Boland M P, Foster S J, O'Neill L A
Department of Biochemistry, Trinity College, Dublin, Ireland.
J Biol Chem. 1997 May 16;272(20):12952-60. doi: 10.1074/jbc.272.20.12952.
The anthracycline antibiotic, daunorubicin, can induce programmed cell death (apoptosis) in cells. Recent work suggests that this event is mediated by ceramide via enhanced ceramide synthase activity. Since the generation of ceramide has been directly linked with the activation of the transcription factor, NFkappaB, this was investigated as a novel target for the action of daunorubicin. Here we describe how treatment of HL-60 promyelocytes and Jurkat T lymphoma cells with daunorubicin results in the activation of the transcription factor NFkappaB. The effect of daunorubicin was evident following 1-2 h treatment, which was in contrast to the time course of activation obtained with the cytokine, tumor necrosis factor, where NFkappaB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IkappaB degradation and increased the expression of an NFkappaB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFkappaB-linked reporter gene, suggesting a synergy between these two agents in this response. These events were sensitive to the iron chelator, deferoxamine mesylate (desferal), and the anti-oxidant and metal chelator pyrrolidine dithiocarbamate. A structurally related compound, mitoxantrone, which, unlike daunorubicin, is unable to undergo redox cycling in cells, also activated NFkappaB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFkappaB activation at a range of concentrations previously reported to block apoptosis induced by this drug. However, this agent could inhibit increases in ceramide induced by daunorubicin, in addition to blocking ceramide synthase activity from HL-60 cells which was activated in response to daunorubicin treatment. These data therefore suggest that the effect of daunorubicin on NFkappaB is unlikely to involve ceramide, but may involve reactive oxygen species generated as a result of endogenous cellular processes rather than reductive metabolism of the drug. As NFkappaB may be involved in apoptosis, this effect may be an important aspect of the cellular responses to this agent.
蒽环类抗生素柔红霉素可诱导细胞发生程序性细胞死亡（凋亡）。近期研究表明，这一过程是由神经酰胺通过增强神经酰胺合酶活性介导的。由于神经酰胺的生成与转录因子NFκB的激活直接相关，因此将其作为柔红霉素作用的新靶点进行了研究。在此，我们描述了用柔红霉素处理HL-60早幼粒细胞和Jurkat T淋巴瘤细胞如何导致转录因子NFκB的激活。柔红霉素处理1-2小时后其作用明显，这与细胞因子肿瘤坏死因子激活的时间进程不同，肿瘤坏死因子在细胞刺激后几分钟内即可检测到NFκB的激活。活化复合物主要含有p50和p65/RelA亚基成分。柔红霉素还诱导IκB降解并增加与NFκB相关的报告基因的表达。此外，发现该药物能强烈增强肿瘤坏死因子诱导与NFκB相关报告基因的能力，表明这两种药物在该反应中有协同作用。这些事件对铁螯合剂甲磺酸去铁胺（去铁敏）以及抗氧化剂和金属螯合剂吡咯烷二硫代氨基甲酸盐敏感。一种结构相关的化合物米托蒽醌，与柔红霉素不同，它不能在细胞中进行氧化还原循环，也能以吡咯烷二硫代氨基甲酸盐敏感的方式激活NFκB。神经酰胺合酶的特异性抑制剂伏马菌素B1在先前报道的能阻断该药物诱导凋亡的一系列浓度下，对柔红霉素诱导的NFκB激活没有影响。然而，该药物除了能阻断柔红霉素处理后HL-60细胞中被激活的神经酰胺合酶活性外，还能抑制柔红霉素诱导的神经酰胺增加。因此，这些数据表明柔红霉素对NFκB的作用不太可能涉及神经酰胺，而可能涉及内源性细胞过程产生的活性氧，而非药物的还原代谢。由于NFκB可能参与凋亡，这种作用可能是细胞对该药物反应的一个重要方面。
