Inhibition Autophagy Promotes Dimethyl Celecoxib(DMC)-induced Apoptosis In Nasopharyngeal Carcinoma Cells

Objective:Nasopharyngeal carcinoma is one of the most common cancers with poor prognosis due to its high degree of malignancy,advanced patients,recurrence and metastasis,and radio/chemotherapy resistance,therefore,it is urgent to find new and reliable therapeutic strategies for nasopharyngeal cancer.Over the past years,a lot of studies have shown that 2,5-dimethylcelecoxib(DMC),a derivative of celecoxib,has a good anti-tumor effect in many tumors.However,The anticancer effect of DMC has not been reported in nasopharyngeal carcinoma.Autophagy is a double-edged sword in cancer treatment.On the one hand,it induces autophagic cell death.On the other hand,it is now more and more recognized that protective autophagy induced by chemotherapy or radiotherapy can inhibit tumor cells apoptosis.Therefore,our mainly aim is to explore whether DMC can be an effectively anticancer drug in nasopharyngeal carcinoma both in vitro and in vivo and investigate the effect of DMC on apoptosis and autophagy regulation in nasopharyngeal carcinoma.This will provide new and more effective strategies for DMC in nasopharyngeal carcinoma.Method:1.Cell viability assay:The inhibition ratios of DMC-treated CNE-2/CNE-2R cells at different concentrations(0,20,40,60,80,100μM)was detected by MTT assay for 24h,48h,and 72h;the cells survival after different DMC concentrations(0,20,40μM)treatment for 12 days was detected by Clone formation assay.2.In vivo experiment:The tumor size and weight of nude mices were observed after DMC(0,20,40 mg/kg)treatment in subcutaneous tumor models.3.Apoptosis analysis:The apoptosis ratio of cells treated with different drugs,includes DMSO,DMC(20,40μM),CQ(20μM),and DMC+CQ(20+20μM)groups,was tested by Flow cytometry.The expression of Bax,Bcl2,cleaved PARP,caspase3,cleaved caspase3 and survivin were detected by western blotting after the cells treated with different drugs,includes DMSO,DMC(20,40μM),CQ(20μM),and DMC+CQ(20+20μM)groups.4.Autophagosomes detection:The protein expression of LC3A/B and P62 after different treatment were tested by western blotting in vitro and in vivo.The LC3A/B fluorescent spots after different treatment were tested by immunofluorescence.Transmission electron microscopy were used to observe the change of double-layered acidic autophagic vesicles in cells after DMC treatment.5.Autophagic flow detection:mRFP-GFP-LC3 adenovirus infected CNE-2/CNE-2R and these cells were observed the dynamic autophagic flux by the LeiCa laser confocal after DMC,RAPA,CQ,DMC+CQ treatment.Result:1.MTT showed that DMC effectively inhibits the proliferation of CNE-2/CNE-2R cells,and the inhibition ratio was more obvious with the increase of concentration and time prolong,their IC50 respectively at 48h was 43.71μM,49.24μM.Similarly,the results of Cloning formation experiments showed that the number of clones after DMC treatment was significantly lower than that of the control(p<0.05).2.The vivo experiment results show DMC greatly decrease the size of tumor and reduce the tumor weight compared with the control.3.The flow apoptotic and western blotting results showed there is an increase with apoptosis cells with the increase of DMC concentration.Compared with the DMC group and the CQ group alone,the apoptosis rate of the combination group between DMC and CQ was greatly increased,which was statistically significant.The western blotting results showed that the Bcl2/Bax ratio decreased with the increase of DMC concentration,while the cleaved PARP,caspase3 and cleaved caspase3 increased.Moreover,compared with the DMC group and the CQ group alone,the Bcl2/Bax ratio and survivin were downregulated and cleaved PARP was upregulated in the combination group between DMC and CQ.4.The protein levels of LC3A/B and P62 were increased both in vitro and in vivo.The LC3 fluorescence spots were increased significantly after DMC treatment compared with the control(p<0.05).3-MA inhibits the LC3A/B and P62 protein levels induced by DMC.While CQ further promotes the LC3A/B and P62 protein levels induced by DMC.Consistently,3-MA could inhibit the LC3 puncta induced by DMC,while CQ could further promote the LC3 puncta induced by DMC.5.The transmission electron microscopy results reveal that DMC could increase the double-layered acidic autophagic vesicles in the cells.6.We observed that the autophagic flux is increased after DMC and RAPA treatment,the autophagic flux and is blocked after CQ treatment.In addition,DMC could remit the blocked autophagic flux by CQ.Conclusion:1.DMC inhibit the proliferation both in vitro and in vivo and induce apoptosis in CNE-2/CNE-2R cells.2.DMC promotes autophagosome accumulation by inducing autophagy and triggers the autophagy flux in CNE-2/CNE-2R cells.3.Inhibition autophagy promotes the DMC-induced apoptosis in CNE-2/CNE-2R cells.