Therapeutic Effect Of Celecoxib Combined With Sorafenib On Renal Cell Carcinoma

?Objective?:To investigate the therapeutic effects of celecoxib combined with sorafenib on renal cell carcinoma cells and xenograft in nude mice model and to observe the inhibitory effects of combination therapy.?Methods?:1.In vitro:(1)ACHN cells were treated by sorafenib after different treat times.The changes of COX-2 protein levels were detected by western blotting(WB).To explore the impact of COX-2 protein level on sorafenib sensitivity,we knocked down COX-2 by small interfering RNA(siRNA)in ACHN cells.COX-2 levels were evaluated by real-time quantitative polymerase chain reaction(RT-qPCR)and WB.Cells survival was detected by CCK-8 assay,and cells apoptosis percentages were measured by AnnexinV-PI staining.(2)Renal carcinoma cells were treated with different concentrations of celecoxib and sorafenib alone or a combination treatment with low dosage of celecoxib and sorafenib.Cells survival was detected by CCK-8assay,and cells apoptosis percentages were measured by AnnexinV-PI staining.2.In vivo:ACHN cells were inoculated into nude mice subcutaneously to produce tumor-bearing models.Xenograft tumor mice were randomly divided into a negative control group,a celecoxib group,a sorafenib group,and a combination treatment group.Tumor measurements were taken every 2 days,and tumor volume(V)was calculated using the ellipsoid formula V=D(d~2)/2,where D is the largest and d is the smallest diameter of the tumor.At the end of the experiments,the animals were killed,and the tumor growth curves were plotted.Immunohistochemistry was used to detect the expression of Ki-67 in all groups of nude mice.?Results?:1.In vitro experiments:Western blotting showed that COX-2 protein levels were significantly elevated after sorafenib treatment.The cytotoxicity of sorafenib was greatly attenuated when cellular COX-2 expression was reduced by small interfering RNA(siRNA).CCK-8 results showed that celecoxib or sorafenib dose-dependently inhibited renal carcinoma cells growth.The inhibitory effect of sorafenib and celecoxib using together was much better than that of using sorafenib alone.Flow cytometry results showed that celecoxib could obviously promote the cells apoptosis induced by sorafenib.2.In vivo experiment:The growth curve of transplanted tumors in nude mice showed that celecoxib and sorafenib could inhibit the tumor volume.The combination treatment of sorafenib and celecoxib significantly slowed the tumor growth of ACHN xenografts than single drug treatment group.The results of immunohistochemistry showed that celecoxib and sorafenib could inhibit the number and proportion of Ki-67positive cells.The combination treatment of sorafenib and celecoxib significantly inhibited the number and proportion of Ki-67 positive cells of ACHN xenografts than single drug treatment group.?Conclusions?:1.Sorafenib can promote the expression of COX-2 in renal carcinoma cells.The cytotoxicity of sorafenib was attenuated when cellular COX-2expression was reduced by small interfering RNA(siRNA)2.Celecoxib combined with sorafenib can effectively inhibit cells proliferation and induce the apoptosis of renal carcinoma cells.The combination therapy has synergistic effect.