Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan, R.O.C.
Int J Oncol. 2010 Jun;36(6):1477-84. doi: 10.3892/ijo_00000634.
The molecular mechanism and possible signaling pathway of apigenin-induced cytotoxicity and apoptosis in human lung cancer cells has not been reported. We investigated the role of ROS, Ca2+, caspases and Bax proteins and mitochondria membrane potential in apigenin-induced apoptosis in A549 cells. Cells were incubated with different concentrations of apigenin then cell morphological changes, DNA damage, cell viability and apoptosis were determined by Comet assay, and flow cytometric analysis. Sub-G1 phase was also examined. Western blot analysis was used to determined the levels of Bax and Bcl-2 and apoptosis associated proteins, and confocal laser microscope for examining the translocation of associated protein after exposed to apigenin. The results indicated that apigenin induced morphological changes, decreased percentage of viable cells and induced apoptosis dose- and time-dependently. DAPI staining and Comet assay also confirmed that apigenin-induced DNA condensation and damage. The levels of caspase-3, -8 and -9 involved in apigenin-induced apoptosis indicating caspase-dependent pathway was induced by apigenin. Western blotting showed that apigenin promoted cytochrome c levels and also induced dysfunction of mitochondria leading to the release of cytochrome c, AIF and Endo G, causing the activation of caspase-9 and -3, then apoptosis in A549 cells.
柚皮素诱导人肺癌细胞毒性和凋亡的分子机制及可能的信号通路尚未报道。我们研究了 ROS、Ca2+、半胱天冬酶和 Bax 蛋白以及线粒体膜电位在柚皮素诱导 A549 细胞凋亡中的作用。用不同浓度的柚皮素孵育细胞，然后通过彗星试验和流式细胞术分析测定细胞形态变化、DNA 损伤、细胞活力和细胞凋亡。还检测了亚 G1 期。Western blot 分析用于测定 Bax 和 Bcl-2 及凋亡相关蛋白的水平，共聚焦激光显微镜用于检查暴露于柚皮素后相关蛋白的易位。结果表明，柚皮素诱导形态变化，降低活细胞比例，并呈剂量和时间依赖性诱导凋亡。DAPI 染色和彗星试验也证实了柚皮素诱导的 DNA 浓缩和损伤。柚皮素诱导凋亡涉及的半胱天冬酶-3、-8 和 -9 表明柚皮素诱导了依赖半胱天冬酶的途径。Western blot 显示柚皮素促进细胞色素 c 水平，并导致线粒体功能障碍导致细胞色素 c、AIF 和 Endo G 释放，从而激活 caspase-9 和 -3，然后导致 A549 细胞凋亡。
