The Role Of Autophagy In Bufalin-induced Apoptosis And Aerobic Glycolysis Of Hepatocellular Carcinoma Cells

Background:Autophagy is a highly evolutionarily conserved life process that commonly exists in eukaryotic cells,and is closely related to the occurrence or progression of tumors.Taking autophagy as a target for anti-tumor drugs is one of the advanced research areas of tumor therapy.Bufalin is one of the main active ingredients of the traditional Chinese medicine Chan Su,which can inhibit the proliferation and induce apoptosis of hepatocellular carcinoma cells through multiple pathways and multiple targets.In previous preliminary experiments,it was observed through electron microscopy and Western-blot that Bufalin could inhibit the formation of autophagosomes in human hepatocellular carcinoma SMMC-7721 cells,and reduced the expression levels of the autophagy marker proteins Beclin 1,LC3-?.In addition,Bufalin could inhibit aerobic glycolysis of human hepatocellular carcinoma cells.So,what role does autophagy play in Bufalin-induced apoptosis and aerobic glycolysis of human hepatocellular carcinoma cells? This study selects human hepatocellular carcinoma cell line SMMC-7721 and Bel-7402,and uses the signal pathways related to the Beclin 1 complex system to study the effect of Bufalin on human hepatocellular carcinoma cell autophagy,and clarifies that the role of autophagy in the hepatocellular carcinoma cell death.Objective:In this study,human hepatocellular carcinoma cells SMMC-7721 and Bel-7402 were used as research objects,and a tumor-bearing mouse model of hepatocellular carcinoma cells was established.Inhibit autophagy and active autophagy to observe the effects of Bufalin on the proliferation,apoptosis,and aerobic glycolysis of human hepatocellular carcinoma cells,and to clarify the role and status of autophagy in Bufalin-induced human hepatocellular carcinoma cell death.The human gene expression chip was used to explore the possible mechanism of Bufalin-induced human hepatocellular carcinoma cell death,and to provide experimental basis and ideas for future anti-tumor research of Bufalin.Methods:Part I : The effect of Bufalin on the proliferation,apoptosis,autophagy,and aerobic glycolysis of human hepatocellular carcinoma cellsMTT assay was used to detect the effects of Bufalin at different concentrations on proliferation inhibition rates in four different human hepatocellular carcinoma cell lines SMMC-7721,Hep G2,Hep3 B,and Bel-7402.Apoptosis was detected by Annexin V-FIFC / PI double staining in human hepatocellular carcinoma SMMC-7721,Bel-7402 cells.Transmission electron microscopy was used to detect the formation of autophagosomes of human hepatocellular carcinoma SMMC-7721 cells treated with Bufalin at different concentrations after 24h;p-EGFP-LC3 plasmid transfection of human hepatocellular carcinoma SMMC-7721 cells was observed the effect of Bufalin on LC3 expression under a fluorescent microscope;Western-blot was used to detect the expression of cell autophagy marker proteins LC3-?/?,Beclin 1,P62,ATG5;q RT-PCR was used to detect the expression of GLUT1,LDHA,HK2 and Beclin 1 m RNA,the key proteins of aerobic glycolysis.Part II : The role of autophagy in Bufalin-induced human hepatocellular carcinoma cell proliferation,apoptosis and aerobic glycolysisMTT,Annexin V-FIFC / PI double staining,Western-blot,q RT-PCR were used to detect autophagy inhibitor CQ,autophagy activator RAPA alone and combined with 100 n M Bufalin on inhibition of proliferation,apoptosis,and aerobic glycolys in human hepatocellular carcinoma SMMC-7721 cells after 24 h.Human gene expression chip was used to detect diseases and signal pathways related to the effect of Bufalin.Part III: Role of ROS and mitochondrial dynein-related protein Drp1 in Bufalin-induced human hepatocellular carcinoma cell deathFlow cytometry was used to detect Bufalin intervention in human hepatocellular carcinoma SMMC-7721 cell ROS levels;MTT,Annexin V-FIFC / PI double staining,Western-blot,q RT-PCR were used to detect ROS inhibitor NAC alone and in combination with 100 n M Bufalinon inhibition of proliferation,apoptosis,and aerobic glycolys in human hepatocellular carcinoma SMMC-7721 cells after 24 h.The Drp1 knockdown and over-express plasmid were constructed,and SMMC-7721 cells were infected after virus packaging.q RT-PCR was used to detect the effects of NAC alone and in combination with 100 n M Bufalin on HK2,Beclin 1 m RNA expression in SMMC-7721 cells.Part IV :In vivo study of the effects of Bufalin and NAC on the proliferation,apoptosis,autophagy,and aerobic glycolysis of human hepatocellular carcinoma cellsHuman hepatocellular carcinoma SMMC-7721 cell-bearing nude mouse model was constructed.After nude mice became tumor,they were randomly divided into negative control group,Bufalin group [1.5 mg /(kg · d)],NAC group[300 mg /(kg · d)] and Bufalin + NAC group,continuous administration for 10 days.The tumor volume was measured,and the expression of aerobic glycolysis-related proteins and Beclin 1,Cleaved Caspase-3 proteins in each group were detected by immunohistochemistry.Results:Part I : The effect of Bufalin on the proliferation,apoptosis,autophagy,and aerobic glycolysis of human hepatocellular carcinoma cells1.Bufalin has a certain inhibitory effect on the proliferation of four hepatocellular carcinoma cells,and its inhibitory effect is dose-dependent;with the same concentration of drug intervention,Bufalin inhibits the proliferation of SMMC-7721 and Bel-7402 cells stronger than Hep G2,Hep3 B cells.Flow cytometry results showed that Bufalin could induce apoptosis of human hepatocellular carcinoma SMMC-7721 and Bel-7402 cells in a dose-dependent manner.2.The treatment of SMMC-7721 cells with Bufalin reduced the autophagy level in a dose-dependent manner after 24 h.After Bufalin effect on SMMC-7721 cells for 24 h,the cytoplasmic LC3 dots were lower than that of the blank control group.Western-blot results showed that the ratio of LC3-II / I and Beclin 1 decreased with the increase in the dose of Bufalin.q RT-PCR results showed that Bufalin could reduce the expression levels of GLUT1,LDHA,HK2,Beclin 1 in SMMC-7721 cells.Part II : The role of autophagy in Bufalin-induced human hepatocellular carcinoma cell proliferation,apoptosis and aerobic glycolysis1.CQ and RAPA combined with Bufalin could enhance the effect of Bufalin on the proliferation of human hepatocellular carcinoma SMMC-7721 and apoptosis induction.The effect of CQ combined with Bufalin is stronger than that of RAPA combined with Bufalin;2.CQ attenuated the effect of Bufalin on the expression of HK2 m RNA in SMMC-7721 cells,while RAPA enhanced the effect of Bufalin on the expression of HK2 m RNA in SMMC-7721 cells;Tab-Beclin 1 induced Beclin1 expression and attenuated the inhibitory effect of Bufalin on the expression level of HK2 m RNA in SMMC-7721 cells;3.Human gene expression chip results IPA analysis showed that Bufalin could significantly activate the TNFR2 Signaling pathway in SMMC-7721 cells,and the upstream regulatory factor TNF was predicted to be strongly activated.Part III: Role of ROS and mitochondrial dynein-related protein Drp1 in Bufalin-induced human hepatocellular carcinoma cell death1.Bufalin could induce ROS production in human hepatocellular carcinoma SMMC-7721 cells in a dose-dependent manner;NAC combined with Bufalin intervention attenuated the effect of Bufalin on the proliferation of human hepatocellular carcinoma SMMC-7721 and apoptosis induction;2.NAC combined with Bufalin could enhance the effect of Bufalin on the conversion of LC3-II to LC3-I and the expression levels of Beclin 1,ATG5 protein,and increase the expression level of P62 protein;NAC combined with Bufalin could enhance the effect of Bufalin on HK2 m RNA expression level;3.Drp1 knockdown could attenuate the effect of Bufalin on the expression levels of Beclin1 and HK2 m RNA in SMMC-7721 cells;Drp1 knockdown increased the expression of Beclin1 and HK2 m RNA in cells after NAC combined with Bufalin.Part IV :In vivo study of the effects of Bufalin and NAC on the proliferation,apoptosis,autophagy,and aerobic glycolysis of human hepatocellular carcinoma cells1.Bufalin significantly inhibited the growth of human hepatocellular carcinoma tumor-bearing mice subcutaneous tumors,while NAC could promote the growth of human hepatocellular carcinoma tumor-bearing mice subcutaneous tumors;combined with NAC,it weakened the inhibitory effects of Bufalin on human hepatocellular carcinoma tumor-bearing mice subcutaneous tumors;2.Bufalin inhibited the expression of GLUT1,LDHA,HK2,PKM2,Beclin1,and induced the up-regulation of Cleaved Caspase-3 expression.Conclusion:1.Bufalin inhibits human hepatocellular carcinoma SMMC-7721 cell proliferation,autophagy,aerobic glycolysis,and induces apoptosis;2.inhibition of autophagy could enhance the effect of Bufalin on human hepatocellular carcinoma SMMC-7721 cell apoptosis;3.The ROS produced by Bufalin induced a negative regulation of autophagy inhibition in hepatoma cells,and Drp1 participate in this regulation;4.Bufalin inhibit the expression of HK2 by acting on Drp1 to inhibit the expression of Beclin1,thereby inhibiting aerobic glycolysis of hepatocellular carcinoma cells.