Rhopaladins’ Analogue Induces Apoptosis Via Regulating The PI3K/AKT/mTOR Signaling Pathway In Cervical Cancer Cells

Background: The incidence and mortality of cervical cancer are increasing year by year,and it ranks fourth for both incidence and mortality.With the development of new drugs and the improvement of drug delivery methods,chemotherapy is expected to become the main means to treat cervical cancer,which can be used before or after cervical cancer,and can also be combined with radiotherapy.Therefore,the research of new drugs is very significant for the treatment of cervical cancer.Objective: The purpose of this study was to investigate the effects of Rhopaladins’ analogs 4-benzylidene-5-pyrrolidones on the proliferation and apoptosis of cervical cancer cell lines and the correlation of apoptosis with PI3K/AKT/mTOR signaling pathway in vitro.Methods:1.New compounds Rhopaladins’ analogs 2-benzoyl-4-benzylidene-5-pyrrolidines(RPDP A-C)were synthesized by multi-component one-pot method.MTT assay was used to explore the cytotoxicities of these compounds on cervical cancer CaSki cells,and the drug concentration was determined.Flow cytometry was used to detect the effect of RPDPB on apoptosis of CaSki cells,and RT-q PCR was used to detect the effect of RPDPB on the m RNA of E6/E7 of CaSki cells.2.RPDPB was used as the lead compound,and was optimized through molecular docking model,then we synthesized the Rhopaladins’ analog 2-styryl-4-benzylidene-5-pyrrolidine(compounds 6a-6j).The molecular docking method was used to calculate the fit of rapamycin target protein(FKBP12-mTOR complex protein)with rapamycin and compound 6e,respectively.3.Cytotoxicities of compounds 6a-6j on cervical cancer cell lines C-33A,CaSki,SiHa,HeLa,liver cancer Hep G2 and normal hepatocyte L02 was detected by MTT assay.At the same time,cisplatin was used as positive control;the effects of compound 6e on apoptosis of cervical cancer cell lines C-33A,CaSki and SiHa were detected by flow cytometry;RT-q PCR was used to detect the effects of compound 6e on the expression of Bcl-2,Bax,p53,caspase-3 and E6/E7 m RNA;Western blot was used to detect the effects of compound 6e on Bcl-2,Bax,p53,Caspase-3,E6/E7 and PI3K/Akt/mTOR pathway proteins.Results:1.Effects of 2-benzoyl-4-benzylidene-5-pyrrolidones on cervical cancer CaSki cellsThree Rhopaladins analogues 2-benzoyl-4-benzylidene-5-pyrrolidine(RPDP A-C)were synthesized in 85%,81% and 78% yields respectively;the MTT assay showed that RPDP A-C could inhibit the proliferation of cervical cancer CaSki cells;Flow cytometry showed that RPDPB induced apoptosis in a dose-dependent manner;PCR showed that RPDPB could down-regulate the m RNA expression of E6/E7.2.Synthesis of 2-styryl-4-benzylidene-5-pyrrolidine(compounds 6a-6j)The structure of RPDPB was optimized by molecular simulation,and the new compounds 6a-6j were synthesized;the docking results of compound 6e and mTOR showed that the bond energies of compound 6e and mTOR were similar to those of rapamycin and mTOR,compound 6e is expected to be a potential substitute for rapamycin.3.Effects of compounds 6a-6j on cervical cancer cell lineThe MTT assay showed that compound 6e and 6j could significantly inhibit the proliferation of cervical cancer cell lines C-33 A,CaSki,SiHa,HeLa and liver cancer cells Hep G2,and had low cytotoxicity to normal liver cells L02;the apoptosis results showed that compound 6e induced apoptosis in C-33 A,CaSki and SiHa cells with a dose-dependent manner;RT-q PCR and Western blot experiments showed that compound 6e could down-regulate Bcl-2 expression and increase Bax,p53 and Caspase-3 expression;Meanwhile,for PI3K/AKT/mTOR pathway,compound 6e can down-regulate the expression of PI3 K,AKT,p-AKT、mTOR and p-mTOR protein.In addition,compound 6e can also inhibit the expression of HPV 16 E6/E7 in CaSki and SiHa cells.Conclusion:1.Rhopaladins’ analogues 2-benzoyl-4-benzylidene-5-pyrrolidines(RPDP A-C)can inhibit the proliferation and induce apoptosis of CaSki cells,which may be related to the down-regulation of E6/E7 m RNA.2.Rhopaladins’ analogue 2-styryl-4-benzylidene-5-pyrrolidone(compounds 6)can inhibit the proliferation and promote the apoptosis of cervical cancer cell lines,and its mechanism can be related to PI3K/AKT/mTOR pathway,and its clear mechanism needs further experimental verification.