Department of Biology, University "Roma Tre", Rome, Italy.
Apoptosis. 2011 Sep;16(9):940-9. doi: 10.1007/s10495-011-0619-8.
The BH3-only Bcl-2 subfamily member Bim is a well known apoptosis promoting protein. However, the mechanisms upstream of mitochondrion membrane permeability by which Bim is involved in apoptosis have been poorly investigated, particularly in response to agents capable of interfering with the cytoskeleton architecture and arresting cells in mitosis. Based on the observation that Bim is sequestered on the microtubule-array by interaction with the light chain of dynein, we have investigated upon depolymerisation, whether Bim could be involved in the commitment of apoptosis. With this purpose H460 Non Small Lung Cancer Cells (NSLC) were treated with the microtubule damaging agent combretastatin-A4 (CA-4) (7.5 nM; 8-48 h), and various parameters were investigated. Upon treatment, cells arrested in mitosis and died through a caspase-3-dependent mitotic catastrophe. Transient knock down of Bim drastically reduced apoptosis, indicating that this protein was involved in cell death as induced by microtubules disorganisation. In response to increasing conditions of microtubules depolymerisation, we found that the protein level of Bim was strongly upregulated in a time-dependent manner at transcriptional level. Furthermore, Bim was released from microtubule-associated components. Bim was translocated to mitochondria, even in a condition of protein synthesis inhibition, where it showed a markedly increased interaction with Bcl-2. In turn, the fraction of Bax bound to Bcl-2 decreases in response to treatment, thereby indicating that Bim possibly promotes Bax release from the pro-survival protein Bcl-2. Overall, we demonstrated that Bim is required for the CA-4-induced cell death in the H460 lung cancer cell line via activation of the mitochondrial signalling pathway. Defining the contribution of Bim to the mechanism of apoptosis may offer some different clues in view of developing new strategies for chemotherapy with CA-4, underlining the relevance of the cytoskeleton integrity in the apoptotic response.
BH3 仅 Bcl-2 亚家族成员 Bim 是一种众所周知的促进凋亡的蛋白质。然而，Bim 参与细胞凋亡的线粒体膜通透性的上游机制尚未得到充分研究，特别是对于能够干扰细胞骨架结构并使细胞在有丝分裂中停滞的药物。基于 Bim 通过与动力蛋白轻链相互作用而被隔离在微管阵列上的观察结果，我们研究了微管解聚后，Bim 是否参与细胞凋亡的启动。为此，我们用微管破坏剂 combretastatin-A4（CA-4）（7.5 nM；8-48 h）处理 H460 非小细胞肺癌（NSCLC）细胞，并研究了各种参数。治疗后，细胞在有丝分裂中停滞并通过 caspase-3 依赖性有丝分裂灾难死亡。Bim 的瞬时敲低大大减少了细胞凋亡，表明该蛋白参与了微管解聚诱导的细胞死亡。随着微管解聚条件的增加，我们发现 Bim 的蛋白水平在转录水平上以时间依赖性方式强烈上调。此外，Bim 从微管相关成分中释放出来。Bim 易位到线粒体，即使在蛋白质合成抑制的情况下，它与 Bcl-2 的相互作用也明显增加。反过来，Bax 与 Bcl-2 结合的部分在治疗后减少，这表明 Bim 可能促进 Bax 从生存蛋白 Bcl-2 中释放。总的来说，我们证明了 Bim 通过激活线粒体信号通路，在 H460 肺癌细胞系中是 CA-4 诱导细胞死亡所必需的。定义 Bim 在 CA-4 诱导的细胞凋亡机制中的作用可能会为开发 CA-4 化疗的新策略提供一些不同的线索，同时强调细胞骨架完整性在凋亡反应中的相关性。
