The Influence And Mechanism Of Bufadienolides By Inhibiting The AKT Signaling Pathway In Human Cervical Cancer

Background and purpose:Although some progress has been made in itsscreening,vaccination,diagnosis and treatment,cervical cancer is still the second leading cause of cancer-related death in women.Cervical cancer incidence in China gradually increased because of many reasons such as large population,significant disparities between the rich and the poor,lacking widespreadscreening for cervical cancer and the increasing prevalence of HPV infection?especially in young women?.Toad venomhas been wildly used as a traditional Chinese medicine in the treatment of superficial infection,edema,pain,heart failure,skin problems and skin cancer.Emerging evidence from recent studies suggests that toad venom has also been proven to have anti-inflammatory,anti-tumor,analgesic,anesthetic and other pharmacological effects.Toad venom may have a good effect on the inhibition of cervical cancer.Experimental approach:CCK-8 test was used to detect the effects of bufadienolides on the growth of cervical cancer HeLa and SiHa cells at different times.To observe the cell cycle arrest of HeLa and SiHa cells treated bufadienolides for 48h,the cells were detected by flow cytometry using Cycletest^TM Plus DNA Reagent Kit.AnnexinV-FITC/PI double staining flow cytometry was used to detect the apoptosis rate of HeLa and SiHa cells exposed to bufadienolides.The expressions of p-AKT,p-cdc2,bcl-2,BAX,caspase 3,Beclin 1,LC2B,P62 and mTOR were detected by Western blot p-P70S6K,p-GSK-3?protein expression changes.Results:?1?In two cervical cancer cell lines,HeLa and SiHa,CCK-8 test showed that different concentrations of bufadienolides have significant killing effect on tumor cells at different times,and cell death rate In a dose-time-dependent manner.?2?Through the flow cytometry cycle experiments showed:treatment of bufadienolides cervical cancer cells HeLa and SiHa 48h,the cell cycle arrest in the G₂/M phase.Apoptosis experiments showed that bufadienolides treatment of cervical cancer cells,the rate of apoptosis increased.?3?The expression of p-cdc2 decreased,while the expression of caspase 3 and bcl-2 decreased,the expression of BAX and BAX/bcl-2increased.Induce apoptosis to inhibit cell proliferation.?4?The expression of Beclin 1and P62 decreased and the expression of LC3B increased.It was demonstrated that inhibition of cell proliferation by bufadienolides may increase autophagy.?5?The expression levels of p-AKT,mTOR,p-P70S6K and p-GSK3?were decreased while the expression levels of AKT and P70S6K were unchanged,indicating that bufadienolides-induced apoptosis and autophagy increase were achieved through the inhibition of AKT pathway.Conclusions and Implications:In this study,we found that bufadienolides is a potent inhibitor of cervical cancer cells.The mechanism is related to the effective inhibition of tumor cell proliferation and the induction of tumor cell's apoptosis and autophagy.Our study also shows these phenomena may have occurred through by inhibiting Akt/mTOR/P70S6K and Akt/GSK-3?signaling pathway.Therefore,we demonstrated that bufadienolides may serve as a new strategy for potentailclinical therapy of cervical cancer.