Total Saponins Of Gynostemma Pentaphyllum - Induced Apoptosis In Murine Leukemia L1210 Cells And Its Mechanism

Gypenosides (Gyp) are major components in Gynostemma pentaphyllum Makino. Gyp can used for treatment of hepatitis,hyperlipoproteinemia,cancer,cardiovascular caspase.Gyp has been shown to have anti-inflammatory,anti-thrombotic,anti-oxidative.Among them,Gyp has been demonstrate capable of inhibiting the growth of various cancer cells in a dose and time dependent manner. These studies showed that the inhibitory effects were related to cell proliferation,apoptosis,cell cycle arrest and intend to elucidate potential molecular mechanisms involved in the apoptosis inducing effect of the agent.Now experimental results were as follows.1.These studies first observed the inhibitory effect of Gyp on murine leukemia L1210 cell lines. Cultured L1210 cells were treated with Gyp at various concentrations of 100～500μg/mL for 12 h,24 h,48 h and 72 h respectively. Cell proliferation was measured with MTT assay and cell cycle was determined by flow cytometry(FCM). The results showed that Gyp was capable of inhibiting the proliferation of L1210 cancer cells in a dose and time dependent manner. The results of FCM analysis showed that percentage of cells in S phase was increased in comparison with control cells treated with Gyp. Next, we observed morphologic changes of cells under invert microscope and fluorescence microscope(DAPI),The morphological changes of apoptosis charactered cell shrinkage with a condensed nucleoplasm.DNA damage of murine leukemia L1210 cells were detected by single cell gel electrophoresis, DNA fragments were increased in a dose-dependence manner. At the same time, we detected the cell apoptosis rate with Annexin-V /7-AAD labeling in FCM analysis. Several different biochemical changes have been proposed to be the essential event that commits a cell to undergo apoptosis. FCM analysis detected loss of mitochondrial membrane potential by Rh123 and increase in the intracellular calcium level by Fluo-3/AM. The results showed loss of mitochondrial membrane potential and increase in the intracellular calcium level of L1210 cell were both in a time-dependent manner. The further assessed ROS after staining DCFH-DA,ROS-specific dye, by FCM. Low concentration of Gyp might eliminate ROS, Gyp concentrations at 350,500μg/mL,the highest production of ROS was induced. 2.The above study demonstrated that the apoptotic-inducing effect of Gyp on murine leukemia L1210 cell lines had closed relationship with mitochondrial apoptosis pathway. The studies used antioxidative(NAC) to demonstrate the effect of reactive oxygen species(ROS) on the cell proliferation inhibitory, cell cycle arrest and mitochondrial membrane potential. First, the studies used MTT assay that NAC at 1 mM pretreated for 1 h,the results showed that the inhibitory effect of Gyp wasprevented by pretreatment with NAC.The flow cytometry analysis showed that the reduction of the mitochondrial membrane potential and cell cycle arrest by Gyp both blocked significantly by pretreatment of NAC.The cell proliferation inhibitory, cell cycle arrest and loss of mitochondrial membrane potential results from ROS generation could dissolved by NAC. Previous studies suggested that ROS which acted as signaling intermediates played an important role in mitochondrial dysfunction. Laser scanning confocal microscope observed the subcellular localization of cytochrome c and Bax. The results showed cytochrome c redistribution from mitochondrial to cytoplasm, Bax translocation and from cytoplasm to mitochondrial. L1210 cell were treatment with Gyp for 12h,24h,48h,72h,the levels of caspase -9,3 increased were detected by western all in time dependent manner.