SiRNA-mediated COX-2Gene Silencing To Enhance The Chemosensitivity And Inhibit The Prpliferation And Invasion On KB/VCR Cell Lines

Oral cancer is one of the most common malignant tumors in head and neck cancers, accounting for more than90%of oral cancer.In recent years, epidemiological studies indicate the incidence of oral cancer is gradually increasing, and tend to be younger, have become one of the major threats to human health and disease.Today,surgical resectioncombined with radiationtherapyremainsthe primary treatmentforearly stagehead and neckcancer. Chemotherapy is still one of the important adjuvant treatments for OSCC.However,With long-term use of chemotherapy drugs, cancer chemotherapy sensitivity decreased, leading to the development of multidrug resistance (MDR).The currentstudy suggests thatthe multidrug resistancemay be associated withP-glycoprotein(P-glycoprotein, P-gp).The enhanced efflux of chemotherapeutics mediated by P-glycoprotein (P-gp) is focused in the research on the mechanism of drug resistance.P-gp, encoded by MDR-1gene, is a phosphorylated transmembrane glycoprotein originating from ATP-binding Cassette superfamily (ABC).P-gp consists of1280amino acid residues corresponding to a molecular weight of170kd, therefore it is called P-170.P-gp molecule consists of two parts, each of which contains six transmembrane domain and the nucleotide binding domain. P-gp, a transmembrane pump with ATP enzyme activity, is able to pump a substantial amount of compounds, especially hydrated cation compounds, from intracellular to extracellular sites, inducing a decline in intracellular concentrations of P-gp substrate.P-gp exerts different functions in various cells and tissues. P-gp in the intestine mainly affects drug absorption, influences drug elimination and metabolism in kidney and liver, which exerts an impact on drug distribution in the circulating system and the central nervous system. This lowers intracellular drug content to protect the body from drug-induced damages. Research on human cancers show that the expression of P-gp is negatively correlated with the sensitivity of cancer cells towards survival rate of cancer patients and antitumor drugs. Some research show that MDR-1varies in different individuals. However, genetic polymorphism of MDR-1exerts no effect on drug transport and disease progress. With contacting anti-cancer medicine for a long time, MDR-1is induced to significant amplification and yields a substantial amount of P-gp expression.Cycloxygenase (COX) is a rate-limiting enzyme during the conversion of arachidonic acid to prostaglandin (PG) which contains two isoforms:COX-1and COX-2. COX-1is expressed constitutively in most tissues and plays a role in the production of PGs that control normal physiological processes. COX-1maintains self-balancing functions, for instance, renal vascular expansion, gastric cell protection and agglutination-promoting prostaglandin thromboxane production via blood platelet. Several studies have supported the involvement of prostanoids in the pathogenesis of cancer. Invitro studies have demonstrated that growth factors, tumor promoters, and oncogenes induce prostanoids synthesis. In vivo, the metabolism of arachidonic acid, via the COX pathway, has been found to be enhanced in various human tumors and it is now widely accepted that this is due to the induction of COX-2enzyme.COX-2is an inducible enzyme, which is induced by various inflammatory and mitogenic stimuli. These findings have been confirmed analyzing many tumors including pancreas, skin, gastric, bladder, lung, head, and neck cancers, suggesting that COX-2, but not COX-1, may play a pivotal role in tumor formation and growth. More recent studies have confirmed carcinogen promoter agent can stimulate or induce the overexpression of COX-2, and tumorigenesis plays an important role. It is now clear that the tumorigenesis involves COX-2overexpression in most cases, it is not clear if the molecular mechanisms are responsible for this overexpression.Recent studies have found, COX-2and P-gp expression showed a significant positive correlation. Our previous phase study indicated that, after the COX-2inhibitor celecoxib treatment KB/VCR cells, not only can enhance the sensitivity of oral cancer chemotherapy with VCR, but also inhibited the expression of P-gp and it is activity. Further research also showed that:Celecoxib can affect the cell cycle of KB/VCR cells and enhance their apoptosis. Although COX-2inhibitors have been shown to be effectively used for the prevention and treatment of tumors, but long-term use of these drugs remains risk and the bleeding tendency in the gastrointestinal and cardiovascular. There for, to explore a new method to inhibit COX-2expression is important.In this study, we use siRNA silencing COX-2in KB/VCR cells to investigate the relationship between chemosensitivity and the expression of P-gp/MDR-1. And explore the mechanism of COX-2siRNA silencing enhanced the sensitivity of chemotherapeutic in KB/VCR cells. On the other hand we want to study the siRNA silencing COX-2in KB/VCR invasive and affect cell growth.In order to provide experimental evidence for tumor gene silencing therapy. Chapter I siRNA-mediated COX-2gene silencing to enhance the chemosensitivity and regulate P-gp on KB/VCR cell lines.Objective:To investigate the effect of small interfering RNA-mediated COX-2gene silencing to enhance the chemosensitivity on KB/VCR cell lines. Methods: KB/VCR cells at the logarithmic phase were collected. The KB/VCR cells were divided into3groups, which were COX-2siRNA group(Transfection with COX-2siRNA), negative control group(Transfection with negative siRNA), blank control group(Complete medium without any treatment). The expressions of COX-2and MDR-1were examined with RT-PCR, after treatment with the above for48h. Flow cytometry was used to evalate the amount of Rho-123accumulated in KB/VCR cells, after treatment with the above for48h. MTT assay was used to analyze the proliferation of KB/VCR cells, after treatment with the above for48h. Results:(1) The RT-PCR results showed that COX-2siRNA significantly down-regulated the expression of COX-2mRNA and MDR-1mRNA, compared with those in negative control group and blank control group (2) The intracellular fluorescence intensity of Rho-123in COX-2siRNA group were siginificantly higher than those in negative control group and blank control group.(3) The growth inhibition of KB/VCR cells in COX-2siRNA group was significantly higher than negative control group and blank control group (P<0.01). Conclusion:siRNA-mediated COX-2gene silencing can enhance the VCR against KB/VCR cells. The mechanism probably correlates with the down regulation of MDR-1gene and inhibit the function of P-gp.Chapter II siRNA-mediated COX-2Gene Silencing Inhibits the Proliferation and Invasion of KB/VCR Cell LinesObjective:To investigate the effect of small interfering RNA-mediated COX-2gene silencing to change the proliferation and invasion of KB/VCR cell lines. Methods:KB/VCR cells at the logarithmic phase were collected. The KB/VCR cells were divided into3groups, which were COX-2siRNA group(Transfection with COX-2siRNA), negative control group(Transfection with negative siRNA), blank control group(Complete medium without any treatment).The transcription of COX-2gene was examined with RT-PCR, after treatment with the above for48h. The protein expression of COX-2was determined by Western blotting, after treatment with the above for48h. MTT assay was used to analyze the proliferation of KB/VCR cells, after treatment with the above for48h. The cell invasion was evaluated using a transwell assay, after treatment with the above for48h. Results: COX-2mRNA and protein levels were significantly reduced in COX-2siRNA group (P<0.01), compared with those in negative control group and blank control group. The growth inhibition of KB/VCR cells in COX-2siRNA group was significantly higher than other groups(P<0.01), compared with those in negative control group and blank control group. Meanwhile, The invasion of KB/VCR cells in COX-2siRNA group was decreased remarkably, compared with those in negative control group and blank control group. Conclusion:siRNA-mediated COX-2gene silencing canincrease the growth inhibition in KB/VCR cells and down regulate the cell invasion.In conclusion, our in vitro study indicates that the siRNA-mediated COX-2gene silencing enhances the drug toxicity of VCR to KB/VCR cell, which correlates with the events of P-gp expression down-regulation and P-gp function inhibition of KB/VCR cells. On the other hand, siRNA-mediated COX-2gene silencing can increase the growth inhibition in KB/VCR cells and down regulate the cell invasion.This study provides a better understanding of the mechanisms by which COX-2affects MDR incidence in carcinoma cells and COX-2gene silencing increases the sensitivity of cancer cells to chemotherapy.