Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells - A possible mechanism contributing to peripheral neuropathy and cellular toxicity following proteasome inhibition
Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells - A possible mechanism contributing to peripheral neuropathy and cellular toxicity following proteasome inhibition
复制标题
DOI：
10.4161/cc.7.7.5625
复制
发表时间：
2008-04-01
期刊：
影响因子：
4.3
通讯作者：
Fojo, Tito
中科院分区：
文献类型：
--
作者：
Poruchynsky, Marianne S.;Sackett, Dan L.;Fojo, Tito
Bortezomib （Velcade((R))）是一种蛋白酶体抑制剂，已被FDA批准用于治疗多发性骨髓瘤（MM）。虽然有效，但由于病因不明的周围神经毒性，其使用受到阻碍。由于蛋白酶体抑制剂改变了蛋白质的降解，我们推测调节微管（MT）稳定性的蛋白质可能在处理后受到影响，并通过比较微管蛋白在聚合(P)和可溶性(S)组分之间的分布来检测细胞中的微管聚合。我们观察到，使用五种蛋白酶体抑制剂处理SY5Y和KCNR[神经母细胞瘤]，HCN2和8226 [MM]细胞后，MT聚合增加；总a-微管蛋白在“P”部分的基线比例在41 - 68%之间，治疗后增加到55 - 99%。在神经细胞系HCN1A和HCN2中观察到乙酰化α -微管蛋白（一种稳定mt的翻译后标记物）的增加，并且在去除蛋白酶体抑制剂后持续了144小时。细胞周期分析显示，三种细胞系在处理后的G(2)M期增加了50 - 75%。与紫杉醇处理的细胞相比，蛋白酶体抑制剂处理的细胞的免疫荧光定位研究没有显示微管束，这表明MT的稳定是通过一种非直接药物结合的机制实现的。我们检测了微管相关蛋白的水平，并观察到微管相关蛋白MAP2在HCN2细胞中使用蛋白酶体抑制剂后增加了1.4 - 3.7倍。这些数据为临床观察到的神经毒性提供了一个合理的解释，并提出了微管稳定有助于细胞毒性的可能性。
Bortezomib (Velcade((R))), a proteasome inhibitor, is approved by the FDA for the treatment of multiple myeloma (MM). While effective, its use has been hampered by peripheral neurotoxicity of unexplained etiology. Since proteasome inhibitors alter protein degradation, we speculated that proteins regulating microtubule (MT) stability may be affected after treatment and examined MT polymerization in cells by comparing the distribution of tubulin between polymerized (P) and soluble (S) fractions. We observed increased MT polymerization following treatment of SY5Y and KCNR [neuroblastoma], HCN2 and 8226 [MM] cells, using five proteasome inhibitors; the baseline proportion of total a-tubulin in 'P' fractions ranged from similar to 41 - 68%, and increased to similar to 55 - 99% after treatment. Increased acetylated alpha-tubulin, a post-translational marker of stabilized MTs, was observed in the neural cell lines HCN1A and HCN2 and this was sustained up to 144 hours after the proteasome inhibitor was removed. Cell cycle analysis of three cell lines after treatment, showed similar to 50 - 75% increases in the G(2)M phase. Immunofluorescent localization studies of proteasome inhibitor treated cells did not reveal microtubule bundles in contrast to paclitaxel treated, suggesting MT stabilization via a mechanism other than direct drug binding. We examined the levels of microtubule associated proteins and observed a 1.4 - 3.7 fold increase in the microtubule associated protein MAP2, in HCN2 cells following treatment with proteasome inhibitors. These data provide a plausible explanation for the neurotoxicity observed clinically and raise the possibility that microtubule stabilization contributes to cytotoxicity.