二甲双胍诱导胃癌细胞MNK-45发生自噬的作用

Effects of metformin in inducing autophagy of gastric cancer MNK-45 cells

    摘要
  • 摘要: 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(LC3bⅠ)、Ⅱ型LC3b(LC3bⅡ)、自噬相关蛋白(beclin1)、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 〖HTSS〗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-45 24 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%、17.6%±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LC3Ⅰ的相对表达量比值分别为1.65±0.08和0.79±0.03，beclin1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(t=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。

     

    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 〖HJ*8〗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 IC 50 value of metformin on gastric cancer MNK -45 cells was 17 mmol/L. Gastric cancer MNK -45 cells were intervened by metformin 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, beclin1, AKT, p AKT, mTOR, p mTOR, P70s6k, p P70s6k of the 2 groups were detected by Western blot. The measurement data were presented as ±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 LC3Ⅱ/LC3Ⅰ 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±0.21 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± 0.11 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.
    Conclusion: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 metformin, and then the autophagy of gastric cancer MNK -45 cells happens.

     

    • HTML全文
    • 参考文献(0)
    • 相关文章
    • 施引文献
  • 资源附件(0)

/

返回文章
返回