Effects of metformin in inducing autophagy of gastric cancer MNK-45 cells
10.3760/cma.j.issn.1673-9752.2014.09.010
- VernacularTitle:二甲双胍诱导胃癌细胞MNK-45发生自噬的作用
- Author:
Xuechao XU
;
Yumin LI
;
Tao LIU
;
Wenting HE
- Publication Type:Journal Article
- Keywords:
Gastric neoplasms;
Metformin;
Autophagy;
Apoptosis
- From:
Chinese Journal of Digestive Surgery
2014;13(9):716-721
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the mechanisms of metformin in inducing autophagy of gastric cancer MNK-45 cells.Methods Human gastric cancer MNK-45 cells in logarithmic growth phase were incubated in the culture plates,and were divided into the intervention group [gastric cancer MNK-45 cells were intervened by metformin at different concentrations (2,4,8,16,32,64 mmol/L) for 24,48,72 hours] and the control group (gastric cancer MNK-45 cells were cultured in the DMEM medium).The inhibition rate of gastric cancer MNK-45 cells was detected by MTT method.The IC50 value of metformin on gastric cancer MNK-45 cells was 17 mmol/L.Gastric cancer MNK-45 cells were intervened by metforrnin at 17 mmol/L for 48 hours in the experimental group.Gastric cancer MNK-45 cells in the control group were cultured in DMEM medium at 17 mmol/L for 48 hours.The apoptosis of the gastric cancer MNK-45 cells of the 2 groups were detected by flow cytometry.The mRNA expressions of Bax and Bcl-2 of the 2 groups were detected by RT-PCR.The protein expressions of type Ⅰ LC3b,type Ⅱ LC3b,beclinl,AKT,p-AKT,mTOR,p-mTOR,P70s6k,p-P70s6k of the 2 groups were detected by Western blot.The measurement data were presented as (x) s,and were analyzed using the one-way ANOVA or repeated measures ANOVA.Data of the 2 groups were compared using the t test.Results The inhibition rates of gastric cancer MNK-45 cells were 3.0％ ± 1.1％,8.6％ ± 1.7％,15.9％ ± 1.6％,26.1％ ± 3.4％,37.5％ ± 2.3％,49.7％± 3.6％ after intervention by metformin at concentrations of 2,4,8,16,32,64 mmol/L for 24 hours,5.2％± 1.9％,10.4％±2.1％,26.9％± 1.6％,49.5％± 1.6％,59.1％±2.0％,82.1％±2.2％ after intervention by metformin at concentrations of 2,4,8,16,32,64 mmol/L for 48 hours,and 9.5％ ± 2.2％,17.6％ ± 1.4％,30.6％ ± 2.6％,63.2％ ± 2.6％,78.9％ ± 1.4％,93.3％ ± 2.7％ after intervention by metformin at concentrations of 2,4,8,16,32,64 mmol/L for 72 hours.There were significant differences in the inhibition rates among the 6 groups at the same time points (F =155.174,728.229,743.826,P ＜ 0.05),and significant differences were also observed within the same group at different time points (F =39.420,58.692,166.125,30.383,117.517,311.642,P ＜ 0.05).The apoptosis rates of gastric cancer MNK-45 cells in the experimental group and the control group were 25.4％ ± 1.7％ and 6.9％ ± 0.5％,with significant difference between the 2 groups (t =18.378,P ＜0.05).The relative mRNA expressions of Bax/Bcl-2 mRNA in the experimental group and the control group were 1.88 ± 0.16 and 1.00 ± 0.00,with significant difference between the 2 groups (t =9.743,P ＜ 0.05).The relative protein expressions of LC3 Ⅱ/LC3 Ⅰ and beclin 1 in the gastric cancer MNK-45 cells were 1.65 ± 0.08 and 1.47 ± 0.06 in the experimental group and 0.79 ± 0.03 and 0.56 ± 0.06 in the control group,with significant difference between the 2 groups (t =18.023,18.283,P ＜ 0.05).The relative protein expressions of AKT and P70s6k in the gastric cancer MNK-45 cells were 0.80 ±0.14 and 0.97 t0.21 in the experimental group and 0.96 ±0.17 and 1.37 ±0.23 in the control group,with no significant difference between the 2 groups (t =2.103,1.699,P ＞0.05).The relative protein expressions of mTOR and p-mTOR were 0.58 ± 0.l 1 and 0.57 ±0.15 in the experimental group and 1.88 ±0.23 and 2.36 ±0.25 in the control group,with significant difference between the 2 groups (t =11.293,10.979,P ＜ 0.05).No p-AKT and p-P70s6k expression was detected in the experimental group,and the expressions of p-AKT and p-P70s6k in the control group were 1.00 ± 0.00 and 1.00 ± 0.00,respectively.Conclusions Metformin could induce autophagy,inhibit proliferation and promote apoptosis of gastric cancer MNK-45 cells.The mechanism may be associated with the inhibition of mTOR expression and the expression of mTOR downstream proteins p-P70s6k by mefformin,and then the autophagy of gastric cancer MNK-45 cells happens.
