The Mechanism Of Babao Dan On Inhibiting Proliferation, Inducing Apoptosis And Autophagy Of Gastric Cancer Cells By Regulating The MTOR Signaling Pathway

Aim:To study on Babao Dan(BBD)in vitro and in vivo,the effect of BBD on the growth of gastric cancer cells was to explore three aspects: cell proliferation,cell apoptosis and cell autophagy,providing a theoretical basis for the clinical treatment of BBD against gastric cancer.Methods:1.In vitro study:(1)Gastric cancer cells(AGS and MGC80-3)were cultured and treated with BBD at different concentrations(0,0.25,0.5,and 1 mg/mL)for 24 h.Cell morphology was observed under the microscope;Trypan blue was used to analyze changes in cell number;And cytotoxic effects was detected by LDH method after BBD intervention for 24 h;Then,the influence of BBD on colony formation ability and cell cycle of gastric cancer cells was tested by colony formation experiment and flow cytometry assay,respectively;The expression of PCNA,Survivin,Cyclin D1,CDK4,p21 about proliferative proteins was detected by Western blot(WB);(2)Hoechst 33258 staining and Annexin V-APC staining were used to detect cell apoptosis;The changes of MMPs were determined by JC-1 staining through flow cytometry;The apoptotic protein levels(Bax,Bcl-2,Fas and FasL)were detected by WB;The activation of caspase-3/-8/-9 was detected by quantitative colorimetry;(3)The LC3-Ⅰ,LC3-Ⅱ,p62,Beclin-1 about autophagy protein levels were analyzed by WB;(4)Activation of p-JNK1/2/3、p-p38、p-ERK1/2 proteins was detected by Bio-plex assay;The expression of these proteins(PI3K、p-AKT、p-mTOR、p-AMPKα)were detected by WB.2.In vivo study: Twelve male nude mice were randomly divided into two groups:Control group(normal saline)and BBD group(0.25 g/kg)daily by gavage,respectively.Subcutaneous injection(MGC80-3 cells)was used to establish subcutaneous tumor transplantation model of gastric cancer in nude mice,and BBD was given for intervention by gavage.(1)One month later,the nude mice were sacrificed and the tumors were removed for weighing,photographing and following-up experiments on the growth of transplanted tumor of gastric cancer;(2)TUNEL assay was used to detect apoptosis in tumor tissues;(3)Ki-67 was explored using IHC analysis;Anti-proliferative activity of BBD was assayed by detectingdifferent mark moleculars by WB analysis such as Survivin,PCNA and related-proliferation factors including Cyclin D1,CDK4,p21 and and key related-apoptotic proteins including Bax,Bcl-2 and cleaved-caspase-3;autophagy regulatory molecules including LC3-Ⅰ,LC3-Ⅱ,p62,Beclin-1;(4)Meanwhile,the phosphorylate proteins(JNK1/2/3,p38,ERK1/2)level and activation of the proteins(PI3K、p-AKT、p-mTOR、p-AMPKα)in tumor tissues were to examine changes in related signaling pathways by Bio-plex and WB.Results:1.In vitro study:(1)BBD reduced cell number and the growth of gastric cancer cells;LDH assays demonstrated BBD increased cytotoxicity(P<0.01);Colony formation experiments illustrated that BBD inhibited the colony formation in AGS and MGC80-3(P<0.05);Cell cycle experiments showed that BBD inhibited G1/S conversion of cell cycle(P<0.01);WB assay showed that BBD up-regulated p21 while down-regulated PCNA,Survivin,Cyclin D1 and CDK4 protein expression via inhibiting the proliferation of gastric cancer cells(P<0.05);(2)Hoechst 33258 and Annexin V-APC staining showed that BBD promoted apoptosis of gastric cancer AGS and MGC80-3 cells(P<0.01);JC-1 staining also showed that BBD led to a gradual decrease in MMPs(P<0.01);WB experiment showed that BBD up-regulated the expression of Bax,Fas and FasL and down-regulated the expression of Bcl-2 apoptotic protein(P<0.05);BBD induced cell apoptosis by stimulating caspase-3/-8/-9activity(P<0.05);(3)WB results showed that BBD increased LC3-I and LC3-II transformation and the expression of Beclin-1 and decreased the expression of p62 to induce cell autophagy(P<0.05);(4)Bio-plex showed BBD inhibited the activation of MAPK(p-JNK1/2/3,p-p38,p-ERK1/2)in AGS and MGC80-3 cells(P<0.05);WB results illustrated that the expression level of PI3 K in cells was decreased,the ratio of p-AKT/AKT and p-mTOR/mTOR expression level was decreased,and the ratio of p-AMPKα/AMPKαexpression level was increased in AGS and MGC80-3 cells(P<0.05).2.In vivo study:(1)BBD could significantly reduce tumor weight in mice with gastric cancer cell tumor transplantation(P<0.05),but had no significant effect on the body weight of mice before and after BBD treatment(P>0.05);(2)TUNEL analysis showed that BBD could induce apoptosis of transplanted tumor cells in gastric cancer(P<0.01);(3)The detection results of IHC showed that BBD decreased the expression of Ki-67 protein in tumor tissues(P<0.01);The results of WB showed that BBD significantly promoted the expression of Bax,cleaved-caspae-3,p21 proteins,LC3-I and LC3-II transformation,Beclin-1 and markedly reduced the expression of Bcl-2,PCNA,Survivin,Cyclin D1,CDK4,p62 proteins in tumor tissues(P<0.05);(4)Bio-plex and WB indicated that BBD inhibited the expression of p-JNK1/2/3,p-p38,p-ERK1/2 proteins in tumor tissues(P<0.05);WB results showed that BBD down-regulated the expression of PI3 K protein,the ratio of p-AKT/AKT and p-mTOR/mTOR,up-regulated the ratio of p-AMPKα/AMPKα in tumor tissues(P<0.05).Conclusions:Generally,BBD had a significant inhibitory effect on gastric cancer in vivo and in vitro,which can inhibit the proliferation,induce apoptosis and autophagy on gastric cancer cells.BBD inhibit the activation of MAPK(p-JNK1/2/3,p-p38,p-ERK1/2),PI3K/AKT and promoted the activation of AMPK by regulating the mTOR signaling pathway in gastric cancer.