Mott Justin L, Bronk Steve F, Mesa Ruben A, Kaufmann Scott H, Gores Gregory J
Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905, USA.
Mol Cancer Ther. 2008 Aug;7(8):2339-47. doi: 10.1158/1535-7163.MCT-08-0285.
Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1. The purpose of this study is to determine if obatoclax sensitizes human cholangiocarcinoma cells to apoptosis. The human cholangiocarcinoma cell lines, KMCH, KMBC, and TFK, were employed for these studies. Protein expression was assessed by immunoblot and protein-protein interactions detected by coprecipitation of the polypeptide of interest with S-tagged Mcl-1. Activation of Bak and Bax was observed by immunocytochemistry with conformation-specific antisera. Obatoclax induced minimal apoptosis alone; however, it increased apoptosis 3- to 13-fold in all three cancer cell lines when combined with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Obatoclax did not alter cellular expression of Bid, Bim, Puma, Noxa, Bak, Bax, Mcl-1, or cFLIP. Mcl-1 binding to Bak was readily identified in untreated cells, and this association was disrupted by treating the cells with obatoclax. Additionally, Bim binding to Mcl-1 was markedly decreased by obatoclax treatment. We also identified alterations in Bak and Bax conformation following treatment with obatoclax plus Apo2L/TRAIL but not with either Apo2L/TRAIL or obatoclax alone. In conclusion, obatoclax releases Bak and Bim from Mcl-1 and sensitizes human cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Obatoclax is a potentially promising adjunctive agent for the treatment of this cancer.
人胆管癌通过过表达Mcl-1逃避凋亡。药物obatoclax（GX15-070）可抑制包括Mcl-1在内的Bcl-2家族抗凋亡成员。本研究的目的是确定obatoclax是否使人胆管癌细胞对凋亡敏感。这些研究采用了人胆管癌细胞系KMCH、KMBC和TFK。通过免疫印迹评估蛋白质表达，并通过将感兴趣的多肽与S标签的Mcl-1共沉淀来检测蛋白质-蛋白质相互作用。用构象特异性抗血清通过免疫细胞化学观察Bak和Bax的激活。Obatoclax单独诱导的凋亡极少；然而，当与Apo2L/肿瘤坏死因子相关凋亡诱导配体（TRAIL）联合使用时，它在所有三种癌细胞系中使凋亡增加了3至13倍。Obatoclax未改变Bid、Bim、Puma、Noxa、Bak、Bax、Mcl-1或cFLIP的细胞表达。在未处理的细胞中很容易鉴定出Mcl-1与Bak的结合，用obatoclax处理细胞可破坏这种结合。此外，obatoclax处理显著降低了Bim与Mcl-1的结合。我们还发现用obatoclax加Apo2L/TRAIL处理后Bak和Bax的构象发生了改变，但单独用Apo2L/TRAIL或obatoclax处理则未出现这种改变。总之，obatoclax使Bak和Bim从Mcl-1中释放出来，并使人胆管癌细胞对Apo2L/TRAIL诱导的凋亡敏感。Obatoclax是一种治疗这种癌症的潜在有前景的辅助药物。
