Mensah-Osman Edith, Al-Katib Ayad, Dandashi Mahmoud, Mohammad Ramzi
Division of Hematology and Oncology, Department of Internal Medicine, Karmanos Cancer Institute at Wayne State University School of Medicine, Detroit, MI 48201, USA.
Int J Oncol. 2003 Dec;23(6):1637-44.
We have previously reported that XK469 inhibited topoisomerase (topo) IIbeta, in Waldenstrom's macroglobulinemia cell line (WSU-WM) however the inhibition alone is not sufficient to induce apoptosis. In this study, the apoptotic potential of XK469 and its mechanism in WSU-WM cell line was investigated. Exposure of WSU-WM cells to XK469 caused a decrease in viable cell number in a dose-dependent manner. In addition, XK469 caused the activation of caspase 3 resulting in subsequent cleavage of PARP. These events were preceded by the release of cytochrome c from the mitochondria to the cytosol. Simultaneous exposure of cells to cyclosporin A prevented the release of cytochrome c to cytosol and reduced the loss of viability. XK469 caused the activation of p53 with up-regulation of p53-dependent proteins such as Bax, p21, Gadd 45 and cyclin B1 in association with G2M arrest. The addition of ubiquitin carboxyl terminal hydrolase (UCH-L1) inhibitor (NaBH4) inhibited up-regulation of p53 and p53 related molecules by XK469 and reduced the loss of viability. Pre-incubation with NOK-1, a monoclonal antibody that prevents Fas-Fas ligand interaction and is inhibitory to Fas signaling interfered with XK469 induced activation of caspase 8 and also reduced the loss of viability. Simultaneous exposure of all three inhibitors (cyclosporin A, NaBH4 and NOK-1) abrogated the toxicity of XK469 by 95%. These data define multiple sequences of biochemical events that mediate cell death induced by XK469. Our study suggests a complex mechanistic cascade of XK469-mediated apoptosis that involves Fas signaling pathway, ubiquitination, p53 activation and cytochrome c release.
我们之前报道过，XK469可抑制华氏巨球蛋白血症细胞系（WSU-WM）中的拓扑异构酶（topo）IIβ，然而单纯的抑制作用并不足以诱导细胞凋亡。在本研究中，我们对XK469在WSU-WM细胞系中的凋亡潜力及其机制进行了研究。将WSU-WM细胞暴露于XK469会导致活细胞数量呈剂量依赖性减少。此外，XK469会导致半胱天冬酶3激活，进而导致聚（ADP-核糖）聚合酶（PARP）随后被切割。这些事件之前有线粒体中的细胞色素c释放到细胞质中。细胞同时暴露于环孢素A可阻止细胞色素c释放到细胞质中，并减少细胞活力丧失。XK469导致p53激活，同时上调p53依赖性蛋白，如Bax、p21、生长停滞和DNA损伤诱导蛋白45（Gadd 45）以及细胞周期蛋白B1，并伴有G2/M期阻滞。添加泛素羧基末端水解酶（UCH-L1）抑制剂（NaBH4）可抑制XK469对p53及其相关分子的上调作用，并减少细胞活力丧失。用NOK-1（一种可阻止Fas-Fas配体相互作用并抑制Fas信号传导的单克隆抗体）预孵育会干扰XK469诱导的半胱天冬酶8激活，也会减少细胞活力丧失。同时暴露于所有三种抑制剂（环孢素A、NaBH4和NOK-1）可消除XK469 95%的毒性。这些数据确定了介导XK469诱导细胞死亡的多个生化事件序列。我们的研究表明，XK469介导的凋亡涉及一个复杂的机制级联反应，包括Fas信号通路、泛素化、p53激活和细胞色素c释放。
