Queen's University Belfast, Centre for Cancer Research and Cell Biology, Belfast, UK.
BMC Cancer. 2010 Jul 20;10:380. doi: 10.1186/1471-2407-10-380.
Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded.
We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation.
Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced.
Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）激活细胞外凋亡途径是一种治疗癌症的新的治疗策略，目前正在临床评估中。鉴定耐药的分子生物标志物可能在预测抗肿瘤的临床活性方面发挥重要作用。线粒体 1 型电压依赖性阴离子通道（VDAC1）在调节凋亡中的作用备受争议。迄今为止，其在调节细胞外凋亡途径中的功能作用尚未被正式排除。
我们在非小细胞肺癌细胞中进行了 VDAC1 的稳定和瞬时 RNAi 敲低，并通过孵育细胞与死亡配体 TRAIL 主要刺激细胞外凋亡途径。我们使用体外凋亡和细胞活力测定以及凋亡标志物的 Western blot，证明 TRAIL 诱导的毒性依赖于 VDAC1。共聚焦显微镜和线粒体分离用于确定线粒体对于 caspase-8 激活的重要性。
在这里，我们表明，无论是稳定还是瞬时敲低 VDAC1，都足以拮抗非小细胞肺癌（NSCLC）细胞中 TRAIL 介导的凋亡。具体而言，VDAC1 是在线粒体中将 procaspase-8 加工为完全活性的 p18 形式所必需的。尽管 VDAC1 表达对于 TRAIL 依赖的内在凋亡途径的激活是必需的，但 VDAC1 的缺失并不改变线粒体对外源 caspase-8 切割的 BID 诱导的线粒体去极化的敏感性。此外，外源性 VDAC1 的表达恢复了在其内源性 VDAC1 已被选择性沉默的细胞中对 TRAIL 的凋亡反应。
VDAC1 的表达对于 caspase-8 的完全加工和激活是必需的，并支持线粒体在通过死亡受体途径调节凋亡信号中的作用。
