Pseudo-ginsenoside Rh2 Induces Protective Autophagy in Hepatocellular Carcinoma HepG2 Cells

Background: Pseudo-ginsenoside-Rh2 (pseudo-G-Rh2), a novel derivative of ginseno-side Rh2, is reported to exert a pro-apoptotic effect on various malignancies. However, whether this anti-cancer action of pseudo-G-Rh2 involves autophagy remains to be determined and ex-plored. Objective: The objective of this study was to investigate the pseudo-G-Rh2-induced apoptosis and autophagy and the underlying mechanism. Methods: In the present study, the MTT assay was used for evaluating cell viability, and the lactate dehydrogenase (LDH) assay was performed to assess cell toxicity. Autophagy evaluation was per -formed using monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM). The levels of autophagy-associated and apoptosis-associated proteins were determined us -ing Western blotting. The Annexin V-FITC/propidium iodide (PI) assay was used to assess apopto-sis. Results: The Annexin V-FITC/PI assay revealed that the percentage of apoptotic cells in HepG2 cells at concentrations 0, 20, 40, and 60 mu M was 3.75%+/- 1.37%, 5.70%+/- 1.04%, 12.30%+/- 2.10%, and 34.26%+/- 4.73%, respectively. Pseudo-G-Rh2 was observed to significantly increase the expres-sions of BAX, cleaved-caspase-3, and cleaved-caspase-9, while it decreased the Bcl-2 expression. MDC and TEM analysis revealed that pseudo-G-Rh2 at concentrations 20, 40, and 60 mu M signifi-cantly facilitated the accumulation of autophagosomes and autolysosomes within the HepG2 cells. Moreover, pseudo-G-Rh2 significantly increased the expressions of LC3 II/LC3 I and Beclin-1 and decreased the expression of p62. The Annexin V-FITC/PI assay also revealed that in comparison to the pseudo-G-Rh2 group, the concurrent treatment with pseudo-G-Rh2 and an autophagy inhibitor (CQ or 3-MA) significantly induced distinct apoptosis. In addition, pseudo-G-Rh2 activated AMPK and inhibited the PI3K/Akt/mTOR pathway in a concentration-dependent manner. Pseu-do-G-Rh2 is similar to the current patents, which enhanced its anti-cancer activity by combining with autophagy inhibitors. Conclusion: Pseudo-G-Rh2 could induce protective autophagy in HepG2 cells, at least in part, via AMPK and the PI3K/Akt/mTOR pathway.