| Colorectal cancer(CRC)is one of the most common malignancies tumors in digestive tract,and its global incidence has been increasing year by year,which has caused a huge economic burden to families and society.Surgical treatment is a radical cure for early colorectal cancer,while chemotherapy and radiotherapy are used to improve 5-year survival in patients with advanced colorectal cancer.However,due to primary or secondary chemotherapy resistance,especially the emergence of multi-drug resistance(MDR)of tumor cells is one of the main reasons leading to the failure of chemotherapy,so that the effect of chemotherapy is not good,and the prognosis of patients is poor.Therefore,it is an urgent problem to overcome multi-drug resistance and improve the effect of chemotherapy in colorectal cancer.Multidrug resistance means that when a drug acts on a tumor to make it resistant,the tumor develops cross-resistance to a variety of antitumor drugs that are not exposed to it,that are structurally independent,and that have different mechanisms.The mechanisms of MDR production are complex and diverse,but the activation of transporters is the most important.The transporters studied in this study include P-glycoprotein(P-gp)and multi-drug resistance related protein 2(MRP-2),both of which belong to the ABC family.P-gp is encoded by ABCB1 gene,while MRP-2 is encoded by ABCC2 gene.Currently,it has been found that the activation of PI3K/Akt signaling pathway plays a regulatory role in the process of multi-drug resistance in colorectal cancer.The PI3K/Akt pathway(Akt or PKB pathway)is a tyrosine kinase cascade signaling pathway and one of the cell survival pathways.GSK3 beta(glycogen synthase kinase 3 beta)as one of the main factors of its downstream,PI3K/Akt pathway activation leads to phosphorylation at GSK3 beta serine 9 sites,thus inhibiting activity of GSK3 beta,by regulating cell proliferation,cell growth,cell apoptosis,cell cycle,and so on,and then participate in the occurrence and development of tumor cells,and is a key regulatory factor of tumor cells.It has been reported that increased GSK3-Ser9 phosphorylation can up-regulate the expression of P-gp and promote the development of multidrug resistance in cholangiocarcinoma.However,the correlation between GSK3 beta and ABC transporters in colorectal cancer has rarely been reported.Objective:To investigate the role of the PI3K/Akt signaling pathway in regulating the expression of ABC transporter through the downstream GSK3? pathway in the multidrug resistance of colorectal cancerMethod:1 The experiment was divided into three groups: colorectal cancer cells HCT-15 as the control group,HCT-15+GSK3? inhibitor(Indirubin-3'-monoxime,HY-19807),HCT-15 +PI3K/Akt pathway inhibitor(GDC-0032,HY-13898)as the experimental group,respectively.2 The expressions of AKT,p-Akt,total GSK3?,p-GSK3?-Ser9 and ABC transporter(P-gp,MRP2)in the three groups of cells were detected by Western blotting.3 Changes in the expression of ABC transporter mRNA in cells of each group were detected by qRT-PCR.4 CCK-8 method was used to detect the 50% inhibition concentration of oxaliplatin on the three groups of cells,and the inhibition rate and drug resistance index were calculated.5 The effects of different inhibitors on cell migration ability and cell cycle were detected by cell scratch assay and cell cycle assay.6 Statistical analysis was performed using SPSS19.0 statistical software,and the experimental results were obtained.Result:1 Western Blot experiment results.1.1 GSK3 beta inhibitor(HY-19807)to act on human colon cancer HCT-15 cells lead to the expression of p-GSK3?-Ser9 increased,and the expression of P-gp and MRP-2 was significantly higher than that of the untreated HCT-15 group,and the difference was statistically significant(P<0.05).AKT and p-Akt expression change in the upstream of GSK3? is not obvious,there were no statistically significant differences(P >0.05).1.2 After PI3K/Akt pathway inhibitor(HY-13898)was applied to human colon cancer HCT-15 cells,the expression levels of p-GSK3?-Ser9 and p-AKT were decreased,while the expression levels of Akt were increased.The relative expression levels of P-gp and MRP2 protein were significantly lower than those of the control group,with statistically significant differences(p <0.01).1.3 After application of different inhibitors compared with control group the expression of total GSK3? does not change significantly in the two experimental group,there were no statistically significant differences(P >0.05).2 Experimental results of real-time fluorescence quantitative PCR(qRT-PCR).2.1 After the action of HY-19807 on HCT-15 cells,the expressions of ABCB1 mRNA and ABCC2 mRNA were significantly higher than those of the control group,with statistically significant differences(P<0.01).2.2 After the action of HY-13898 on HCT-15 cells,the relative expressions of ABCB1 mRNA and ABCC2 mRNA were significantly lower than those of the control group(P<0.01).3 Effects of different inhibitors on the sensitivity of oxaliplatin in HCT-15 cells3.1 After HY-19807 was applied to HCT-15 cells,the inhibitory concentration(IC50)of oxaliplatin on HCT-15 cells increased from(19.83±0.8)?g/mL to(27.15±1.57)?g/mL,and the drug resistance index was 1.36,the difference was statistically significant(P<0.01).3.2 After HY-13898 was used to block the PI3K/Akt signaling pathway of HCT-15 cells,the IC50 of oxaliplatin on HCT-15 cells decreased from(19.83±0.8)?g/m L to(9.93±1.77)?g/m L,and the drug resistance index was 0.5,the difference was statistically significant(P<0.01).4 Cell scratch test results4.1 Cell scratch 24 h,48h after observation under the inverted microscope,HCT-15 cells 24 h migration rate(23.61±0.99)%,48 h migration rate(34.76±10.75)%.4.2 After the application of HY-19807 on HCT-15 cells,the 24 h migration rate was(21.63±3.79)%,and the 48 h migration rate was(19.90±4.05)%.Compared with the control group,the cell migration ability was not significantly promoted,and the difference was not statistically significant(P>0.05).4.3 After HY-13898 was applied to HCT-15 cells,the 24 h migration rate was(12.63±0.52)%(P<0.01)and 48 h migration rate(17.76±7.83)%(P<0.05).Compared with the control group,cell migration was inhibited,especially after 24 h,and the difference was statistically significant.5 Effects of different inhibitors on cell cycle of CRC5.1 HCT-15 cells HCT-15 cells as control group without any treatment.Flow cytometry analysis showed that the proportion of G1 phase cells was(59.61±0.73)% and that of S phase cells was(23.38±0.74)%.5.2 After HCT-15 cells were treated with HY-19807 for 2 hours,compared with the control group,the percentage of cells in G1 phase was(45.69±0.96)% and significantly reduced,while the percentage of cells in S phase was(39.77±0.92)% and significantly increased,and the difference was statistically significant(P =0.000).5.3 After the treatment of HCT-15 cells with HY-13898 for 24 hours,compared with the control group,the percentage of cells in the G1 phase was(84.17±1.35)% and increased significantly,while the percentage of cells in the S phase was(6.90±0.59)% and decreased significantly,and the difference was statistically significant(P=0.000).Conclusion:1 GSK3? inhibitor can increase the expression of p-GSK3?-Ser9,P-gp and MRP2 protein in HCT-15 cells,and PI3K/Akt pathway inhibitor can reduce the expression of p-GSK3?-Ser9,P-gp and MRP2 protein in HCT-15 cells.It has been confirmed from the protein level that the phosphorylation of GSK3?-Ser9 can regulate the expression of multi-drug resistance related proteins in colorectal cancer.2 GSK3? inhibitor can increase the expression of ABCB1 mRNA and ABCC2 mRNA in HCT-15 cells.PI3K/Akt pathway inhibitor can reduce the expression of ABCB1 mRNA and ABCC2 mRNA in HCT-15 cells.It has been proved from the gene level that GSK3?-Ser9 phosphorylation can regulate the expression of multi-drug resistance related protein genes in colorectal cancer.3 After the application of GSK3? inhibitor,the sensitivity of HCT-15 cells to oxaliplatin decreased,while PI3K/Akt pathway inhibitor can enhance the sensitivity of HCT-15 cells to oxaliplatin,and the phosphorylation level of GSK3?-Ser9 affects the sensitivity of HCT-15 cells to oxaliplatin.4 The PI3K/Akt pathway regulates the expression of ABC transporter through the GSK3? pathway in HCT-15 cells,and the phosphorylation level of GSK3?-Ser9 can affect the proliferation of colorectal cancer cells. |
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