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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
Dr. KARTHIK B
1.
NAME OF THE CANDIDATE
POSTGRADUATE
AND ADDRESS
DEPT OF ORAL PATHOLOGY
COORG INSTITUTE OF DENTAL SCIENCES,
VIRAJPET-571 218
2.
NAME OF THE INSTITUTION
COORG INSTITUTE OF DENTAL SCIENCES,
VIRAJPET-571 218
3.
COURSE OF THE STUDY &
SUBJECT
M.D.S –ORAL PATHOLOGY
4.
DATE OF ADMISSION TO
THE COURSE
31st MAY 2008
5.
TITLE OF THE TOPIC:
Immunohistochemical determination of inducible nitrous oxide synthase in oral squamous
cell carcinoma. A comparative study in tobacco habituers and nonhabituers.
6.
BRIEF RESUME OF THE INTENDED WORK.
6.1 NEED FOR THE STUDY
The causal relation between tobacco use and oral squamous cell carcinoma has long
been established. It is only now that the interaction of compounds present in tobacco and
the connective tissue components is being investigated. Tobacco smoke extract has been
found to alter the connective tissue of skin and lung by the increased production of matrix
metalloproteinases and decreased collagen production, and this property may help in the
metastasis of oral squamous cell carcinoma.
Nitric oxide (NO) is a diatomic free radical molecule which is generated by a family of
three enzymes called Nitrous oxide synthase (NOS): NOS1, NOS2 and NOS3. NOS2 or
inducible Nitrous oxide synthase (iNOS) is over-expressed in many malignant tumors
including oral squamous cell carcinoma and is shown to facilitate tumor growth and
metastasis.3 Nitric oxide produced by iNOS, upregulates matrix metalloproteinases, which
degrade the extracellular matrix and aid in angiogenesis. iNOS expression has been
found to be more in lungs of rats exposed to cigarette smoke and in lung carcinomas of
smokers, implicating inos expression as a result of tobacco exposure.1
In the light of causative relationship between tobacco and oral squamous cell
carcinoma and the association between NO/ NOS pathway with tumor growth, the effect
of tobacco on NO/NOS pathway is an area that has seldom been studied.
This study will examine the expression of iNOS in oral squamous cell carcinoma in
tobacco habituers and non-habituers in an attempt to understand the role of tobacco in the
behaviour of oral squamous cell carcinomas.
6.2 REVIEW OF LITERATURE
The study was done to compare the levels of iNOS/NO in nonsmall–cell lung cancer
tissue of smokers and non-smokers. The expression was higher in lung cancer tissue of
smokers than that of non-smokers. In smokers the levels of caspase-3 activity was
suppressed which may have lead to the proliferation and growth of lung cancer cells.1
The association between NOS3 and Myeloperoxidase (MPO) polymorphisms and
breast cancer risk by cigarette smoking among post-menopausal women was examined. It
was found that the variant NOS3 genotype associated highly in smokers and significantly
increased in smokers who smoked more than 10/day.2
NOS activity has been detected in tumour cells of various histogenetic origins and has
been associated with tumor grade, proliferation rate and expression of important signaling
components associated with cancer development such as oestrogen receptor. Nitric oxide
has both genotoxic and angiogenic properties. Increased NO generation in a cell may
select mutant P53cells and contribute to tumor angiogenesis by upregulating vascular
endothelial growth factor (VEGF).3
Immunochemistry was used to study iNOS expression in 48 patients with either
extracapsular or encapsulated matastasis in head and neck cancer. iNOS expression was
significantly associated with extracapsular spread than in encapsulated group. It was
concluded that iNOS protein seems to be associated with extracellular spread and
invasion in head and neck cancer.4
A study was done which measured iNOS mRNA ,iNOS protein and NO in oral
squamous cell carcinoma, oral dysplasias and normal epithelium. RT-PCR was used to
quantify and compare iNOS mRNA levels in these tissue specimens.iNOS protein levels
in specimens was evaluated by immunohistochemistry. It revealed increased levels of
iNOS expression in oral squamous cell carcinoma than dysplasia and normal epithelium.5
The hypothesis that oxides of nitrogen in cigarette smoke solution may exert a
negative feedback mechanism upon NO release from epithelial and basophilic cells was
investigated in-vitro. Cigarette smoke solution inhibited both NO production and
degranulation in a dose dependent manner and also decreased expression of NOS mRNA
and protein expression. N-acetyl-cystiene, partially reversed the inhibition of betahexosaminadase release suggesting cigarette smoke solution may act via oxidative free
radicals.6
6.3 OBJECTIVES OF THE STUDY
1. To compare and correlate iNOS expression in oral squamous cell carcinoma in
tobacco habituers and nonhabituers.
2. To correlate iNOS expression with clinical staging of oral squamous cell
carcinoma in tobacco habituers and nonhabituers.
3. To evaluate the association of tobacco and the biologic behavior of oral
squamous cell carcinoma.
7.
MATERIALS AND METHOD
The materials used for this study are :
o Paraffin embedded biopsy specimen of oral squamous cell carcinoma, in habituers
and nonhabituers.
o Leica RM 2125 soft tissue microtome
o Adherent or silicone coated glass slides
o Xylene
o Graded alcohol
o 0.5% Methanoic peroxide
o Tris buffer saline
o Distilled water
o Primary antibody to iNOS (anti iNOS sc 651 Santa cruz biotechnology)
o Secondary detection system (polymer HRP label system)
o Trypsin/ Proteinase K
o Concentrated Hydrochloric acid
o Samsung M 183 DN microwave oven
o Labomed CXR III microscope
o Staining chamber
o Electronic weight balance
o Micropipettes
o Necessary glassware
o Diamino-benzidine (DMB)
o Hematoxylin and eosin
Method: Immunohistochemistry
7.1 SOURCE OF DATA
Material for the present study will consist of archival tissue specimen from the
Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences,
Virajpet. The study sample size will consist of oral squamous cell carcinoma, 20 each in
habituers and nonhabituers.
7.2 METHOD OF COLLECTION OF DATA
1.
Formalin fixed paraffin embedded tissue blocks are the source of material.
2.
Study group will consist of those patients who are nonhabituers and have been
diagnosed as
squamous cell carcinoma from the biopsy specimens of their oral
lesions
3.
The patients who are habituers and have got oral squamous cell carcinoma make
the control group
4.
Sections obtained from the primary tumor of both study and the control group is
stained by hematoxylin and eosin and reassessed.
5.
The sections will be stained using polyclonal antibody against iNOS [Santa Cruz
Biotechnology]
6.
Sections will be placed onto positively charged slides heated upto 50ºC for 40
minutes and will be dewaxed in xylene for 10 minutes and then will be rehydrated
in alcohol solutions and placed 5% hydrogen peroxide in absolute alcohol and
finally water to complete rehydration
7.
For antigen retrieval the slides will be treated in microwave oven and treated with
avidin block.
8.
The sections will be then rinsed with Tris–buffered saline (TBS) and incubated
with secondary detection system (polymer HRP/label system) and slides will be
treated with diamino-benzidine (DMB) and will be counter stained with Mayer’s
Hematoxylin .
9.
Scoring will be done according to the criteria recommended by Brennen PA et al.
7.3 Does the study require any investigation or investigations or interventions to be
conducted on patients or other humans & animals, If so, please describe briefly
No
7.4 Has the ethical clearance been obtained from your institution in case of 7.3?
No
8.
LIST OF REFERENCES
1. Chen GG, Lee TW, Xu H, Yip JHY, Li M, Mok TSK et al. Increased inducible
nitric oxide synthase in lung carcinoma of smokers. Cancer 2008; 112(2): 372381.
2. Yang J, Ambrosone CB , Hong C, Ahn J, Rodriguez C, Thun MJ et al.
Relationships between polymorphisms in NOS3 and MPO genes, cigarette
smoking and risk of post-menopausal breast cancer. Carcinogenesis 2007;
28(6):1247-1253.
3. Xu W, Liu LZ, Loizidou M, Ahmed M, Charles IG. The role of nitric oxide in
cancer. Cell Res 2002; 12(5-6):311-320..
4. Brennan PA, Dennis S, Poller D, Qunitero M, Puxeddu R, Thomas GJ. Inducible
nitric oxide synthase: Correlation with extra capsular spread and enhancement of
tumor cell invasion in head and neck squamous cell carcinoma. Head Neck 2008;
30: 208-14.
5. Connelly ST, Macabeo-Ong M, Dekker N, Jordan RCK, Schmidt BL. Increased
nitric oxide levels and iNOS over expression in oral squamous cell carcinoma.
Oral Oncol 2005; 41: 261-267.
6. Wie XM, Kim HS, Kumar RK, Heywod GJ, Hunt JE, Mcneil HP et al. Effects of
cigarette smoke on degranulation and NO production by mast cells and epithelial
cells. Respir Res 2005; 6: 108.
9.
Signature of the candidate
10.
Remarks of the guide
11.
Name & Designation of (in block letters)
11.1 Guide
DR. SUDHEENDRA U S
(READER)
11.2 Signature
11.3 Co- guide (if any)
11.4 Signature
11.5 Head of the Department
11.6 Signature
DR. SHAILESH KUDVA
(PROFESSOR AND HEAD)\
12.
12.1 Remarks of the Chairman & Principal
12.2 Signature
DR. SEQUEIRA PETER SIMON
(PRINCIPAL)