Abstract: in 2012 and death due to cancer is

Abstract:

Cancer is one of the most leading cause of mortality around the Globe, the incidence of cancer is about 14.1 million has been reported in 2012 and death due to cancer is about 8.2 million worldwide in which thirty-three percent of death is due to smoking and tobacco. Oral cancer is any kind of malignant neoplasm that is may be present in oral cavity like on the floor of mouth, on the lip and on the tongue. Oral Cancer is arising due to the mutation in mainly in proto-oncogene, tumor suppressor gene (p53) and activation of PI3K pathway which activates mTOR pathway and leads to Cancer. Metformin a member of biguanide is used to treat mainly for non –insulin dependent diabetes mellitus some reports showing its beneficial role of metformin in different type of cancer. The basic understanding towards how metformin affect the altered signaling is still unclear this study is to know the basic mechanism of metformin in oral cancer biology and to determine its therapeutic efficacy in oral cancer.     

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Introduction:

Metformin is an oral antihyperglycemic agent that is used for the treatment of Non-insulin dependent diabetes mellitus metformin which sensitize the insulin receptor and it increases the uptake of peripheral glucose and utilization (1).Metformin regulates blood glucose level by decreasing the amount of blood sugar which is produced by liver and absorption by intestine (2). Metformin a biguanide a drug that is used to treat hyperglycemia and inhibit liver gluconeogenesis and increase the uptake of glucose in muscles. (Zhou2001).

Oral Cavity cancer includes cancer of squamous cell carcinoma, Verrucous carcinoma, lymphomas and tumors of oropharyngeal tissue. Oral squamous cell cancer (OSCC) is the most predominant cancer of oral cavity which accounts for about 90% of  Oral cancer. Oral squamous cell carcinoma which includes the lip and tongue tissue 3 329 full). Tobacco use is of the main cause of squamous cell carcinoma chewing tobacco and smoking is responsible for oral cancer in india (warnakulasuriya) apart from tobacco Human papillomavirus is also playing role in development of oral cancer. https://doi.org/10.1093/jnci/djg107).

Current therapy of Oral squamous cancer carcinoma  is dependent upon the severity of the cancer early stage cancer is treated with chemotherapy but advanced cancer is treated by multidirectional approaches and include resection of large tumors and chemotherapy

  Oral cancer is caused due to the mutation is some specific gene those help in cell cycle regulation the proto-oncogene which helps in normal cell division is unregulated by conversion of proto-oncogene in to oncogene which affect normal cellular process of division (3) in various studies it has been observed that due to the up regulation of genes involved in cell cycle like Cyclin D1, CDK, AMPK pathway altered during the oral cancer (Oncogene (2008) 27, 3576–3586). recent studies also show correlation of mTORC I in oral cancer. mTOR pathway

Origin of proposal and rationale of the study:

The current cancer therapy is based on surgery and radiotherapy, chemotherapy, biological or immune therapy, hormonal therapy which aim to inhibit cell proliferation 2. Cisplatin, cisplatinum, or cis-diamminedichloroplatinum (II), is a well-known chemotherapeutic drug. It has been used for treatment of oral cancers.  Its mode of action is it inhibits DNA repair mechanisms, which causes DNA damage, and subsequently inducing apoptosis in cancer cells. However, its side effects lead to this drug a less preferred drug for cancer so new approaches need to be done to cure oral cancer. https://doi.org/10.1016/j.ejphar.2014.07.025

Hypothesis:

Metformin which is already used as a hyperglycemic agent have showing promising effect against different type of cancer remodeling of this drug for cancer treatment may reduce the dependency of other chemotherapy which has several side effect and also painful to the patients. Metformin may inhibit the growth of oral cancer by decreasing the expression of various gene which tumors in their growth 

 

 

 

 

Review of literature: Cancer is a group disease which promotes uncontrolled regulation of cell cycle. Cancer is a leading disease in all around of the world the incidence of cancer is high in India according to the data of National institute of cancer preventation and research India   over seven lakh new case registered with cancer with a prevalence of 2.5 million cases. Oral cancer is most frequent cancer in men and third most frequent in both male and female. Around 80% of oral cancer is due by tobacco use. (3,4) The hyperglycemic drug metformin has been successfully used for treatment of type II diabetes and there is increasing evidence that indicating metformin is an anticancer agent that exhibit chemotherapeutic activities (1-4). It has been observed  that cancer rates in diabetics using metformin are lower than in patients using other insulin sensitizing agents (2,5-12). In report, the overall survival rate  of type II diabetic patients with colorectal cancer was 76.9 months for individuals treated with metformin compared to 56.9 months for those receiving other diabetic medications and this represented a 30% improvement in overall survival (6). A comparable study in pancreatic cancer patients showed that diabetics using metformin had a 32% lower risk of death and longer overall survival than diabetics using other drugs (10). This latter paper recommended using metformin as a supplemental  therapy for treatment of pancreatic cancer patients (10). The clinical application of metformin as an anticancer agent also showed in some reports. Both Invivo and Invitro result showing that metformin induces apoptosis and restrict the growth of various types of cancer. Some studies showing that metformin induces AMPK pathway which inhibit the expression of mTORC I and it block mTOR pathway which leads to cell for apoptosis

              Metformin anticancer properties have contributed by several mechanism such as- 1Activation of AMPK Pathway 2. By cell cycle arrest and induction of apoptosis via activation of p53 3. Inhibition of mTOR pathway by blocking action of mTORC I protein which is play important role in mTOR signaling

The major effect of metformin on cancer cell is mediated by AMP-activated protein kinase that play significant role in cell metabolism. AMPK upregulated  by metformin which subsequently inhibit mammalian target of rapamycin complex I and that downregulated the mTOR pathway in cancerous cells. AMPK pathway is a ser/thr kinase which is responsible for cell growth during starvation condition it is activated in cancerous cell which activates apoptosis.

Apart from AMP kinase dependent pathway metformin has role in cell cycle regulation by blocking the cell cycle between G? to G1 phase and without inducing apoptosis it blocks cell cycle progression 38 Metformin also induced cell death by activation of caspase pathway 39Metformin reduce the level of cyclin D in cancer cell which shows its antiproliferative activity towards cancer.    

Specific objective:

1.     To investigate anti proliferative activity of metformin on oral cancer cell lines.

·         MTT assay (Cytotoxicity)

·         Annexin V-FITC apoptosis assay

     2. To investigate the signaling pathway that modulates the growth of oral cancer on    metformin exposure

·         Check the expression levels of Cyclin D1, CDK, AMPK, mTOR, p70RSK and p53

·         Expression of EMT markers to check metastatic growth reduction

·          RNA Isolation and Real-Time Polymerase Chain Reaction- of different protein                 contributing in the growth and proliferation of cancer and altered pathway will be performed to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA.

·         Immunoblotting can be performed for the various proteins of the signaling pathway including both cytoplasmic and nuclear protein and the proliferation regulating and

Work plan

The study will be performed for following two objectives. The experimental set up for each objective is as follows.

Objective # 1: To investigate anti proliferative activity of metformin on oral cell lines.

Cell proliferation assay (Cell Viability (MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide) Assay)

 To measure cytotoxicity, oral cancer cells will plate  in a 96-well plate. After treatment with metformin, viable cells will stain with MTT. The media will then remove, and Formosan crystals dissolve in dimethyl sulfoxide (DMSO). Measure the absorbance at 570 nm

Ø  Both time dependent and dose dependent study will be performed after cytotoxic assessment.

 

Annexin V- FITC apoptosis assay:

To measure cell death rate Annexin V-FITC dye which binds to phosphotidyl serine which present on the surface of cell during apoptosis. At the onset of apoptosis phosphotidyl serine translocated to external surface and serve as a signal for phagocytes so redistribution of phosphotidyl serine on the outer surface of cell binds with annexin V and its conjugate can be used for the detection of apoptosis because they interact strongly and specifically with exposed phosphotidyl  serine.

·         Both time dependent and dose dependent study will be performed

Objective #2: To investigate the signaling pathway that modulates the growth of oral cancer on metformin exposure

For the investigation of signaling oral squamosal  cancer cells we will be checked the Expression of following proteins: Check the expression of various proliferation markers like Cyclin D1 CDK, AMPK, p53, p70RSK and mTOR  at m-RNA ( using RT-PCR) as well as protein level (Western blotting).

Western Blotting

Western blotting will be used to evaluation and validation of differential proteins for in our study. Sample will be separated into  SDS gel and then electro transferred onto nitrocellulose membrane . Membranes will be incubated with corresponding primary Finally, reactivity will be visualized by horseradish peroxidase conjugated antihuman immunoglobulin as secondary antibody using gel imaging system.

RT-PCR

RNA extracted from sample and used for synthesis of cDNA by Reverse transcriptase. RT-PCR will perform in a fluorescent quan­titative PCR using specific primers for  proliferation primers and then  Agarose gel electrophoresis will be used for the separation of amplified.

Expected Outcome:

Despite of advancement in medical field there is limited option available for cancer patients day by day due to unhealthy life style and also the bad habits causes disease like cancer so to cure cancer some novel therapy against cancer is needed. Present approved therapies are costly as well as painful; chemotherapy and radiotherapy against oral cancer have many side effects to patients and advance stage of cancer involved removal of tumor that impact to patient economy also so some new therapeutic agents needed in current situation.

Metformin which is an old drug and used for treatment of diabetes mellitus type II now showing some good results against cancer so by repurposing of metformin we can used this drug against oral tumors. Epidemiological studies already showing promising effect of metformin according to reports metformin reduce the risk of cancer in diabetes patients significantly. Both In vivo and In vitro studies indicating that positive results and confirming that metformin has anti proliferative activities. So the combination of present drug and metformin may show some synergistic effect against cancer growth and reduce its aggressiveness. Metformin showing reduce expression of those gene which have role in oral carcinogenesis so it inhibit mTOR pathway and cyclin D1 expression in other type of cancer so this study should give details study of metformin in oral cancer and it also try to elucidate the signaling involved in oral cancer.