The Correlation Between The Sensitive Effect Of Celecoxib On The Chemotherapy Of Oral Cancer KB/VCR Cells And Cell Cycle Arrest

Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasm in the head and neck squamous cell carcinomas (HNSCC), accounting for over ninety percent of oral cancers in this region. Recent epidemiological datas indicated that the incidence of OSCC has elevated gradually and the patients have tended to be younger. Currently surgery is the main therapeutic method for patients of OSCC in early stage, assist with chemoradiotherapy which is main for the advanced. Chemotherapy is still one of the important adjuvant treatments for OSCC. However, the decreased sensitivity of cancer cells towards chemotherapeutics after exposure to chemotherapy drugs for long time is the major reason for poor effect, leading to the development of multidrug resistance (MDR). The enhanced efflux of chemotherapeutics mediated by P-glycoprotein (P-gp) is focused in the research on the mechanism of drug resistance.P-gp is one of ATP-binding cassette (ABC) superfamily transport members which is encoded by MDR1gene and located on plasma membrane, and is also the first efflux permeability phosphated glycoprotein. So far it still serves as the characteristics of ABC superfamily. P-gp could transfer the chemotherapeutics out of extracellular backwards with the activity of ATP, thereby reducing the target drug intracellular concentration and limiting their cytotoxicity. It is able to pump a mount of substrates including anthracycline, polypeptide antibiotics, alkaloid and some dyestuffs. P-gp has an abroad distribution in human and was first identified in cancer cells, but later studies have demonstrated that P-gp is also expressed in normal tissue cells including liver, kidney, intestine, pancreas, adrenal gland, capillary endothelial in blood-brain barrier and blood-testis barrier, choroid plexus, placental trophoblastic. Studies have showed that P-gp involved in the absorption and excretion of endogenous substances. The functions of P-gp include:(1) limiting the intestinal absorption of drugs;(2) suppressing the pathway of sensitization once the drugs get into systemic circulation, consequently making the tissue cell insensitive;(3) promoting drug clear and metabolism. In physiological conditions, P-gp plays a role in protection of organism, for example it maintains the blood-brain barrier and transports the exogenous substances in endothelial cells to capillary lumen in order to protect the brain; P-gp in the kidney plays a role in drug excretion; P-gp in the gastrointestinal tract could reduce the absorption of exogenous toxin. In pathological conditions, P-gp is over-expressed in the tissue, consequently inducing MDR. The drugs would combine with P-gp when they get in touch with cancer cells, and then P-gp pumps the substrates out of extracellular backwards utilizing the energy of ATP hydrolysis, thereby reducing the intracellular drug concentration and the cytotoxicity of chemotherapeutics was decreased or even dissolved, thus the tumor cells become drug resistance. The research on human malignancy confirmed that MDR is closely related to the level of P-gp on plasma membrane. It is considered that the expression level of P-gp correlates with the extent of drug resistance in distinct MDR cell lines while the expression is very low in drug-sensitive cells.Cycloxygenase (COX) is the key rate-limiting enzyme in the conversion of arachidonic acid to prostaglandin (PG) which contains two isoforms:COX-1and COX-2. COX-1is expressed in normal tissues and is a "housekeeping" enzyme responsible for various physiological functions. COX-1maintains self-balancing functions, for instance gastric cell protection, renal vascular expansion and agglutination-promoting prostaglandin thromboxane production via blood platelet. Moreover, COX-2is expressed in seminal vesicle, kidney and certain regions of the brain while it is expressed very low or not detected in normal tissues. COX-2can be stimulated by mitogens, cytokines, growth factors, carcinogens and participates in the development of neoplasms and pathogenesis such as cell apoptosis, immunological surveillance, angiogenesis, invasion, metastasis and cell differentiation. The action mechanism of COX-2in the development and progression of neoplasms approximately includes:(1) carcinogens activation;(2) induction of cancer cell proliferation;(3) suppression of apoptosis;(4) stimulation of angiogenesis;(5) enhancement of tumor invasiveness and metastasis;(6) promotion of escape from immune surveillance. Studies have showed that the expression of COX-2significantly correlates with overall survival and prognostic parameter such as tumor size, degrees of differentiation, metastasis situation of lymph node and tumor stage. COX-2is a potential molecular target in prevention and therapy of oral cancer. In addition, it has been demonstrated recently COX-2significantly correlates with the expression of P-gp. Some studies have indicated elevated expression of COX-2seemed to stimulate expression of P-gp. Therefore, it is speculated that the expression of P-gp could been regulated by COX-2in tumor tissue, resulting in decreased sensitivity of cancer cells to chemotherapeutics and multidrug resistance, and COX-2inhibition could block the expression of P-gp and enhance the sensitivity to anticarcinogen.Studies have showed that selective COX-2inhibitor could reduce oncogenesis, growth and metastasis of many neoplasms in vivo and vitro. Celecoxib could inhibit tumor cell proliferation through suppression of COX-2activity. However it is showed that COX-2independent pathway may play a role in antiproliferation of chemotherapeutics, the mechanism of which contains suppression of cell cycle process, induction of apoptosis and restrain of angiogenesis. Studies have also showed that COX-2inhibitors enhance the cytotoxicity of chemotherapeutics. A number of studies have confirmed the combination of COX-2inhibitors with chemotherapeutics significantly suppress the growth of cancer cells in vivo and vitro and COX-2inhibitors increase the sensitivity of chemotherapy partly through inhibiting the expression and activity of P-gp/MRP1. In this study we investigate the correlation between the enhanced effect of celecoxib on the chemotherapy sensitivity of KB/VCR cell lines resistant to vincristine (VCR) and down-regulation of P-gp and the regulation of cell cycle related protein Cyclin D1and p21WAF1/CIP1, to further explore the development of chemotherapeutics resistance in oral cancer and the molecular mechanism of multidrug resistance reversion by COX-2inhibitors based on the previous studies.Objective Investigate the antiproliferation and cell cycle distribution effect of celecoxib and vincristine in KB/VCR cells, and whether celecoxib could enhance the antiproliferation effect of vincristine and change cell cycle distribution of KB/VCR cells. Method KB/VCR cells are cultured in general. The logarithmic phase cells are taken into experiment. There are4groups:(1) celecoxib group:the concentrations of celecoxib are10,20,40and80μM, respectively;(2) VCR group:the concentrations of VCR are0.375,0.75,1.5and3μM, respectively;(3) combined treatment group: preliminary experiment has demonstrated the lower than20μM concentration of celecoxib has no effect on the growth of KB/VCR cells, so10μM concentration of celecoxib combined with1.5μM concentration of VCR is employed for this group;(4) control group:complete medium without any treatment. MTT assay was used to determine the growth inhibition rate and the difference between combined treatment group and separate treatment group was calculated after treatment with the above for24,48,72h. Flow cytometry was employed to analyze the distribution of cell cycle after treatment for24h, according to the introductions of detection kit while the difference of cell number in each phase was calculated. Results MTT assay showed10μM concentration of celecoxib has no effect on the growth of KB/VCR cells (F=1.961, P=0.196) after treatment for24,48,72h, however the growth inhibition rate was increasing in a dose-dependent and time-dependent manner with the enhanced concentrations and prolonged time (all P<0.05). Moreover,0.375μM concentration of VCR has no effect on the growth of KB/VCR cells (F=0.275, P=0.766) after treatment for24,48,72h, however the growth inhibition rate was increasing in a dose-dependent and time-dependent manner with the enhanced concentrations and prolonged time (all P<0.05).10μM celecoxib significantly enhanced the toxicity of VCR against the KB/VCR cells in a time-dependent manner. The growth inhibition rate of KB/VCR cells was37.53±2.05%,46.67±3.17%and54.02±1.53%respectively when treated with celecoxib plus VCR for24,48,72h, which were significantly higher than those treated with celecoxib or VCR alone (all P<0.01). Low concentration of celecoxib could result in cell cycle arrest. It blocked the transition from G1phase to S phase, leading to an increased number in Go/Gi phase, a decreased number in S phase and a corresponding change of cells in G2/M phase, and the effect on G0/G1phase cycle arrest was enhancing in a dose-dependent with the enhanced concentrations (all P<0.01). Each concentration of VCR could result in cell cycle arrest. It blocked the cells in G2/M phase, leading to an decreased number in S phase and a change of number in G0/G1phase (all P<0.05). The combination of celecoxib and VCR regulated the distribution of cell cycle. It blocked the transition from G1phase to S phase. Compared to the VCR group, the number of G0/G1phase cells increased in combined treatment group (56.08±0.46%) accompanied with the decreased number in S phase and G2/M phase (S:22.83±0.20%; G2/M:21.09±0.66%)(P<0.05). Conclusion Both celecoxib and VCR suppressed the growth of KB/VCR cells and resulted in cell cycle arrest, but celecoxib could enhance the antiproliferation effect of VCR. Celecoxib significantly enhanced the toxicity of VCR against the KB/VCR cells in a time-dependent manner which may in part be mediated through cell cycle arrest. Objective Investigate the correlation between the down-regulation of P-gp and the regulation of cell cycle related protein Cyclin D1and p21WAF1/CIPI by celecoxib in order to realize the potential molecular mechanism of sensitization effect of the COX-2inhibitor. Method KB/VCR cells are cultured in general. The logarithmic phase cells are taken into experiment. There are4groups:(1) celecoxib group:the concentration of celecoxib is10μM;(2) VCR group:the concentration of VCR is1.5μM;(3) combined treatment group:10μM concentration of celecoxib combined with1.5μM concentration of VCR is employed;(4) control group:complete medium without any treatment. Western blot was performed to detect the expression of P-gp and the cell cycle related proteins Cyclin D1and p21WAF1/CIP1after treatment for24h. Results Compared to the VCR group, the expression of P-gp and Cyclin D1were significantly lower while p21WAF1/CIPI Was higher after the treatment of celecoxib plus VCR. Conclusion Selective COX-2inhibitor celecoxib enhanced the chemotherapy sensitivity of oral cancer KB/VCR cells via down-regulating the expression of P-gp, Cyclin D1and up-regulating the expression of p21WAF1/CIP1.In conclusion, the enhanced sensitivity of KB/VCR cells to VCR by selective COX-2inhibitor celecoxib may in part be mediated through the down-regulation of P-gp and cell cycle arrest due to modification of Cyclin Dl and p21WAF1/CIP1expression.