΜ + IR
10 μΜ + IR
0
2
4
6
8
10
% YH2AX
Untreated 1 μΜ 5 μΜ 10 μM
RT 1 μΜ + IR 5 μΜ + IR 10 μΜ + IR
ns
B
.
A
.
B.
A.

43
death in the standalone NEO212 treatmentsets compared to the concurrently treated groups. Additionally,
the rate of DNA damage was much lower in these cell lines, and like cell death, there was no significant
increase with NEO212 + IR. Moreover, the DNA damage markers at low concentrations of NEO212 alone
are more than the concurrently treated group, as observed with 5 µM. (Fig 3.22)
From these data, C666.1 reacts differently to concurrent NEO212 + IR treatment and is less
sensitive than the TW1 cell line. Moreover, which concentrations would work best to observe this synergist
effect of NEO212 + IR in C666.1 cells is still being determined. Hence, our results are inconclusive
concerning the radiosensitization of C666.1 to IR by NEO212.

44
Chapter 4: Discussion
At present, nasopharyngeal carcinoma is known to be predominantly hard to treat owing to the
unique positioning of the tumor at the back of the throat. Due to this geographical feature of the cancer,
surgical procedures for treatment and prognosis are almost impossible. Despite the availability of different
treatment options, none have been able to combat patients' long-term survival, metastatic reoccurrence,
and overall disease prognosis. The use of combination therapies has been made famous but fails to
overcome advanced stages of the disease, resulting in off-target toxicities and decreased progression-free
survival rates. Additionally, nasopharyngeal carcinoma inherently has two subtypes, EBV-positive and EBVnegative tumors. The presence of the Epstein-Barr virus (EBV) in specific cell lines poses a subsequent
obstacle owing to its influence on tumor malignancy and cancer progression.
In this study, we explore the effect of NEO212 as a robust radio-sensitizer for both EBVnegative and positive cell lines, namely TW1 and C666.1. The objective is to determine clinical relevance
that can achieve a synergistic effect between NEO212 and ionizing radiation (IR). Our conceptualization of
the study originated from the development of radioresistance in certain NPC cell lines, which is suspected
to be the reason behind distant metastasis and disease recurrence post-treatment (17). The reason behind
resistance development is still unclear, but studies have shown that several molecular and cellular
aberrations could play a role. This also includes the prevalence of EBV infection in certain cell lines (3).
Multiple studies have demonstrated the effectiveness of combining IR with other small-molecule drugs,
allowing the synergism between the two modalities to generate radio-sensitized cells (29, 35 – 37).
NEO212 has proven to be a successful small-molecule drug for the treatment of various
cancer models, including nasopharyngeal carcinoma and glioblastoma (2, 25 – 29, 40). NEO212 has also
been established as a clinically relevant radiosensitizer in TMZ-resistant gliomas. Moreover, NEO212 has
shown increased cellular uptake compared to other therapeutic molecules like TMZ. In addition, NEO212

45
is also seen to optimally circumvent the MGMT-resistance and DNA repair mechanisms observed in TMZresistant gliomas (29). With these promising results from previous studies, we considered NEO212, a TMZPOH conjugate drug, a viable therapeutic strategy for radio-sensitizing NPC cells by observing synergistic
effects with IR.
First, the results of this study demonstrate that ionizing radiation alone can significantly
decrease cell viability. IR works in a dose-dependent manner for TW1 and C666.1 cell lines. We showed
that the optimal dose of IR for the TW1 cell line was 2.4 Gy with a range between 1 Gy and 2 Gy, while the
optimum dose to reach 50% cell death for the EBV-positive C666.1 cell line was 3.7 Gy, nearly twice as
much, ranging between 2 Gy and 4 Gy (Fig 3.1, Fig 3.2, and Fig 3.3) In summary, we concluded that the
C666.1 cells had more radioresistance than the TW1 cells, thus requiring a higher dose of IR to reach its
IC50. This further leads to validating the effect of EBV infection on the development of radioresistance in
NPC cell lines because the EBV-infected cell line C666.1 indicated more resistance to IR than the TW1 cell
line, which is absent of the EBV infection.
Next, we used the colony formation assay to determine colony survival rates to
demonstrate the IC50 of NEO212 in TW1 and C666.1 cell lines. We observed that in contrast to the
response achieved with IR, the C666.1 cell line was more sensitive to NEO212 than the TW1 cell line (Fig
3.4, Fig 3.5, Fig 3.6). For the TW1 cell line, the IC50 with NEO212 was observed to be approximately 32 µM.
On the other hand, the C666.1 cell line showed a much lower IC50 between 4 µM and 13 µM. Previous
research demonstrates the effect of NEO212 on the activation of the lytic cycle of EBV in the C666.1 cell
line; it is shown that NEO212 causes reactivation of the EBV lytic cycle in C666.1 validated by the expression
of lytic cycle markers like Zta and Ea-D (44).
Secondly, we were able to establish the effectiveness of concurrent chemo-radiotherapy
with NEO212 and IR in the TW1 and C666.1 cell lines. We used the formulated comparative analysis to

46
identify the difference between the individual drugs’ dose effect and the combined effect produced via
combination therapy. Our results showed that the application of IR to NEO212 treated samples resulted in
an increase in cell death in both TW1 and C666.1 cell lines. In the TW1 cell line, implementing a constant
IR of 4 Gy over a range of NEO212 concentrations negligibly affected the IC50, decreasing it from 31 µM to
30 µM. Further analysis showed that an increase in IR from 4 Gy to 6 Gy produced a more significant
decrease in its IC50 from 31 µM to 12 µM hand in hand with the observed increase in cell death(Fig 3.7, Fig
3.8, Fig 3.9); for the C666.1 cell line, a similar pattern was observed, however, in the case of these results
by taking into account the radioresistance of the cell line and implementing higher doses of IR such as 6 Gy
and 8 Gy, we observed robust cell death with the IC50 lowered from 8 µM to 0 µM. We attribute this scale
of cellular death to increasingly high doses of IR concurrently administered with a range of NEO212
concentrations (20 µM to 100 µM) (Fig 3.10, Fig 3.11, Fig 3.12).
With these results, our data also generated values for the fold sensitization of NEO212 with IR for
TW1 and C666.1 cell lines. Determining the synergism of two similar drug mechanisms is based on the
difference in their ability as individual treatments and combined therapies. According to the quantitative
analysis of cell survival, the effect of combination therapy is either >1, an antagonistic effect, <1, a
synergistic effect, and =1, an additive effect. Synergistic effects indicate that the two therapies can combine
and produce a more pronounced detrimental impact on the cells than the probable outcome of their
potencies (45). We demonstrated that at deficient concentrations of NEO212 and a constant dose of 4 Gy
IR, NEO212 synergized with IR, thus producing an increased cytotoxic effect, as observed in our previous
results with concurrent therapy. We also demonstrated that the synergistic effect was lost at higher
concentrations of NEO212. Moreover, an antagonistic effect was observed. (Fig 3.13)
In the case of C666.1 cells, we could not observe the synergistic effect of NEO212 and IR.
Moreover, the results demonstrated an antagonistic effect even at concentrations as low as 20 µM.

47
Considering these results and our previous understanding of the sensitivity of the C666.1 cell line to
NEO212, we attributed this antagonistic effect to the unfavorably high concentration of both NEO212 and
IR. (Fig 3.14) Furthermore, on comparative analysis of the two cell lines, NEO212 in the TW1 cell line
showed synergism owing to the lenient conditions of NEO212 and IR but antagonism in the case of the
C666.1 due to the high treatment conditions. (Fig 3.15) With the help of scientific literature, it was easy to
identify that the root cause of the unfavorable results was the undeniably high doses of treatment; it has
been mentioned that for two therapeutic modalities to synergize, their doses should be lowered. This is
owed to the fact that higher doses result in one mode of treatment overpowering the other, thus
preventing the two treatments from synergizing. In compliance with scientific literature, these results
provide the basis of treatment conditions to observe synergism between NEO212 and IR.
Even though we could demonstrate the synergism between NEO212 and IR in the TW1 cell
line, we wanted to optimize the therapeutic targets by complying with low doses of treatment to allow
better synergism between the two modalities in both TW1 and unresponsive C666.1 cell lines. Our results
demonstrated that NEO212 is a robust radiosensitizer of TW1 cells when administered at low doses (below
30 µM) and a constant IR dose of 4 Gy (Fig 3.16). We demonstrated that the IC50 of NEO212 was lowered
from 30 µM to 11 µM when administered concurrently with IR, thereby providing an increased cytotoxic
effect on the cells. Thus, we could show the effects of NEO212 as a radiosensitizer in the TW1 cell line.
For the C666.1 cell line, we considered the cells' radiosensitivity and considerably reduced
the dose of NEO212 and IR to prevent one from overpowering the other, hoping to produce the synergism
observed in the TW1 cell line. However, according to our results, NEO212 could not synergize with IR even
at low doses ranging from 1 µM to 30 µM. In addition, the results proved to be inconclusive due to the
100% cell survival observed for C666.1 with this treatment condition. (Fig 3.17)

48
Considering previous research, we assume that the C666.1 cell line's MGMT-positive
feature explains the lack of NEO212's synergy with IR. O6-methylguanine–DNA methyltransferase—MGMT
is a robust DNA damage repair protein known to cause TMZ resistance in MGMT-positive cancers such as
glioblastoma. MGMT is responsible for the de-alkylation at the O6-guanine position, thereby creating
resistance to methylating drugs such as TMZ. It has also been said that MGMT can contribute to
radioresistance irrespective of its O6-methylation reversal properties. However, the underlying
mechanisms are not fully understood (29, 46). We demonstrated that C666.1 cells are positive for MGMT
expression, while TW1 cells lack the expression of the MGMT protein. (Fig 3.18) This MGMT-positive
feature, along with the inherent EBV infection in C666.1 cells, could be the reason NEO212 is unable to
synergize with IR.
Furthermore, previous research demonstrated that NEO212 can overcome MGMT resistance in
NPC cell lines by repeated treatments. This is because the strong alkylating effect of NEO212 is observed
to decrease the expression of MGMT. Thus, repeated treatments produce better cytotoxicity due to the
reduced MGMT protein post-initial NEO212 treatment (2). In addition to the inherent viral infection and
MGMT-positive feature of C666.1, our single-treatment condition was insufficient to decrease MGMT
expression, synergize, and inhibit cell survival.
Considering our positive results for TW1 and the inconclusive nature of NEO212’s effect
on C666.1, we sought to demonstrate the molecular effect that NEO212 had with IR on the cells. We
showed that NEO212 could cause DNA damage at low concentrations with IR in the TW1 NPC cells,
observed by previous bands of DNA-damage marker ƳH2AX in our western blot. (Fig 3.19) These results
were indicative that NEO212 + IR causes cell death due to DNA damage. Additionally, following our previous
results concerning C666.1, we observed an increased effect of DNA damage in the standalone treatment
of NEO212 at 10 µM compared to the NEO212 + IR at the same concentration. (Fig 3.20) These results

49
further indicate the lack of synergism between NEO212 + IR and solely demonstrate the sensitivity of
C666.1 to NEO212 alone.
To further validate that these results observed were strikingly different for the two cell
lines, we relied on the same treatment conditions and observed cell death concerning DNA damage via
FACS analysis. Consistent with the western blot results, we demonstrated that NEO212 + IR generated
increased cell death than the standalone NEO212 treatment, with 20 µM + IR showing the most significant
increase in cell death. The rate of cell death in the TW1 cell line was also concurrent with the amount of
DNA damage demonstrated by the percentage of DNA damage marker ƳH2AX. (Fig 3.21) Additionally, the
C666.1 data for the FACS analysis proved inconsistent and inconclusive due to the observance of similar
cell death patterns in both NEO212 alone and concurrent IR. Moreover, the levels of DNA-damage marker
ƳH2AX were increasing low in the C666.1 cell line and showed no significant increase with concurrent
treatment. (Fig 3.22)
In conclusion, our data displayed that NEO212, when implemented at low concentrations
of <30 µM with a constant IR dose of 4 Gy, acted as a robust radiosensitizer in NPC TW1 cell lines. The
results of the MTT and CFA assays and the Western blot and FACS analysis support the idea that NEO212
synergizes with IR at clinically relevant concentrations, thereby radio-sensitizing the cells and positively
increasing its cytotoxic effect. We also observed that this phenomenon of NEO212 was not consistent with
the C666.1 cell line, which is known to be inherently infected with EBV and MGMT-positive; by our results
for the C666.1 cell line as well as previously cited data, we believe that the reason for our inconclusive
results amounts to three keys shortcomings of the experimental set up: (1) NEO212 is known to cause
activation of the lytic cycle in EBV-positive cell lines such as C666.1 (44), EBV is also known to be connected
to the radioresistance and metastatic reoccurrence of NPC thus standing as a significant obstacle for the
C666.1 cells, (2) C666.1 cell is positive for the DNA-repair protein MGMT, studies demonstrate that MGMT
is responsible for TMZ-resistance and possibly contribute to radioresistance as well. This further creates an

50
additional level of fine-tuning when it comes to the therapeutic outcomes of C666.1 cells, and (3) For
MGMT-resistance and NEO212, it has been stated that in MGMT-positive cells, NEO212 can overcome this
resistance with the help of repeated doses. However, in our cohort of C666.1 cells, the treatment
conditions were subjected solely to a single dose, thus preventing the ability of NEO212 to actively diminish
cellular MGMT and exert its entire cytotoxic potential.
Although our results have shown promising effects for the radiosensitization of TW1 NPC
cells and produced insightful outcomes for treating the peculiar C666.1 cell line, further studies are
required to determine the true radio-sensitization potential of NEO212 in NPC cells. This includes, but is
not limited to, further in vivo experiments concerning concurrent treatment of NEO212 + IR. More
importantly, we need to analyze the striking effect of repeated treatments on the radio-sensitization of
NPC cells and the mechanisms by which EBV and MGMT-positive cell lines, like C666.1, can be
radiosensitizers.

51
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Abstract (if available)

Abstract Nasopharyngeal carcinoma (NPC) is a head and neck tumor difficult to treat surgically. Current treatments work for early-stage tumors but are less effective for late-stage NPC, leading to poor outcomes. Chemoradiotherapies often cause off-target toxicity and reduced patient outcomes, primarily due to NPC cell line radio-resistance. This study evaluates NEO212, a novel perillyl alcohol-temozolomide conjugate, as a radiosensitizer for NPC cell lines with ionizing radiation (IR). We hypothesize that NEO212 will enhance IR efficacy at clinically relevant concentrations. We also assess its effects on Epstein-Barr Virus (EBV) and O6-methylguanine-DNA methyltransferase (MGMT) negative and positive NPC cell lines TW1 and C666.1, respectively.

We used cell proliferation, viability, cell death, and apoptosis assays, as well as methylthiazoletetrazolium assays, colony formation assays, Western blots, and fluorescence-activated cell sorting to determine the effects of NEO212 and IR. Results showed both NPC cell lines were susceptible to NEO212. The MGMT and EBV-positive C666.1 cell line was slightly more radioresistant and chemo sensitive than TW1. NEO212 was a robust radiosensitizer for TW1, with the synergism most effective at lower NEO212 doses. However, C666.1 showed inconclusive results, likely due to the DNA-damage repair protein MGMT. Overall, NEO212 shows promise as a radiosensitizer for NPC and a potential alternative to current therapies. Further studies are needed to optimize conditions for synergism between NEO212 and IR in MGMT and EBV-positive NPC cell lines and to validate these findings in vivo and clinical settings.

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Creator Dsouza, Megan Anika (author)

Core Title Radiosensitization of nasopharyngal carcinoma cells by NEO212, a perillyl alcohol-temozolomide conjugate drug

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2024-08

Publication Date 07/31/2024

Defense Date 07/31/2024

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

Tag cancer therapy,chemoradiation,chemotherapy,EBV,nasopharyngeal carcinoma,NEO212,OAI-PMH Harvest,perillyl alcohol,Radiation,radiosensitization,Radiotherapy,synergism,temozolomide

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Advisor Schönthal, Axel H. (committee chair), Minea, Radu O. (committee member), Swenson, Steve (committee member), Tahara, Stanley (committee member)

Creator Email madsouza@usc.edu,meganikadsouza@gmail.com

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