Signal Transduction Mechanism Of Ginsenoside Rh₂ On Apoptosis Of Murine MFC Gastric Cancer Cells

Ginsenosides are major effective ingredients of the root of Panax ginseng C.A.Meyer. In past decades,several different biological effects of them have been reported. Among them, ginsenoside Rh₂ (G-Rh₂) with a dammarane skeleton has been demonstrated capable of inhibiting the growth of various cancer cells in a dose and time dependent manner. The previous studies showed that the inhibitory effects were related to apoptosis inducing effect of Rh₂ on cancer cells. In this study, we firstly investigate the inhibitory effect of Rh₂ on murine gastric cells and intend to elucidate potential molecular mechanisms involved in the apoptosis-inducing effect of the agent.Firstly, we observed the inhibitory effect of ginsenoside Rh₂ on murine gastric cancer cells. Cultured MFC cells were treated with Rh₂ at various concentrations of 3,10, 30 mg·L^-1 for 1 h, 2 h, 4 h and 6 h respectively. Cell proliferation was measured with methylthiazolyl tetrazolium (MTT) assay and cell cycle was determined by flow cytometry (FCM). The results clearly showed that Rh₂ was capable of inhibiting the proliferation of gastric cancer cells in a dose and time dependent manner. The results of FCM analysis showed that percentage of cells in G0/G1 phase was increased in comparison with control cells treated with Rh₂. We further observed the expression of cell cycle regulatory protein cyclin D1 and p27^kip1 by various methods including flow cytometry, immunocytochemistry and western blotting. As expected, G0/G1 arrest was accompanied by an alteration of the level of cyclin D1 and p27^kip1. Next, we ovserved morphologic changes of cells under invert microscope and fluorescence microscope (Hoechst33258). The morphological changes of apoptosis charactered cell detachment, membrane blebbing, cell shrinkage with a condensed cytoplasm, and vesicle formation were observed on MFC cells treated with Rh₂. At the same time, we detected the cell early apoptosis rate with Annexin-V labeling in FCM analysis and the DNA fragmentation which measured by agarose gel electrophoresis. The apoptotic cell ratio and DNA fragmentation were both increased in a dose-dependence manner. Above preliminary results showed that the growth inhibitory effect of Rh₂ on murine gastric cancer cells is involved in cell cycle arrest and apoptosis inducing effect.Several different biochemical changes have been proposed to be the essential event that commits a cell to undergo apoptosis. These events include increase in the intracellular calcium level, loss of mitochondrial membrane potential, cytochrome c redistribution and caspase activation. To examine whether intracellular calcium ion might be involved in the Rh₂-induced apoptosis, we tested intracellular calcium ion with Fluo-3/AM, a calcium-specific dye by laser confocal micrograph. The results indicated that the content of calcium ion in the cells increased markly in tested cells as compared to the control cells. To test whether the apoptosis inducing effect of Rh₂ was associated with mitochondrial dysfunction, we assessed△ψmt after staining with Rhodamine 123 using the laser confocal micrograph. The intensity of red fluorescence decreased significantly within 1 h after Rh₂ treatment and diminished markedly thereafter. We found here that mitochondrial depolarization was involved in during Rh₂-induced apoptosis on gastric cancer cells. Furthermore, Western blotting was used to assess the expression of caspase-9 and caspase-3 on MFC cells.As results showed,the expression of caspase-3 and caspase-9 were both increased similarly within 30 min of Rh₂ treatment, indicating two kinases were up-regulated during Rh₂-induced apoptotic cell death.The above study demonstrated that the apoptotic-inducing effect of Rh₂ on murine gastric cancer cells had closed relationship with mitochondrial apoptosis pathway. Previous studies suggested that the reactive oxygen species (ROS) which acted as signaling intermediates played an important role in mitochondrial dysfunction. When ROS generation exceeds the antioxidative capacity of cells, oxidative stress can occur and cause cell death. ROS also are linked with the signaling pathways leading to cell death through the MAPK pathway. JNK is one of important members of MAPKs family. We designed a serious of experiments to shed light on the ROS and JNK participate in mitochondrial membrane depolarization and apoptosis after Rh₂ treatments. Firstly, we determined by MTT assay that NAC at 3 mM pretreated for 2 h and SP600125 at 5μm pretreated for 2 h are optical. MTT results also showed that the inhibitory effect of Rh₂ was prevented by pretreatment with NAC and SP600125. The flow cytometry analysis showed that the reduction of the mitochondrial membrane potential and the increase of early apoptosis rate induced by Rh₂ were both blocked significantly by pretreatment of NAC and SP600125. They all provided evidences that ROS and JNK participated in mitochondrial membrane depolarization and apoptotic progression. We further assessed ROS after staining DCFH-DA, a reactive oxygen species-specific dye, by flow cytometry and the confocal micrograph. Our data showed ROS gradually increased, peaking at 30 min after treatment of the cells with Rh₂, and sharply decreasing thereafter. It is likely that decreasing of ROS at 2 h is due to the dying cells. We concluded that the distubination of mitochondrial electron transport system eventually resulted in gernation of ROS in the gastric cancer cells, and the ROS release might constitute a positive feedback mechanism that can promote the neighboring mitochondrial release of ROS. Next, using Western blot, we detected the activities of some kinases including JNK, c-Jun, ATF2, caspase-3, Bcl-2, Bax, p53 after the addition of NAC and SP600125. Result showed that the addition of antioxidants attenuates Rh₂-induced JNK activation and apoptosis, indicating that ROS was involved in the up-regulation of JNK activity during apoptosis induced by Rh₂ on MFC cells. These results suggested that ROS is one of the important signaling molecules for activation JNK and depolarization of the mitochondrial membrane potential in Rh₂-induced apoptosis. The results also showed that the SP600125, the JNK inhibitor, suppressed the Rh₂-induced apoptosis, suggesting that the Rh₂-induced activation of JNK is involved in the apoptotic induction. Rh₂-induced translocation of Bcl-2/Bax to mitochondria and the expression of p53 were significantly suppressed,the phosphorylation of transcriptional factor c-Jun, ATF2 decreased,as comprared to that of cells treated with Rh₂ alone, indicating that JNK cascade plays an important role on the expression of the transcriptional factor c-Jun, ATF2, the expression of Bcl-2, Bax and p53.