DHA-inhibited proliferation through the PTEN/PI3K/Akt pathway in gastric cancer SGC7901 cells
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摘要:
目的 探讨二氢青蒿素(dihydroartemisinin, DHA)通过PTEN/PI3K/Akt通路对人胃癌细胞株SGC7901细胞周期的影响及其分子机制。 方法 不同浓度(6.25、12.5、25、50、100μmol/L)DHA作用SGC7901细胞24、48、72h后, 细胞计数法检测SGC7901细胞增殖的情况。不同浓度DHA作用SGC7901细胞24 h后, 流式细胞术测定细胞周期的分布; RT-PCR和Western blotting分别测定cyclin D1、P27的mRNA和蛋白的表达水平; Western blotting测定PTEN、PI3K、p-Akt的表达水平。分别以PTEN特异性小干扰RNA(PTEN-siRNA)及无关序列对照siRNA(non-specific siRNA, NS-siRNA)转染细胞, 加入100μmol/L DHA, 作用SGC7901细胞24 h后, Western blotting测定cyclin D1、P27、PTEN、PI3K、p-Akt的表达水平。 结果 DHA剂量和时间依赖性抑制SGC7901细胞的增殖, 使细胞周期阻滞于G1期(P < 0.05)。RT-PCR和Western blotting分析结果显示, 100μmol/L DHA作用SGC7901细胞24 h后, cyclin D1 mRNA和蛋白表达显著下降, P27 mRNA和蛋白表达显著上升(P < 0.05)。PTEN的蛋白表达显著增加, PI3K和p-Akt的表达水平逐渐下降(P < 0.05)。敲低PTEN表达后, DHA对PI3K和p-Akt的表达水平的影响明显减弱, 与此同时, cyclin D1表达水平升高, P27表达有所下降(P < 0.05)。 结论 DHA通过抑制PTEN/PI3K/Akt信号通路的活化, 影响细胞增殖相关基因cyclin D1和P27的表达, 进而使细胞阻滞于G0/G1期, 抑制人胃癌SGC7901细胞的增殖。 Abstract:Objective This study aims to elucidate the mechanism of anti-proliferation induced by dihydroartemisinin(DHA) on human gastric cancer SGC7901 cells through the PTEN/PI3K/Akt pathway. Methods SGC7901 cells were treated with DHA at various concentrations(6.25, 12.5, 25, 50, and 100 μmol/L) in different lengths of time(24, 48, and 72 h). We detected the changes in proliferation through cell count. The cell cycles were measured through flow cytometry. The cells were treated with 100 μmol/L DHA and were cultured for 24 h. The expressions of cyclin D1 and P27 were detected through reverse transcription-polymerase chain reaction and Western blot analysis, respectively. The expressions of PTEN, PI3K, and p–Akt were also detected through Western blot analysis. The PTEN expression was downregulated by the RNAi technology. The cells were subsequently treated with 100 μmol/L DHA for 24 h, followed by a Western blot analysis for the expressions of PTEN, PI3K, p–Akt, cyclin D1, and P27. Results Resultsof the cell count showed that DHA greatly inhibited the growth and proliferation of SGC7901 cells in a dose-and time-dependent manner(P < 0.05). The DHA inhibited the proliferation of SGC7901 cells through the induced cell cycle G1phrase arrest. The expressions of cyclin D1, PI3K, and p–Akt were downregulated, and the expressions of P27 and PTEN were upregulated after DHA treatment(P < 0.05). The DHA-elicited decrease in cyclin D1, PI3K, and p-Akt expression was significantly induced, and the DHA-elicited increase in P27 and PTEN expression was significantly reduced(P < 0.05) when the SGC7901 cells were transfected with PTEN-specific siRNA to block the endogenous PTEN expression induced by DHA. Conclusion DHA induces the cell cycle G0/G1 phase arrest through the regulation of cyclin D1 and P27 expression by activating the PTEN/ PI3K/Akt signaling pathway in human gastric cancer SGC7901 cells. -
Key words:
- dihydroartemisinin /
- gastric cancer /
- proliferation /
- Akt signal transduction pathway /
- cell cycle
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图 1 DHA对SGC7901细胞cyclin D1、P27 mRNA（A）和蛋白（B）表达的影响
Figure 1. Effects of DHA on mRNA (A) and protein (B) levels of cyclin D1 and P27
1: Control group; 2: DHA group
图 2 DHA对SGC7901细胞PTEN/PI3K/Akt信号通路的影响
Figure 2. Effects of DHA on the PTEN/PI3K/Akt signaling pathway
1: Control group; 2: DHA group
图 3 DHA对转染PTEN siRNA的SGC7901细胞中PTEN/PI3K/Akt通路及相关基因的影响
Figure 3. Effects of PTEN siRNA on the PTEN/PI3K/Akt pathway and re⁃ lated gene expression
1: NS-siRNA control group; 2: NS-siRNA+DHA group; 3: PTEN-siRNA group; 4: PTEN-siRNA+DHA group
表 1 DHA作用后SGC7901细胞周期分布的比较（%，x±s）
Table 1. Comparison of the SGC7901 cell cycle distributions after treatment with DHA(%, x±s)
表 2 DHA对SGC7901细胞cyclin D1、P27 mRNA和蛋白表达的影响（n=6，x±s）
Table 2. Effects of DHA on mRNA and protein levels of cyclin D1 and P27 in SGC7901 cells(n=6, x±s)
表 3 DHA对SGC7901细胞PTEN/PI3K/Akt信号通路的影响（n=6，x±s）
Table 3. Effects of DHA on the PTEN/PI3K/Akt signaling pathway(n=6, x±s)
表 4 DHA对转染PTEN-siRNA后SGC7901细胞中PTEN/PI3K/Akt通路及相关基因表达的影响（n=6，x±s）
Table 4. Effects of PTEN siRNA on the PTEN/PI3K/Akt pathway and related gene expression(n=6, x±s)
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