Division of Gastroenterologic and General Surgery, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Cancer Res. 2010 Mar 1;70(5):1960-9. doi: 10.1158/0008-5472.CAN-09-3535. Epub 2010 Feb 16.
Apoptosis induction by BH3 mimetics is a therapeutic strategy for human cancer. These mimetics exert single-agent activity in cells "primed" for cell death. Primed cells are dependent upon antiapoptotic Bcl-2 proteins for survival and are characterized by the ability of the BH3 mimetic to induce cytochrome c release from their isolated mitochondria. Our aim was to examine the single-agent activity of obatoclax, a BH3 mimetic in cholangiocarcinoma cell lines. In clonogenic assays, inhibition of colony formation was observed by obatoclax treatment. Despite single-agent activity by obatoclax, the mitochondria from these cells did not release cytochrome c after incubation with this BH3 mimetic. However, immunofluorescence and cell fractionation studies identified Bax activation and translocation to mitochondria after treatment with obatoclax. shRNA targeted knockdown of Bax doubled the IC50 for obatoclax but did not abrogate its cytotoxicity, whereas knockdown of Bak did not alter the IC50. In a cell-free system, obatoclax induced an activating conformational change of Bax, which was attenuated by a site-directed mutagenesis of a previously identified protein activation site. Finally, the drug also elicited a significant in vivo response in a rodent model of this disease. In conclusion, single-agent obatoclax treatment results in Bax activation, which contributes, in part, to cell death in cholangiocarcinoma cells. These data indicate that BH3 mimetics may also function as direct activators of Bax and induce cytotoxicity in cells not otherwise primed for cell death.
BH3 模拟物诱导细胞凋亡是治疗人类癌症的一种策略。这些模拟物在“为细胞死亡做好准备”的细胞中具有单一药物活性。“做好准备”的细胞依赖抗凋亡 Bcl-2 蛋白生存，其特征是 BH3 模拟物能够诱导细胞色素 c 从其分离的线粒体中释放。我们的目的是研究 BH3 模拟物 obatoclax 在胆管癌细胞系中的单一药物活性。在集落形成实验中，obatoclax 处理观察到集落形成抑制。尽管 obatoclax 具有单一药物活性，但在用这种 BH3 模拟物孵育后，这些细胞的线粒体并未释放细胞色素 c。然而，免疫荧光和细胞分级分离研究鉴定出 Bax 在 obato 克拉治疗后被激活并转移到线粒体。靶向 Bax 的 shRNA 敲低使 obatoclax 的 IC50 增加了一倍，但并未消除其细胞毒性，而 Bak 的敲低则没有改变 IC50。在无细胞系统中，obatoclax 诱导 Bax 的激活构象变化，该变化被先前鉴定的蛋白激活位点的定点突变所减弱。最后，该药物在这种疾病的啮齿动物模型中也产生了显著的体内反应。总之，单一药物 obatoclax 治疗导致 Bax 激活，这在一定程度上导致胆管癌细胞死亡。这些数据表明，BH3 模拟物也可能作为 Bax 的直接激活剂发挥作用，并在未为细胞死亡做好准备的细胞中诱导细胞毒性。
