Celastrus Orbiculatus Extracts Inhibit The Metastasis Through PI3K/Akt/mTOR Signaling Pathway In Human Gastric Cancer MGC-803 Cells

Celastrus orbiculatus grows in large numbers in many provinces of China,and has been used as an anti-inflammatory and anti-rheumatism drug since the Qing Dynasty.In order to protect the roots of plants and maintain ecological balance,we used plant stems to extract anticancer active ingredients.In recent years,we have revealed the biological function of the antineoplastic activity of the C.orbiculatus extracts(COE),including the effective inhibition of COE on the expression of mTOR protein in hepatocellular carcinoma cells,however,its mechanisms have not been completely understood.Gastric cancer is a serious threat to human health,with high morbidity and mortality.About 70 million people died of gastric cancer each year,accounting for more than half of the number of deaths in digestive system tumors.Due to early clinical symptoms are not obvious,the early diagnosis and treatment rate is low.In clinical practice,although we have identified many factors that affect the curative effects of GC,the invasion and metastasis of GC cells are two most influential ones.Mammalian target of rapamycin(mTOR)is the key protein that regulates cell growth and metabolism.It has been found that the expression of mTOR protein in patients with GC increased abnormally.GC cells obtain the ability to metastasize to lymph nodes by undergoing the epithelial mesenchymal transition(EMT).In addition to tumorigenesis,aberrant EMT activation,which regulates the sensitivity of gastric cancer cells to cisplatin.The mechanisms have not clearly underlying the Celastrus orbiculatus extracts(COE)inhibited the expression of the mammalian target of rapamycin(mTOR)in human gastric cancer cells.In this study,we first constructed a human gastric cancer MGC-803 cell with low mTOR expression.To study whether COE could target mTOR to inhibit the invasion and migration of gastric cancer cells,and whether the mechanism of action was related to the PI3K/Akt/mTOR signaling pathway.The study will be elaborated by three parts,as follow.Part I Abnormal Expression of mTOR and EMT Related Proteins in Gastric Carcinoma and Construction of MGC-803/mTOR-CellsObjective:To observe the expressions of mTOR、p-mTOR and EMT related proteins in human gastric cancer tissue samples.And then construct the human gastric cancer MGC-803 cell with low mTOR expression.Methods:The expressions of mTOR、p-mTOR and EMT related proteins(E-cadherin,N-cadherin and Vimentin)in human gastric cancer tissue were observed by immunohistochemistry.The MGC-803/mTOR-cells were constructed by knockdown of mTOR using lentivirus infection technique.24 hours after infection,the cells were visualized under an inverted fluorescence microscope to detect fluorescence intensity.The expression of mTOR protein was detected by western blot respectively.Results:Compared with the adjacent normal tissue,the expression of proteins mTOR and p-mTOR in gastric cancer cells were increased significantly,indicating that the mTOR signaling pathways were abnormally activated in gastric cancer cells.The results showed that E-cadherin was decreased,but the expression of N-cadherin and Vimentin were significantly increased in gastric carcinoma.24 hours after infection,the green fluorescent protein(GFP)was observed obviously in the MGC-803/mTOR-cells under microscope.Compared with the wild type MGC-803 cells,the western blot results showed that mTOR protein was significantly decreased in MGC-803/mTOR-cells(P<0.001).Conclusions:In gastric cancer tissue samples,mTOR and p-mTOR expressions were increased.E-cadherin expression was decreased,N-cadherin and Vimentin expressions were significantly increased.The construction of human gastric cancer MGC-803/mTOR" cells is successful.Part Ⅱ Effects of Celastrus Orbiculatus extracts on the proliferation,the invasion and migration in MGC-803/mTOR-cellsObjective:To detect the effect of COE on the proliferation,migration and invasion of MGC-803/mTOR-cells.Methods:The human gastric carcinoma MGC-803/mTOR’ cells of logarithmic phase were added with different concentrations of COE(10,20,40,80,160,320μg/mL)for 24,48,or 72 h.Effect of COE on the proliferation of MGC-803/mTOR’ cells were detected by MTT assay.The cells were treated with various concentrations(20,40,80 μg/mL)of COE or 5-fu(50 μg/mL)for 24h.The invasion of MGC-803/mTOR-cells was detected by transwell invasion assay.The migration of MGC-803/mTOR-cells was detected by transwell migration assay and cell scratch test.Results:The results showed that the COE inhibited the proliferation of the cells significantly(P<0.05).The IC50 value of COE for 24 h was approximately 129.17p.g/mL.In order to eliminate the effect of cytotoxicity on the experimental results,the subsequent concentration of COE were set to 20,40,80 μg/mL for 24 h.Compared with the control group,the invasion and migration ability of MGC-803/mTOR" cells in human gastric cancer was significantly reduced in a concentration-dependent manner(P<0.05).Conclusion:COE inhibits the proliferation,invasion and migration ability of human gastric cancer MGC-803/mTOR-cells.Part Ⅲ COE Inhibit the Metastasis through Attenuating PI3K/Akt/mTOR Signaling Pathway in MGC-803/mTOR CellsObjective:To investigate whether the COE inhibited the metastasis through mTOR signaling pathway in human gastric cancer MGC-803/mTOR-cells.Methods:The MGC-803/mTOR-cells were treated with various concentrations(20、40、80μg/mL)of COE or 5-fu(50 μg/mL)for 24h.We analyzed the effect of the extract of COE on the EMT in MGC-803/mTOR-cells by Western Blotting assay.To further explore the molecular mechanisms for COE inhibition on the invasion and metastasis in MGC-803/mTOR" cells,the key proteins related to mTOR signaling pathway were detected using Western Blotting assay.Results:The results showed that the expression of E-cadherin protein was increased,and the expression of N-cadherin and Vimentin were decreased simultaneously in the MGC-803/mTOR-cells(P<0.05).With the increase of COE concentration,the protein expression level of E-cadherin is further increased.At the same time,the expression of N-cadherin and Vimentin decreased more obviously.The p-4EBP1,4EBP1,p-P70S6k,P70S6k,p-mTOR,mTOR,PI3K and Akt proteins were reduced in dose-dependent manners in MGC-803/mTOR-cells(P<0.05).Conclusions:The experimental results were consistent with the expectation that COE inhibited the invasion and metastasis through PI3K/Akt/mTOR signaling pathway.