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

CHEN Zi-han YAN Qiu-ying GU Jun-fei PU Wen-yuan SHEN Zheng-jie CHENG Hai-bo MENG Qing-hai SUN Dong-dong

陈姿含, 闫秋莹, 顾俊菲, 濮文渊, 沈政洁, 程海波, 孟庆海, 孙东东. 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[J]. 南京中医药大学学报, 2023, 39(1): 21-31. doi: 10.14148/j.issn.1672-0482.2023.0021
引用本文: 陈姿含, 闫秋莹, 顾俊菲, 濮文渊, 沈政洁, 程海波, 孟庆海, 孙东东. 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[J]. 南京中医药大学学报, 2023, 39(1): 21-31. doi: 10.14148/j.issn.1672-0482.2023.0021
CHEN Zi-han, YAN Qiu-ying, GU Jun-fei, PU Wen-yuan, SHEN Zheng-jie, CHENG Hai-bo, MENG Qing-hai, SUN Dong-dong. Berberine Activates Autophagy to Induce Apoptosis of Colorectal Cancer Cells in A Caspase-Dependent Manner[J]. Journal of Nanjing University of traditional Chinese Medicine, 2023, 39(1): 21-31. doi: 10.14148/j.issn.1672-0482.2023.0021
Citation: CHEN Zi-han, YAN Qiu-ying, GU Jun-fei, PU Wen-yuan, SHEN Zheng-jie, CHENG Hai-bo, MENG Qing-hai, SUN Dong-dong. Berberine Activates Autophagy to Induce Apoptosis of Colorectal Cancer Cells in A Caspase-Dependent Manner[J]. Journal of Nanjing University of traditional Chinese Medicine, 2023, 39(1): 21-31. doi: 10.14148/j.issn.1672-0482.2023.0021

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

doi: 10.14148/j.issn.1672-0482.2023.0021
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    Corresponding author: 孟庆海, 男, 讲师, 主要从事中药复方研究, E-mail: junmoyan@163.com孙东东, 男, 教授, 主要从事中医药抗癌及方剂治法配伍的研究, E-mail: sundd@njucm.edu.cn
  • 摘要:   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。

     

  • Figure  1.  Berberine induced autophagy and inhibited cell viability in HCT116 cells

    Note: (A) The chemical structure of berberine. (B, C) The cell morphology was observed under the light microscope. (D-E) The cell viability of HCT116 cells treated with berberine. (F) Autophagic vesicles of berberine-treated HCT116 cells. The red arrows indicated possible autophagosomes. (G-H) The cell viability of HCT116 cells treated with berberine. (J) The cell viability of SW620 cells treated with berberine. (K, L) LC3B positive cells and the number of GPF-LC3B or mCherry-LC3B positive cells in figure G. (M, N) Expression of LC3 A/B in berberine treated HCT116 cells. The scale bar in blue color was 1 μm. The scale bar in black and white color was 50 μm. n=10 in B-E, n=5 in F, K-L, n=3 in G-J, M, N. In the indicated comparison, *P < 0.05, * *P < 0.01, * * *P < 0.001.

    Figure  2.  Berberine inhibits cell viability by activating the autophagic flux in HCT116 cells

    Note: (A, B) Cell morphology and viability of HCT116 cells were pre-treated with 3-MA (10 mmol · L-1), CQ (20 μmol · L-1) or BafA1 (10 nmol · L-1) for 1h and then treated with or without berberine (20 μmol · L-1) for 6 h. (C-E) The AAV-mCherry-GFP-LC3B transfected HCT116 cells were pre-treated with 3-MA (10 mmol · L-1), CQ (20 μmol · L-1) or BafA1 (10 nmol · L-1) for 1 h and then treated with or without berberine (20 μmol · L-1) for 6 h, and the number of GPF-LC3B positive cell in C and mCherry-LC3B positive cell in C were determined. (F, G) The AAV-mCherry-GFP-LC3B transfected HCT116 cells were treated with Rapamycin (10 nmol · L-1) and with or without berberine (20 μmol · L-1) for 6 h, the number of GPF-LC3B positive cells in E and the number of mCherry-LC3B positive cell in E were determined. The scale bar in black and white color was 50 μm. n=10 in A, n=5 in B-G. In the indicated comparison, *P < 0.05, **P < 0.01, * * *P < 0.001.

    Figure  3.  Berberine induced caspase-dependent apoptosis in HCT116 cells

    Note: (A, B) Images of the cell cloning experiment were taken after berberine was applied to the HCT116 cell, and the colony number in A was counted. (C, D) Flow cytometry was performed after berberine was applied to HCT116 cells, and the apoptosis rate was based on the results of C. (E, F) Images of the cell cloning experiment were taken after HCT116 cells were treated with or without berberine and Z-VAD-FMK for 48 h and the colony number of E were counted. (G, H) Flow cytometry was performed after HCT116 cells were treated with or without berberine and Z-VAD-FMK for 48 h and the apoptosis rate was calculated based on the results of G. (I) Tunel staining was performed after HCT116 cells were treated with or without berberine and Z-VAD-FMK for 48 h. The scale bar in white color was 50 μm. n=5. In the indicated comparison, ***P < 0.001.

    Figure  4.  Autophagy mediated berberine induced apoptosis in HCT116 cells

    Note: (A, B) Images of the cell cloning experiment were taken after HCT116 cells were treated with or without berberine and CQ and the colony number were counted. (C, D) Flow cytometry was performed after HCT116 cells were treated with or without berberine and CQ, and the apoptosis rate was calculated based on the results of C. (E, F) Images of the cell cloning experiment were taken in HCT116 cells with control siRNA or ATG5 siRNA transfection followed with or without berberine treatment, and the colony number was counted. (G) Flow cytometry was performed in HCT116 cells with control siRNA or ATG5 siRNA transfection followed with or without berberine and the apoptosis rate was calculated based on the results of G. n=5. In the indicated comparison, ***P < 0.001.

    Figure  5.  Berberine promoted autophagy dependent apoptosis in HCT116 xenograft tumor mice

    Note: (A, B) The body weight and tumor volume of HCT116 xenograft tumor mice with oxaliplatin or berberine administration. (C) The tumor weight of HCT116 xenograft tumor mice with oxaliplatin or berberine treatment. (D) The tumor growth inhibition rate of berberine. (E)HE staining was performed in tumor, heart, liver, lung, and kidney from HCT116 xenograft tumor mice with oxaliplatin or berberine treatment. (F-K) Immunohistochemical staining of p-mTOR, ATG5, ATG7, cleaved-caspase3, and cleaved-caspase8 was performed in tumors from HCT116 xenograft tumor mice with oxaliplatin or berberine treatment. And the level of proteins in tumor from mice were calculated based on the result of E. (L, M) Tunel staining was performed in tumor from HCT116 xenograft tumor mice with oxaliplatin or berberine treatment., and Tunel positive area in tumor from mice were calculated based on the result of K. The scale bar in black and white color was 50 μm. n=5. In the indicated comparison, **P < 0.01, ***P < 0.001.

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  • 收稿日期:  2022-06-20
  • 网络出版日期:  2023-01-18
  • 发布日期:  2023-01-10

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