Li Shi-Sheng, Tang Qing-Lai, Wang Shu-hui, Chen Yue-Hong, Liu Jia-Jia, Yang Xin-Ming
Department of Otolaryngology, Head and Neck Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
Tumori. 2011 Nov-Dec;97(6):762-70. doi: 10.1177/030089161109700614.
Despite progress in treatment techniques, the five-year survival rate of nasopharyngeal carcinoma (NPC) is disappointing. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. Given the antiapoptotic functions of Bcl-2 and Akt, we examined the effects of targeting these pathways alone or simultaneously on TRAIL apoptosis in NPC cell lines.
We first tested the cytotoxic effect of TRAIL and the expression of death receptors, Bcl-2, Akt, and p-Akt on four NPC cell lines by MTT and Western blotting, respectively. Small interfering RNAs (siRNAs) targeting Bcl-2 and PI3-K inhibitor (LY294002) were used alone or combined with TRAIL in the cell lines and cytotoxicity was examined by MTT. Apoptosis rates, mitochondrial transmembrane potential, and apoptotic pathway signals were detected by flow cytometric analysis, DiOC6(3) assays, and Western blotting after the various combination treatments on CNE-2, the cell line that was most resistant to TRAIL.
Although no direct correlation between the sensitivity to TRAIL and the relative expression levels of Bcl-2 and activated Akt was found in the NPC cell lines examined, siRNA mediated the downregulation of Bcl-2 and LY294002-induced inactivation of Akt, increasing the sensitivity of all examined NPC cell lines to TRAIL. Synergistic enhancement of TRAIL-mediated cytotoxicity was observed in combination treatment of Bcl-2 siRNA and LY294002 compared to cells treated with each treatment alone. The synergistic effects were mediated through increased apoptotic signaling of the mitochondrial pathway, as was evident from the more increased mitochondrial depolarization, activation of caspase-9 and caspase-3, and suppression of XIAP.
This study provides proof of principle that TRAIL combined with simultaneously targeting the Bcl-2 and Akt signaling pathways may have potential as a novel future treatment strategy for NPC.
尽管治疗技术有所进步，但鼻咽癌（NPC）的五年生存率仍不尽人意。肿瘤坏死因子相关凋亡诱导配体（TRAIL）可在大多数肿瘤细胞中选择性诱导凋亡，同时不损伤正常细胞。鉴于Bcl-2和Akt的抗凋亡功能，我们研究了单独或同时靶向这些通路对NPC细胞系中TRAIL诱导凋亡的影响。
我们首先分别通过MTT法和蛋白质免疫印迹法检测了TRAIL对四种NPC细胞系的细胞毒性作用以及死亡受体、Bcl-2、Akt和磷酸化Akt的表达。针对Bcl-2的小干扰RNA（siRNAs）和PI3-K抑制剂（LY294002）单独或与TRAIL联合应用于细胞系中，并通过MTT法检测细胞毒性。在对TRAIL最具抗性的CNE-2细胞系进行各种联合处理后，通过流式细胞术分析、DiOC6(3)检测和蛋白质免疫印迹法检测凋亡率、线粒体跨膜电位和凋亡通路信号。
在所检测的NPC细胞系中，虽然未发现对TRAIL的敏感性与Bcl-2和活化Akt的相对表达水平之间存在直接相关性，但siRNA介导了Bcl-2的下调，LY294002诱导了Akt的失活，增加了所有检测的NPC细胞系对TRAIL的敏感性。与单独使用每种处理的细胞相比，在Bcl-2 siRNA和LY294002联合处理中观察到TRAIL介导的细胞毒性的协同增强。协同效应是通过线粒体途径凋亡信号的增加介导的，这从线粒体去极化增加、caspase-9和caspase-3的激活以及XIAP的抑制中明显可见。
本研究提供了原理证明，即TRAIL联合同时靶向Bcl-2和Akt信号通路可能作为一种新的未来NPC治疗策略具有潜力。
