Roles Of Autophagy In Apoptosis Induced By Pseudo-Ginsenoside-Rh2 In Hepatocellular Carcinoma HepG2 Cells

Pseudo-ginsenoside-Rh2 was a novel compound,a derivative of ginsenoside（G）-Rh2.Our previous study showed that Pseudo-G-Rh2 could induce mitochondria-mediated apoptosis on lung adenocarcinoma A549 cells and gastric cancer SGC cells.However,there was no research about the effect of Pseudo-ginsenoside-Rh2 on hepatoma carcinoma HepG2 cells.Object:To investigate the inhibitory effect of Pseudo-G-Rh2 on HepG2 hepatoma cells and molecular mechanism.Methods:Pseudo-G-Rh2 treated hepatoma carcinoma HepG2 cells in vitro.MTT assay was used to evaluate cell viability.Lactate dehydrogenase（LDH）assay was used to assess cell toxicity.Monodansylcadaverine（MDC）staining and transmission electron microscopy（TEM）were used to evaluate autophagy.Annexin V-FITC/propidium iodide（PI）assay was used to evaluate apoptosis.Western blotting was used to detect the level of autophagy-associated（Beclin-1,LC3Ⅱ/Ⅰ,p62）,apoptosis-associated proteins（BAX,Bcl-2,caspase-3,caspase-9）and pathway-associated proteins（AMPK,PI3K,Akt,mTOR）.Result:1.The effect of Pseudo-G-Rh2 on cell activity and apoptosis:1)Pseudo-G-Rh2 inhibited hepatoma carcinoma HepG2 cells proliferation in a dose- dependent manner.The IC₅₀ value was 68.8μM for 24 h.2)Pseudo-G-Rh2 increased the LDH activity on hepatoma carcinoma HepG2 cells supernatant in a dose-dependent manner.3)Pseudo-G-Rh2 induced apoptosis on hepatoma carcinoma HepG2 cells.4)Pseudo-G-Rh2 increased the expression of BAX,cleaved-caspase-3/9,and decreased theexpression of Bcl-2.2.The effect of Pseudo-G-Rh2 on autophagy:1)As shown in MDC staining and TEM,Pseudo-G-Rh2 increased the accumulation of autophagosomes and autolysosomes.2)Pseudo-G-Rh2 increased the expression of Beclin-1,LC3Ⅱ/Ⅰ,and decreased the expression of p62.3)Compared with the Pseudo-G-Rh2 group,Pseudo-G-Rh2 combined with chloroquine（CQ） increased the expression of LC3Ⅱ/Ⅰ.4)As shown in Annexin V-FITC/PI assay,compared with the Pseudo-G-Rh2 group,Pseudo- G-Rh2 combined with CQ increased the expression of cleaved/pro-caspase-3/9 and th apoptosis radio significantly on HepG2 cells5)Compared with the Pseudo-G-Rh2 group,Pseudo-G-Rh2 combined with 3-methyladenine（3-MA）decreased the expression of LC3Ⅱ/Ⅰ.6)Compared with the Pseudo-G-Rh2 group,Pseudo-G-Rh2 combined with 3-MA increased the expression of cleaved/pro-caspase-3/9 and the apoptosis radio on HepG2 cells.3.The effects of Pseudo-G-Rh2 on autophagy-associated pathways:Pseudo-G-Rh2 increased the expression of p-AMPK and decreased the expression of p-PI3K,p-Akt,p-mTOR.Conclusion:Pseudo-G-Rh2 induces HepG2 cells apoptosis and protective autophagy,at last in part,via AMPK/PI3K/Akt/mTOR pathway.