SSPH I, a Novel Anti-Cancer Saponin, Inhibits Autophagy and Induces Apoptosis via ROS Accumulation and ERK1/2 Signaling Pathway in Hepatocellular Carcinoma Cells.

INTRODUCTION: Saponin of Schizocapsa plantaginea Hance I (SSPH I), a novel bioactive phytochemical isolated from the rhizomes of Schizocapsa plantaginea, has been demonstrated to exhibit anti-cancer activity against various tumors in preclinical studies. However, the molecular mechanisms involved in the suppression of hepatocellular carcinoma (HCC) are poorly understood. The present study aimed at analyzing the effects of SSPH I on autophagy and apoptosis in vitro. METHODS: MTT and colony forming assays were used to detect cell viability and cell proliferation. Hoechst 33,258 staining and flow cytometry were used to determine apoptosis and ROS production. The apoptosis and autophagy-related protein expression levels were evaluated via Western blot assay. Characteristics of autophagy and apoptosis were observed by transmission electron microscopy. Lysosomal activity was stained with Lyso-Tracker Red and Magic Red Cathepsin B. RESULTS: The results showed that SSPH I exhibited potent anti-cancer activity and proliferation in HepG2 and BEL-7402 cells and inhibited HepG2 cells through inhibiting autophagy and promoting apoptosis. The mechanistic study indicated that the inhibition of autophagy of SSPH I was mediated by blocking autophagosome-lysosome fusion. Additionally, we found that SSPH I could mediate the activation of MAPK/ERK1/2 signaling pathway, and the use of NAC (ROS inhibitor) and U0126 (MEK1/2 inhibitor) converted the effect of SSPH I on apoptosis and autophagy in HepG2 cells. CONCLUSION: These data suggest that SSPH I induces tumor cells apoptosis and reduces autophagy in vitro by inducing ROS and activating MAPK/ERK1/2 signaling pathway, indicating that SSPH I might be a novel agent for the treatment of HCC.

Saponins isolated from Schizocapsa plantaginea inhibit human hepatocellular carcinoma cell growth in vivo and in vitro via mitogen-activated protein kinase signaling.

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G2/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.