LY294002抑制PI3K/AKT/mTOR信号通路对胃癌细胞MGC-803的影响
Effects of inhibiting PI3K/AKT/mTOR signaling pathway by LY294002 on gastric cancer MGC-803 cells
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摘要:
目的 探讨磷脂肌醇3-激酶(PI3K)特异性抑制剂LY294002对胃癌细胞MGC-803的PI3K/丝-苏氨酸蛋白激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路活性及细胞株生物学行为的影响。
方法 12.5、25、50 μmol/L LY294002作用于胃癌细胞MGC-803(实验组)，分别应用实时荧光定量PCR和Western blot检测PI3K/AKT/mTOR通路中关键信号分子PTEN、PI3K、AKT、mTOR、P70S6K、4EBP的mRNA和蛋白的表达情况；同时通过MTT实验、流式细胞术，测定LY294002对胃癌细胞MGC-803的增殖、细胞周期和凋亡的影响。
结果 实验组的PI3K、PTEN、AKT、mTOR、4EBP、P70S6K基因的mRNA相对表达量与对照组(二甲基亚砜)相比，差异有统计学意义(P < 0.05)，且AKT、mTOR、4EBP、P70S6K的表达量随着LY294002浓度升高而逐渐下降。LY294002对MGC-803细胞AKT、p-mTOR、p-70S6K、p-AKT蛋白的表达有明显抑制作用，且p-mTOR、p-70S6K、p-AKT蛋白的表达随着LY294002浓度升高而逐渐下降。MTT实验结果显示，LY294002对MGC-803细胞的增殖有明显的抑制作用，增殖率随LY294002浓度升高而逐渐降低(F=26.15，P < 0.01)。细胞周期实验结果显示，加入LY294002的MGC-803细胞相比于对照组细胞的G0/G1期比例较高(F=358.594，P < 0.001)。凋亡实验显示，LY294002组的凋亡率高于对照组且凋亡率随LY294002浓度增加而增加(F=4.929，P < 0.05)。
结论 LY294002作用于MGC-803细胞，可使其PI3K/AKT/mTOR信号通路关键信号分子受到不同程度的抑制，AKT、mTOR、4EBP、P70S6K基因的mRNA和磷酸化蛋白的表达量随LY294002浓度升高呈下降趋势，且LY294002可降低细胞增殖率、改变细胞周期比例、提高细胞凋亡率。
Abstract:Objective To investigate the effects of phosphoinositide 3-kinase (PI3K) inhibitor LY294002 on the activity of PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, as well as relevant biological behavioural alteration of gastric cancer MGC-803 cells.
Methods Human gastric cancer MGC-803 cells were treated with LY294002 at 12.5, 25, and 50 μmol/L (experimental groups). The mRNA expressions of key signal molecules in PI3K/AKT/mTOR signaling pathway including PTEN, PI3K, AKT, mTOR, P70S6K, and 4EBP, as well as related protein expressions, were detected by quantitative real-time PCR and Western blot, respectively. The growth-inhibiting effects of LY294002 on MGC-803 cells were detected by MTT assay. The effects on cell cycle and cell apoptosis were detected by flow cytometry.
Results Compared with the control (DMSO) group, LY294002 significantly regulated the mRNA expressions of PI3K, PTEN, AKT, mTOR, 4EBP, and P70S6K (P < 0.05), and the mRNA expression levels of AKT, mTOR, 4EBP, and P70S6K were declined with LY294002 concentration increasing. LY294002 significantly down-regulated the protein expression of AKT, p-mTOR, p-70S6K, and p-AKT, which showed a declining trend with LY294002 concentration increasing. The MTT assay results showed significant inhibiting effects of LY294002 on MGC-803 cells' proliferation as the proliferation rate increased with LY294002 concentration reducing (F=26.15, P < 0.01). The results of flow cytometry showed that the proportions of G0/G1 cells (F=358.594, P < 0.001) and the apoptosis rates (F=4.929, P < 0.05) of the LY294002 groups were higher than those of the control group in a dose-dependent manner.
Conclusion LY294002 may inhibit the key signaling molecules of PI3K/AKT/mTOR signaling pathway in MGC-803 cells to varied degrees. The mRNA expressions of AKT, mTOR, 4EBP, and P70S6K and related phosphorylated proteins show a declining pattern with LY294002 concentration increasing. Besides, LY294002 could decrease cell proliferation rate, change the proportion of cell cycle, and increase apoptosis rate.
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