| Objective: The incidence rate of colorectal cancer is high in the world.The incidence rate of digestive tract cancer is only next to that of gastric cancer and esophageal cancer.A small number of patients with colorectal cancer can be treated by surgery in the early stage,but the reality is that most of the patients have lost the opportunity of surgery when they are found to be diagnosed,so they enter the long process of chemotherapy,radiotherapy,targeted treatment and immunotherapy.At present,the chemotherapy of colorectal cancer is still based on 5-fluorouracil,combined with platinum and irinotecan.The target includes the treatment of large molecule monoclonal antibody and small molecule tyrosinase inhibitor.In this paper,the inhibition of 5-FU and Apatinib on the proliferation of HT-29 cell line was studied.Based on the fact that Apatinib can inhibit the proliferation of colon cancer cells by inhibiting the PI3K/Akt signaling pathway related proteins,further explore the therapeutic effect and possible molecular mechanism of Apatinib in combination with5-FU for colon cancer,provide a multi-line,effective and better tolerated treatment plan for colon cancer patients,explore more valuable pathways,and guide future drug use.Methods:1.Ht-29 colorectal cancer cell lines were cultured,and the cells were divided into control group,5-Fu group,Apatinib group and combined group.In addition to the control group,5-Fu,Apatinib and 5-Fu + Apatinib were added in different concentration gradients.Apatinib and 5-Fu were used to treat ht-29 in colorectal cancer cell line with single drug and double drug.2.MTT method was used to detect the decreased proliferation effect of Apatinib and5-Fu in ht-29 colorectal cancer cell lines at different concentrations for 48 h after single or combined administration.The concentration of Apatinib and 5-Fu with the most obvious inhibitory effect on cell proliferation was selected as the dominantconcentration.Chen-talalay median effect method was used to calculate the effect of the two drugs combined.3.Logarithmically grown cells were taken and the cell cycle and apoptosis of colorectal cancer cell line ht-29 were detected by flow cytometry after treatment with the dominant concentration of Apatinib and 5-Fu monotherapy.4.The logarithmically growing cells were taken and the proliferation,cloning and formation ability of colorectal cancer cell line ht-29 cells were detected by plate cloning experiment after the dominant concentration of Apatinib and 5-Fu monotherapy and combined treatment.5.Logarithmically grown cells were taken and the expression of PI3 K,p-pi3 k,Akt and p-akt in intestinal cancer cell line ht-29 cells treated with Apatinib and 5-Fu monotherapy at the dominant concentration were detected by Western blot.Results:1.MTT results suggested that compared with the control group,the inhibition rate of cell proliferation in the combined group,the Apatinib group,and the 5-Fu group increased after 48 h of cell treatment,with statistically significant differences(P<0.05).Compared with the Apatinib group and the 5-Fu group,the inhibition rate of cell proliferation in the combined group was significantly increased after 48 h of treatment(P<0.05).The inhibition of Apatinib 80 mol/L+ 5-Fu100 mol/L group was the most significant.The CI value was calculated by chou-talalay median effect method when the inhibition rate of cell proliferation was 0.50 effect caused by the interaction of Apatinib and 5-Fu for 48h: CI=0.88<1.2.Flow cytometry showed that compared with the Apatinib80 mol/L group(41.6±2.1)and the 5-Fu100 mol/L group(43.9 ± 1.8),the Apatinib80 mol/L+ 5-Fu100mol/L combined group(56.1 ± 1.5)significantly increased the apoptosis rate,with statistically significant differences(P<0.001).3.The clone formation experiment showed that compared with the Apatinib80 mol/L group with 61.71±1.72 cells and the 5-Fu100 mol/L group with 57.75±4.21 cells,the Apatinib80 mol/L+ 5-Fu100 mol/L group with 36.12 ± 2.16 cells significantly decreased the cell formation ability,and the difference was statistically significant(P<0.001).4.Western blot results showed that there was no statistically significant difference in the expression of Akt and PI3 K proteins in the control group,apatinib group,5-Fu group and the combined group(P>0.05).The relative expression levels of p-pi3 k protein in Apatinib group and combined group were 0.61 ± 0.10 and 0.80 ± 0.05,respectively.The relative expression levels of p-akt in Apatinib group and combined group were 0.10±0.01 and 0.11±0.01,respectively.Compared with the blank group and the 5-Fu group,the expression levels of p-pi3 k and p-akt were significantly reduced in the Apatinib group and the dual-drug group,with statistically significant differences(p <0.01).Conclusion:1.Apatinib monotherapy and 5-Fu monotherapy inhibited the proliferation of ht-29 colorectal cancer cell lines.2.Combined use of Apatinib and 5-Fu can significantly inhibit the proliferation of ht-29 colorectal cancer cell lines,and combined use of the two drugs can synergistically inhibit the proliferation of ht-29 colorectal cancer cell lines.3.5-Fu and Apatinib alone and in combination can promote the apoptosis of ht-29 cell line of colorectal cancer,and the combination of two drugs is more effective in inducing the apoptosis.4.Apatinib single drug can effectively block the phosphorylation of Akt into p-akt,block the pathway where it is located and prevent further transmission,weaken the proliferation of intestinal cancer cells and induce apoptosis.There was no significant synergistic effect on the expression of p-akt when the two drugs were combined,and the synergistic effect of the two drugs may be achieved by other mechanisms. |
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