Study On The Effect Of RCE-4,active Ingredient Of Reineckia Carnea,in Combination With Celecoxib On Proliferation Of Cervical Cancer Ca Ski Cells And Its Mechanism

Objectives: Cervical cancer,ranking forth for both incidence and mortality rate,is one of the most threatening cancer among women.The therapeutic methods of cervical cancer patients in the first and middle stage is mainly local surgery,supplemented by radiotherapy and chemotherapy.For the poor prognosis of advanced,metastatic,and recurrent patients,chemotherapy is the main therapeutic method,and cytotoxic drugs in combination with molecular targeting drugs is a promising therapy for them.RCE-4,a steroidal saponin isolated from Reineckia carnea,has been proved to have anti-cervical cancer effect in Ca Ski cells,but the effect in vivo still need to be enhanced.Therefore,this subject aims to explore the effect of RCE-4 in combination with Celecoxib on anti-cervical cancer Ca Ski cells and its mechanism.Methods: The MTT assay and Calcu Syn V2.0 software were used to detect cell proliferation and calculate the combination index(CI);the expression levels of various proteins were analyzed using western blot assay;mitochondrial membrane potential(MMP)was assessed using JC-1 staining;acridine orange/ethidium bromide(AO/EB)double-fluorescence staining was used to detect the apoptosis of Ca Ski cells;a co-immunoprecipitation(Co-IP)assay was used to analyze the relative content of Bcl-2-Beclin 1 complex in Ca Ski cells.Results: The results demonstrate that the combination of RCE-4 and NSAIDs increases the inhibition of Ca Ski cells compared to the single-RCE-4 group,and Celecoxib provided the best synergistic effect among the four NSAIDs tested,with a CI of 0.32.The combination of RCE-4 and celecoxib significantly down-regulated the expression of cyclooxygenase-2(COX-2)and nuclear transcription factor-κB(NF-κB),and promoted the expression of non-steroidal anti-inflammatory drugs activity gene-1(NAG-1).In addition,autophagy induced by RCE-4 was markedly inhibited in combination with celecoxib,which was associated with down-regulation of the expression of microtubule-associated protein 3(LC3)-II,Beclin 1,p62 and autophagy-related gene(ATG)3/4B/5/7/14.RCE-4-induced apoptosis was significantly enhanced by altering the depolarization of mitochondrial membrane potential and the expression of B cell lymphoma-2(Bcl-2),B cell lymphoma-xl(Bcl-xl),Bcl-2 associated X protein(Bax),Bcl-2/Bcl-xl-associated death promoter(Bad)and cleaved cysteinyl aspartate specific proteinase(cleaved-caspase)3/7/9.Furthermore,the formation of the Bcl-2-Beclin 1 complex was significantly inhibited in Ca Ski cells treated with RCE-4 in combination with celecoxib.Conclusions: RCE-4 combined with Celecoxib had a better anti-cervical cancer effect in Ca Ski cells,compared with RCE-4 treated only,possibly through inhibiting the formation of Bcl-2-Beclin1 complex,down-regulating the proteins expression of Beclin1 as well as Bcl-2,and thus inhibiting RCE-4 induced cytoprotective autophagy and enhancing RCE-4 induced apoptosis on Ca Ski cells.Moreover,the clinical dosage of RCE-4 is expected to decrease in the future.But the specific mechanism and pathway of inflammatory,autophagy,and apoptosis related proteins regulated by RCE-4 in combination with Celecoxib need to be further studied.