Research On The Anti-cancer Effect Of Gyp On Human Colorectal Cancer SW-480 And SW-620 Cells And Its Synergistic Effect With 5-FU

Gypenosides(Gyp),a popular folk medicine in the China,is the major components in extracts from Gynostemma pentaphyllum Makino.It exist mainly as dammarane type-triterpene glycosides.Gyp had been known for its wide beneficial effects for treating hepatitis,hyperlipoproteinemia and cardiovascular disease.Studies have shown that Gyp has an activity of anti-inflammatory,anti-thrombotic,antioxidative and anti-cancer actions.In the present study,the cytotoxicity,cell cycle arrest,anti-migration of SW-480,SW-620 cells induced by Gyp were investigated.Role of reactive oxygen species(ROS)in Gyp induced cell death was analyzed by intracellular ROS generation and ROS scavenger.On the other hand,Gyp increase the effect of colorectal cancer clinical drug,5-FU,were also investigated.These results may provide evidences for the role of Gyp as a potent anti-colorectal cancer agent in clinical application.Now experimental results were as follows.1.These studies first observed the inhibitory effect of Gyp on human colorectal cancer,SW-480,SW-620 cells.Cultured SW-480,SW-620 cells were treated with Gyp at various concentrations for 24 h and 48 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 SW-480,SW-620 cancer cells in a dose and time dependent manner.The results showed that Gyp can induce cell cycle arrest at the G0/G1 phase in SW-480 cells.In SW-620 cells,the result showed no significant in cell cycle arrest.Next,we observed morphologic changes of cells under invert microscope and fluorescence microscope.The morphological changes of apoptosis charactered cell shrinkage with a condensed nucleoplasm.DNA fragment of cells were detected by flow cytometry following PI staining,DNA fragments were increased in a dose-dependence manner.At the same time,we detected the cell apoptosis rate with Annexin V-PE/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 Rh 123 and increase in the ROS level by DCFH-DA.The results showed loss of mitochondrial membrane potential and increase in ROS.The study also showed that Gyp could exert an inhibitory effect on cell migration in vitro and serious microfilament network collapse as well as the significant decrease in the number of microvilli.2.The above study demonstrated that the apoptotic-inducing effect of Gyp on human colorectal cancers SW-480,SW-620 cells had closed relationship with mitochondrial apoptosis pathway.The studies used antioxidative(NAC)to demonstrate the effect of reaetive oxygen species(ROS)on the cell proliferation inhibitory,cell apoptosis rate,DNA fragment and mitochondrial membrane potential.First,the studies used MTT assay that NAC pretreated for 1 h,the results showed that the inhibitory effect of Gyp was prevented by pretreatment with NAC.The flow cytometry analysis showed that the reduction of the mitochondrial membrane potential,DNA fragment,cell apoptosis and anti-migration by Gyp both blocked significantly by pretreatment of NAC.The loss of mitochondrial membrane potential results from ROS generation could dissolved by NAC in SW-620 cells,but not in SW-480 cells.The results in this study implied that ROS play an important role in Gyp induced cell toxicity and apoptosis and the mitochondria damage may be upstream of ROS generation post Gyp treatment in SW-480 cells.Meanwhile,in the SW-620 cells,ROS generation may be upstream of the mitochondria damage post Gyp treatment.3.The study first tested the 5-FU alone on SW-620 cell proliferation inhibition.The result showed no significant difference between different drug concentrations,24 h cell inhibitory effect is not obvious.After joint Gyp,cell proliferation of SW-620 cells was significantly decreased.The next experiments was to choose 5-FU for 10 μg/ml with Gyp for 70 μg/ml.The cell apoptosis rate was increased in SW-620 cells,with Annexin V-PE/7-AAD labeling in FCM analysis.The morphological changes of apoptosis charactered cell shrinkage with a condensed nucleoplasm.DNA damage of SW-620 cells were detected by single cell gel electrophoresis,DNA fragment were increased.