School of Medicine and Public Health, University of Newcastle, Callaghan, NSW, Australia.
Cell Death Dis. 2013 Jun 6;4(6):e655. doi: 10.1038/cddis.2013.192.
Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF(V600E) melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF(V600E) melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF(V600E) melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.
过去的研究表明，组蛋白去乙酰化酶（HDAC）和突变 BRAF（v-Raf 鼠肉瘤病毒致癌基因同源物 B1）抑制剂协同杀死具有 BRAF 激活突变的黑色素瘤细胞。然而，涉及的机制仍不太清楚。在这里，我们报告 HDAC 和 BRAF 抑制剂的组合通过诱导坏死杀死 BRAF(V600E)黑色素瘤细胞。用 HDAC 抑制剂 suberoylanilide hydroxamic acid（SAHA）或 panobinostat（LBH589）和 BRAF 抑制剂 PLX4720 联合治疗激活了半胱天冬酶级联，但半胱天冬酶对于杀伤似乎是可有可无的，因为抑制半胱天冬酶并不总是阻止细胞死亡的诱导。当大多数垂死的细胞对 Annexin V 呈阳性时，它们立即获得碘化丙啶阳性，表明诱导坏死。这得到了半胱天冬酶非依赖性的高迁移率族蛋白 B1 的释放的支持，并且通过质膜破裂和核和细胞质内容物的丢失进一步得到巩固，如透射电子显微镜分析所示。值得注意的是，坏死抑制剂 necrostatin-1 或受体相互作用蛋白激酶 3（RIPK3）的小干扰 RNA（siRNA）敲低均不能抑制细胞死亡，这表明 RIPK1 和 RIPK3 不会导致 HDAC 和 BRAF 抑制剂组合在 BRAF(V600E)黑色素瘤细胞中诱导坏死。重要的是，SAHA 和临床上可用的 BRAF 抑制剂 vemurafenib 即使在抑制 caspase-3 的情况下，也能在小鼠模型中协同抑制 BRAF(V600E)黑色素瘤异种移植物的生长。总之，这些结果表明，HDAC 和 BRAF 抑制剂的联合治疗可以绕过经典的细胞死亡途径杀死黑色素瘤细胞，这在黑色素瘤的治疗中可能具有治疗优势。
