Shou Yan, Li Nianyu, Li Li, Borowitz Joseph L, Isom Gary E
Neurotoxicology Laboratory, Department of Medicinal Chemistry and Molecular Pharmacology, Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907-1333, USA.
J Neurochem. 2002 May;81(4):842-52. doi: 10.1046/j.1471-4159.2002.00880.x.
Cyanide induces apoptosis through cytochrome c activated caspase cascade in primary cultured cortical neurons. The underlying mechanism for cytochrome c release from mitochondria after cyanide treatment is still unclear. In this study, the roles of endogenous Bcl-2 proteins in cyanide-induced apoptosis were investigated. After cyanide (100-500 microm) treatment for 24 h, two pro-apoptotic Bcl-2 proteins, Bcl-X(S) and Bax were up-regulated as shown by western blot and RT-PCR analysis. The expression levels of two antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-X(L), remained unchanged after cyanide treatment, whereas the mRNA levels of Bcl-X(S) and Bax began to increase within 2 h and their protein levels increased 6 h after treatment. NF-kappaB, a redox-sensitive transcription factor activated after cyanide treatment, is responsible for the up-regulation of Bcl-X(S) and Bax. SN50, which is a synthetic peptide that blocks translocation of NF-kappaB from cytosol to nucleus, inhibited the up-regulation of Bcl-X(S) and Bax. Similar results were obtained using a specific kappaB decoy DNA. NMDA receptor activation and reactive oxygen species (ROS) generation are upstream events of NF-kappaB activation, as blockade of these two events by MK801, l-NAME or PBN inhibited cyanide-induced up-regulation of Bcl-X(S) and Bax. Up-regulation of pro-apoptotic Bcl-X(S) and Bax contributed to cyanide-induced cytochrome c release, because SN50 and a specific Bax antisense oligodeoxynucleotide significantly reduced release of cytochrome c from mitochondria as shown by western blot analysis. It was concluded that NF-kappaB-mediated up-regulation of Bcl-X(S) and Bax is involved in regulating cytochrome c release in cyanide-induced apoptosis.
氰化物通过细胞色素c激活的半胱天冬酶级联反应在原代培养的皮质神经元中诱导细胞凋亡。氰化物处理后线粒体释放细胞色素c的潜在机制仍不清楚。在本研究中，研究了内源性Bcl-2蛋白在氰化物诱导的细胞凋亡中的作用。经氰化物（100 - 500微摩尔）处理24小时后，蛋白质免疫印迹和逆转录-聚合酶链反应分析显示，两种促凋亡Bcl-2蛋白Bcl-X(S)和Bax上调。两种抗凋亡Bcl-2蛋白Bcl-2和Bcl-X(L)的表达水平在氰化物处理后保持不变，而Bcl-X(S)和Bax的mRNA水平在处理后2小时内开始增加，其蛋白水平在处理后6小时增加。NF-κB是一种在氰化物处理后被激活的氧化还原敏感转录因子，负责Bcl-X(S)和Bax的上调。SN50是一种合成肽，可阻止NF-κB从细胞质转运到细胞核，抑制了Bcl-X(S)和Bax的上调。使用特异性κB诱饵DNA也获得了类似结果。N-甲基-D-天冬氨酸（NMDA）受体激活和活性氧（ROS）生成是NF-κB激活的上游事件，因为MK801、L-硝基精氨酸甲酯（l-NAME）或N-叔丁基-α-苯基硝酮（PBN）对这两个事件的阻断抑制了氰化物诱导的Bcl-X(S)和Bax上调。促凋亡Bcl-X(S)和Bax的上调导致了氰化物诱导的细胞色素c释放，因为蛋白质免疫印迹分析显示，SN50和特异性Bax反义寡脱氧核苷酸显著减少了线粒体中细胞色素c的释放。得出的结论是，NF-κB介导的Bcl-X(S)和Bax上调参与调节氰化物诱导的细胞凋亡中的细胞色素c释放。
