A

MAJOR PROJECT REPORT

ON

DEVELOPMENT OF ORAL CANCER ANIMAL MODEL USING

CARCINOGEN (4NQO) AND STUDY THE ROLE OF SOC INHIBITOR

Submitted in partial fulfillment of the requirement for the award of the degree of

Bachelor of Technology

In

Biotechnology

Of

DR. A. P. J. ABDUL KALAM TECHNICAL UNIVERSITY

(Formerly Uttar Pradesh Technical University)

Submitted By: Supervised By:

PRIYANKA VERMA (1706854033) Dr. ANUJ KUMAR SINGH
PRIYA RAO (1706854030) Assistant Professor,
Department of Biotechnology

DEPARTMENT OF BIOTECHNOLOGY
MEERUT INSTITUTE OF ENGINEERING & TECHNOLOGY, MEERUT
2017-2021
 CERTIFICATE
Date: …………

This is to certify that Ms. Priyanka Verma and Ms. Priya Rao, Roll No.
1706854033 and 1706854030 of B. Tech. (Biotechnology) VIII semester has carried
out a project work on DEVELOPMENT OF ORAL CANCER ANIMAL
MODEL USING CARCINOGEN (4NQO) AND STUDY THE ROLE OF SOC
INHIBITOR under my supervision for the partial fulfilment of the award of the
degree of Bachelor of Technology in Biotechnology at Department of Biotechnology,
Meerut Institute of Engineering & Technology (MIET), Meerut (Affiliated to Dr.
A.P.J. Abdul Kalam Technical University, Lucknow). The above said project is a
bonafide record of work done by him during the session 2020-21.

Supervisor:

Dr. ANUJ KUMAR SINGH

Assistant Professor,
Department of Biotechnology

i
 DECLARATION

We hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material which to a substantial extent has been
accepted for the award of any other degree or diploma of the university or other
institute of higher learning.

Name : Priyanka Verma

Roll No. : 1706854033

Name : Priya Rao

Roll No. : 170685430

ii
 ABSTRACT

According to the World Health Organization’s (WHO’s) report of GLOBAN 2018,

oral squamous cell carcinoma is the 16th most common cancer worldwide. Various
models have been investigated to induce the oral carcinoma, the carcinogenic 4NQO
mice model has proven to be successful although it takes long time for development
of tumor. Development of oral cancer was investigated in Swiss albino mice with
4NQO. We had grouped mice in three groups, first group was provided with DMSO
water without 4NQO and the remaining groups were provided with 4NQO to the mice
in drinking water. After 8-10 weeks, we stopped providing 4NQO water to the groups
and after another week we had injected synta66 (SOC inhibitor) intraperitoneally to
the third group mice and other remaining groups were controlled. After treatment the
mice were sacrificed and further histological studies were carried for the
determination cancer development and to study effect of synta66 in the mice model.
Synta66 is a selective pore inhibitor of the Orai1 channel (store operated calcium
channel) which decreases the migration in breast cancer Commotion in the expression
and function of certain ion channels leads to the cancer hallmarks. The dysregulated
SOCC helps in tumor development and malignant transformation of cells which leads
to high proliferation rate, altered apoptosis, cell migration and invasion. It was
observed that 4NQO induced the carcinogenic mice model and synta66 decreases the
proliferation as the Ki67 (proliferative marker) expression reduced in mice model. In
conclusion, the results of our study demonstrate that by using carcinogen (4NQO) to
successfully develop oral cancer in mouse after the development of tumor we treated
it with synta66 (SOC inhibitor) shows anti-cancerous role in oral cancer and seems
suitable for therapeutic research applications.

iii
 ACKNOWLEDGEMENT

For the completion of this work. First of all, we would like to thank the almighty God
without whose blessings this work would have never been completed.

We sincerely express our thanks with deep sense of gratitude to Dr. Nitin Sharma,
Professor (HOD, Department of Biotechnology, MIET) for constant
encouragement, invaluable guidance and all possible help which will be great
inspiration for us throughout our life.

We would like to convey our deep thanks to Dr. Anuj Kumar Singh (Assistant
Professor, Department of Biotechnology, MIET) a teacher and a guide, without
whose cooperation and help it would not possible for us to complete this project work
successfully.

We would like to thanks to Dr. Ashima Kathuria (Associate Professor,

Department of Biotechnology, MIET) and Ms. Vandana Thakur (Assistant
Professor, Department of Pharmacy, MIET) for guidance and all possible help.

We would also like to thanks to Mr. Manoj (Animal maintaining facility,

Department of Pharmacy, MIET) and the lab assistant Mr. Jitendra Kumar, Mr
Yasir and Mr Gajendra (Department of Biotechnology, MIET) for their
contribution made in making available all the requirements.

We would also like to thank our junior Yugal Gupta for helping us during project
work.

We can never express feeling to our parents, brother, sister and friends who gave us
help in each task of our work and giving us strong emotional support.

Thanks again to all

Priyanka Verma (1706854033)

Priya Rao (1706854030)

iv
 TABLE OF CONTENTS

1. INTRODUCTION 1-5

 Oral cancer

 Ion channel

 SOC inhibitors

 Ki67

 Objective

2. REVIEW OF LITERATURE 6-11

3. MATERIALS AND METHODOLOGY 11-16

 Development of mouse model with carcinogen

 Treatment with synta66

 Histopathology

a. H&E

b. IHC

4. RESULTS AND DISCUSSION 17-26

5. CONCLUSION AND FUTURE DIRECTION 27-28

6. REFERENCES 29-30

v
 LIST OF FIGURES

Figure No.: Title

 Fig. 1: Chemical structure of 4NQO (4-nitroquinoline 1-oxide)

 Fig. 2: diagrammatical representation of treatment process
 Fig. 3: Mice sucking 4NQO solution in drinking water

 Fig. 4: Injecting synta66 intraperitoneally

 Fig. 5.1: Untreated tongue of mice observed under 10X magnification

 Fig. 5.2: Untreated tongue of mice observed under 20X magnification
 Fig. 6.1: Treated tongue of mice with carcinogen(4NQO) observed under 10X
magnification

 Fig. 6.2: Treated tongue of mice with carcinogen(4NQO) observed under 20X
magnification
Fig. 7.1: Treated with synta66 in 4NQO induced cancer mice model observed
under 10X magnification

 Fig. 7.2: Treated with synta66 in 4NQO induced cancer mice model observed
under 20X magnification
 Fig. 8: Untreated controlled tongue of mice observed under 20X magnification
 Fig.9: Treated tongue of mice with carcinogen (4NQO) observed under 20X
magnification
 Fig.10: Treated with synta66 in 4NQO induced cancer mice model observed
under 20X magnification

vi
 ABBREVIATIONS

1. 4NQO - 4-Nitroquinoline 1-oxide

2. DMSO - Dimethylsulfoxide
3. SOCC – Store Operated Calcium Channel
4. VGCC – Voltage Gated Calcium Channel
5. LGCC - Ligand-Gated Calcium Channels
6. TRP- Transient Receptor Potential
7. SOCE - Store Operated Calcium Entry
8. PM – Plasma membrane
9. ER – Endoplasmic Reticulum
10. GBM - Glioblastoma
11. H&E- Hematoxylin-eosin
12. IHC – Immunohistochemistry
13. OSCC-Oral Squamous Cell Carcinoma

vii
Chapter 1: Introduction

1
 INTRODUCTION

Oral cancer

Oral cancer also known as mouth cancer or oral squamous cell carcinoma is the 16 th
most common cancer worldwide [1]. Oral cancer is caused due to the uncontrolled
growth of cells. If the cancer has spread to close tissues, organs, lymph nodes then the
5-year survival rate drops to 65%. The major risk factors for causing oral cancer are
tobacco, alcohol consumption, Human papillomavirus (HPV16 and 18), radiation, UV
light, chewing areca or betel nuts, poor nutrition and chronic trauma [2] [3]. The risk
of developing oral cancer is 3 times more in smokers compared to the non-smokers
and the risk of oral cancer also increased by 87% in those who never smoked but
exposed to an environment of cigarette smoke compared to those who neither smoke
nor exposed [3]. The common symptom of oral cancer varies in patients’ age, sex, and
the location of the tumor inside oral cavity. However, most of the cancerous patches
are skinny, irregular and blend of red and white patches within the mouth [4].
Appearance of rough crusts or worn areas on the lip, loosening of teeth, bleeding of
gums, constant ear ache, hoarseness in throat, issue in breathing, chewing and
swallowing the food, modification in voice, weight loss. Oral cancer can be prevented
by avoiding smoking, no consumption of tobacco product, paan and alcohol, avoid
sun exposure on the lower lip, HPV vaccination and by high consumption of fruits
and vegetables which reduce 40-50% in risk of oral cancer [4].

Ion channel

Ion channels are integral membrane proteins that have an aqueous pore through
which, once it is open, precise ions can move freely within these compartments.
Recirculation of ions amid cellular compartments as a result of channels’ opening can
affect a plenty of cellular processes and functions covering from electrical excitation

2
to locomotion. Amid these processes also are those that are critical for retaining
normal tissue homeostasis, such as cell proliferation, migration, and apoptosis. Ion
channels are divided onto two broad classes, voltage-gated channels (VGC) and
ligand-gated channels (LGC) [5].

Calcium channel is an ion channel which is selectively permeable to the calcium ion.
Calcium ion channel have various different type of channel like voltage gated
channel, ligand gated channel, TRP channel (transient receptor potential), stored
operated calcium channel, among these TRP channel and SOC channel is of great
importance. Stored operated calcium channel placed within the cytoplasmic
membrane of all non-excitable cells. The concept of SOCE was first given by James
Putney in 1986, SOCE get activated when calcium ion release from the endoplasmic
reticulum and mitochondria. It has been shown that the tumor proliferation in
different human cancers occur due to the SOCE remodelling [6], two essential
proteins like STIM1on the endoplasmic reticulum act as calcium ion sensor and
ORAI1 on the cytoplasmic membrane act as pore forming protein, these play a very
important role in stored operated calcium entry [7]. The SOC inflow controls various
physiological functions (such as apoptosis, cell proliferation and migration) and
pathological functions (such as SCID (severe combined immunodeficiency disease),
cardiovascular and pulmonary diseases, and cancer of the breast, colon, and,
esophagus). Co-expression of Orai1 and STIM1 is enough to redevelop the function
of store-operated calcium channel. Overexpression of the STIM1 and STIM2 gene
leads to the invasion, migration of the cancerous cell in breast cancer and correlate
with the poor survival rate, and the increased amount Orai1 has also seen in the breast
cancer. Hence, SOCE is the primary mechanism for the entry of calcium ion in most
different kinds of cancer [8].

SOC inhibitors

The SOCE inhibitors Synta66 had a dramatic effect on the cytoplasmic Ca2+
concentration ([Ca2+] cyt). Little specific pharmacological inhibitors in MDA-MB-231

3
basal B breast cancer cells, Synta66 decreased the migration of MDA-MB-468 basal
A breast cancer cells, as reflected in a significant increase in the percentage of cells
with levels of migration less than 10 μm with complete media re-addition [9].

Ki67

The expression of Ki67 is strongly associated with tumor cell proliferation and
growth, and is widely used in routine pathological investigation as a proliferation
marker. Ki67 immunostain was analysed in all cases and was correlated to the grade
of dysplasia and grade of differentiation of oral squamous carcinomas and display
high values and intensity at the tumoral invasion front (IP 62.3%). According to the
result, Ki67 proving to be beneficial in assessing tumor aggressiveness. Ki67 has a
prognostic value allowing the identification of aggressive forms, highly proliferative
by oral carcinomas [10].

4
OBJECTIVE

1. Generation of chemically induced oral cancer mice model

2. Treatment of oral cancer animal models with SOC inhibitor

3. Effect of SOC inhibitor on oral cancer.

5
Chapter 2: Review of Literature

6
 Review of literature

Oral cancer also known as mouth cancer or oral squamous cell carcinoma is the 16th
most common cancer worldwide with about 354,864 new cases and 177,384 deaths
based on the World Health Organization’s (WHO’s) report of GLOBAN 2018. Oral
cancer is two times more prevalent in men than women. According to this report for
oral cancer 246,420 new cases and 119,693 deaths of male, and 108,444 new cases
and 57,691 deaths of female have been found in 2018 [11]. In India, oral cancer 2nd
most common cancer whereas 1st among males with 92,011 new cases and 4th among
females with 27,981 new cases in 2018 [12]. Oral cancer has overall 5-year survival
rate for patients with an early diagnosis of oral cavity and pharynx cancers is 84%.
Oral cancer are often diagnosed through physical examination, biopsies (punch
biopsy, fine or core needle biopsy) of the affected area, endoscopy and imaging
investigation like X-ray of mandible, maxillary sinuses, and chest, CT-scan, MRI,
PET-scan are done when it spread to distant body parts. The Union for International
Cancer Control Tumor, Nodes, Metastasis (TNM) staging system is widely used for
staging oral cancer, TNM estimate the degree of spread of cancer from its initial
position. Oral cancer is treated by removal of squamous cell carcinoma from oral
cavity and neck through surgery followed by chemotherapy or radiotherapy to destroy
remaining cancerous cells [13]. Most of the OSCC cases are diagnosed in advanced
stage which leads to survival at 5 year less than 50% and cure of 30% [2]. Oral cancer
can be prevented by avoiding smoking, no consumption of tobacco product, paan and
alcohol, avoid sun exposure on the lower lip, HPV vaccination and by high
consumption of fruits and vegetables which reduce 40-50 percent in risk of oral
cancer. It can also be prevented by conducting self-examination once a month and to
meet the dentist in regular basis [13].

7
Ion channel may be crucial for carcinogenesis first hinted in the late 1980s. It was
observed that cancer cell lines act abnormal patterns of ion channel functional
expression and that pharmacological blockade of few channels can prohibit cancer
cell growth in early 1950. This intense research not only confirmed the role of ion
channels in defining pathological features of major cancer hallmarks, but provided
solid evidences that, in fact, these hallmarks can be viewed as pathological states
largely caused by disturbed expression and function of certain ion channels. Such
view is in line with the definition of channelopathy, although to emphasize specific
relevance to cancer the use of the term oncochannelopathy would be more
appropriate. It is proposed that cancer hallmark for each particular cancer type is
regarded as oncochannelopathy of specific channel, which aberrant expression/
function contributes to this hallmark via dysregulation of certain cellular processes.
Opening of plasma membrane ion channels is tightly regulated by their intrinsic
gating mechanisms, which in turn are controlled by the presence of specific
endogenous or exogenous physical or chemical factors. When open, ion channels
selectively pass through certain ion species down its electrochemical gradient which
affects such basic cellular characteristics as 1) membrane potential due to generation
of transmembrane ionic current, 2) cell volume due to coupled transmembrane
movement of water to compensate for altered intracellular tonicity, and 3) the state of
intracellular signaling pathways due to changes in ion-effector molecule interaction.
Any commotion in these orderly changes resulting from ion channel dysfunction
would seriously impair cell proliferation, differentiation, apoptosis, and motility and
promote the development of one or more oncochannelopathy in the form of certain
cancer hallmark.

Calcium is a universal signaling ion regulating a plethora of cellular processes and

functions including those that determine whether the cell has to live, multiply, move,
or die. There are two classes of Ca2+-permeable channels, plasma membrane (PM)
and ER membrane ones. PM Ca2+-permeable channels in turn consist of seven major
subclasses distinguished by the principal mechanism of their activation: 1) VGCC 2)
LGC 3) store-operated channels (SOC), 4) transient receptor potential (TRP)
channels[5].

8
SOC is a generic name for the channel underlying the major Ca2+ entry mechanism in
non-excitable cells, termed store-operated Ca2+ entry (SOCE). This type of channel
opens only when ER Ca2+ store becomes depleted. Documented evidences have
shown that calcium (Ca2+) regulation through store-operated Ca2+ (SOC) channels
(SOCC) is a monitoring apparatus in many cancers such as breast cancer, lung cancer,
cervical cancer and hepatocellular carcinoma. The dysregulated SOCC helps in tumor
development and malignant transformation of cells which leads to high proliferation
rate, altered apoptosis, cell migration and invasion. The SOCC are controlled by three
plasma membrane proteins (Orai-1, Orai-2 and Orai-3) and two endoplasmic
reticulum (ER) membrane proteins stromal interaction molecules (STIM)-1 and
STIM-2. Recent findings added more explanation to the mechanism of Ca2+ release-
activated Ca2+ (CRAC), i.e., when the release of stored Ca2+ deplete the store of ER,
STIM-1 changes its confirmation and gives a signal to Orai-1 protein for opening the
gate at the plasma membrane and allow the extracellular Ca2+ to enter inside the cell.
Role of SOCC and the key members of SOCC have not been extensively studied in
oral cancer. Report shows that SOCE, Orai-1 and Orai-2 expression were significantly
elevated in cancer cells compared to normal epithelial cells. The knockdown
repressed cancer progression and metastasis-associated proteins and augmented the
expression of antitumor related genes. It has shown that by inhibiting SOCE on cancer
cell proliferation and migration with the help of 2-APB, La3+/LaCl3 and SKF96365,
chemical inhibitors of SOCE. The inhibitor, 2-APB, was considered as the most
reliable blocker for SOCE whereas La3+ was reported as a blocker of the capacitive
Ca2+ entry. SKF96365 is the most selective and recently identified inhibitor and has
been successfully studied in various cancers [14].

9
Recent report shows that Synta66 is a selective pore inhibitor of the Orai1 channel
that mediates its effects by binding close to the selectivity filter, and an efficient
inhibitor of SOCE in GBM cells. Synta66 action on the Orai1 pore was determined by
complete inhibition of ectopically expressed STIM1/Orai1 currents, whilst Orai1
mutants exhibiting reduced Ca2+ selectivity were less sensitive to Synta66 inhibition.
No significant inhibition of GBM cell viability by Synta66 was observed. Synta66 is
thus a valuable tool for the evaluation of the contribution of Orai1 channels to cancer
cell progression[15].The SOCE inhibitors Synta66 had a dramatic effect on the
cytoplasmic Ca2+ concentration ([Ca2+]cyt). Little specific pharmacological inhibitors
in MDA-MB-231 basal B breast cancer cells, Synta66 decreased the migration of
MDA-MB-468 basal a breast cancer cells, as reflected in a significant increase in the
percentage of cells with levels of migration less than 10 μm with complete media re-
addition [9].

Several animal models for oral squamous cell carcinoma are used including hamster,
rat and mouse models, with each model having its own advantages and disadvantages.
The introduction of 4-nitroquinoline-1-oxide (4-NQO) for inducing tumors in rats, it
has been extensively used in both rats and mouse and can useful for analyzing the
effects of anti-tumor agents The 4NQO oral squamous cell carcinoma model is a
lengthy multi-step process in which eventual invasive squamous cell carcinoma is
attained after several months[16]. 4-NQO, a water-soluble quinoline derivative,
produces a spectrum of preneoplastic and neoplastic lesions in the oral cavity,
especially tongue, of rats following 4-NQO application in drinking water. Oral lesions
produced by 4-NQO are comparable to human lesions, because many ulcerated and
endophytic or exophytic tongue tumors and dysplasia develop when 4-NQO in the
drinking water is given to rats or 4-NQO is applied topically to the oral mucosa.
Using 4-NQO-induced rat oral carcinogenesis model, we have reported several
candidates for chemopreventive agents against oral malignancy [17].

10
Fig 1. Chemical structure of 4NQO (4-nitroquinoline 1-oxide) [18]

11
Chapter 3: Materials and Methods

12
 Materials and Methods:

Development of mouse model with carcinogen:

18 6-9 weeks male Swiss albino mice weight between 20-35gm, purchased from
AIIMS Medical facilities, were used for the studies with a carcinogen (4NQO). The
experiments were approved by IAEC/MIET/2021/04. All the mice were kept in the
required condition with 12 hours light/dark cycle in the animal house. All mice were
sustained on a normal meal. Mice were separated into 3 groups, 6 mice/group in
different cages. Initially they were kept in quarantine for a week, after that they were
given the 4NQO treatment to them. A stock solution of carcinogen 4NQO was
prepared by dissolving 4NQO powder in DMSO at 100mg/ml and stored at -20ºC.
After quarantine period, 1% DMSO in drinking water given to the controlled group1
mice and other two remaining groups were given 4NQO stock solution diluted in the
drinking water (100µg/ml) and it was stored in dark colour bottle, and the water got
changed weekly. Mice were allowed to approach to the drinking water all the time
during treatment period. After 9 weeks we stopped delivering the carcinogen to the
mice and replaced it with normal drinking water, and observe sign of sickness and
weight loss. Body weight of the mice was measured in the beginning and in the end of
the experiment. All the mice were attentively supervised on daily basis.

Treatment with synta66:

After 9 weeks treatment with 4NQO, mice were given drinking water for a week, and
after that group-3 mice were injected with synta66 (5mg/kg) intraperitoneally once a
week and remaining two groups were controlled. This treatment was given for two
time (2 doses), after the treatment all the mice were sacrificed by application of
thiopental sodium 3 times of the dose of anesthesia. The tongue of mice were
dissected and preserved in 10% formalin.

13
 Fig.2: diagrammatical representation of treatment process

Histopathology

H&E: After the treatment mice were sacrificed and tongue was removed and
fixed in freshly made 4% buffered formalin. Then the tongue was sectioned and
placed in cassettes to dehydrate the tissue in ascending grade of alcohol to remove
formalin and water. After that tissue were embedded in paraffin wax. After paraffin-
embedding, serial sections of 3-10 micron thick were made and one in five subsequent
sections was stained with hematoxylin-eosin.

14
 IHC: Immunohistochemical analysis was performed on serial sections for
Ki67. Ki67 is a proliferative marker. In fixation, 10% neutral buffered formalin for 24
hours at room temperature. Sectioning paraffin coating for removal of water in the
slide and then de-paraffinization and hydration. Heat induced epitope retrieval is the
most widely used in Antigen retrieval, add blocking solution at room temperature
after that add primary antibody, antibody dilution by protein blocking solution then
incubate slides 30-60 min. at room temperature and add a small amount of water into
the incubation box. Add secondary antibody and incubate slide for 30-60 min at room
temperature and then wash slide for 3-5 min. After that add substrate for desolation
and clearing and washing slide with distilled water. Scan the slides and validate the
results under a microscope.

15
Chapter 4: Result and Discussion

16
 RESULT

1. Generation of chemically induced oral cancer mice model.

Swiss albino mice were segregated into different cages according to their
group before administrating the 4NQO doses. Mice were kept 4mice/cage in
quarantine for a week to let them familiar with each oth er. After a week they
were administered with 4NQO solution leaving group 1 controlled. Regular
meals were given to the mice and observe their behaviour on daily basis. Body
weight of the mice was measured in the beginning and in the end of the experiment.
After the 9 weeks treatment, we observe certain weight loss, loss of appetite ,
fatigue and swelling in their mouth.

Fig 3: Mice sucking 4NQO solution in drinking water

17
2. Treatment of oral cancer animal models with SOC inhibitor

 No. of doses - 2

 Route - Intraperitoneally

 Weight – Table for the weight of mice and standard deviation:

S.No. Before After 8 After 1st After 2nd Before

4NQO weeks dose of dose of dissection
treatment 4NQO synta66 synta66
treatment

Group-1 17* 29.675* 32.425* 33.5* 34.35*

(Control) ±1.4142 ±4.1015 ±2.7861 ±2.6457 ±2.6463
- 4NQO
-Synta66
Group-2 32.3 31.2425 30.275 30.225 28.25
+ 4NQO ±4.0191 ±3.3830 ±3.3018 ±4.8044 ±7.6264
-Synta66
Group-3 28.15 31.425 33.275 32.55 33.2
+ 4NQO ±3.0435 ±1.3326 ±1.6938 ±2.0338 ±1.5853
+Synta66
*Group 1 was given 1% DMSO in drinking water

 Behaviour – All the mice were observed on daily bases until the end of
experiment. All mice were healthy in the beginning of the experiment and
their health disturbed as the consume 4NQO in drinking water. After 8 weeks
of 4NQO treatment, as the doses of synta66 given to them their health
improved.

Fig 4: Injecting synta66 intraperitoneally

18
Histological finding:

After the treatment and slide preparation, observe the slide under the
microscope with different magnification (10X, 20X).

H&E (Haematoxylin and Eosin):

Fig. 5.1: Untreated tongue of mice observed under 10X magnification

Fig.5.2: Untreated tongue of mice observed under 20X magnification

19
Fig.6.1: Treated tongue of mice with carcinogen (4NQO) observed under 10X magnification

Fig.6.2: Treated tongue of mice with carcinogen (4NQO) observed under 20X magnification

20
Fig.7.1: Treated with synta66 in 4NQO induced cancer mice model observed under 10X
magnification

Fig.7.2: Treated with synta66 in 4NQO induced cancer mice model observed under 20X
magnification

21
IHC (immunohistochemistry with Ki67):

Fig 8: Untreated controlled tongue of mice observed under 20X magnification

Fig 9: Treated tongue of mice with carcinogen (4NQO) observed under 20X magnification

22
 Fig 10: Treated with synta66 in 4NQO induced cancer mice model observed under 20X
magnification

For Histological findings,

With help of H&E staining, it was observed that in fig 6 (Treated tongue of mice
with a carcinogen (4NQO)), there was some abnormal growth of cells, and
infiltration of cells was observed compared to fig 5 (Untreated tongue of mice
model). Similarly, as for fig 7 (Treated with synta66 in 4NQO induced cancer
mice model) was compared to fig 6 (Treated tongue of mice with a carcinogen
(4NQO)), it was observed that there were some reduction in the growth of abnormal
cells and no infiltration of cells were observed.

23
With the help of immunohistochemistry with Ki67, Ki67 is a proliferative marker.
The Ki67 was highly expressed in Fig 9 (Treated tongue of mice with a
carcinogen (4NQO)) as compared to fig 8 (Untreated controlled tongue of mice),
which indicates the proliferation of cancerous cells and development of oral cancer.
Similarly, the Ki67 expression was reduced in fig 10 (Treated with synta66 in
4NQO induced cancer mice model) as compared to fig 9 (Treated tongue of
mice with a carcinogen (4NQO)), which indicates the reduction of proliferation of
cancerous cells in the oral cancer mice model.

24
 DISCUSSION

In the recent study, it has been demonstrated that 4-NQO-induced pre-malignant and
malignant lesions in oral cavity in mice not only pathologically and morphologically
mimicked human oral cancer and also study the role of synta66 (SOC inhibitor). 4-
NQO is a synthetic carcinogen first investigated by Nakahara et al. [19]. Various
models have been investigated to induce the oral carcinoma, the carcinogenic 4NQO
mice model has proven to be successful although it takes long time for development
of tumor. Development of oral cancer was investigated in Swiss albino mice with
4NQO. We have grouped mice in three groups, first group was provided with DMSO
water without 4NQO and the remaining groups were provided with 4NQO to the mice
in drinking water. After 9 weeks, we stopped providing 4NQO water to the groups
and after another week we have injected synta66 (SOC inhibitor) intraperitoneally to
the third group mice and other remaining groups were controlled. After 9 weeks all
the mice group 2&3 were weigh and observe that there were some weight loss which
indicates that there might be chance for tumor development, due to tumor formation
the mice would not been able to take up meals which result into weight loss. We can
consider weight loss as a symptom for tumor formation. After treatment of synta66 all
the mice were sacrificed by application of thiopental sodium 3 times of the dose of
anesthesia. The tongue of mice were dissected and preserved in 10% formalin and
done histopathological studies. Examination of tissue sections from mice treated with
4-NQO in the drinking water revealed pathological evidence of carcinogenesis. All
tongues of mice exhibited multiple lesions, including carcinomas.

Recent studies shows that Synta66 is a selective pore inhibitor of the Orai1 channel
that mediates its effects by binding close to the selectivity filter, and an efficient
inhibitor of SOCE in GBM cells and also decreased the migration of MDA-MB-468
basal a breast cancer cells. In recent study, it has been showed that SOCE candidate
proteins Orai-1 and Orai-2 have significant roles in oral cancer. SOCE, Orai-1 and

25
Orai-2 expressions were significantly elevated in cancer cells compared to normal
epithelial cells. The influx of Ca2+ through SOCC is the primary Ca2+ supply route
for non-excitable cells. Our study with the chemical inhibitors — synta66 suggested
that SOCE regulates oral cancer cell migration and proliferation.

In our study, we found that the expression of Ki67 was overexpressed in 4NQO-
induced mice in correlation with the untreated mice, which shows that cell
proliferation arises. But the expression of Ki67 was reduced after the treatment with
synta66 in 4NQO- induced mice in correlation with the controlled 4NQO- induced
mice. This pathological observation suggests that there was development of oral
cancer mice model with the help of synthetic carcinogen (4NQO). Recent study
showed that Orai-1 and Orai-2 are involved in positive regulation of oral cancer.
Further, this is the first report which shows that Orai-1 and Orai-2 are overexpressed
in oral cancer cells and tissues and play a critical role in cellular proliferation,
survival, migration and metastasis [14]. As we know synta66 inhibit the store
operated calcium entry and reduce the expression Orai-1 and Orai-2 in the cancer cell
and regulate the oral cancer cell migration and proliferation. This study plays a very
important role for finding new target which help exploitable therapeutic targets.

26
Chapter 5: Conclusion and Future Direction

27
 Conclusion and future direction

Our study showed that synta66 inhibit the SOCE and reduce the expression of Orai-1
and Orai-2 in the cancer cell and regulate the cell proliferation and migration. It was
observed that 4NQO induced the carcinogenic mice model and synta66 decreases the
proliferation as the Ki67 (proliferative marker) expression reduced in mice model. In
conclusion, the results of our study demonstrate that by using carcinogen (4NQO) to
successfully develop oral cancer in mouse after the development of tumor we treated
it with synta66 (SOC inhibitor) shows anti-cancerous role in oral cancer and seems
suitable for therapeutic research applications. Our data indicated that the 4-NQO-
induced oral cancer is the useful model in this; it mimics many features and molecular
events observed for human OSCC development. Hence, oral cancer mice model
provide a preclinical study platform and it will make a significant contribution to the
development of early diagnosis and therapeutic effect on OSCC. As there is few study
has done on synta66 and, no further study has done on oral cancer. Our experiment
strongly suggests that such an approach may result in potential novel and clinically
exploitable therapeutic targets.

28
 REFERENCES:

[1] ―Cancer today.‖ http://gco.iarc.fr/today/home (accessed Sep. 07, 2020).

[2] C. Rivera, ―Essentials of oral cancer,‖ Int. J. Clin. Exp. Pathol., vol. 8, no. 9, pp. 11884–
11894, Sep. 2015.

[3] ―Essentials of oral cancer.‖ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637760/

(accessed Jul. 23, 2021).

[4] ―Oral Cancer: Symptoms, Causes, Treatments, and More.‖

https://www.webmd.com/oral-health/guide/oral-cancer (accessed Jul. 23, 2021).

[5] N. Prevarskaya, R. Skryma, and Y. Shuba, ―Ion Channels in Cancer: Are Cancer
Hallmarks Oncochannelopathies?,‖ Physiol. Rev., vol. 98, no. 2, pp. 559–621, Apr.
2018, doi: 10.1152/physrev.00044.2016.

[6] C. Dubois et al., ―Remodeling of channel-forming ORAI proteins determines an

oncogenic switch in prostate cancer,‖ Cancer Cell, vol. 26, no. 1, pp. 19–32, Jul. 2014,
doi: 10.1016/j.ccr.2014.04.025.

[7] S. Yang, J. J. Zhang, and X.-Y. Huang, ―Orai1 and STIM1 are critical for breast tumor
cell migration and metastasis,‖ Cancer Cell, vol. 15, no. 2, pp. 124–134, Feb. 2009, doi:
10.1016/j.ccr.2008.12.019.

[8] E. Varghese et al., ―Anti-Cancer Agents in Proliferation and Cell Death: The Calcium
Connection,‖ Int. J. Mol. Sci., vol. 20, no. 12, p. E3017, Jun. 2019, doi:
10.3390/ijms20123017.

[9] I. Azimi et al., ―Pharmacological inhibition of store-operated calcium entry in MDA-

MB-468 basal A breast cancer cells: consequences on calcium signalling, cell migration
and proliferation,‖ Cell. Mol. Life Sci. CMLS, vol. 75, no. 24, pp. 4525–4537, Dec.
2018, doi: 10.1007/s00018-018-2904-y.

[10] L. P. Dragomir, C. Simionescu, A. Stepan, I. M. Dragomir, and M. R. Popescu,

―P53,p16 and Ki67 immunoexpression in oral squamous carcinomas,‖ p. 5.

29
[11] ―Cancer today.‖ http://gco.iarc.fr/today/home (accessed Jun. 26, 2020).
[12] ―356-india-fact-sheets.pdf.‖ Accessed: Jun. 25, 2020. [Online]. Available:
https://gco.iarc.fr/today/data/factsheets/populations/356-india-fact-sheets.pdf

[13] R. Sankaranarayanan, K. Ramadas, H. Amarasinghe, S. Subramanian, and N. Johnson,

―Oral Cancer: Prevention, Early Detection, and Treatment,‖ in Cancer: Disease Control
Priorities, Third Edition (Volume 3), H. Gelband, P. Jha, R. Sankaranarayanan, and S.
Horton, Eds. Washington (DC): The International Bank for Reconstruction and
Development / The World Bank, 2015. Accessed: Jun. 25, 2020. [Online]. Available:
http://www.ncbi.nlm.nih.gov/books/NBK343649/

[14] A. K. Singh et al., ―Orai-1 and Orai-2 regulate oral cancer cell migration and
colonisation by suppressing Akt/mTOR/NF-κB signalling,‖ Life Sci., vol. 261, p.
118372, Nov. 2020, doi: 10.1016/j.lfs.2020.118372.

[15] L. Waldherr et al., ―Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by

Direct Inhibition of the Ca2+ Selective Orai1 Pore,‖ Cancers, vol. 12, no. 10, p. 2876,
Oct. 2020, doi: 10.3390/cancers12102876.

[16] R. A. L. Schoop, M. H. M. Noteborn, and R. J. Baatenburg de Jong, ―A mouse model

for oral squamous cell carcinoma,‖ J. Mol. Histol., vol. 40, no. 3, pp. 177–181, 2009,
doi: 10.1007/s10735-009-9228-z.

[17] K. Yoshida et al., ―Dietary garcinol inhibits 4-nitroquinoline 1-oxide-induced tongue

carcinogenesis in rats,‖ Cancer Lett., vol. 221, no. 1, pp. 29–39, Apr. 2005, doi:
10.1016/j.canlet.2004.08.016.

[18] ―4-nitroquinoline N-oxide (CHEBI:16907).‖

https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:16907 (accessed Jul. 23,
2021).

[19] W. Nakahara, F. Fukuoka, and T. Sugimura, ―Carcinogenic action of 4-nitroquinoline-

N-oxide,‖ Gan, vol. 48, no. 2, pp. 129–137, Jun. 1957.

30