Bcl-2 down-regulation is a novel mechanism of paclitaxel resistance
Bcl-2 down-regulation is a novel mechanism of paclitaxel resistance
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DOI：
10.1124/mol.64.1.51
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发表时间：
2003-07-01
影响因子：
3.6
通讯作者：
Scambia, G
中科院分区：
文献类型：
--
作者：
Ferlini, C;Raspaglio, G;Scambia, G
紫杉烷类通过抑制微管动力学发挥作用；在这项研究中，我们研究了线粒体作为紫杉烷类的额外靶点。我们在有或没有刺激线粒体呼吸状态的紫杉烷存在下孵育分离的线粒体。结果表明，在刺激呼吸状态后，它们迅速引起 Deltapsi(m) 的损失。为了评估 [C-14] 紫杉醇与分离的线粒体的结合，沉淀线粒体蛋白质，产生 18.6 +/- 2.1 cpm/杯蛋白质。刺激呼吸状态后，[C-14]紫杉醇的结合增加至163.2 +/- 46.7 cpm/杯蛋白质。 Bcl-2免疫沉淀后的CPM值比对照抗体高62.8倍，从而表明Bcl-2参与紫杉醇结合。然后，我们建立了一组对单独增加剂量的紫杉醇或对高剂量的紫杉醇/环孢素A A2780 TC细胞具有抗性的A2780细胞系。在这两种情况下，Bcl-2 表达持续下调，而 Bcl-2 家族其他成员（例如 Bax 和 Bcl-x）的水平在紫杉醇耐药细胞系中没有变化。当 A2780TC 细胞稳定转染 Bcl-2 构建体时，紫杉醇敏感性部分恢复，从而支持 Bcl-2 下调在维持耐药性中的直接作用。最后，我们通过免疫组织化学在一小部分卵巢癌紫杉醇耐药患者中检查了 Bcl-2，我们注意到在这种临床环境中，相对于药物敏感肿瘤中发现的表达水平，该蛋白被下调。这些发现表明，Bcl-2 是紫杉烷类的另一个细胞内靶标，并且其下调与紫杉烷类耐药有关。
Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of Deltapsi(m) after stimulation of the respiratory state. To evaluate the binding of [C-14] paclitaxel to isolated mitochondria, mitochondrial proteins were precipitated yielding 18.6 +/- 2.1 cpm/mug of protein. After stimulation of the respiratory state, binding of [C-14] paclitaxel increased up to 163.2 +/- 46.7 cpm/mug of protein. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.