Johnsen John I, Pettersen Ingvild, Ponthan Frida, Sveinbjørnsson Baldur, Flaegstad Trond, Kogner Per
Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden.
Int J Oncol. 2004 Dec;25(6):1849-57.
The majority of high-risk neuroblastomas lack the expression of caspase-8 due to gene silencing which suggest a mechanism for the selection of tumour cells that are refractory to multiple cytotoxic drugs including tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Inhibitors of DNA methyltransferases and IFN-gamma induce expression of caspase-8, and sensitise some neuroblastoma cells to TRAIL-mediated apoptosis. Here we demonstrate that a combination of cytostatic drugs with IFN-gamma and TRAIL synergistically induces neuroblastoma cell death, which may have implications for future therapy of children with neuroblastoma. Treatment of neuroblastoma cells with IFN-gamma induced caspase-8 expression in all cell lines investigated. In five of the neuroblastoma cell lines (SHEP-1, SK-N-AS, SK-N-FI, SH-SY-5Y and Kelly), IFN-gamma promoted TRAIL-mediated cleavage of caspase-8, initiating a caspase cascade involving caspase-7 and PARP followed by apoptosis. IFN-gamma-mediated facilitation of apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk and the caspase-8 specific inhibitor zIEDT-fmk, indicating an important role of caspase-8 in mediating sensitation by IFN-gamma in neuroblastoma cells. In three of the cell lines [SK-N-BE(2), SK-N-DZ and IMR-32] caspase-8 expression was induced by IFN-gamma, but the cells were still resistant to TRAIL-mediated apoptosis. The pattern of basal TRAIL receptor expression, decoy receptors, FLIP and FADD could not be correlated with resistance or sensitivity to TRAIL-induced apoptosis. Importantly, treatment of neuroblastoma cell lines with cytostatic drugs increased apoptosis in the TRAIL-sensitive cell lines whereas the resistant cell lines were susceptible to TRAIL-mediated apoptosis in the presence of the anticancer drugs. The mechanism of the increased susceptibility to apoptosis might results from drug-mediated up-regulation of the death receptors DR4 and DR5.
大多数高危神经母细胞瘤由于基因沉默而缺乏半胱天冬酶 - 8的表达，这提示了一种肿瘤细胞选择机制，这些肿瘤细胞对包括肿瘤坏死因子相关凋亡诱导配体（TRAIL）在内的多种细胞毒性药物具有抗性。DNA甲基转移酶抑制剂和γ干扰素可诱导半胱天冬酶 - 8的表达，并使一些神经母细胞瘤细胞对TRAIL介导的凋亡敏感。在此我们证明，细胞生长抑制剂与γ干扰素和TRAIL联合使用可协同诱导神经母细胞瘤细胞死亡，这可能对未来神经母细胞瘤患儿的治疗具有重要意义。用γ干扰素处理神经母细胞瘤细胞可在所有研究的细胞系中诱导半胱天冬酶 - 8的表达。在五个神经母细胞瘤细胞系（SHEP - 1、SK - N - AS、SK - N - FI、SH - SY - 5Y和Kelly）中，γ干扰素促进了TRAIL介导的半胱天冬酶 - 8的裂解，启动了一个涉及半胱天冬酶 - 7和聚（ADP - 核糖）聚合酶（PARP）的半胱天冬酶级联反应，随后导致细胞凋亡。γ干扰素介导的凋亡促进作用被泛半胱天冬酶抑制剂zVAD - fmk和半胱天冬酶 - 8特异性抑制剂zIEDT - fmk抑制，这表明半胱天冬酶 - 8在介导γ干扰素使神经母细胞瘤细胞致敏中起重要作用。在三个细胞系[SK - N - BE(2)、SK - N - DZ和IMR - 32]中，γ干扰素诱导了半胱天冬酶 - 8的表达，但这些细胞对TRAIL介导的凋亡仍具有抗性。基础TRAIL受体表达、诱饵受体、FLIP和FADD的模式与对TRAIL诱导凋亡的抗性或敏感性无关。重要的是，用细胞生长抑制剂处理神经母细胞瘤细胞系可增加TRAIL敏感细胞系中的细胞凋亡，而在抗癌药物存在的情况下，抗性细胞系对TRAIL介导的凋亡敏感。凋亡易感性增加的机制可能是药物介导的死亡受体DR4和DR5的上调。
