Department of Pathology II, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan.
Anticancer Res. 2010 Sep;30(9):3381-90.
Sulforaphane (SFN), which is present in cruciferous vegetables, induces growth arrest and/or cell death in cancer of various organs. The involvement of autophagy in the SFN-induced apoptotic death of human breast cancer cells was investigated.
Cell proliferation and viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion assay. Flow cytometry, immunofluorescence, electron microscopy, and Western blot analysis were used for detection of apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors.
SFN dose- and time-dependently retarded the growth and induced cell death in MCF-7 and MDA-MB-231 human breast cancer cells. In MDA-MB-231 cells, 30 μM SFN caused S and G2/M cell-cycle arrest associated with increased p21WAF1 and p27KIP1 levels and decreased cyclin A, cyclin B1 and CDC2 levels. Cell death was due to apoptosis with increased caspase-3 and lowered BCL-2 levels. In addition, the SFN-treated cells exhibited autophagy, as characterized by the appearance of autophagic vacuoles by electron microscopy, the accumulation of acidic vesicular organelles by flow cytometry, and the punctuate patterns of microtubule-associated protein 1 light chain 3 (LC3) by fluorescein microscopy. The levels of LC3-I and -II proteins (processed forms of LC3-I) and LC3 mRNA were increased. Treatment with autophagy inhibitor bafilomycin A1 (but not 3-methyladenine) with SFN significantly enhanced apoptosis, which was accompanied by increases in the level of BAX and the cleavage of caspase-3 and poly(ADP-ribose)polymerase (PARP)-1 and decreases in the mitochondrial membrane potential (ΔΨm).
These results indicate a cytoprotective role of autophagy against SFN-induced apoptosis and that the combination of SFN treatment with autophagy inhibition may be a promising strategy for breast cancer control.
存在于十字花科蔬菜中的萝卜硫素（SFN）可诱导多种器官的癌症生长停滞和/或细胞死亡。研究了自噬在 SFN 诱导人乳腺癌细胞凋亡死亡中的作用。
通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定和台盼蓝排斥试验评估细胞增殖和活力。流式细胞术、免疫荧光、电子显微镜和 Western blot 分析用于检测细胞凋亡和自噬，自噬抑制剂用于评估自噬的作用。
SFN 剂量和时间依赖性地延缓 MCF-7 和 MDA-MB-231 人乳腺癌细胞的生长并诱导细胞死亡。在 MDA-MB-231 细胞中，30μM SFN 导致 S 和 G2/M 细胞周期停滞，与 p21WAF1 和 p27KIP1 水平升高以及细胞周期蛋白 A、细胞周期蛋白 B1 和 CDC2 水平降低有关。细胞死亡是由于 caspase-3 增加和 BCL-2 水平降低而导致的凋亡。此外，SFN 处理的细胞表现出自噬，特征为电镜下出现自噬空泡、流式细胞术下酸性囊泡细胞器积累以及微管相关蛋白 1 轻链 3（LC3）荧光显微镜下点状模式。LC3-I 和 -II 蛋白（LC3-I 的加工形式）和 LC3 mRNA 的水平增加。SFN 联合自噬抑制剂巴弗洛霉素 A1（而非 3-甲基腺嘌呤）治疗可显著增强凋亡，同时增加 BAX 水平，切割 caspase-3 和多聚（ADP-核糖）聚合酶（PARP）-1，并降低线粒体膜电位（ΔΨm）。
这些结果表明自噬对 SFN 诱导的凋亡具有细胞保护作用，SFN 治疗联合自噬抑制可能是控制乳腺癌的一种有前途的策略。
