二甲双胍诱导胃癌细胞MNK-45发生自噬的作用
이갑쌍고유도위암세포MNK-45발생자서적작용
Effects of metformin in inducing autophagy of gastric cancer MNK-45 cells
  • 万方数据
    中华消化外科杂志 중화소화외과잡지
    CHINESE JOURNAL OF DIGESTIVE SURGERY
    2014年 9期 716-721 ,共6页

    徐学超%李玉民%刘涛%何雯婷

    서학초%리옥민%류도%하문정

    胃肿瘤%二甲双胍%自噬%凋亡 胃腫瘤%二甲雙胍%自噬%凋亡
    위종류%이갑쌍고%자서%조망
    Gastric neoplasms%Metformin%Autophagy%Apoptosis
    目的 探讨二甲双胍诱导胃癌细胞MNK-45发生自噬的作用.方法 取对数生长期的人胃癌细胞MNK-45,接种于培养板中:采用不同浓度二甲双胍(2、4、8、16、32、64 mmol/L)分别干预24、48、72 h并作为不同浓度药物干预组,用等量DMEM培养基处理为对照组,MTT法检测肿瘤细胞的抑制率,计算二甲双胍对胃癌细胞的半数凋亡浓度(IC50)值为17 mmol/L.分别采用17 mmol/L二甲双胍(实验组)和等量DMEM培养基(对照组)处理胃癌细胞MNK-45 48 h.流式细胞仪检测两组细胞的凋亡情况;RT-PCR检测两组细胞中Bax和Bcl-2的mRNA表达水平;Western blot检测Ⅰ型微管相关蛋白轻链(LC3b Ⅰ)、Ⅱ型LC3b(LC3bⅡ)、自噬相关蛋白(beclinl)、AKT、磷酸化AKT(p-AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR (p-mTOR)、核糖体蛋白s6激酶(P70s6k)、磷酸化P70s6k (p-P70s6k)蛋白的表达水平.计量资料采用(x)±s表示,多组间比较采用单因素方差分析,重复测量数据采用重复测量的方差分析,两组比较采用t检验.结果 MTT法检测不同浓度二甲双胍(2、4、8、16、32、64 mmol/L)分别干预胃癌细胞MNK-4524 h后细胞抑制率分别为3.0％±1.1％、8.6％±1.7％、15.9％±1.6％、26.1％±3.4％、37.5％±2.3％、49.7％±3.6％,干预48 h后细胞抑制率分别是5.2％±1.9％、10.4％±2.1％、26.9％±1.6％、49.5％±1.6％、59.1％±2.0％、82.1％±2.2％,干预72 h后细胞抑制率分别是9.5％±2.2％、l7.6％±1.4％、30.6％±2.6％、63.2％±2.6％、78.9％±1.4％、93.3％±2.7％,6组不同浓度二甲双胍干预细胞同时间点细胞抑制率比较,差异有统计学意义(F=155.174,728.229,743.826,P＜0.05);相同浓度二甲双胍干预细胞不同时间点细胞抑制率比较,差异有统计学意义(F=39.420,58.692,166.125,30.383,117.517,311.642,P＜0.05).流式细胞仪检测实验组和对照组胃癌细胞MNK-45的凋亡率分别为25.4％±1.7％和6.9％±0.5％,两组比较,差异有统计学意义(t=18.378,P＜0.05).RT-PCR检测实验组和对照组胃癌细胞MNK-45中凋亡相关基因Bax/Bcl-2 mRNA相对表达量的比值分别为1.88±0.16和1.00±0.00,两组比较,差异有统计学意义(t=9.743,P＜0.05).Western blot检测实验组和对照组MNK-45细胞中自噬蛋白LC3Ⅱ与LC3 Ⅰ的相对表达量比值分别为1.65±0.08和0.79±0.03,beclinl蛋白的相对表达量分别为1.47±0.06和0.56 ±0.06,两组比较,差异有统计学意义(t=18.023,18.283,P＜0.05);AKT蛋白分别为0.80 ±0.14和0.96 ±0.17,P70s6k蛋白分别为0.97 ±0.21和1.37 ±0.23,两组比较,差异无统计学意义(=2.103,1.699,P＞0.05);mTOR蛋白分别为0.58 ±0.11和1.88 ±0.23,p-mTOR蛋白分别为0.57±0.15和2.36 ±0.25,两组比较,差异有统计学意义(t=11.293,10.979,P＜0.05);实验组胃癌细胞MNK-45中p-AKT和p-P70s6k不表达,对照组分别为1.00±0.00和1.00±0.00.结论 二甲双胍能够诱导胃癌细胞MNK-45发生自噬,抑制其增殖,并促进其凋亡;其作用机制可能是二甲双胍抑制了mTOR表达从而影响mTOR下游蛋白p-P70s6k的表达水平,诱导胃癌细胞发生自噬.
    목적 탐토이갑쌍고유도위암세포MNK-45발생자서적작용.방법 취대수생장기적인위암세포MNK-45,접충우배양판중:채용불동농도이갑쌍고(2、4、8、16、32、64 mmol/L)분별간예24、48、72 h병작위불동농도약물간예조,용등량DMEM배양기처리위대조조,MTT법검측종류세포적억제솔,계산이갑쌍고대위암세포적반수조망농도(IC50)치위17 mmol/L.분별채용17 mmol/L이갑쌍고(실험조)화등량DMEM배양기(대조조)처리위암세포MNK-45 48 h.류식세포의검측량조세포적조망정황;RT-PCR검측량조세포중Bax화Bcl-2적mRNA표체수평;Western blot검측Ⅰ형미관상관단백경련(LC3b Ⅰ)、Ⅱ형LC3b(LC3bⅡ)、자서상관단백(beclinl)、AKT、린산화AKT(p-AKT)、포유동물뢰파매소파단백(mTOR)、린산화mTOR (p-mTOR)、핵당체단백s6격매(P70s6k)、린산화P70s6k (p-P70s6k)단백적표체수평.계량자료채용(x)±s표시,다조간비교채용단인소방차분석,중복측량수거채용중복측량적방차분석,량조비교채용t검험.결과 MTT법검측불동농도이갑쌍고(2、4、8、16、32、64 mmol/L)분별간예위암세포MNK-4524 h후세포억제솔분별위3.0％±1.1％、8.6％±1.7％、15.9％±1.6％、26.1％±3.4％、37.5％±2.3％、49.7％±3.6％,간예48 h후세포억제솔분별시5.2％±1.9％、10.4％±2.1％、26.9％±1.6％、49.5％±1.6％、59.1％±2.0％、82.1％±2.2％,간예72 h후세포억제솔분별시9.5％±2.2％、l7.6％±1.4％、30.6％±2.6％、63.2％±2.6％、78.9％±1.4％、93.3％±2.7％,6조불동농도이갑쌍고간예세포동시간점세포억제솔비교,차이유통계학의의(F=155.174,728.229,743.826,P＜0.05);상동농도이갑쌍고간예세포불동시간점세포억제솔비교,차이유통계학의의(F=39.420,58.692,166.125,30.383,117.517,311.642,P＜0.05).류식세포의검측실험조화대조조위암세포MNK-45적조망솔분별위25.4％±1.7％화6.9％±0.5％,량조비교,차이유통계학의의(t=18.378,P＜0.05).RT-PCR검측실험조화대조조위암세포MNK-45중조망상관기인Bax/Bcl-2 mRNA상대표체량적비치분별위1.88±0.16화1.00±0.00,량조비교,차이유통계학의의(t=9.743,P＜0.05).Western blot검측실험조화대조조MNK-45세포중자서단백LC3Ⅱ여LC3 Ⅰ적상대표체량비치분별위1.65±0.08화0.79±0.03,beclinl단백적상대표체량분별위1.47±0.06화0.56 ±0.06,량조비교,차이유통계학의의(t=18.023,18.283,P＜0.05);AKT단백분별위0.80 ±0.14화0.96 ±0.17,P70s6k단백분별위0.97 ±0.21화1.37 ±0.23,량조비교,차이무통계학의의(=2.103,1.699,P＞0.05);mTOR단백분별위0.58 ±0.11화1.88 ±0.23,p-mTOR단백분별위0.57±0.15화2.36 ±0.25,량조비교,차이유통계학의의(t=11.293,10.979,P＜0.05);실험조위암세포MNK-45중p-AKT화p-P70s6k불표체,대조조분별위1.00±0.00화1.00±0.00.결론 이갑쌍고능구유도위암세포MNK-45발생자서,억제기증식,병촉진기조망;기작용궤제가능시이갑쌍고억제료mTOR표체종이영향mTOR하유단백p-P70s6k적표체수평,유도위암세포발생자서.
    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.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문