Chawla-Sarkar Mamta, Leaman Douglas W, Jacobs Barbara S, Borden Ernest C
Center for Drug Discovery and Development, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
J Immunol. 2002 Jul 15;169(2):847-55. doi: 10.4049/jimmunol.169.2.847.
All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.
所有人类黑色素瘤细胞系（通过膜联蛋白V和TUNEL检测评估）对TRAIL/Apo2L蛋白诱导的凋亡具有抗性。TRAIL/Apo2L激活了半胱天冬酶-8和半胱天冬酶-3，但未观察到随后的凋亡事件，如聚（ADP-核糖）聚合酶裂解和DNA片段化。为了探究细胞对凋亡抗性的分子机制，分析了黑色素瘤细胞系中凋亡调节因子（凋亡蛋白酶相关因子-1、FLIP、半胱天冬酶-8、半胱天冬酶-9、半胱天冬酶-3、细胞凋亡抑制因子、Bcl-2或Bax）的表达；未观察到相关性。IFN-β在黑色素瘤细胞系中诱导TRAIL/Apo2L表达，且其与凋亡诱导相关。由于IFN-β诱导了其他与凋亡相关的基因产物，因此推测一个或多个IFN刺激基因可能使细胞对TRAIL/Apo2L敏感。在用TRAIL/Apo2L处理之前，将黑色素瘤细胞系用IFN-β处理16 - 24小时。无论它们对单独一种细胞因子的敏感性如何，超过30%的细胞在联合处理后发生凋亡。IFN-β和TRAIL/Apo2L联合诱导凋亡与半胱天冬酶-9的协同激活、线粒体膜电位降低以及聚（ADP-核糖）聚合酶的裂解相关。在敏感的WM9、A375或WM3211细胞中观察到IFN-β和TRAIL/Apo2L处理后X连锁凋亡抑制因子的裂解，但在抗性的WM35或WM164细胞中未观察到。因此，与单独使用任何一种细胞因子相比，体外IFN-β和TRAIL/Apo2L联合处理在黑色素瘤细胞系中具有更强的凋亡和抗生长作用。
