小檗碱通过激活自噬诱导caspase依赖性凋亡发挥抗结直肠癌作用

  目的  探究小檗碱通过自噬依赖性凋亡发挥抗结直肠功效的潜在作用机制。  方法  使用HCT116细胞和移植瘤模型小鼠进行小檗碱抗肿瘤药效和可能作用机制的研究。体外研究中, 通过利用CCK8实验评价小檗碱对结直肠癌细胞活力的影响, 使用细胞克隆实验评价小檗碱对结直肠癌细胞增殖的影响, 同时借助流式细胞术和TUNEL染色法对小檗碱诱导结直肠癌细胞凋亡作用进行研究。透射电镜和mCherry-GFP-LC3B腺病毒转染细胞用于检测细胞内自噬流变化。通过对小鼠组织进行HE染色、免疫组化染色和TUNEL染色, 评价小檗碱体内抗结直肠癌作用。  结果  体外研究结果表明, 小檗碱能够抑制结直肠癌细胞活力, 诱导细胞凋亡, 同时提高细胞内LC3B水平。使用自噬抑制剂3-MA、CQ和BafA1进行干预, 自噬抑制剂能够促进HCT116细胞生长, 抵消小檗碱诱导的结直肠癌细胞凋亡作用。雷帕霉素增强小檗碱上调HCT116细胞中LC3B水平的作用, 同时Z-VAD-FMK或ATG siRNA能够废除小檗碱诱导结直肠癌细胞凋亡的作用。在体研究结果表明, 小檗碱能够降低肿瘤体积和重量, 引起肿瘤组织发生凋亡。进一步的体内作用机制研究结果表明, 小檗碱显著抑制p-mTOR表达, 同时显著上调ATG5、ATG7、cleaved-caspase3和cleaved-caspase8表达。  结论  小檗碱能够通过诱导结直肠癌细胞发生自噬, 促进其发生caspase依赖性凋亡, 进而抑制结直肠癌细胞增殖。 

Berberine Activates Autophagy to Induce Apoptosis of Colorectal Cancer Cells in A Caspase-Dependent Manner

  OBJECTIVE  To investigate the potential mechanism of berberine in autophagy-mediated apoptosis of colorectal cancer.  METHODS  HCT116 cells and a mouse model of xenograft tumor were used to detect the tumor suppressor effect of berberine and its potential mechanism. CCK8 assay was used to investigate the cell viability. Cell cloning assay was used to detect the effect of berberine on cell growth, and the influence of berberine on cell apoptosis was evaluated by using flow cytometry assay and TUNEL staining. The transmission electron microscope and mCherry-GFP-LC3B adenovirus translocation assays were used to observe the change of autophagy in cells. HE, immunohistochemical and TUNEL staining of mouse tissue were used to estimate the effect of berberine on colorectal cancer in vivo.  RESULTS  Berberine inhibited the viability of tumor cells, and increased the apoptosis rate and LC3B level in HCT116 cells. 3-MA, CQ or BafA1 promoted the growth of HCT116 cells and cancelled the effect of berberine on apoptosis. Rapamycin increased the effect of berberine on up-regulating LC3B level in HCT116 cells, while Z-VAD-FMK or ATG5 siRNA abolished the effect of berberine on inducing cancer cells apoptosis. In vivo, berberine reduced tumor volume and tumor weight, and caused apoptosis in tumor tissues. In mouse tumor tissues, p-mTOR expression was significantly inhibited by berberine, while ATG5, ATG7, cleaved-caspase3 and cleaved-caspase8 were significantly up-regulated.  CONCLUSION  Berberine induces autophagy and promotes caspase-dependent apoptosis of HCT116 cells, thereby inhibiting cell proliferation of colorectal cancer.