Chow Jyh-Ming, Shen Shing-Chuan, Wu Chin-Yen, Chen Yen-Chou
Section of Hematology-Oncology, Department of Internal Medicine, Taipei Municipal Wan-Fang Hospital, Taipei Medical University, Taipei, Taiwan.
Apoptosis. 2006 Nov;11(11):1999-2011. doi: 10.1007/s10495-006-0085-x.
In the present study, we found that baicalein (BE), but not its glycoside baicalin (BI), induced apoptosis in human leukemia HL-60 and Jurkat cells, but not in primary murine peritoneal macrophages (PMs) or human polymorphonuclear (PMN) cells, by the MTT assay, LDH release assay, and flow cytometric analysis. Activation of the caspase 3, but not caspase 1, enzyme via inducing protein processing was detected in BE-induced apoptosis. The ROS-scavenging activity of BE was identified by the anti-DPPH radical, DCHF-DA, and in vitro plasmid digestion assay, and none of chemical antioxidants including allpurinol (ALL), N-acetyl-cystein (NAC), and diphenylene iodonium (DPI) affected BE-induced apoptosis in HL-60 cells. This suggests that apoptosis induced by BE is independent of the production of ROS in HL-60 cells. Interestingly, the apoptotic events such as DNA ladders formation and activation of the caspase 3 cascade were significantly blocked by TPA addition in the presence of membrane translocation of PKCalpha, and TPA-induced protection was reduced by adding the PKC inhibitors, GF-109203X and staurosporin. TPA addition induces the phosphorylation of JNKs and ERKs, but not p38, protein in HL-60 cells, and incubation of HL-60 cells with JNKs inhibitor SP600125, but not ERKs inhibitor, PD98059 or the p38 inhibitor SB203580, suppressed the protective effect of TPA against BE-induced apoptotic events including DNA ladders, apoptotic bodies, caspase 3 and D4-GDI protein cleavage in according with blocking JNKs protein phosphorylation. In addition, PKC inhibitor GF-109203X treatment blocks TPA-induced ERKs and JNKs protein phosphorylation, which indicates that activation of PKC locates at upstream of MAPKs activation in TPA-treated HL-60 cells. Additionally, a loss in mitochondrial membrane potential with a reduction in Bcl-2 protein expression, the induction of Bad protein phosphorylation, and translocation of cytochrome c from mitochondria to the cytosol were observed in BE-treated HL-60 cells, and these events were prevented by the addition of TPA. GF-109203X and SP600125 suppression of TPA against cytochrome c release induced by BE was identified. This suggests that activation of PKC and JNKs participate in TPA's prevention of BE-induced apoptosis via suppressing mitochondrial dysfunction in HL-60 cells.
在本研究中，我们发现黄芩素（BE）而非其糖苷黄芩苷（BI）可诱导人白血病HL-60和Jurkat细胞凋亡，但不能诱导原代小鼠腹腔巨噬细胞（PMs）或人多形核（PMN）细胞凋亡，通过MTT法、乳酸脱氢酶释放法和流式细胞术分析得以证实。在BE诱导的凋亡中，检测到通过诱导蛋白加工激活了半胱天冬酶3而非半胱天冬酶1。通过抗DPPH自由基、DCHF-DA和体外质粒消化试验鉴定了BE的活性氧清除活性，包括别嘌呤醇（ALL）、N-乙酰半胱氨酸（NAC）和二苯基碘鎓（DPI）在内的化学抗氧化剂均不影响BE诱导的HL-60细胞凋亡。这表明BE诱导的凋亡与HL-60细胞中活性氧的产生无关。有趣的是，在PKCalpha膜转位存在的情况下，添加佛波酯（TPA）可显著阻断诸如DNA梯状条带形成和半胱天冬酶3级联激活等凋亡事件，并且添加PKC抑制剂GF-109203X和星形孢菌素可降低TPA诱导的保护作用。添加TPA可诱导HL-60细胞中JNKs和ERKs而非p38蛋白的磷酸化，用JNKs抑制剂SP600125而非ERKs抑制剂PD98059或p38抑制剂SB203580孵育HL-60细胞，可抑制TPA对BE诱导的凋亡事件的保护作用，包括DNA梯状条带、凋亡小体、半胱天冬酶3和D4-GDI蛋白裂解，这与阻断JNKs蛋白磷酸化一致。此外，PKC抑制剂GF-109203X处理可阻断TPA诱导的ERKs和JNKs蛋白磷酸化，这表明在TPA处理的HL-60细胞中PKC的激活位于MAPKs激活的上游。另外，在BE处理的HL-60细胞中观察到线粒体膜电位丧失，Bcl-2蛋白表达降低，Bad蛋白磷酸化诱导以及细胞色素c从线粒体转运至胞质溶胶，添加TPA可阻止这些事件发生。已鉴定出GF-109203X和SP600125可抑制TPA对BE诱导的细胞色素c释放的作用。这表明PKC和JNKs的激活通过抑制HL-60细胞中的线粒体功能障碍参与TPA对BE诱导凋亡的预防。
