Autophagy Inhibition Enhances Celecoxib-induced Apoptosis In Osteosarcoma

Purpose: Osteosarcoma(OS)is the most prevalent bone malignancy in childhood and adolescence,with highly aggressive and early systemic metastases.Although great progress has been made in surgical treatment along with chemotherapy,the 5-year overall survival rate for OS has not been significantly improved.Current chemotherapy regimens have only progress slowly,particularly during the last 30 years.Therefore,it is still urgent to develop novel therapeutic approaches for osteosarcoma treatment.Research content: The research focus on the antitumor activity of celecoxib,a selective COX-2 inhibitor,in two OS cell lines 143 B and U2 OS in vitro and in vivo.Furthermore,we explored whether autophagy inhibition via inhibitors(CQ,SAR405)or silencing Atg5 expression enhances the anticancer effect of celecoxib.Methods: Colony formation assay and CCK-8 were used to analyze the cell growth,the migration ability of cells was examined using 24-well Transwell chambers,cell cycle and apoptosis analysis were tested with Flow cytometry.Apoptosis and autophagy related components were tested with western blot,immunofluorescence,fluorescent mitochondrial probe JC-1 test and other technique.Result: We showed that celecoxib inhibits OS cell growth,causes G0/G1-phase arrest,modulates apoptosis and autophagy and reduces migration in OS cells.In addition,the results of fluorescent mitochondrial probe JC-1 test indicated that the mitochondrial pathway mediates celecoxib-induced apoptosis.Significantly,the autophagy inhibitor CQ combined with celecoxib causes greater cell proliferation inhibition and apoptosis.Pharmacologic inhibition of autophagy with another potent autophagy inhibitor SAR405 also enhances celecoxib-mediated suppression of cell viability.These results were confirmed with shRNAs targeting the autophagy-related gene Atg5.In OS tumor xenografts in vivo,celecoxib also presents antitumor activity.Taken together,our results shed light on the function and mechanism of antitumor action of celecoxib for treatment of OS patients.Conclusion: Gene silencing of Atg5 or treatment with autophagy inhibitor CQ or SAR405 markedly enhances cell proliferation inhibition and promotes apoptosis induced by celecoxib.