Celastrus Orbiculatus Extract Suppresses Migration And Invasion Of MGC-803 Cells By Inhibiting Prohibitin

Gastric cancer is one of the most common malignancies in the world,ranking the second in the world.In China,the morbidity of gastric cancer of 2015 was 15.8%,and the mortality was 17.6%,ranking the second in the incidence of cancer.The 5-year overall survival rate after radical resection in patients with early stage gastric cancer is 90%,but the early detection rate is low,and about 80%of patients in China have been in advanced stage at presentation..At present,the clinical therapy of gastric cancer are limited,the overall survival rate of pure surgical resection is about 20%,and the radiotherapy and chemotherapy fail to have significant survival advantages.Invasion and metastasis in early stage are the important death cause of patients with gastric cancer,and the process of invasion and metastasis is complex,chiefly including changes in cell adhesion properties,the degradation of extracellular matrix,EMT,tumor angiogenesis,etc.,and is regulated by the many factors.Prohibitin(PHB)is a evolutionarily highly conserved protein and play an important role in regulating cellular energy metabolism,proliferation,apoptosis,nuclear transcription.The abnormal expression of PHB exist in many types of malignant tumors,and studies have found that PHB is closely related to the invasion and metastasis of tumor cells.Celastrus Orbiculatus Thunb.,also named Guo Shanfeng,daemonorops,etc.Its stem,root,leaf,fruit,seeds can all be used as medicine.In vivo and vitro,Celastrus Orbiculatus ethylacetate extract(COE)have Antineoplasmic activity.COE can inhibit the proliferation of tumor cells,induce apoptosis,inhibit ihe nvasion and migration of tumor cells and so on,thereby inhibiting the growth and metastasis of various tumor cells.Studies have found that COE can significantly inhibit the invasion and metastasis of gastric cancer cell and the occurrence of EMT.After screening of target protein by proteomic technology,PHB is one of the four functional proteins protein.Therefore,this study constructed human gastric cancer MGC-803/PHB" cells,and discussed the effect of different concentrations of COE on MGC-803/PHB-cells.It was found that COE could inhibit the invasion and migration of gastric cancer cells by regulating PHB,and the molecular mechanism may be related to the c-Raf/ERK signaling pathway.The study will be elaborated by three parts,as follows.Part I Effects of COE on expression of PHB in MGC-803 cellsObjective:To investigate the effects of COE on expression of PHB in MGC-803 cells.Methods:The human gastric carcinoma MGC-803 cells were divided into negative control group,COE group(10,20,40?g/mL)and positive control groups(5-fu 32?g/mL,JLC 0.4 mg/mL).After 24 h,western blotting was used to measure the expression of PHB in human gastric cancer MGC-803 cells.Results:COE treatment MGC-803 cells for 24 h decreased the expression of PHB in a dose-dependent manner.(P<0.05).Conclusion:COE inhibits the expression level of PHB in human gastric cancer MGC-803 cells.Part II Construction of MGC-803/PHB-cells and Effects of PHB silencing on invasion and migration of MGC-803 cells Objective:To construct the human gastric cancer MGC-803/PHB-cells and to observe effects of PHB silencing on invasion and migration of MGC-803 cells.Methods:The MGC-803 cells were infected with concentrated virus according to the manufacturer's instructions.Cells were then grown for 48 hours at 37°C,5%C02.The cells were visualized under a fluorescence microscope(Nikon,Chiyoda-Ku,Tokyo,Japan)to detect transfection efficiency and the PHB expression in the infected cells was validated by Western Blotting assay.The invasion of cells was measured by transwell invasion assay.The migration of cells was detected by transwell migration assay and cell scratch test.Western Blotting analysis indicated the protein expression of E-cadherin,Vimentin,N-cadherin,MMP-2,MMP-9 and c-Raf/ERK signaling pathways.Results:Western blotting results indicated that PHB protein of LV-PHB-RNAi group was significantly decreased in MGC-803 cells compared with Vehicle group and wild-type cells.Compared with the control groups,the LV-PHB-RNAi group displayed a decrease in the protein expression of Vimentin,N-cadherin,MMP-2,and MMP-9,however,increased expression of E-cadherin was observed.Furthermore,PHB silencing significantly reduced the levels of c-Raf,ERK,and p-ERK.Conclusion:The construction of gastric carcinoma MGC-803/PHB' is successful.PHB silencing inhibited the invasion and migration of MGC-803 cells in a dose-dependent manner.And the mechanism may be related to c-Raf/ERK signaling pathway.Part III Effects of COE on invasion and migration of MGC-803/PHB-cellsObjective:To observe effects of COE on invasion and migration of MGC-803 cells.Methods:Control MGC-803/PHB-cells exhibited active growth in vitro,whereas cells treated with various concentrations of COE for 24,48,or 72 h.Effect of COE on the proliferation of MGC-803/PHB-cells were detected by MTT assay.Transwell invasion assay were used to measure the invasion of MGC-803/PHB-cells.Cell scratch test and transwell migration assay were used to measure the migration of MGC-803/PHB-cells.After 24h,western blot was used to detect the EMT-related proteins(E-cadheri,Vimentin,N-cadherin),MMP-2,MMP-9 and the key proteins of c-Raf/ERK pathway.Results:COE treatment inhibited the viability of MGC-803/PHB-cells by the MTT assay.The COE concentrations of 10,20,and 40 ?g/mL were used in subsequent experiments to exclude the cytotoxic effect of COE on cell invasion and migration.COE treatment for 24 h inhibited the invasion and migration of MGC-803 cells in a dose-dependent manner,COE decreased the expression of Vimentin,N-cadherin,MMP2,MMP9,c-Raf,ERK1/2 protein in a concentration-dependent manner,and increased the expression of N-cadherin,p-ERK1/2 protein.Conclusion:COE inhibited the invasion and migration of MGC-803/PHB-cells in a dose-dependent manner.And the mechanism may be related to c-Raf/ERK signaling pathway.