Experimental study on metformin enhancing the sensitivity of colon cancer cells to 5-FU through CXCR4/Akt signaling pathway
10.3760/cma.j.cn115355-20240718-00360
- VernacularTitle:二甲双胍通过CXCR4/Akt信号通路增强结肠癌细胞对5-FU敏感性的实验研究
- Author:
Hongxia YAN
1
;
Linxiu HE
;
Xinfeng CAI
;
Yixun ZHANG
Author Information
1. 山西省肿瘤医院 中国医学科学院肿瘤医院山西医院 山西医科大学附属肿瘤医院药学部，太原 030013
- Publication Type:Journal Article
- Keywords:
Colorectal neoplasms;
Metformin;
Fluorouracil;
Receptors, CXCR4;
X-ray repair cross complementing protein 1;
Signal transduction
- From:
Cancer Research and Clinic
2024;36(12):919-927
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of metformin on the chemosensitivity of colon cancer cells to 5-fluorouracil (5-FU) and its possible mechanism.Methods:Colon cancer cell lines HCT-116 and HT-29 were cultured in vitro, and the CCK-8 method was used to determine cell viability after intervention with different concentrations (1.25, 2.5, 5, 10, 20, 40, 80 mmol/L) of metformin hydrochloride (MET-HCl) or different concentrations (1.25, 2.5, 5, 10, 20, 40 μmol/L) of 5-FU. The survival rate and 48-hour half maximal inhibitory concentration ( IC50) of the two drugs were calculated. The combined index (CI) of MET-HCl and 5-FU was calculated using the Chou-Talalay model, and the concentrations of MET-HCl and 5-FU in subsequent experiments were determined based on the concentration of the two drugs when the inhibition rate (Fa) was 0.5. The CCK-8 method and Annexin V-FITC/PI flow cytometry were used to detect the survival rate and apoptosis rate of HCT-116 cells or HT-29 cells treated with the two drugs alone and in combination at experimental concentrations, respectively. Cells treated with drug free culture flnid were used as the control group. CXCR4 specific small interfering RNA (siRNA) was transfected into HCT-116 cells and HT-29 cells, and the decreased ( P < 0.05) expression of CXCR4 protein detected by Western blotting indicated successful knockdown of CXCR4. CCK-8 method was used to detect the survival rate of cells with CXCR4 knockdown by two drugs alone and in combination, and the untransfected cells added drug free culture fluid were served as the si-control group. Western blotting was used to detect the expression of proteins related to the CXCR4/Akt signaling pathway in HCT-116 cells or HT-29 cells treated with two drugs alone or in combination. Results:CCK-8 assay showed that MET-HCl and 5-FU decreased the viability of HCT-116 and HT-29 cells in a concentration-time-dependent manner; at 48 h, IC50 of MET-HCl and 5-FU in HCT-116 cells were (13.0±5.8) mmol/L and (16.9±7.2) μmol/L, respectively, and IC50 in HT-29 cells were (8.6±2.8) mmol/L and (9.7±3.1) μmol/L, respectively. When the cell inhibition rates of HCT-116 cells or HT-29 cells detected by CCK-8 method were 50%, 75% and 90% after 48 h of treatment, the corresponding CI values of HCT-116 cells were 0.48, 0.37 and 0.25, and HT-29 cells were 0.57, 0.51 and 0.49, suggesting that the combination of the two drugs had a synergistic effect. Based on Fa = 0.5, the experimental concentrations of MET-HCl and 5-FU were determined to be 10 mmol/L and 5 μmol/L. CCK-8 method showed that after HCT-116 cells or HT-29 cells were treated with 10 mmol/L MET-HCl, 5 μmol/L 5-FU alone or in combination for 48 h, the cell viability of each drug group was lower than that of the control group (all P < 0.05), there was no statistically significant difference in cell viability between MET-HCl alone and 5-FU alone (both P > 0.05), and the cell viability of the combination of the two drugs was lower than that of the two drugs alone (both P < 0.01). After CXCR4 was knocked down, the cell viability of each drug group was lower than that of the si-control group (all P < 0.05), but there was no significant difference in cell viability between the two drugs alone and the combination of the two drugs (all P > 0.05). Flow cytometry showed that after 24 h of treatment, the apoptosis rate of HCT-116 cells or HT-29 cells treated with MET-HCl alone or the combination of the two drugs was higher than that of the control group (all P < 0.05), but there was no statistically significant difference in the apoptosis rate of cells treated with 5-FU alone compared to the control group (all P > 0.05); the apoptosis rate of HCT-116 cells treated with the combination of the two drugs was higher than that of the two drugs alone (both P < 0.05); the apoptosis rate of HT-29 cells treated with the combination of the two drugs was higher than that of 5-FU alone ( P < 0.05), but there was no significant difference with MET-HCl alone ( P > 0.05). Western blotting showed that the relative expression levels of CXCR4, p-Akt and XRCC1 proteins in HCT-116 or HT-29 cells treated with MET-HCl alone and in combination of two drugs were lower than those in the control group (all P < 0.05), but there was no statistically significant difference in the relative expression levels of the 3 proteins in the two cell lines treated with 5-FU alone compared to the control group (all P > 0.05); the relative expression levels of the 3 proteins in the two cell lines treated with MET-HCl alone and in combination of two drugs were lower than those treated with 5-FU alone (all P < 0.05), the relative expression levels of CXCR4 and p-Akt proteins in the two cell lines treated with the combination of two drugs were lower than those treated with MET-HCl alone (all P < 0.05), and the relative expression level of XRCC1 protein in HCT-116 cells treated with the combination of two drugs was lower than that in HCT-116 cells treated with MET-HCl alone ( P < 0.05), but there was no significant difference in the relative expression level of XRCC1 protein in HT-29 cells treated with the combination of two drugs and MET-HCl alone ( P > 0.05). Conclusions:MET-HCl may reduce the expression of XRCC1 through the CXCR4/Akt signaling pathway, thereby enhancing the sensitivity of colon cancer cells to 5-FU.
