二甲双胍对人胃癌SGC-7901/L-OHP细胞奥沙利铂耐药逆转作用的研究

Reversal effect of metformin on drug resistance of oxaliplatin in human gastric cancer SGC-7901/L-OHP cells

目的探讨奥沙利铂(L-OHP)耐药的人胃癌细胞(SGC-7901/L-OHP)在联合应用二甲双胍(DMBG)后增殖及凋亡的变化,并探讨其可能的作用机制。方法首先使SGC-7901细胞在持续接触并梯次增加L-OHP浓度的条件下培养,建立SGC-7901/L-OHP细胞株,四甲基偶氮唑盐(MTT)法检测、计算DMBG及L-OHP单独使用时对SGC-7901/L-OHP的细胞增殖抑制率及半数抑制浓度(IC_(50)),计算DMBG联合应用L-OHP后细胞增殖抑制率、IC_(50)及逆转指数;流式细胞仪检测分析联合用药前后SGC-7901/L-OHP细胞周期分布及细胞凋亡的变化,逆转录-聚合酶链式反应(RT-PCR)法检测细胞周期调节因子Cyclin D1 mRNA表达,Western-blot法检测Cyclin D1蛋白表达。结果 (1)MTT法检测结果:DMBG作用于SGC-7901/L-OHP细胞48 h后IC_(50)为(320.13±2.15)μg/ml,L-OHP单独作用于SGC-7901/L-OHP细胞48 h后IC_(50)为(69.12±0.27)μg/ml,联用DMBG后的IC_(50)降至(21.07±2.15)μg/ml,差异有统计学意义(P<0.05),逆转指数为3.28倍。(2)流式细胞仪检测结果:联合用药后各组细胞周期的构成情况有统计学差异(P<0.05),L-OHP+DMBG组SGC-7901/L-OHP细胞停留在G1期的比例最高,相应的停留在G2期及S期的细胞比例最低;细胞凋亡率分别为空白组(6.51±3.24)%,DMBG组(9.25±1.27)%,L-OHP组(17.63±0.31)%,DMBG+L-OHP组(47.13±3.26)%;依空白组、DMBG组、L-OHP组、DMBG+L-OHP组之序,凋亡率递增(P<0.01),组间两两(除空白组与DMBG组间)比较差异均有统计学意义(P<0.05,P<0.01)。(3)Cyclin D1 mRNA及蛋白检测结果:联用DMBG后Cyclin D1 mRNA及蛋白表达均较联合用药前明显下调(P均<0.05)。结论 L-OHP与DMBG联合应用能改变SGC-7901/L-OHP细胞周期分布,逆转SGC-7901/L-OHP细胞对L-OHP的耐药性,促进SGC-7901/L-OHP细胞凋亡。

Objective To investigate the changes of proliferation and apoptosis of Oxaliplatin-resistant human gastric cancer cells（SGC-7901/L-OHP） after combination with metformin（DMBG）,and explore its possible mechanism.Methods SGC-7901/L-OHP cell lines were established by persistent contact and gradually increasing of L-OHP concentration on SGC-7901 cells. MTT assay was used to determine and calculate the cell proliferation inhibition rate and IC50 of SGC-7901/L-OHP when DMBG and L-OHP were used alone or combined,as well as to calculate the reversal index when DMBG combined with L-OHP used. Cell cycle phase distribution and apoptosis of SGC-7901/L-OHP cells were detected by flow cytometry before and after medication; the expression of Cyclin D1 mRNA（cell cycle regulators） was detected by real-time reverse transcription polymerase chain reaction（RT-PCR）; the expression of Cyclin D1 protein was determined by Western blot method. Results（1） At 48 hours after medication,MTT results showed that IC50 were（320. 13 ± 2. 15） μg/ml for DMBG contacting SGC-7901/L-OHP cells,（69. 12 ± 0. 27） μg/ml for L-OHP in single use and（21. 07 ± 2. 15） μg/ml after combination of DMBG and L-OHP,respectively. There was a significant difference between alone medication and combined medication（P 〈0. 05）. The reversal index was 3. 28 times.（2） Flow cytometry showed that there were significant differences in compositions of cell cycle in each group after combined use of drugs（P 〈0. 05）. In the L-OHP ＋ DMBG group,the proportion of SGC-7901/L-OHP cells staying in G1 phase was the highest, and the corresponding proportion of cells staying in G2 phase and S phase was the lowest. After combined use of drugs,the apoptosis rates were（6. 51 ± 3. 24） % in blank control group,（9. 25 ± 1. 27）% in DMBG group,（17. 63 ± 0. 31） % in L-OHP group and（47. 13 ± 3. 26） % in DMBG ＋ L-OHP group,respectively,and according to control group,DMBG group,L-OHP group,DMBG ＋ L-OHP group of ascending order（P 〈0. 01）. There were significant differences in pairwise comparisons except for intra-group of blank control group and DMBG group（P 〈0. 05,P 〈0. 01）.（3） The expressions of Cyclin D1 mRNA and Cyclin D1 protein significantly decreased after combination of drugs compared with pre-medication（all P 〈0. 05）. Conclusion Application of Oxaliplatin combined with Melbine can change the distribution of SGC-7901/L-OHP cell cycle,reverse the drug-resistance of SGC-7901/L-OHP to L-OHP,and promote the apoptosis of SGC-7901/L-OHP cells.