Kittiratphatthana Natthawan, Kukongviriyapan Veerapol, Prawan Auemduan, Senggunprai Laddawan
Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand.
J Pharm Pharmacol. 2016 Sep;68(9):1184-92. doi: 10.1111/jphp.12586. Epub 2016 Jun 23.
To investigate the apoptosis-inducing effect and underlying mechanisms of luteolin in cholangiocarcinoma (CCA) cells.
Cell viability was determined by sulphorhodamine B. Apoptosis was detected using acridine orange/ethidium bromide dye staining and annexin V/PI staining followed by flow cytometry. The effect of luteolin on the oxidative status of CCA cells was evaluated by measuring intracellular reactive oxygen species (ROS) levels using the dihydroethidium method and quantifying glutathione levels. The mitochondria transmembrane potential (ΔΨm) was examined through JC-1 staining. The protein levels were determined by Western blot. Caspase activity was determined using specific fluorogenic substrates.
Luteolin decreased KKU-100 CCA cells' viability by induction of apoptosis. Luteolin treatment increased ROS production and decreased glutathione levels. These changes were associated with the decrease of Nrf2, γ-glutamylcysteine ligase and heme oxygenase-1 proteins. Moreover, luteolin induced mitochondrial depolarization, which was accompanied by the release of cytochrome c and a decrease of Bcl-2 and Bcl-XL proteins. Pretreatment with antioxidants, 4-hydroxy-TEMPO and N-acetyl-L-cysteine significantly prevented luteolin-induced CCA cell death and loss of ΔΨm. In addition, luteolin induced the activation of caspase-9 and caspase-3.
Luteolin exerts its pro-apoptotic action partly through generating intracellular ROS that then contributes to the activation of mitochondria-mediated apoptotic cell death.
研究木犀草素对胆管癌细胞（CCA）的凋亡诱导作用及其潜在机制。
采用磺酰罗丹明B法测定细胞活力。使用吖啶橙/溴化乙锭染色和膜联蛋白V/碘化丙啶染色，随后进行流式细胞术检测细胞凋亡。通过二氢乙锭法测量细胞内活性氧（ROS）水平并定量谷胱甘肽水平，评估木犀草素对CCA细胞氧化状态的影响。通过JC-1染色检测线粒体跨膜电位（ΔΨm）。采用蛋白质印迹法测定蛋白质水平。使用特异性荧光底物测定半胱天冬酶活性。
木犀草素通过诱导凋亡降低了KKU-100 CCA细胞的活力。木犀草素处理增加了ROS的产生并降低了谷胱甘肽水平。这些变化与Nrf2、γ-谷氨酰半胱氨酸连接酶和血红素加氧酶-1蛋白的减少有关。此外，木犀草素诱导线粒体去极化，伴随着细胞色素c的释放以及Bcl-2和Bcl-XL蛋白的减少。用抗氧化剂4-羟基-TEMPO和N-乙酰-L-半胱氨酸预处理可显著预防木犀草素诱导的CCA细胞死亡和ΔΨm的丧失。此外，木犀草素诱导了半胱天冬酶-9和半胱天冬酶-3的激活。
木犀草素部分通过产生细胞内ROS发挥其促凋亡作用，进而促进线粒体介导的凋亡性细胞死亡的激活。
