| BackgroundDespite numerous studies on the diagnosis and treatment of pancreatic cancer in recent years,pancreatic cancer still ranks as the most deadly malignancy among all tumors with a low 5-year survival rate(approximately 9%).About half of pancreatic cancer patients cannot receive surgical treatment due to distant metastasis at the first diagnosis,and the limited effect of chemotherapy makes the treatment unable to achieve the desired effect.Therefore,it is of great significance to find effective treatment for pancreatic cancer,improve the quality of life of pancreatic cancer patients,and prolong the survival of pancreatic cancer patients.The study found that about 80% of the cancer before symptoms of patients with pancreatic cancer is elevated fasting glucose or diabetes,and in epidemiological study found that patients with pancreatic cancer tumors growth and a positive linear relationship with blood sugar,diabetes in pancreatic cancer may exist,and the mechanism is still in the exploration,and control blood sugar has become an important method in the comprehensive treatment of pancreatic cancer..In recent years,epithelial mesenchymal transformation has been considered to play an important role in tumor development.When epithelial-derived tumor cells show mesenchymal cell phenotype,the expression of e-cadherin,the most important adhesion molecule between tumor cells,is significantly down-regulated or even lost,which significantly increases the mobile ability of tumor cells,leading to enhanced invasion and metastasis ability of tumor cells.Objective1.To determine the effects of different glucose concentrations on epithelial mesenchymal transformation and cell cycle of pancreatic cancer cells,and whether changes in cell cycle can be caused by changing the expression of pi3k-akt pathway protein.2.The effects of different hypoglycemic drugs on the epithelial mesenchymal transformation of pancreatic cancer cells,as well as on the expression of cell cycle-relatedproteins and proteins in the PI3K-Akt pathway were determined.MethodsIn this study,we selected a total of three cell lines as experimental subjects,namely,normal pancreatic ductal epithelial cell H6c7 and human pancreatic ductal epithelial cancer cell Panc-1,and transfected H6c7-k-ras cell line with k-ras mutation.During the study,three cell lines were cultured in medium with different glucose concentrations for 36 hours,then cell cycle was detected by flow cytometry,and changes in protein expression levels of relevant important marker molecules e-cadherin,Vimentin,p53,p21,cdc2,Cyclin B1,PI3 K,Akt,p-Pi3 k and p-Akt were detected by western blot assay.On the basis of the above experiments,insulin,metformin and exendin-4 of different concentrations were added to the medium and Panc-1 cells were cultured for 36 h according to the experimental requirements,and the above protein imprinting experiment was repeated.Result1.Western blot analysis of protein expression levels of EMT-related marker molecules in three types of cells at different glucose concentrations showed that high glucose inhibited and promoted E-cadherin and Vimentin,respectively,in the three types of cells.Compared with H6c7 cells,H6c7-k-ras and PANC-1 cells had lower glucose concentrations for epithelial mesenchymal transformation.2.Flow cytometry was used to detect the cell cycle changes of the three types of cells at different glucose concentrations,and the results showed that the cell cycle of H6c7 was not affected by glucose concentrations(P>0.05).However,high concentration of glucose significantly reduced the proportion of G0/G1 phase cells in H6c7-k-ras cells and Panc-1 cells(P<0.05),and simultaneously increased the proportion of G2/M phase cells(P<0.05).3.Western blot was used to detect the protein expression levels of cell cycle regulatory factors in three cells at different glucose concentrations.The results showed that glucose concentration had no significant effect on the protein expression of p53,p21,cdc2 and Cyclin B1 in H6c7 cell(P>0.05).However,high concentration of glucose significantly reduced the protein expression of p53 and p21 in H6c7-k-ras and Panc-1 cells(P<0.05),while increased the protein expression of cdc2 and Cyclin B1(P<0.01).4.Western blot was used to detect the protein expression level of PI3K-Akt signalingpathway in three different cell lines under different glucose concentration culture conditions.The results showed that glucose concentration did not significantly affect the protein expression of PI3 K,p-PI3 K,Akt and p-Akt in H6c7 cells(P>0.05).In H6c7-k-ras cells,glucose concentration did not affect the protein expression of PI3K(P>0.05),but high glucose concentration increased the protein expression of Akt(P<0.05),and the protein expression of p-PI3 K and p-Akt increased more significantly(P<0.01),with statistical difference.In Panc-1,the expression of PI3 K,p-Pi3 k,Akt and p-Akt increased with high glucose concentration(P<0.05),but the effect on p-PI3 K and p-Akt was more significant(P<0.01).5.After insulin,metformin and exendin-4,protein expression levels of EMT-related marker molecules in Panc-1 cells in high-concentration glucose culture were detected by Western blot.The results showed that insulin and exendin-4 had no significant effect on the protein expression of E-cadherin and Vimentin.Metformin can promote the expression of E-cadherin and inhibit the expression of Vimentin.6.After insulin,metformin and exendin-4 were used,protein expression levels of panc-1 cell cycle in culture with high glucose concentration were determined by Western blot.The results showed that metformin increased the protein expression of p53(P<0.05)and decreased the protein expression of cdc2 and Cyclin B1(P<0.05).Exendin-4 can reduce the protein expression of cdc2 and cyclin B1(P<0.05),and has no significant effect on p53 and p21.Insulin inhibited the protein expression of p53(P<0.05)and increased the protein expression of cdc2 and cyclin B1(P<0.05).7.Western blot was used to detect the protein expression level of pi3k-akt signaling pathway in panc-1 cells in high-concentration glucose culture after the action of insulin,metformin and exendin-4.The results showed that none of the drugs affected the expression of PI3 K and Akt,but insulin significantly increased the protein expression of P-PI3 k and P-Akt(P<0.05),while metformin and exendin-4 significantly inhibited the protein expression of P-PI3 k and P-Akt(P<0.05).ConclusionIn summary,high glucose concentration can promote the epithelial mesenchymal transformation of pancreatic cancer cells and promote the proliferation cycle of pancreatic cancer cells by up-regulating the expression of related proteins in the PI3K-Akt signaling pathway.Metformin can inhibit the epithelial mesenchymal transformation of pancreaticcancer cells,up-regulate the expression of p53 protein,down-regulate the expression of cdc2 and cyclin B1 proteins,and inhibit the protein expression of PI3K-Akt signaling pathway.Exendin-4 has no obvious effect on epithelial mesenchymal transformation of pancreatic cancer cells,but can down-regulate the protein expression of cdc2 and cyclin B1,and inhibit the protein expression of PI3K-Akt signaling pathway.Insulin has no effect on the epithelial mesenchymal transformation of pancreatic cancer cells,but can down-regulate the protein expression of p53,up-regulate the protein expression of cdc2 and cyclin B1,and promote the protein expression of PI3K-Akt signaling pathway. |
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