Mechanisms Research Of Reversal Of 5-fluorouraci Resistance By Epigallocatechin Gallate In Gastric Cancer Drug Resistance Cell Line

Source and Background:More than 80%of patients were advanced gastric cancer in China,once confirmed by pathological diagnosis.Chemotherapy has important role in the comprehensive treatment of advanced gastric cancer.However,drug resistance,such as 5-FU,is an important reason for tumor recurrence and metastasis in advanced gastric cancer.It is necessary to study the mechanism of 5-FU resistance and find a way to reverse the resistance.Objective and significance:Epigallocatechin gallate(EGCG)has vital role in anti-tumor and chemotherapeutic drug sensitization.However,whether EGCG could reverse the resistance of gastric cancer to 5-FU and the mechanisms remain unclear.The aim of this study was to establish a gastric cancer cell line SGC7901/5-FU,which is resistant to the drug of 5-FU,and to explore the mechanism of EGCG in reversing the drug resistance to SGC7901/5-FU.Which may provide evidence for the use of EGCG as a 5-FU chemosensitizer in adjuvant therapy for gastric cancer.Research methods:5-FU drug-resistant cell line SGC7901/5-FU was established by the method of high dose repeated pulse combined with the method of increasing drug concentration.MTT assay and colony formation assay were performed to detect the proliferation activity of parental cells and drug-resistant cells after drug treatment,calculate the half inhibitory concentration IC50 value and resistance index RI.Western blot was used to detect drug resistance-related proteins in gastric cancer resistant cell line.On the basis of above,the SGC7901/5-FU cells were divided into different groups according to EGCG as the main treatment factor,MTT and clone formation assay were used to detect the cell proliferation activity,and the apoptosis rate was detected by flow cytometry.Western blot was used to detect the expression of apoptosis-related proteins.The expression of drug-resistant proteins was detected by RT-PCR and Western blot.The expression of signal pathway resistance-related proteins was also detected by Western blot.In addition,we also established SGC7901/5-FU nude mice transplanted tumor model and then EGCG as the main factor grouping to observe the transplanted tumor growth inhibition on nude mice,The expressions of apoptosis-related proteins MDR1 and P-gp,and signal pathway resistance-related factors such as VEGF and p-TFAP2A were detected by immunohistochemistry in the transplanted tumor.All experiments were repeated 3 times.All P values were two-sided and P values less than 0.05 was considered to be statistically significant.Research results and process:The IC50 value of the drug resistant cell line was significantly higher than that of the parental cell line.The drug resistance index RI was 15.6.The expression of drug resistance-related protein ABCG2、P-GP.MDR-1 和 GST-π were significantly increased in the drug resistant cell line.The proliferation activity of the drug-resistant cell line was higher than that of the parental cell line in different 5-FU concentration(P<0.05).Compared with the 5-FU group,the IC50 of 5-FU + EGCG treated group cells was significantly decreased,and the resistance index RI decreased to 0.12.The proliferation activity of the drug-resistant cell line were significantly decreased after the adding of EGCG under different 5-FU concentrations.The apoptotic rates of control group,5-FU group,EGCG group and EGCG combined with 5-FU group were(3.0 ± 1.0)%,(7.0 ± 1.3)%,(6.0 ± 1.2)%and(18.0 ± 1.4)%,respectively.The apoptotic rate of 5-FU + EGCG group was significantly higher than that of other three groups(F = 129.5,P = 0.0000).Western blot results showed that the expression of apoptosis-related proteins MDR-1,P-gp,VEGF and p-TFA2A were significantly decreased in EGCG-treated cells,and the expression of apoptotic protein PARP and pro-apoptotic protein Bax were significantly increased(P<0.05).The expression of Survivin and Bcl-2 were significantly decreased(P<0.05).The results of RT-PCR reported that the expression of MDR-1 and P-gp was also decreased in EGCG-treated cell.The level of VEGF was down-regulated in EGCG-treated cell detected with ELISA.Animal experiments confirmed that EGCG could significantly inhibit the growth of gastric cancer resistant cell line SGC7901/5-FU transplanted tumor in nude mice,and the expression of MDR-1,P-gp,VEGF and p-TFAP2A in 5-FU+EGCG group were significantly decreased by immunohistochemistry(P<0.05).main conclusion:1,The cell line SGC7901/5-FU,which is resistant to 5-FU,could be established with the method of high dose repeated pulse combined with increasing drug concentration.2,The gastric cancer cell line SGC7901/5-FU transplanted tumor model could be successfully established in nude mice.3,EGCG could reverse the drug resistance of gastric cancer resistant cell line SGC7901/5-FU,and its mechanisms might through the following ways to achieve,1.inhibiting the expression of MDR-1 and P-gp,2.up-regulating the PARP/Survivin and Bax/Bcl-2 protein expression ratios,3..inhibiting the expression of VEGF and down-regulating TFAP2A/VEGF signaling pathway.