Gypenoside L Inhibits Proliferation Of Liver And Esophageal Cancer Cells By Inducing Senescence

The purpose of the experiment: Gynostemma pentaphyllum Thunb Makino is a grass climbing plant of the genus Cucurbitaceae and Gynostemma.It is a precious dualuse plant for folk medicine in China.It is widely distributed,safe and non-toxic.Gynostemma can regulate the immune system and exert various functions,such as antihypertensive,anti-hyperlipidemic,anti-diabetes and anti-fatty liver.Therefore,it has extremely high medicinal and health care development value.Our previous experiments have showed that Gypenoside L,a saponin isolated from G.pentaphyllum,has potent anti-hepatocarcinoma tumor and anti-esophageal cancer activity,as it triggered cell death via inhibiting autophagy and inducing vacuolation.However,the detailed mechanisms still remained to be clarified.Therefore,in our works,we further investigated the possible molecular mechanisms of Gyp-L toward liver and esophageal cancer cells.Methods: 1.Study on the anti-tumor activity of Gyp-L: Two cell lines of liver cancer and esophageal cancer were used in this experiment.The effect of Gyp-L on growth and proliferation of tumor cells were determined by the CCK8 Kit Assay.Morphology and number of these cells were also observed before and after Gyp-L treatment;2.Study on the anti-tumor mechanism of Gyp-L:(1)using EDU staining assay to detect cell proliferation,?-galactosidase staining to detect cellular senescence and using quantitative real-time PCR to detect the mRNA expression levels of senescenceassociated cytokines,such as IL-6,IL-1a,TIMP1,CYCL1 and CYCL2;(2)Flow cytometry assay was used to determine the effect of Gyp-L on cell cycle;(3)Western Blot assay was used to detect the effect of Gyp-L on cell cycle and senescenceassociated proteins,including p18,p21,p27,p38,AKT,ERK,CDK2,CDK4,CDK6,CYCLIND1,etc.;(4)Western Blot detection the effect of Gyp-L on NF-?B signaling pathway;(5)Specific inhibitors of MAPK and NF-?B signaling pathway were used confirm their functions in Gyp-L-induced cellular senescence;(6)CCK8 assay was sed of tumor cells.Results and Conclusion: 1.Gyp-l inhibited cell growth of liver cancer and esophageal cancer in vitro;2.Gyp-L inhibited tumor cell proliferation by inducing cellular senescence;3.Gyp-l caused cell cycle arrest at S phase;4.Gyp-L activated p38,ERK and NF-?B signaling pathways to activate the expression of cellular senescencerelated proteins and cell cycle-related proteins,which in turn,caused cell cycle arrest and inhibited tumor cell proliferation;5.Gyp-l enhanced the sensitivity of tumor cells to first-line clinical drugs,such as cisplatin and 5-fluorouracil.