姜黄素加重结肠癌细胞株HCT-116线粒体损伤和促进细胞凋亡的作用研究

Curcumin aggravates the mitochondria damages of colon cancer cell line HCT-116 and promotes the apoptosis of the cells

摘要:

目的 观察姜黄素对结肠癌细胞株HCT-116细胞增殖和凋亡的影响以及对细胞线粒体形态和功能的改变，并探讨其可能的机制。方法 体外培养结肠癌细胞株HCT-116，给与不同浓度的姜黄素（5、10、20、30 μmol/L）处理，细胞计数试剂盒CCK-8观察细胞增殖的变化并筛选出最佳作用浓度。流式细胞术检测细胞凋亡的改变。线粒体膜电位检测试剂盒JC-1检测细胞内线粒体膜电位的变化。电镜和Mito Tracker Deep Red染色观察细胞内线粒体形态结构的变化。三磷酸腺苷（adenosine triphosphate，ATP）和活性氧（reactive oxygen species，ROS）检测试剂盒检测细胞ATP和ROS的变化。分光光度计检测与凋亡密切相关的casppase-3和caspase-9活性变化。最后，Western blot检测结肠癌细胞内caspase-3和caspase-9蛋白水平的变化。结果 姜黄素处理细胞株HCT-116细胞后，细胞的增殖水平受到抑制，且呈浓度-时间依赖性，其半数最大抑制浓度（half maximal inhibitory concentration，IC₅₀）为13 μmol/L。姜黄素不仅增加早期凋亡的结肠癌细胞的比例，还使细胞内线粒体膜电位水平下降，且呈浓度依赖性。电镜结果显示姜黄素处理后，细胞内线粒体出现明显的线粒体肿胀，线粒体嵴肿胀、消失、膜破裂和空泡等。Mito Tracker Deep Red染色后显示线粒体数量减少且呈碎片状。细胞内的ATP生产明显下降。另外，姜黄素还增加细胞内caspase-3和caspase-9酶活性和蛋白水平的表达。结论 姜黄素抑制结肠癌HCT-116细胞的增殖，并促进细胞的凋亡，其机制与姜黄素加重线粒体形态和功能的损伤有关。

Abstract:

Objective To observe the effects of curcumin on the proliferation and apoptosis of colon cancer cell line HCT-116 as well as on the changes in mitochondrial morphology and function，and to explore the potential mechanism.Methods Colon cancer cell line HCT-116 was cultured in vitro and treated with different concentrations of curcumin（5，10，20，and 30 μmol/L）. Cell count kit-8 was used to observe the changes in cell proliferation，and the optimal concentration was identified. Flow cytometry was used for determination of the changes in cell apoptosis. Mitochondrial membrane potential assay kit JC-1 was applied to determine the changes in intracellular mitochondrial membrane potential. Electron microscopy and Mito Tracker Deep Red staining were used for observing the changes in mitochondrial morphology and structure. The changes in cellular adenosine triphosphate（ATP） and reactive oxygen species（ROS） were assessed by the corresponding assay kits. The changes in the activities and protein levels of apoptosis-related caspase-3 and caspase-9 enzymes were measured by spectrophotometry and western blotting，respectively.Results After curcumin treatment，the proliferation of HCT-116 cells was significantly inhibited in a concentration-time dependent manner，and the half maximal inhibitory concentration was 13 μmol/L. Curcumin not only remarkably increased the proportion of cells in early apoptosis，but also significantly reduced the intracellular mitochondrial membrane potential level in a concentration dependent manner. The electron microscopy results showed that curcumin caused obvious mitochondrial swelling in cells，including crista swelling，disappearance，membrane rupture，and vacuoles. Mito Tracker Deep Red staining showed a significant decrease in mitochondria number and fragmentation appearance. The intracellular ATP production was significantly decreased. In addition，curcumin also increased the activities and protein expression of caspase-3 and caspase-9 enzymes in cells.Conclusion Curcumin inhibits the proliferation of colon cancer HCT-116 cells and promotes cell apoptosis. The mechanism is related to the aggravation of mitochondrial morphology and function damage by curcumin.