藤梨根乙醇提取物通过AKT/PUMA通路诱导结直肠癌细胞凋亡

Ethanol Extract of Tengligen Induces Apoptosis of Colorectal Cancer Cells through AKT/PUMA Pathway

    摘要
  • 摘要:
      目的  研究藤梨根乙醇提取物(TLG)对结直肠癌细胞的作用及其抗肿瘤机制。
      方法  利用MTT检测TLG对人结直肠腺癌细胞HCT-15、LoVo的毒性, 细胞平板克隆实验检测TLG对结直肠癌细胞HCT-15、LoVo增殖的影响, Hoechst-33258染色法和流式细胞术检测TLG对结直肠癌细胞凋亡的影响, Western blot实验检测TLG对HCT-15中相关蛋白Caspase-3、PARP、P65、PUMA、AKT、GSK-3β表达的影响, 双荧光素酶报告基因实验检测TLG对HCT-15中相关启动子活性的影响。
      结果  结直肠癌细胞HCT-15、LoVo经TLG处理后, 与对照组相比, 细胞活力以时间及剂量依赖性下降(P < 0.01，P < 0.001), 转染AKT cDNA、PUMA siRNA或GSK-3β抑制剂处理后能够恢复增殖(P < 0.001);不同浓度TLG处理后, 细胞凋亡率提高(P < 0.01, P < 0.001), 转染AKT cDNA或GSK-3β抑制剂处理后能够遏制TLG诱导的凋亡(P < 0.01，P < 0.001);Western blot显示HCT-15细胞中的p-AKT/AKT、p-GSK-3β/GSK-3β蛋白表达量随TLG浓度上升而减少(P < 0.01), Cleaved-Caspase-3/Caspase-3、Cleaved-PARP/PARP、p65、PUMA蛋白表达量随TLG浓度上升而增加(P < 0.05, P < 0.01，P < 0.001), 同时转染AKT cDNA、PUMA siRNA或GSK-3β抑制剂处理后能够阻滞TLG对下游蛋白Cleaved-Caspase-3/Caspase-3、Cleaved-PARP/PARP、p65、PUMA的影响(P < 0.05, P < 0.01，P < 0.001);报告基因结果显示NF-κB、PUMA启动子活性随TLG浓度上升而增强(P < 0.05, P < 0.01), 同时转染AKT cDNA或GSK-3β抑制剂处理后恢复(P < 0.01, P < 0.001)。
      结论  TLG可以显著诱导结直肠癌细胞凋亡, 发挥较强的抑制肿瘤作用, 其机制可能与AKT/PUMA信号通路有关。

     

    Abstract:
      OBJECTIVE  To investigate the effect and antitumor mechanism of ethanol extract of Tengligen (TLG) on colorectal cancer cells.
      METHODS  The toxicity of TLG on human colorectal adenocarcinoma cells (HCT-15, LoVo) was detected by methyl thiazolyl tetrazolium (MTT). Cell plate cloning assay was used to detect the effect of TLG on colorectal cancer cell proliferation. Hoechst-33258 staining and flow cytometry were used to detect the effect of TLG on colorectal cancer cell apoptosis. Western blot assay was used to detect the effect of TLG on the related proteins Caspase-3, PARP, p65, PUMA, AKT, GSK-3β in HCT-15. Dual luciferase reporter assay was performed to detect the effect of TLG on the activity of related promoter in HCT-15.
      RESULTS  Treatment of colorectal cancer cells (HCT-15, LoVo) with TLG resulted in a time- and dose-dependent decrease (P < 0.01, P < 0.001) in cell viability compared with control cells, and transfection with AKT cDNA, PUMA siRNA or GSK-3β inhibitor treatment was able to restore proliferation (P < 0.01). After treatment with different concentrations of TLG, the rate of apoptosis was increased (P < 0.01, P < 0.001), and cells transfected with AKT cDNA, PUMA siRNA, or GSK-3β inhibitor was able to suppress TLG induced apoptosis (P < 0.01, P < 0.001). Western blot showed that p-AKT/AKT and p-GSK-3β/GSK-3β protein expression in HCT-15 cells decreased (P < 0.001) while Cleaved-Caspase-3/Caspase-3, Cleaved PARP/PARP, p65 and PUMA protein levels increased with the increase of TLG concentrations (P < 0.05, P < 0.01, P < 0.001); simultaneous transfection of AKT cDNA, PUMA siRNA, or GSK-3β inhibitor was able to block the effects of TLG on downstream proteins Cleaved-Caspase-3/Caspase-3, Cleaved PARP/PARP, p65, PUMA (P < 0.05, P < 0.01, P < 0.001). Reporter gene results showed that NF-κB and PUMA promoter activities were enhanced (P < 0.05, P < 0.01) with the increase in TLG concentration, but recovered by simultaneous transfection of AKT cDNA or GSK-3β inhibitor treatment (P < 0.01, P < 0.001).
      CONCLUSION  TLG can significantly induce apoptosis and exert strong tumor suppressive effects in colorectal cancer cells, and the mechanism may be related to the AKT/PUMA signaling pathway.

     

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