The Study Of Apatinib Mesylate With 5-Fluorouracilon On The Proliferation And Apoptosis Of Colon Cancer Cells

Colon cancer is a common malignant tumor of the digestive system.Surgery combined with adjuvant chemotherapy is a commonly accepted treatment mode.The appearance of molecular-targeted drug treatment models in recent years has enriched its treatment options and greatly improved the quality of life and survival time of patients.The application of a new anti-angiogenesis molecular targeting drug,apatinib mesylate,has brought new vitality to the treatment of colon cancer.Objective:The purpose of this study was to observe the effects of apatinib、5-fluorouraciland their combination on the proliferation and apoptosis of human colon cancerHCT-116 cells,and further study the possible mechanism of apatinib combined with 5-fluorouracil inducing apoptosis in HCT-116 cells.Methods:(1)Human colon cancer HCT-116 cells were cultured in vitro and divided into 5 groups:blank control group(only cells without other processing factors);solvent control group(0.1%DMSO);apatinib group(10,20,40,80,160,320 μmol/L);5-fluorouracil(20,40,80,160,320,640μmol/L);Apatinib+5-fluorouracil combined group(135+40 μmol/L,135+ 80μmol/L,135+160μmol/L);after treatment of cells in different drug concentrations and culture for 24 hours,the CCK-8 test was used to detect the proliferation of HCT-116 cells by apatinib,5-fluorouracil,and combination drugs.The effect of activity was then selected for the appropriate combination of apatinib and 5-fluorouracil(135 μmol/L+80μmol/L)for subsequent experiments.(2)After culturing the HCT-116 cells required for the experiment in vitro,the experiments were grouped into the apatinib group(135 μmol/L),5-fluorouracil(80 μmol/L),and the combined group(135 μmol/L+80μmol/L),solvent control group(0.1%DMSO).Flow cytometry was used to detect apoptosis;Real-time PCR and Western Blot were used to detect cell Bax,Bcl-2,PLCyl,ERK1/2,p-PLCyl and p-ERK1/2 gene mRNA and protein expression,respectively..Results:(1)CCK-8 test results showed that both apatinib and 5-fluorouracil can inhibit cell proliferation in vitro,and the inhibition rate increased with increasing concentration,showing a concentration-dependent relationship.The combined use of the two drugs has an additive inhibitory effect.(2)The results of flow cytometry detection of HCT-116 cells showed that the apoptosis rate of the apatinib and 5-fluorouracil alone group was higher than that of the control group,and the apoptosis rate of the combined drug group was higher than that of the single drug experimental group.(3)Real-time PCR results showed that after apatinib and 5-fluorouracil were applied to HCT-116 cells,the expression of pro-apoptotic gene Bax was significantly higher than that of the control group,and the expression of anti-apoptotic gene Bcl-2 was significant.Compared with the control group,the expression of related genes in the combination group was more significant than that in the single drug group.(4)The results of Western Blot showed that,after apatinib and 5-fluorouracil were applied to HCT-116 cells,respectively,compared with the control group,the expression of pro-apoptotic protein Bax increased and the expression of anti-apoptotic protein Bcl-2 decreased.There was no significant change in the expression of PLCyl,ERK1/2 protein;the expression of p-PLCyl and p-ERK1/2 protein in the 5-fluorouracil monotherapy group did not change significantly,but the p-PLCγ1 and p-ERK1/2 protein expression decreased.There was no significant change in the expression of PLCyl,ERK1/2 protein in the combined application group;the increased expression of pro-apoptotic protein Bax,the decreased expression of anti-apoptotic protein Bcl-2,and the expression of p-PLCyl and p-ERK1/2 proteins The decrease was more significant than that of the single drug group.Conclusion:(1)Both apatinib and 5-fluorouracil have inhibitory effects on the proliferation of HCT-116 cells,and they show a concentration-dependent relationship.The combined application of the two drugs in the inhibition of cell proliferation is manifested as a combined effect of drugs.(2)Both apatinib and 5-fluorouracil can induce the apoptosis of HCT-116 cells and inhibit the proliferation of the cells,and the effect is more obvious when the two drugs are combined.Possible mechanism:The combination of apatinib and apatinib with 5-fluorouracil promote the expression of apoptotic protein Bax,inhibit the expression of anti-apoptotic protein Bcl-2,it inhibits the phosphorylation of PLCγ1 and ERK1/2,and blocks the conduction of PLCγ1/ERK1/2 signal pathway.