| Research Purpose:Jianpi Yangzheng Recipe was created by professor liu shenlin,a national famous Chinese medicine doctor in China,based on years of clinical experience and the use of drugs for digestive diseases.On the basis of using this recipe to treat gastric cancer,colorectal cancer,pancreatic cancer and other digestive system tumors,its clinical efficacy is accurate.On the basis of results of clinical practice and previous experimental explorations,our present research aims to study the effect of Jianpi Yangzheng Recipe on apoptosis and Epithelial-mesenchymal transition(EMT)and related protein expressions in gastric cancer cell lines with various differentiated degrees(MGC-803,SGC-7901 and BGC-823),in order to explore its possible mechanism of action via aerobic glycolysis pathway regulated by PKM2 and PI3K/AKt/mTOR signaling pathway,providing a new theoretical basis for the treatment of gastric cancer with traditional Chinese medicine.Research Methods:1 We performed MTT assay to study the inhibitive efficient of Jianpi Yangzheng Recipe with different concentrations on gastric cancer cell lines MGC-803,SGC-7901,BGC-823.2 Flow cytometry was used to detect the effect of Jianpi Yangzheng Recipe with different concentrations on gastric cancer cell lines,MGC-803,SGC-7901 and BGC-823 apoptosis.3 We used the cell cycle detection kit to detect the effect of Jianpi Yangzheng Recipe on the cell cycle of MGC-803,SGC-7901 and BGC-823 in human gastric cancer.4 We observed the effect of Jianpi Yangzheng Recipe on the cell migration ability of three gastric cancer cell lines by cell scratch experiment.5 Cell invasion experiment(Transwell)was used to observe the effect of Jianpi Yangzheng Recipe on the invasion ability of three gastric cancer cells.6 Western blotting assay was conducted to detect apoptosis-related proteins of gastric cancer cells in each group,such as B lymphocytoma-2 gene(Bcl-2),Bcl-2 related X protein(Bax),Caspase-3,Cleaved caspase-3 and EMT-related proteins such as E-cadherin,N-cadherin and Vimentin,also,expression of matrix metalloproteinases,MMP-9 and MMP-2.7 Western blotting was performed to explore the effect of Jianpi Yangzheng Recipe on the expression of PI3K/AKt/mTOR signaling pathway in three gastric cancer cells.8 Lactic acid(LD)test kit was used to detect the effects of Jianpi Yangzheng Recipe with different concentrations on lactic acid production in three gastric cancer cells.9 We used glucose assay kit to detect the effect of Jianpi Yangzheng Recipe on the utilization of glucose in three gastric cancer cell lines.10 Western blotting assay was used to detect the protein expression of glycolytic related proteins,such as pyruvate dehydrogenase kinase 1(PDK1),pyruvate dehydrogenase kinase 4(PDK4)and pyruvate kinase isoenzyme M2(PKM2).11 PKM2-overexpressed lentivirus vector(LV-PKM)was constructed and transfected into three gastric cancer cells,MGC-803,SDC-7901 and BGC-823.Immunofluorescence detection and Western blotting were performed to verify the successful transfection,and a PKM2-overexpressed gastric cancer cell model was established.Negative control group(NC),Jianpi Yangzheng Recipe(8mg/mL)group(NC+JPYZ),PKM2 overexpression group(OE),and The treatment group of Jianpi Yangzheng Recipe(OE+JPYZ)with PKM2-overexpressed cell model,were set.11.1 Western blotting detected the expression levels of Bax and Bcl-2,EMT-related proteins E-cadherin,N-cadherin and Vimentin in gastric cancer cell lines transfected with PKM2 overexpressed lentivirus.11.2 Western blotting was performed to detect the expression levels of PI3K,AKt,mTOR,P-PI3K,P-Akt and P-mTOR in gastric cancer cell lines transfected by PKM2 overexpressed lentivirus.12 The lung metastasis model of gastric cancer in mice was established.The model group,the Jianpi Yangzheng Recipe group,the paclitaxel group,and the Jianpi Yangzheng Recipe combined with paclitaxel group were set.PET-CT imaging was used to detect the inhibition of Jianpi Yangzheng Recipe on lung metastasis,and Western blotting was used to detect the expression of apoptosis-related proteins Bax and Bcl-2,and EMT-related proteins E-cadherin,N-cadherin and Vimentin.Research Results:1 MTT assay showed that the proliferation of MGC-803,SGC-7901 and BGC-823 cells could be effectively inhibited by Jianpi Yangzheng Recipe with different concentrations,and the results were dose-dependent and time-dependent.2 Flow cytometry results indicated that,the apoptosis of GC cells MGC-803,SGC-7901 and BGC-823 could be induced by Jianpi Yangzheng Recipe with different concentrations,which was positively correlated with drug concentrations.3 The results of cell cycle detection suggested that Jianpi Yangzheng Recipe could block the cell cycle S phase of GC cells MGC-803,SGC-7901 and BGC-823,and the degree of block was positively correlated with the drug concentrations.4 The results of cell scratch test showed that the scratch healing rate of the three gastric cancer cells MGC-803,SGC-7901 and BGC-823 significantly decreased under the effect of Jianpi Yangzheng Recipe,suggesting that the cell migration ability was weakened and positively correlated with the drug concentrations.5 The results of cell invasion experiment(Transwell)revealed that the number of cells passing through the cell compartment was significantly reduced after Jianpi Yangzheng Recipe was applied to MGC-803,SGC-7901 and BGC-823 cells,indicating that the cell invasion ability was reduced and showed a certain concentration dependence.6 Western blotting results suggested that the expression levels of apoptosis-related protein Bcl-2 were down-regulated after Jianpi Yangzheng Recipe was treated to MGC-803,SGC-7901 and BGC-823 gastric cancer cells,while the expression levels of Bax and Cleaved Caspase-3 were up-regulated,and the expression levels of EMT-related protein E-cadherin were up-regulated,while the expression levels of N-cadherin,Vimentin,MMP-9 and MMP-2 were down-regulated,all with a certain drug concentration dependence.7 Western blotting results revealed that Jianpi Yangzheng Recipe could effectively inhibit the PI3K/AKt/mTOR signaling pathway,that is,P-PI3K,P-AKt and P-mTOR protein expression was down-regulated after Jianpi Yangzheng Recipe was applied to MGC-803,SGC-7901 and BGC-823 cells,and there was a certain drug concentration dependence.8 The results of lactic acid(LD)test kit showed that the amount of lactic acid produced by the three gastric cancer cells MGC-803,SGC-7901 and BGC-823 significantly decreased under the effect of Jianpi Yangzheng Recipe,which was positively correlated with the drug concentrations.9 The results of glucose assay kit showed that the amount of glucose used by MGC-803,SGC-7901 and BGC-823 gastric cancer cells significantly decreased under the effect of Jianpi Yangzheng Recipe,which was also positively correlated with drug concentration.10 Western blotting experiments indicated that,compared with the blank group,there was no significant statistical difference in the protein expression levels of PDK1 and PDK4 in each drug group,while the expression levels of PKM2 were significantly down-regulated with the increase of drug concentration.11 Immunofluorescence microscopy and Western blotting assay confirmed the successful transfection of PKM2-overexpressed lentivirus,thus establishing a PKM2-overexpressed gastric cancer cell line model.11.1 Western blotting results showed that lentivirus transfection with PKM2 overexpression group(OE)could significantly down-regulate the expression of E-cadherin and Bax proteins,and significantly up-regulate the expression of N-cadherin,Vimentin and Bcl-2 proteins.The effect of Jianpi Yangzheng Recipe on OE group up-regulated the expression of E-cadherin and Bax proteins,and down-regulated the expression of N-cadherin,Vimentin and Bcl-2 proteins.11.2 Western blotting experiments showed that the PKM2 overexpression group(OE)transfected with lentivirus could significantly activate the PI3K/AKt/mTOR signaling pathway,that is,the expressions of P-PI3K,P-Akt,and P-mTOR proteins were significantly up-regulated,while the expressions of P-PI3K,P-Akt,and P-mTOR proteins were significantly down-regulated when the Jianpi Yangzheng Recipe was treated to the OE group.12 In vivo experiments on the lung metastasis model of gastric cancer in mice,PET-CT imaging and Western blotting experiments both showed that Jianpi Yangzheng Recipe and paclitaxel had good synergic effects on lung metastasis of gastric cancer.Research Conclusions:1 Jianpi Yangzheng Recipe can effectively inhibit the proliferation of MGC-803,SGC-7901 and BGC-823 in human gastric cancer cells and induce cell apoptosis by increasing Bax/Bcl-2,and activating Caspase-3.2 Jianpi Yangzheng Recipe can significantly inhibit the migration and invasion of MGC-803,SGC-7901 and BGC-823cells,which may be related to up-regulation of E-cadherin protein expression and down-regulation of N-cadherin,Vimentin,MMP-2 and MMP-9 protein expression.3 Jianpi Yangzheng Recipe can inhibit the activation of PI3K/AKt/mTOR signaling pathway in gastric cancer cells.4 Jianpi Yangzheng Recipe can effectively inhibit the aerobic glycolysis of MGC-803,SGC-7901 and BGC-823cells and the mechanism may be related to the obvious down-regulation of key protein expression of PKM2.5 The expression of N-cadherin,Vimentin and Bcl-2 proteins was up-regulated and E-cadherin and Bax proteins were down-regulated by activating PI3K/AKt/mTOR signaling pathway in gastric cancer cell lines transfected with PKM2 lentivirus.The results showed that the activity of the PI3K/AKt/mTOR signaling pathway in gastric cancer cells was inhibited by Jianpi Yangzheng Recipe,and the mechanism was related to the inhibition of the aerobic glycolysis pathway in cells regulated by PKM2.In conclusion,the mechanism of Jianpi Yangzheng Recipe inducing cell apoptosis and inhibiting EMT of gastric cancer cells may be related to inhibiting the aerobic glycolysis pathway by down-regulating the protein expression of PKM2 and thus inhibiting the activity of PI3K/AKt/mTOR signaling pathway. |
PDF Full Text Request