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

BACKGROUND: Pseudo-ginsenoside-Rh2 (pseudo-G-Rh2), a novel derivative of ginsenoside 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 explored. 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 performed using monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM). The levels of autophagy-associated and apoptosis-associated proteins were determined using Western blotting. The Annexin V-FITC/propidium iodide (PI) assay was used to assess apoptosis. RESULTS: The Annexin V-FITC/PI assay revealed that the percentage of apoptotic cells in HepG2 cells at concentrations 0, 20, 40, and 60 µM 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 expressions 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 µM significantly 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. Pseudo- 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.

Pseudo-Ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway.

Ginsenoside Rh2, a major effective constituent of ginseng, has been suggested to have a pro-apoptotic effect in a variety of cancer cells. Pseudo-Ginsenside-Rh2 (pseudo-G-Rh2) is a novel derivative of ginsenoside Rh2. The aim of the present study was to evaluate the effect of pseudo-G-Rh2 on the apoptosis of lung adenocarcinoma A549 cells. The cytotoxicity of pseudo-G-Rh2 on A549 cells was evaluated using an MTT assay. Apoptosis was detected using DAPI staining and flow cytometry. The expression of apoptosis associated proteins was identified by western blot analysis. The results demonstrated that pseudo-G-Rh2 inhibits the proliferation of A549 cells in a dose-dependent manner. DAPI staining revealed topical morphological changes in apoptotic bodies following pseudo-G-Rh2 treatment. Flow cytometric analysis revealed that the percentage of Annexin V-fluorescein isothiocyanate-positive cells, which are apoptotic, increased with pseudo-G-Rh2 treatment in a dose-dependent manner. Furthermore, treatment with pseudo-G-Rh2 increased the level of reactive oxygen species in A549 cells as well as the activation of caspase-9, caspase-3 and poly ADP-ribose polymerase. Pseudo-G-Rh2 treatment was observed to induce mitochondrial membrane potential loss. Furthermore, the results of western blotting revealed that B-cell lymphoma 2 (Bcl-2) expression was significantly decreased while Bcl-2-associated X protein expression was significantly upregulated in A549 cells with pseudo-G-Rh2 treatment. Pseudo-G-Rh2-induced apoptosis was accompanied by sustained phosphorylation of Ras, Raf, extracellular signal-regulated kinase (ERK) and p53. In conclusion, the results of the present study suggest that pseudo-G-Rh2 induces mitochondrial apoptosis in A549 cells and is responsible for excessive activation of the Ras/Raf/ERK/p53 pathway.

Chemical constituents in acid hydrolysates of total saponins from stems and leaves of Panax ginseng / 中草药

Objective: To study the chemical constituents in the acid hydrolysates of total saponins from the stems and leaves of Panax ginseng. Methods: The chemical constituents were isolated and purified by various chromatographic methods, and their structures were identified by NMR and MS data analysis. Results: Thirty-four compounds were isolated and identified as 3β-acetoxy-12β-hydroxy-20(R), 25-epoxydammarane (1), 3β, 6α-diacetoxy-12β-hydroxy-20(R), 25-epoxydammarane (2), 3β-acetoxy-6α, 12β-dihydroxy-20(R), 25-epoxydammarane (3), 20(R)-panaxadiol (4), isodehydroprotopanaxatriol (5), 3-oxo-6α, 12β-dihydroxy-20(R), 25-epoxydammarane (6), dammar-(E)-20(22)-ene-3β, 12β, 25-triol (7), 6α-acetoxy-3β, 12β-dihydroxy-20(R), 25-epoxydammarane (8), 20(S)-protopanaxadiol (9), 20(R)-protopanaxadiol (10), 20(S)-25-ethoxyl-dammarane-3β, 12β, 20-triol (11), 20(R)-panaxatriol (12), dammar-(E)-20(22), 24-diene-3β, 6α, 12β-triol (13), 27-demethyl-(E, E)-20(22), 23-dien-3β, 12β-dihydroxydammar-25-one (14), 3β, 12β-dihydroxy-22, 23, 24, 25, 26, 27-hexanordamaran-20-one (15), 3β-acetoxy-6α, 12β, 25-trihydroxy-20(S), 24(R)-epoxy-dammarane (16), 20(S)-25-ethoxyl-dammarane-3β, 12β, 20-triol (17), dammar-(E)-20(22)-ene-3β, 6α, 12β, 25-tetrol (18), dammar-(Z)-20(22)-ene-3β, 6α, 12β, 25-tetrol (19), 20(S)-dammarane-3β, 12β, 20, 25-tetrol (20), 20(R)-dammarane-3β, 12β, 20, 25-tetrol (21), 20(S)-protopanaxatriol (22), 20(R)-protopanaxatriol (23), (20S, 24S)-dammarane-20, 24-epoxy-3β, 6α, 12β, 25-tetraol (24), (20S, 24R)-dammarane-20, 24-epoxy-3β, 6α, 12β, 25-tetraol (25), (20R, 24R)-dammarane-20, 24-epoxy-3β, 6α, 12β, 25-tetraol (26), 3β, 6α, 12β-trihydroxy-22, 23, 24, 25, 26, 27-hexanordamaran-20-one (27), 12β-hydroxy-20(R), 25-epoxydammarane-3β-O-β-D-glucopyranoside (28), 20(S)-dammarane-3β, 6α, 12β, 20, 25-pentol (29), 20(R)-dammarane-3β, 6α, 12β, 20, 25-pentol (30), 20(R)-dammarane-3β, 6α, 12β-trihydroxy-20, 25-epoxy-6-O-β-D-glucopyranoside (31), pseudo-ginsenoside-Rh2 (32), 20(S)-ginsenoside-Rh1 (33), and 20(R)-ginsenoside-Rh1 (34). Conclusion: Compounds 1-3, 6, 8, 11, 17, 24-26, and 32 are ginseng triterpenoids isolated from the acid hydrolysates of total saponins from the stems and leaves of P. ginseng for the first time. Pseudo-ginsenoside-Rh2 is a potent antiproliferative agent against human cancer cells.