Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action

Pinostrobin (PN) is a naturally occurring dietary bioflavonoid, found in various medicinal herbs/plants. Though anti-cancer potential of many such similar constituents has been demonstrated, critical biochemical targets and exact mechanism for their apoptosis-inducing actions have not been fully elucidated. The present study was aimed to investigate if PN induced apoptosis in cervical cancer cells (HeLa) of human origin. It is demonstrated that PN at increasing dose effectivity reduced the cell viability as well as GSH and NO2- levels. Condensed nuclei with fragmented chromatin and changes in mitochondrial matrix morphology clearly indicated the role of mitochondria in PN induced apoptosis. A marked reduction in mitochondrial membrane potential and increased ROS production after PN treatment showed involvement of free radicals, which in turn further augment ROS levels. PN treatment resulted in DNA damage, which could have been triggered by an increase in ROS levels. Decrease in apoptotic cells in the presence of caspase 3 inhibitor in PN-treated cells suggested that PN induced apoptosis via caspase dependent pathways. Additionally, a significant increase in the expression of proteins of extrinsic (TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD, Fas/TNFRSF6) and intrinsic pathway (Bad, Bax, HTRA2/Omi, SMAC/Diablo, cytochrome C, Pro-Caspase-3, Cleaved Caspase-3) was observed in the cells exposed to PN. Taken together, these observations suggest that PN efficiently induces apoptosis through ROS mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS mediated mitochondrial damage in HeLa cells.Pinostrobin (PN) 是一种天然存在的膳食生物类黄酮（bioflavonoid），广泛存在于多种药用草本及植物中。尽管诸多同类成分的抗癌潜力已得到证实，但其诱导细胞凋亡的关键生化靶点与确切分子机制尚未完全阐明。本研究旨在探究PN是否可诱导人源宫颈癌细胞（HeLa）发生凋亡。研究证实，随着给药剂量升高，PN可有效降低细胞活力以及谷胱甘肽（GSH）和亚硝酸根离子（NO2-）水平。染色质凝集、核固缩以及线粒体基质形态改变，均明确表明线粒体参与了PN诱导的凋亡过程。PN处理后，线粒体膜电位显著降低、活性氧（ROS）生成增加，提示自由基参与其中并进一步促进ROS水平升高。PN处理可引发DNA损伤，该损伤可能由ROS水平升高所触发。在经PN处理的细胞中加入半胱天冬酶3（caspase 3）抑制剂后，凋亡细胞比例显著降低，表明PN通过半胱天冬酶依赖通路诱导细胞凋亡。此外，经PN处理的细胞中，外在凋亡通路相关蛋白（TRAIL R1/DR4、TRAIL R2/DR5、TNF RI/TNFRSF1A、FADD、Fas/TNFRSF6）与内在凋亡通路相关蛋白（Bad、Bax、HTRA2/Omi、SMAC/Diablo、细胞色素C、Pro-Caspase-3、Cleaved Caspase-3）的表达量均显著上调。综上，上述结果表明，PN可通过活性氧介导的外在与内在依赖型信号通路，以及活性氧介导的线粒体损伤，有效诱导HeLa细胞发生凋亡。