Celecoxib Enhances Chemosensitivity Ofovarian Cancer Cells By Inhibiting Mitochondrial P-gp

Objective To investigate the effects of celecoxib,a cyclooxygenase-2(COX-2)inhibitor,on the chemotherapy response of ovarian cancer cells to paclitaxel and cisplatin and the underlying molecular mechanism related to mitochondrial P-gp.Methods 1.MTT assays were used to detect the growth inhibitory effects of celecoxib monotherapy and in combination with paclitaxel or cisplatin for a short time(48h)in an ovarian cancer cell line A2780 and its paclitaxel-resistant line A2780 T.2.The effect of celecoxib monotherapy and in combination with paclitaxel or cisplatin on the apoptosis of A2780 T cells was detected by flow cytometry;the colony formation assays were used to detected the effect of a long-term(10-14 d)use of celecoxib on the colony formation of A2780 T cells at low(10 μM)and high concentrations(100 μM).3.A2780 T cells were treated with celecoxib of different concentrations.The mitochondrion morphology was evaluated under an electron microscopic.A laser confocal microscopy was employed to detect the distribution of mitochondria and the expression of P-gp.4.The mitochondria in A2780 T and its parental A2780 cells were extracted by density gradient centrifugation.The integrity and purity of the mitochondria were confirmed by immunoblotting(WB)and the function of P-gp to pump out Rho123 was detected by flow cytometry.5.The apoptosis-related proteins caspase-3,Bcl-2,Bax and PARP were detected by WB in cells treated with celecoxib,paclitaxel,and cisplatin alone or in combination.Results 1.In the short-term(48 h)treatment,celecoxib monotherapy at the high concentration(100 μM),but not the low concentration treatment,inhibited the viability of A2780 T and A2780 cells(p<0.05)and enhanced the cytotoxicity of paclitaxel and cisplatin(p<0.05).2.The celecoxib treatment induced apoptosis in A2780 T cells in a dose-dependent manner,and high concentration(100 μM)celecoxib enhanced the apoptosis of A2780 T cells induced by paclitaxel or cisplatin(p<0.01).Moreover,celecoxib at both low concentrations(10 μM)and high concentrations(100 μM)inhibited the colony formation of A2780 T cells(p<0.01,p<0.001).3.The celecoxib treatment induced a pre-apoptotic morphology of mitochondria in A2780 T cells with mitochondria aggregation around the nucleus.4.The purity and integrity of the mitochondria extracted were verified.Compare to the untreatment group(64.77%),the celecoxib treatment decreased the Rho123 efflux rate of mitochondria(31.6%),similar to the effect of cyclosporine A(19.71%),a specific inhibitor of P-gp.5.The celecoxib treatment significantly decreased the P-gp expression in A2780 T cells at both mRNA and protein levels;this inhibition was not dependent of celecoxib concentration or treatment time.The mitochondria P-gp protein was also be decreased by the celecoxib treatment.6.Caspase-3 was significantly activated by celecoxib.PARP was significantly activated when combined with paclitaxel.The Bcl-2 expression was significantly inhibited in cells treated with celecoxib combined with cisplatin.Conclusion Celecoxib enhances the sensitivity of ovarian cancer cells to paclitaxel and cisplatin,which may partially due to the expression and function inhibition of mitochondrial P-gp that leads to imbalance of mitochondrial homeostasis,thus initiating the mitochondrial apoptosis pathway.