Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, Frankfurt 60528, Germany.
Cell Death Differ. 2013 Sep;20(9):1161-73. doi: 10.1038/cdd.2013.45. Epub 2013 Jun 7.
Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.
奥巴妥克拉克斯（GX15-070）是一种抗凋亡 Bcl-2 蛋白的小分子抑制剂，已被报道通过自噬引发细胞死亡。然而，其潜在的分子机制仍不清楚。在这里，我们发现 GX15-070 刺激自噬体膜上的坏死体组装是将 GX15-070 刺激的自噬与坏死性凋亡联系起来的关键事件。GX15-070 主要诱导横纹肌肉瘤细胞发生非凋亡形式的细胞死亡，这从缺乏典型的凋亡特征，如 DNA 片段化或半胱天冬酶激活以及对广谱半胱天冬酶抑制剂 zVAD.fmk 不敏感中显而易见。相反，GX15-070 触发大量自噬体的积累，这对于 GX15-070 诱导的细胞死亡是必需的，因为沉默 Atg5 或 Atg7 阻断自噬体形成会使 GX15-070 介导的细胞死亡。免疫共沉淀研究表明，GX15-070 刺激自噬体膜成分 Atg5 与坏死体成分 FADD、RIP1 和 RIP3 相互作用。这种 GX15-070 诱导的坏死体在自噬体上的组装是一种依赖 Atg5 的方式，因为 Atg5 的敲低会破坏这种复合物的形成。RIP1 是 GX15-070 诱导细胞死亡所必需的，因为通过 shRNA 介导的敲低或 RIP1 抑制剂 necrostatin-1 对 RIP1 的遗传和药理学抑制都会阻断 GX15-070 诱导的细胞死亡。同样，RIP3 的敲低可挽救 GX15-070 介导的细胞死亡和集落形成存活的抑制。有趣的是，RIP1 或 RIP3 的沉默对 GX15-070 刺激的自噬体形成没有影响，这强调了 RIP1 和 RIP3 在自噬诱导的下游介导细胞死亡。值得注意的是，在体内的绒毛尿囊膜模型中，GX15-070 以依赖 RIP1 的方式显著抑制肿瘤生长。总之，GX15-070 通过促进坏死体在自噬体上的组装来引发坏死性凋亡。这些发现为 GX15-070 诱导的非凋亡性细胞死亡的分子机制提供了新的见解。
