Study On The Effect Of Celecoxib Enhancing Chemosensitivity And Inducing Apoptosis Of KB/VCR Cell Lines

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in head and neck cancers, which accounts for about80%of oral and maxillofacial malignant tumors. In recent years, the incidence of oral cancer has significantly increased and is increasing affecting more young patients, and is recognized as one of the key factors threatening human health. Currently oral surgery is still the optimal treatment of oral cancer, while chemotherapy and radiotherapy serves as the important adjuvant therapies for treating oral cancer. However, due to the generation of multidrug resistance (MDR), the sensitivity of cancer cells towards chemotherapeutics is declining. It has been recognized that the emergence of MDR by cancer cells significantly correlates with the over-expression of membrane pump proteins, including P-glycoprotein (P-gp), simultaneously with enhanced role of anti-apoptotic in cancer cells.P-gp, encoded by MDR-1gene, is a phosphorylated transmembrane glycoprotein originating from ATP-binding Cassette superfamily (ABC). Some studies has confirmed that MDR-1varies in different individuals. However, genetic polymorphism of MDR-1exerts no effect on drug transport and disease progress. Following long-term contact with anti-cancer medicine, MDR-1is induced to significant amplification and yields a substantial amount of P-gp expression. 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. When tumor cells encounter antitumor drugs, liposoluble drugs enter cells via concentration gradient effect. Following combining with P-gp, liposoluble drugs are constantly pumped outside, powered by ATP hydrolysis, which leads to continuous decrease in intracellular drug levels. Thus, the toxic effect of drugs on cancer cells is weakened gradually, even losing efficacy and, finally, generating drug resistance of cancer cells. P-gp is not abnormal protein and it exerts different functions in various tissues and cells. P-gp can be expressed in a variety of tissues such as the brain, liver, intestine, kidney, adrenal, and testis, and can also be expressed in peripheral blood leukocytes, including T, B lymphocytes, and natural killer cells. P-gp existing in the intestine mainly affects drug absorption, influences drug metabolism and elimination in liver and kidney, and exerts an impact on drug distribution in the central nervous system and the circulating system, which lowers intracellular drug content to protect organic from drug-induced damages. Studies on human cancer indicate that the expression of P-gp is closely related to the reducing sensitivity of cancer cells towards antitumor drugs.Cyclooxygenase (COX), or prostaglandin endoperoxide synthase (PGES), a rate-limiting enzyme during the conversion process from arachidonic acid to prostaglandin (PG), directly affects PG synthesis and participates in multiple physiological and pathological events. Currently, there are two isforms of COX: 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. Cyclooxygenase-2(COX-2), or prostaglandin endoperoxide synthase-2(PGES-2), is the inducible isform of cyclooxygenase. COX-2is undetectable in most normal tissues (except for the central nervous system, kidneys, and seminal vesicles), but is induced by various inflammatory and mitogenic stimuli. Growth factors (EGF, PDGF), pro-inflammatory cytokines (IL1β, IL2,TNF), tumor promoters, bile acids, endotoxin, nitric oxide and UVB are all stimulator of COX-2expression. It has been demonstrated that the over-expression of COX-2correlates with the incidence and development of malignant tumors, which leads to the incidence of epithelial cells canceration regarding following aspects, oncogenesis, enhanced adherence of extracellular matrix, anti-cellular apoptosis, down-regulating expression of E-cadherin and TGF-β2receptors, and up-regulating expression of Bcl-2protein, etc. In addition, cancer cells with overpression of COX-2can regulate cell growth and differentiation, and produce large numbers of angiogenesis factor and stimulate the formation of blood vessels, ect.Cell apoptosis is an important mechanism that determines cell development and the balance of organization. Apoptosis is similar to the division and differentiation of cells that is regulated by several genes and it is a result of the cascading gene expression. Bcl-2is a kind of oncogenes, which is related to the extension of cell life, tumor progression and the inhibition of apoptosis. Members of the Bcl-2family of proteins are significant regulators of apoptosis and can be divided into discrete groups of molecules, those that are pro-apoptotic (Bax, Bak, Bad, Bid) and those that are anti-apoptotic (Bcl-2, Bcl-x, Mcl). Overexpression of Bcl-2and Bcl-xl is one of the important reasons for oncogenesis and multidrug resistance. Apoptosis is closely related with the balance between the pro-and the anti-apoptotic members of the Bcl-2family. The level of Bcl-2expression is increasing while decreasing of Bax in most tumors. Cell apoptosis can induced by multiple factors can be inhibited through upregulating Bcl-2or downregulating Bax. Otherwise, it can be promoted through upregulating Bax or downregulating Bcl-2.Recent findings indicate that the expression of COX-2is positively correlated with cell apoptosis resistance. Selective COX-2inhibitors can inhibite the growth of various carcinomas and induce apoptosis, and at the same time it can enhance the toxicity of anti-cancer drugs towards carcinoma cells. The existing mechanisms of COX-2inhibitors resisting tumor activity include suppressing the cell division cycle, inducing cell apoptosis, and inhibiting tumor angiogenesis. Studies have shown that through reducing P-glycoprotein, Bcl-xl, and Bcl-2expression and inducing translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol, low concentrations inhibitor of COX-2, celecoxib, activated caspase-3, promoted cells going into apoptosis and enhanced the sensitivity of tumor cells to chemotherapy drugs.Our previous studies have confirmed that Celecoxib, a selective inhibitor of COX-2, enhanced the sensitivity of KB/VCR cells to VCR, and the effect was related to the expression and function of P-gp. Based on the observation, we investigated the correlation between enhancing the sensitivity of KB/VCR cells to VCR and inducing cell apoptosis in the study.Chapter Ⅰ Celecoxib enhanced the inhibitory effect of vincristine on growth of the KB/VCR cellsObjective:To investigate the influence of COX-2selective inhibitor Celecoxib plus VCR upon the proliferation of KB/VCR oral cancer cell lines. Methods: KB/VCR cells at the logarithmic phase were collected, incubated into a96-well plate at a concentration of2×103cells, and cultured for24h. Following the attachment of the cells to the wall, RPMI-1640medium containing vincristine (VCR)(0.375,0.75,1.5and3μmol/L) and control medium without VCR (control group) were supplemented respectively. Following24h,48h and72h culture, MTT method was employed to study the inhibitory effect of VCR on the proliferation and growth of KB/VCR cells. Then the MTT method was employed to study the inhibitory effect of Celecoxib (10,20,40,80μmol/L) on the inhibitory rate of KB/VCR cells growth. In the study, after KB/VCR cells were treated with Celecoxib (10μmol/L), vincristine (VCR)(1.5μmol/L), and Celecoxib puls VCR(10μmol/L+1.5μmol/L) for24h,48h, and72h, MTT assay was used to measure the inhibitory rate of each group. The MTT result indicated:after KB/VCR cells were treated with VCR at different concentration for24h,48h, and72h, the effect of inhibition was increasing in a dose-dependent and time-dependent manner with the increasing of concentration (P<0.01). At a lower dose(0.375μmol/L), no significant difference was found in KB/VCR cell growth inhibition rate after24h,48h, and72h treatment(F=3.040, P=0.098). After KB/VCR cells were treated with Celecoxib at deferent concentration for24h,48h, and72h, the effect of inhibition was increasing in a dose-dependent and time-dependent manner with the increasing of concentration (P<0.01). At a lower dose(0.375μmol/L), no significant difference was found in KB/VCR cell growth inhibition rate after24h,48h, and72h treatment(F=1.133, P=0.364). After24h,48h, and72h treatment, the growth inhibition rate of KB/VCR cells in Celecoxib (10μmol/L) puls VCR (1.5μmol/L) group was significantly higher than that in VCR group and Celecoxib group, existing significant difference(P<0.01). Conclusion:Low doses of Celecoxib puls VCR could significantly enhance the effect of KB/VCR cell growth inhibition.Chapter Ⅱ Celecoxib enhanced the sensitivity of KB/VCR cells to VCR and induced apoptosis of KB/VCR cell linesObjective:To study the mechanism of cyclooxygenase-2(COX-2) inhibitor Celecoxib in enhancing the sensitivity of KB/VCR cells to VCR and inducing apoptosis of KB/VCR cell lines. Methods:KB/VCR cells were divided into several groups, which were VCR groups (0.375,0.75,1.5and3μmol/L), Celecoxib groups (10,20,40and80μmol/L), and VCR puls Celecoxib (1.5μmol/L+10μmol/L). After24h, Annexin V-FITC/PI double labeling method was employed to detect early stage cell apoptosis. Western blot was employed to detect the expression of Bcl-2and Bcl-xl. Results:FCM analysis of AV-FITC/PI-double labeled Annexin revealed that the rate of apoptosis in Celecoxib puls VCR group was significantly high compared with VCR group (P<0.01). The expressions of Bcl-2and Bcl-xl in Celecoxib plus VCR group were markedly lower, compared with other groups(P<0.01). Conclusions: Selective COX-2inhibitor Celecoxib could enhance the sensitivity of KB/VCR to VCR by down-regulating the expression of Bcl-2and Bcl-xl and inducing cell apoptosis.In conclusion, we found small dose Celecoxib could enhance the toxic effect of drugs on KB/VCR cells to VCR. The mechanism is related to enhancing the rate of apoptosis, and down-regulating the expression of Bcl-2and Bcl-xl.