Effects Of Nitric Oxide On Human Oral Squamous Cell Carcinoma Tca8113 Cell Line

Oral and maxillofacial cancer is one of the commolon neoplasms in china, which occurs 5.6% in the malignancies. Radical surgery is the most effective strategy for curing the cancer patients. However, patients with advanced and/or metastatic lesions have poorer prognosis even treated actively, with mean survival of 6 months, 50% patients will be faced with recurrence. Although multiple risk factors are involved in the growth and development of OSCC, the exact molecular mechanisms responsible for mutational events leading to this disease remain enigmatic. It is becoming increasingly evident that oxidative/reductive (redox) DNA damage existingchronic inflammolation plays an important role in oral carcinogenesis.Nitric oxide (NO) is an unstable free-radical molecule involved in a large variety of pathophysiological processes. Because of its redox properties, the role of NO in tumor biology has gained much attention in recent years. Endogenous NO is synthesized by three isoforms of nitric oxide synthase (NOS), each being theproduct of a distinct gene. Inducible nitric oxide synthase (iNOS) is one of three key enzymes generating nitric oxide (NO) from the amino acid 1-arginine. INOS derived NO plays an important role in numerous physiological (e.g. blood pressure regulation, wound repair and host defense mechanisms) and pathophysiological (inflammolation, infection, neoplastic diseases, liver cirrhosis, diabetes) conditions. iNOS is the synthase isoform most commolonly associated with malignant disease. Nevertheless, the role of iNOS during tumor development is highly complex, and incompletely understood. Both promoting and deterring actions have been described, presumably depending upon the local concentration of iNOS within the tumor microenvironment. In particular, pivotal effects such as malingnant transformation, angiogenesis, and metastasis are modulated by iNOS. Hence, therapeutically interference with iNOS activity is of considerable interest, especially in tumors where metastatic activity, host defence mechanisms and the level of differentiation seem to be correlated to iNOS expression, hi a variety of human malignant tumors, e.g. breast, lung, prostate, bladder, colorectal cancer, and malignant melanoma, expression of iNOS can be observed, hi most tumors, there is a higher expression and activity compared to adjacent normal tissue. However, it must be said that that there are many conflicting reports and that increased levels of iNOS are not a ubiquitious finding in human cancer. Although increased expression and activity of NOS were also observed in head and neck squamous cell carcinoma (HNSCC), the role of NO in the growth and metastasis of OSCC is still poorly understood.CDDP has been widely used in the head and neck cancer. With a moderate sensitivity of 44.4% in OSCC reported by in vitro studies, 50% patients will be resistant to this chemoreagent. Therefore, it is feasible to help the phycisions choose a better chemotherapy programm that will be sensitive to cure the cancer so as toimprove the patients'outcome if we may elucidate the possible mechanism of NO induced chemoresistance in CDDP treated patients.The present study consists of three parts, and focuses on studying the dual role of NO and the relative mechanisms in oral carcinogenesis.Section I: Investigations of iNOS induced apoptosis in oral squamous cancer and changes of p53 expressionThis section tried to investigate the effects of apoptosis induced by exogenous NO in Tca8113 cells and the possible mechanism. SNP as NO donor was used to treat the cells. Cytotoxic and apoptotic effects of NO on Tca8113 cells were examined by using MTT assay, acridine orange (AO) staining, Wright-Giemsa staining, agarose gel electrophoresis and flow cytometry, Western blotting was performed for investigating the apoptotic mechanism. Our results observed that NO had a remarkable proliferation inhibiting effect on Tca8113 cells. After being exposed to exogenous NO, Tca8113 cells showed series changes of morphology similar to apoptosis such as cell shrinkage, nuclear condensation, DNA fragmentation, G2/M phase arrest and upregulation of the tumor suppressor P53 protein. Our study demonstrates that exogenous NO has a proliferation inhibition and apoptosis induction effect on Tca8113 cells in concentration and time-dependent manner, P53 protein may be involved in the regulation of apoptosis induced by NO.Section II: Establish a cell line that can stably express iNOSin oral cancer cell lineAims: to establish a cell line that may stably express iNOS in Tca8113 cell line.Methods: PCMV-INOS and control vector PCMV were bestowed from Prof Liu Jing Sheng at china national medical institute. Plasmid pCMV-iNOS was transfected with Tca8113 cell line using lipofectamine, control plasmid was alsotransfected using as a control. We detected the transfection efficiency by eletrophoresis and western blot. The growth curve was assessed by MTT assay. NO concentration was measured using NO enzyme detection kit. Cell cycle was evaluated using flowcytometry.Results: three bands with 307bp, 3628bp and 5409bp were achieved when pCMV-iNOS plasmid was digested by EcoR I, respectively, which was agree with Prof Liu's report. After G418 selection for 2 weeks, a cell clone that highly expressed iNOS was selected and proliferated. We then detected the NO concentration among the iNOS transfectant, control vector transfectant and wild cell line Tea, the concentration was 121.74±39.45, 268.12±87.16 and 115.53±32.24 nmol/L after two days cultivation, respectively. INOS transfectant grow slight quickly than the wild type cell line, however, there was no statistical significance. The proliferation index was 0.5 for pCMV-iNOS/Tca, 0.39 for Tea and 0.32 for pCMV/Tca, the difference reach significance (p<0.05).Conclusions: we successfully established a cell line that could highly express iNOS, pCMV-iNOS/Tca. Furthermore, this transfectant showed higher proliferation behavior than the wild type cell line.Section III: The role of iNOS on CDDP induced apoptosis as well as cell adhesion, migration and carcinogenesis in Tca8113 cell lineAims: To investigate the role of iNOS on CDDP induced cell apoptosis as well as cell adhesion, migration and carcinogenesis in Tca8113 cell line.Methods: CDDP induced apoptosis was detected by DNA fragmentation assay and flow cytometry. Cell migration was evaluated by wound assay. Carcinogenesis was investigated using nude mouse model in vivo. Comparisons of E-cadherin, P27, P53 and iNOS were made by western blot and tissue fluorescent stain.Results: Cell apoptosis using DNA fragmentation assay was detected among the transfectant, control vector transfectant as well as Tca8113 cell line when treated with CDDP. However, the apoptotic rate among them showed no statistical significance using flowcytometry. Furthermore, there was no difference of cell migration among the three groups. The effects of iNOS on tumor growth were evaluated in vivo by using Tca8113 animal model. Results iNOS transfected cells grow quickly than the vector only transfectant and wild type of the cell. Moreover, increased G2/M and S phase cells were observed in iNOS transfectant in comparison with the other two groups. The mean tumor weight in iNOS group was 2.5±0.48g, which was significant higher than that of vector only group(l.l ±0.24g) and wild group (1.3±0.32g). When treated with L-NAME, inhibitor of NOS, iNOS was decreased in expression. However, E-cadherin was not changed. CDDP may induce cell apoptosis either the cells are transfected with NO or not. However, decreased cell adhesion is observed in transfected cells. This result may imply that iNOS may have a trival effect on CDDP induced Tca8113 cell apoptosis, nevertheless, E-cadherin, p27 as well as p53 may play different role on regulating chemoreagent induced cell apoptosis. These results were confirmed by using tissue fluorescent stain.Conclusions: iNOS transfectant showed higher proliferation behavior in vitro and higher potential of tumorgenesis in vivo than the other two types of cells, even though it did not influence cell migration. On the other hand, iNOS did not appear to effect cell apoptosis, E-cadherin, P27 as well as p53 may have different role on regulating cell apoptosis.In conclusion, NO plays a multiple and complex role in oral squamous cell development. We observed that exogenous NO might inhibit cell growth and inducecell apoptosis in Tca8113 cell line. However, when NO was endogenously supplied by cell transfection, interestingly, it does not make effects on cell growth and apoptosis. These results demonstrated the possible role of NO in oral squamous cell apoptosis, furthermore, it also implied that cell microcircumstance may influence the efficiency of chemotherapy. Our results showed that it is advisable to using iNOS for NO provider, which may fully simulate the biological process of NO in vivo. However, this method cannot mimic the concentration of NO in vivo, further investigation on this issue is warranted.