INSERM U865, University of Lyon, Faculté Laënnec, Lyon, France.
Gastroenterology. 2011 Aug;141(2):663-73. doi: 10.1053/j.gastro.2011.04.055. Epub 2011 Apr 30.
BACKGROUND & AIMS: Oxaliplatin sensitizes drug-resistant colon cancer cell lines to tumor necrosis factor-related apoptosis inducing ligand (TRAIL), a death receptor ligand that is selective for cancer cells. We investigated the molecular mechanisms by which oxaliplatin sensitizes cancer cells to TRAIL-induced apoptosis.
We incubated the colon cancer cell lines HT29 and V9P, which are resistant to TRAIL, with TRAIL or with oxaliplatin for 2 hours, followed by TRAIL. Annexin V staining was used to measure apoptosis; RNA silencing and immunoblot experiments were used to study the roles of apoptosis-related proteins. Site-directed mutagenesis experiments were used to determine requirements for phosphorylation of Bcl-xL; co-immunoprecipitation experiments were used to analyze the interactions among Bcl-xL, Bax, and Bak, and activation of Bax.
Oxaliplatin-induced sensitivity to TRAIL required activation of the mitochondrial apoptotic pathway; reduced expression of Bax, Bak, and caspase-9, and stable overexpression of Bcl-xL, reduced TRAIL-induced death of cells incubated with oxaliplatin. Mitochondrial priming was induced in cells that were sensitized by oxaliplatin and required signaling via c-Jun N-terminal kinase and phosphorylation of Bcl-xL. Mimicking constitutive phosphorylation of Bcl-xL by site-directed mutagenesis at serine 62 restored sensitivity of cells to TRAIL. Co-immunoprecipitation experiments showed that oxaliplatin-induced phosphorylation of Bcl-xL disrupted its ability to sequestrate Bax, allowing Bax to interact with Bak to induce TRAIL-mediated apoptosis.
Oxaliplatin facilitates TRAIL-induced apoptosis in colon cancer cells by activating c-Jun N-terminal kinase signaling and phosphorylation of Bcl-xL. Oxaliplatin-induced sensitivity to TRAIL might be developed as an approach to cancer therapy.
奥沙利铂可使耐药结肠癌细胞系对肿瘤坏死因子相关凋亡诱导配体（TRAIL）敏感，TRAIL 是一种对癌细胞具有选择性的死亡受体配体。我们研究了奥沙利铂使癌细胞对 TRAIL 诱导的细胞凋亡敏感的分子机制。
我们用 TRAIL 或奥沙利铂孵育对 TRAIL 耐药的结肠癌细胞系 HT29 和 V9P2 小时，然后再用 TRAIL。用 Annexin V 染色来测量细胞凋亡；用 RNA 沉默和免疫印迹实验来研究凋亡相关蛋白的作用。用定点突变实验来确定 Bcl-xL 磷酸化的要求；用共免疫沉淀实验来分析 Bcl-xL、Bax 和 Bak 之间的相互作用，以及 Bax 的激活。
奥沙利铂诱导的 TRAIL 敏感性需要激活线粒体凋亡途径；Bax、Bak 和 caspase-9 的表达减少，Bcl-xL 的稳定过表达，减少了用奥沙利铂孵育的细胞对 TRAIL 的诱导死亡。奥沙利铂敏感化的细胞中诱导了线粒体启动，这需要通过 c-Jun N 端激酶信号和 Bcl-xL 的磷酸化。通过定点突变在丝氨酸 62 模拟 Bcl-xL 的组成性磷酸化，恢复了细胞对 TRAIL 的敏感性。共免疫沉淀实验表明，奥沙利铂诱导的 Bcl-xL 磷酸化破坏了其与 Bax 结合的能力，使 Bax 与 Bak 相互作用，从而诱导 TRAIL 介导的凋亡。
奥沙利铂通过激活 c-Jun N 端激酶信号和 Bcl-xL 的磷酸化，促进结肠癌细胞中 TRAIL 诱导的细胞凋亡。奥沙利铂诱导对 TRAIL 的敏感性可能作为一种癌症治疗方法。
