Department of Pharmacology, College of Medicine, Catholic University of Daegu, Daegu 705-718, Korea.
Korean J Physiol Pharmacol. 2011 Feb;15(1):1-7. doi: 10.4196/kjpp.2011.15.1.1. Epub 2011 Feb 28.
Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy.
许多抗癌药物以及电离辐射已被证明可诱导自噬，自噬最初被描述为一种蛋白质回收过程，最近有报道称其在各种疾病中发挥关键作用。在 HCT116 人结肠癌细胞中，我们发现姜黄素，一种从植物姜黄中提取的多酚类植物化学物质，可显著诱导微管相关蛋白 1 轻链 3（LC3）-I 向 LC3-II 的转化，并降解自噬体降解的自噬体 1（SQSTM1）标志物。此外，我们发现姜黄素引起 GFP-LC3 形成斑点，这是自噬体的标志物，并且在 GFP-LC3 表达的 HCT116 细胞中 GFP-LC3 和 SQSTM1 蛋白水平降低。进一步证实，用过氧化氢处理细胞会诱导 LC3 转化率增加和 GFP-LC3 和 SQSTM1 水平降低，但在抗氧化剂 N-乙酰半胱氨酸（NAC）存在的情况下，这些变化几乎完全被姜黄素阻断，表明姜黄素导致活性氧（ROS）的产生，导致自噬体的形成和自噬溶酶体的降解。与 NAC 平行，自噬体-溶酶体融合的强效抑制剂巴弗洛霉素 A 也可减少 SQSTM1 的降解，细胞活力测定进一步证实，巴弗洛霉素 A 和 NAC 也部分阻断了姜黄素诱导的细胞死亡。我们还观察到 NAC 消除了姜黄素诱导的细胞外信号调节激酶（ERK）1/2 和 p38 丝裂原激活蛋白激酶（MAPK）的激活，但没有 Jun N-末端激酶（JNK）。然而，ERK1/2 和 p38 MAPK 的激活似乎对姜黄素诱导的自噬没有影响，因为姜黄素诱导的 LC3 蛋白转化和 SQSTM1 降解在 NAC 存在下没有改变。总之，我们的数据表明，姜黄素诱导 ROS 的产生，导致 HCT116 人结肠癌细胞中自噬的激活和伴随的细胞死亡。然而，ROS 依赖性 ERK1/2 和 p38 MAPK 的激活，但不是 JNK，可能不参与姜黄素诱导的自噬。
