Inhibition of Autophagy Potentiates Sulforaphane-Induced Apoptosis in Human Colon Cancer Cells
Inhibition of Autophagy Potentiates Sulforaphane-Induced Apoptosis in Human Colon Cancer Cells
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DOI：
10.1245/s10434-009-0696-x
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发表时间：
2010-02-01
影响因子：
3.7
通讯作者：
Nagawa, Hirokazu
中科院分区：
文献类型：
--
作者：
Nishikawa, Takeshi;Tsuno, Nelson H.;Nagawa, Hirokazu
背景萝卜硫素（Sulforaphane，SUL）是一种天然存在于广泛食用的蔬菜中的异硫氰酸酯，近年来因其通过诱导细胞凋亡抑制肿瘤细胞生长而受到关注。我们研究了SUL诱导人结肠癌细胞自噬的能力，以及抑制自噬是否能增强SUL的促凋亡作用。MTS法和集落形成法检测SUL对细胞增殖的影响。流式细胞术检测细胞凋亡和caspase活性。流式细胞仪检测吖啶橙染色细胞中酸性囊泡细胞器（AVO）的形成。免疫印迹法检测LC 3蛋白。免疫细胞化学法检测LC 3和细胞色素c的定位。通过用相对高浓度的SUL长时间处理细胞观察到促凋亡作用。处理16 h后，观察到AVO的明显形成和LC 3向自噬体的募集，即自噬的特征。用特异性自噬抑制剂（3-methyladenine）处理细胞可增强SUL的促凋亡作用，该作用依赖于半胱天冬酶的激活和细胞色素c向胞浆的释放。目前的结果表明，诱导自噬在结肠癌细胞作为一种保护性反应，对SUL的促凋亡作用，因此，增强的促凋亡作用的自噬抑制。这些发现为自噬抑制剂联合化疗药物治疗结直肠癌提供了前提。
Background. Sulforaphane (SUL), an isothiocyanate naturally present in widely consumed vegetables, particularly broccoli, has recently attracted attention due to its inhibitory effects on tumor cell growth by inducing apoptosis. We investigated the ability of SUL to induce autophagy in human colon cancer cells and whether inhibition of autophagy could potentiate the proapoptotic effect of SUL.Methods. The proliferation of cells treated with SUL was assessed by MTS assay and colony-forming assay. Apoptosis and caspases activity were investigated by flow cytometry. The formation of acidic vesicular organelles (AVOs) was detected in acridine-orange-stained cells by flow cytometry. Western blotting was used for the detection of light chain 3 (LC3). Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry.Results. The proapoptotic effect was observed by treatment of cells with relatively high concentrations of SUL for long periods of time. After 16 h of treatment, evident formation of AVOs and recruitment of LC3 to autophagosomes, features of autophagy, were observed. Treatment of cells with a specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL, which was dependent on the activation of caspases and the release of cytochrome c to the cytosol.Conclusion. The present results demonstrate induction of autophagy in colon cancer cells as a protective reaction against the proapoptotic effect of SUL, and consequently, the potentiation of the proapoptotic effect by autophagy inhibition. These findings provide a premise for use of autophagy inhibitors in combination with chemotherapeutic agents for treatment of colorectal cancer.