Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Korea.
Cell Prolif. 2019 May;52(3):e12577. doi: 10.1111/cpr.12577. Epub 2019 Feb 6.
This study aims to explore the roles of N-myc and caspase-8 in TRAIL-resistant IMR-32 cells which exhibit MYCN oncogene amplification and lack caspase-8 expression.
We established N-myc-downregulated IMR-32 cells using shRNA lentiviral particles targeting N-myc and examined the effect the N-myc inhibition on TRAIL susceptibility in human neuroblastoma IMR-32 cells expressing caspase-8.
Cisplatin treatment in IMR-32 cells increased the expression of death receptor 5 (DR5; TRAIL-R2), but not other receptors, via downregulation of NF-κB activity. However, the cisplatin-mediated increase in DR5 failed to induce cell death following TRAIL treatment. Furthermore, interferon (IFN)-γ pretreatment increased caspase-8 expression in IMR-32 cells, but cisplatin failed to trigger TRAIL cytotoxicity. We downregulated N-myc expression in IMR-32 cells using N-myc-targeting shRNA. These cells showed decreased growth rate and Bcl-2 expression accompanied by a mild collapse in the mitochondrial membrane potential as compared with those treated with scrambled shRNA. TRAIL treatment in N-myc-negative cells expressing caspase-8 following IFN-γ treatment significantly triggered apoptotic cell death. Concurrent treatment with cisplatin enhanced TRAIL-mediated cytotoxicity, which was abrogated by an additional pretreatment with DR5:Fc chimera protein.
N-myc and caspase-8 expressions are involved in TRAIL susceptibility in IMR-32 cells, and the combination of treatment with cisplatin and TRAIL may serve as a promising strategy for the development of therapeutics against neuroblastoma that is controlled by N-myc and caspase-8 expression.
本研究旨在探讨 N-myc 和 caspase-8 在 TRAIL 耐药的 IMR-32 细胞中的作用，这些细胞表现出 MYCN 癌基因扩增且缺乏 caspase-8 的表达。
我们使用靶向 N-myc 的 shRNA 慢病毒颗粒建立了 N-myc 下调的 IMR-32 细胞，并检测了 N-myc 抑制对表达 caspase-8 的人神经母细胞瘤 IMR-32 细胞对 TRAIL 敏感性的影响。
顺铂处理 IMR-32 细胞通过下调 NF-κB 活性增加了死亡受体 5（DR5；TRAIL-R2）的表达，但不增加其他受体的表达。然而，顺铂介导的 DR5 增加未能诱导 TRAIL 处理后的细胞死亡。此外，干扰素（IFN）-γ预处理增加了 IMR-32 细胞中 caspase-8 的表达，但顺铂未能触发 TRAIL 细胞毒性。我们使用靶向 N-myc 的 shRNA 下调了 IMR-32 细胞中的 N-myc 表达。与用乱序 shRNA 处理的细胞相比，这些细胞的生长速度和 Bcl-2 表达降低，同时线粒体膜电位轻度崩溃。在用 IFN-γ 处理后，N-myc 阴性细胞中 caspase-8 的表达下调，TRAIL 处理显著触发了凋亡性细胞死亡。顺铂与 TRAIL 联合治疗可增强 TRAIL 介导的细胞毒性，而 DR5：Fc 嵌合体蛋白的额外预处理则可阻断这种作用。
N-myc 和 caspase-8 的表达参与了 IMR-32 细胞对 TRAIL 的敏感性，顺铂和 TRAIL 的联合治疗可能为开发针对 N-myc 和 caspase-8 表达调控的神经母细胞瘤治疗方法提供一种有前途的策略。
