The Preliminary Study Of Celecoxib Enhances The Sensitivity Of KB/VCR Oral Cancer Cell Lines To Vincristine

Oral cancer is one of the most common malignant tumors in head and neck cancers. In recent years, the incidence of oral cancer has significantly increased and is increasingly affecting more young patients, and is recognized as one of the key factors threatening human health. Oral surgery is the optimal treatment of oral cancer, while chemotherapy serves as one of the important adjuvant therapies for treating oral cancer. Nowadays, the main challenge of chemotherapy is the declining sensitivity of cancer cells towards chemotherapeutics, leading to the occurrence of multidrug resistance (MDR), enabling cancer cells to present resistant capacities against the structure and function of a broad spectrum of chemotherapeutics. 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).P-gp, encoded by MDR-1gene, is a phosphorylated transmembrane glycoprotein originating from ATP-binding Cassette superfamily (ABC). 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, losing efficacy and, finally, generating drug resistance of cancer cells. P-gp exerts different functions in various tissues and 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 cancers indicate that the expression of P-gp is negatively correlated with the sensitivity of cancer cells towards antitumor drugs and survival rate of cancer patients. Some studies suggest 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.Cyclooxygenase (COX), 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 isoforms 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. 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 and UVB irradiation are all stimulators of COX-2expression. It has been demonstrated that the over-expression of COX-2correlates with the incidence and development of malignant tumors, including oral cancer, 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-P2receptors, and up-regulating expression of Bcl-2protein, etc.Recent findings indicate that COX-2expression is positively correlated with P-gp expression in tumor tissues. Besides inhibiting the growth of various carcinomas and inducing apoptosis, COX-2selective inhibitors also 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.In this in vitro experimental study, we investigated the inhibitory effect of Celecoxib on the growth of KB/VCR oral cancer cell lines. We also analyzed the enhancing effect of Celecoxib on the sensitivity of cancer cells to VCR and its relationship to the expression and function of P-gp, which helps understand the underlying mechanism of COX-2inhibitors in enhancing the sensitivity of cancer cells to antitumor drugs.Chapter I Celecoxib enhanced the inhibitory effect of vincristine on the growth of the KB/VCR cellsObjective:This study aims to investigate the influence of COX-2selective inhibitor Celecoxib upon the proliferation of KB/VCR oral cancer cell lines. Methods: KB and 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, PRMI-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 and KB/VCR cells. Then the MTT method was employed to study the inhibitory effect of Celecoxib (10,20,40and80μmol/L) on the proliferation and growth of KB and KB/VCR cells. MTT method was employed to study the inhibitory effect of Celecoxib on KB/VCR cells, which were divided into5groups, which were VCR group (1.5μmol/L), Celecoxib group (10μmol/L), Celecoxib plus VCR group(10μmol/L+1.5μmol/L), Celecoxib plus VCR and PGE2group(10μmol/L+1.5μmol/L+100μg/L) and control group. Jin Zheng Jun’s Method was used to calculate q values, the criterion for evaluating the interaction of Celecoxib and VCR.0.85≤q≤1.15indicates additive effect; q>1.15means synergistic effect; and q<0.85refers to antagonism. Western blot was employed to detect the expression of P-gp. Results: The MTT result indicated:Vincristine (VCR) alone inhibited KB cell proliferation time-and dose-dependently (P<0.01). Inhibitory rates were deferent among VCR at deferent concentration level respectively (P<0.01). Vincristine (VCR) alone inhibited KB/VCR cell proliferation time-and dose-dependently (P<0.01). Inhibitory rates were deferent among VCR at deferent concentration level respectively (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=1.306, P=0.318). VCR in different concentrations and time points, the growth inhibition rate of KB cells were significantly higher than that of KB/VCR cells (P<0.01). Celecoxib alone inhibited KB cell proliferation time-and dose-dependently (P<0.01). Inhibitory rates were deferent among Celecoxib at deferent concentration level respectively (P<0.01). But at a lower dose (10μmol/L), no significant difference was found in KB cell growth inhibition rate after24h,48h and72h treatment (F=0.366, P=0.704). Celecoxib alone inhibited KB/VCR cell proliferation time-and dose-dependently (P<0.01). Inhibitory rates were deferent among Celecoxib at deferent concentration level respectively (P<0.01). But also at a lower dose (10μmol/L), no significant difference was found in KB/VCR cell growth inhibition rate after24h,48h and72h treatment (F=1.769, P=0.225). The growth inhibition rate of KB/VCR cells in VCR plus celecoxib group was significantly higher than that in VCR group, Celecoxib group and Celecoxib+VCR+PGE2group(all P<0.01). All q values (1.160,1.282,1.287) were more than1.15after Celecoxib was combined with VCR treatment for24h,48h and72h, suggesting the combination displays synergistic effect. Western blot showed that the expression of P-gp of KB/VCR cells was significantly higher than that of KB cells (P<0.01). Conclusion:Low doses of Celecoxib could significantly enhance the lethal effect of anticancer drugs VCR on KB/VCR cells, and the combination of Celecoxib and VCR displays synergistic effect. The mechanism may be related to apoptosis and the expression of P-gp. Chapter II Celecoxib enhances the sensitivity of KB/VCR cells to VCR by down-regulating P-gp expressionObjective:This study aims to investigate the mechanism of COX-2inhibitors in enhancing the sensitivity of KB/VCR cells to VCR. Methods:KB/VCR cells were divided into5groups, which were VCR group(1.5μmol/L), Celecoxib group(10μmol/L), Celecoxib plus VCR group(10μmol/L+1.5μmol/L), Celecoxib plus VCR and PGE2group(10μmol/L+1.5μmol/L+100μg/L) and control group. Flow cytometry was used to evaluate P-gp function by detecting the accumulation of active P-gp functional fluorescence substrate within KB/VCR cells. Western blot was employed to detect the expression of P-gp. Results:The expression of P-gp in Celecoxib group and VCR plus Celecoxib group were markedly lower, compared with those in control group and VCR group. The expression of P-gp in Celecoxib+VCR+PGE2group was significantly higher than those in VCR plus Celecoxib group. The Rho123fluorescence intensity of KB/VCR cells in Celecoxib plus VCR group, Celecoxib group and Celecoxib+VCR+PGE2group were significantly higher than those in control and VCR groups (all P<0.01). Conclusion:Celecoxib could enhance the toxicity of VCR against KB/VCR cells. The mechanism probably correlates with the down-regulation of celecoxib on the expression of P-gp.In conclusion, our in vitro study indicates that the COX-2selective inhibitor, Celecoxib, 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. This study provides a better understanding of the mechanisms by which COX-2affects MDR incidence in carcinoma cells and COX-2inhibitor increases the sensitivity of cancer cells to chemotherapy.