Anemoside B4 Induces Apoptosis And Autophagy Via The PI3K/Akt/mTOR Pathway In Hepatocellular Carcinoma

Objective:Apoptosis and autophagy are the main ways in tumor cels death.The PI3K/Akt/mTOR signaling pathway is an important signaling pathway regulating apoptosis and autophagy.Studies have shown that the Chinese medicine Pulsatilla chinensis has anti-tumor effect,and Anemoside B4 is the obvious active ingredients in the extract of dried roots of Pulsatilla Chinensis.This experiment is to investigate the effect of Anemoside B4 on the proliferation of SMMC7721,study the effects of apoptosis and autophagy,explore the possible mechanism from the PI3K/Akt/mTOR signaling pathway.Methods:1、Cell culture:After resuscitation of SMMC7721 cells,cultured in DMEM supplement with 10%FBS and 1%penicillin-streptomycin,and incubated in a humidified incubator with 5%CO₂ at 37℃.2、Cell viability assay:CCK-8 assay and Colony formation assay were used to detect the effect of Anemoside B4 on the proliferation of SMMC7721 cells,after Anemoside B4treated SMMC7721 cells.3、Determination of apoptosis:After treatment,the effect of Anemoside B4 on the apoptosis of SMMC7721 cels was observed by the DAPI,Annexin V-FITC and PI immunofluorescence staining assay,Annexin V-FITC and PI double staining flow cytometry assay.Western blot was used to detect the Bcl-2,Bax,Cleaved Caspase-3 and Cleaved PARP proteins.4、Quantification of autophagy:The effect of Anemoside B4 on the autophagy of SMMC7721 cells was observed by Transmission Electron Microscopy experiment,AO staining assay and MDC staining assay.Western blot was used to detect the LC3-Ⅱ/LC3-Ⅰ、Beclin-1 and p62 proteins,after SMMC7721 cells were exposed to Anemoside B4.After CQ treated SMMC7721 cells to inhibit the autophagy,detected the LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and p62 proteins again.5、Analysis of the proteins related to the PI3K/Akt/mTOR signaling pathway:Western blot assay was performed to analysis the p-AKT,AKT,p-mTOR and mTOR proteins,after treated with Anemoside B4.Results:1、The CCK-8 assay showed that in the same time period,with the increase of the concentrations of Anemoside B4,the inhibition of SMMC721 cell proliferation gradually increased,at the fixed concentration,with the increase of the time,the inhibition increased.Anemoside B4 inhibited SMMC7721 cell proliferation in dose-and time-dependent manner.2、Colony formation assay showed that the number of clones of SMMC721 cells decreased in dose-dependent manner,after SMMC7721 cells treated with 20,40 and 80μM Anemoside B4 for 24 h.3、Immunofluorescence staining assay showed that the cells in the experimental group showed more red fluorescently labeled nuclei and more green fluorescently labeled cell membranes,compared with the control group,after SMMC7721 cells treated with 80μM Anemoside B4 for 24 h.4、Flow cytometry assay showed that the rate of apoptosis in the experimental group was 55.03±2.79%,the control group was 2.33±0.29%（p<0.01）,after SMMC7721cels treated with 80μM Anemoside B4 for 24 h.5、Western blot assay showed that the expression of Bax,Cleaved Caspase-3 and Cleaved PARP protein was gradually increased,the expression of Bcl-2 protein was gradually decreased compared with the control group,after SMMC7721 cells treated with20,40 and 80μM Anemoside B4 for 24 h.6、Transmission electron microscopy showed that double membrane autophagic vacuoles appeared in the experimental group,but not in the control group,after SMMC721 cells treatment with 80μM Anemoside B4 for 24 h.7、AO staining showed,compared with the control group,a large amount of red fluorescent labeled autophagic vesicles appearing in the experimental group,after SMMC7721 cells treated with 80μM Anemoside B4 for 24 h.8、MDC staining showed,numerous yel ow-green fluorescent labeled autophagic vesicles appearing in the experimental group,but not found in the control group,after SMMC721 cells exposed with 80μM Anemoside B4 for 24 h.9、Western blot assay showed that the expression of LC3-Ⅱ/LC3-Ⅰand Beclin-1 proteins gradually were increased,the expression of p62 protein was gradually decreased compared with the control group,after SMMC7721 cells treated with 20,40 and 80μM Anemoside B4 for 24 h.After 24 h treatment of SMMC7721 cells with Anemoside B4and Chloroquine,Chloroquine inhibited the autophagy-inducing effect of Anemoside B4in SMMC7721 cells.10、Western blot assay showed that after treated with 20,40 and 80μM Anemoside B4for 24 h,the expression of p-Akt and p-mTOR proteins were gradually reduced,the Akt and mTOR proteins expression had no significant change compared with the control group.Conclusion:1、Anemoside B4 inhibited SMMC7721 cell proliferation in dose-and time-dependent manners.2、Anemoside B4 induced the apoptosis in SMMC7721 cels by up-regulating the expression of Bax,Cleaved Caspase-3 and Cleaved PARP proteins and down-regulating the expression of Bcl-2 protein.3、Anemoside B4 induced autophagy in SMMC7721 cels and its mechanism was achieved by up-regulating the expression of LC3-II/LC3-I and Beclin-1 proteins and down-regulating the expression of p62 protein.4、Anemoside B4 induced apoptosis and autophagy in SMMC7721 cels by down-regulating the expression of p-Akt/Akt and p-mTOR/mTOR proteins via the PI3K/Akt/mTOR signaling pathway.