Huerta-Yepez Sara, Vega Mario, Jazirehi Ali, Garban Hermes, Hongo Fumiya, Cheng Genhong, Bonavida Benjamin
Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Oncogene. 2004 Jun 24;23(29):4993-5003. doi: 10.1038/sj.onc.1207655.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues and this prompted its potential therapeutic application in cancer. However, not all cancers are sensitive to TRAIL-mediated apoptosis and, therefore, TRAIL-resistant cancer cells must be sensitized first to become sensitive to TRAIL. Treatment of prostate cancer (CaP) cell lines (DU145, PC-3, CL-1, and LNCaP) with nitric oxide donors (e.g. (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate)) sensitized CaP cells to TRAIL-induced apoptosis and synergy was achieved. The mechanism by which DETANONOate mediated the sensitization was examined. DETANONOate inhibited the constitutive NF-kappa B activity as assessed by EMSA. Also, p50 was S-nitrosylated by DETANONOate resulting in inhibition of NF-kappa B. Inhibition of NF-kappa B activity by the chemical inhibitor Bay 11-7085, like DETANONOate, sensitized CaP to TRAIL apoptosis. In addition, DETANONOate downregulated the expression of Bcl-2 related gene (Bcl-(xL)) which is under the transcriptional regulation of NF-kappa B. The regulation of NF-kappa B and Bcl-(xL) by DETANONOate was corroborated by the use of Bcl-(xL) and Bcl-x kappa B reporter systems. DETANONOate inhibited luciferase activity in the wild type and had no effect on the mutant cells. Inhibition of NF-kappa B resulted in downregulation of Bcl-(xL) expression and sensitized CaP to TRAIL-induced apoptosis. The role of Bcl-(xL) in the regulation of TRAIL apoptosis was corroborated by inhibiting Bcl-(xL) function by the chemical inhibitor 2-methoxyantimycin A(3) and this resulted in sensitization of the cells to TRAIL apoptosis. Signaling by DETANONOate and TRAIL for apoptosis was examined. DETANONOate altered the mitochondria by inducing membrane depolarization and releasing modest amounts of cytochrome c and Smac/DIABLO in the absence of downstream activation of caspases 9 and 3. However, the combination of DETANONOate and TRAIL resulted in activation of the mitochondrial pathway and activation of caspases 9 and 3, and induction of apoptosis. These findings demonstrate that DETANONOate-mediated sensitization of CaP to TRAIL-induced apoptosis is via inhibition of constitutive NF-kappa B activity and Bcl-(xL) expression.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）已被证明在诱导癌细胞凋亡方面具有选择性，对正常组织的毒性最小，这促使其在癌症治疗中具有潜在的应用价值。然而，并非所有癌症都对TRAIL介导的凋亡敏感，因此，必须先使TRAIL耐药癌细胞致敏，使其对TRAIL敏感。用一氧化氮供体（如（Z）-1-[2-（2-氨基乙基）-N-（2-氨乙基）氨基]重氮-1,2-二醇盐（DETANONOate））处理前列腺癌细胞系（DU145、PC-3、CL-1和LNCaP）可使前列腺癌细胞对TRAIL诱导的凋亡致敏，并实现协同作用。研究了DETANONOate介导致敏作用的机制。通过电泳迁移率变动分析（EMSA）评估，DETANONOate抑制了组成型核因子κB（NF-κB）活性。此外，DETANONOate使p50发生S-亚硝基化，导致NF-κB受到抑制。化学抑制剂Bay 11-7085对NF-κB活性的抑制作用与DETANONOate一样，使前列腺癌细胞对TRAIL凋亡致敏。此外，DETANONOate下调了受NF-κB转录调控的Bcl-2相关基因（Bcl-(xL)）的表达。使用Bcl-(xL)和Bcl-x κB报告系统证实了DETANONOate对NF-κB和Bcl-(xL)的调控作用。DETANONOate抑制野生型中的荧光素酶活性，对突变细胞无影响。NF-κB的抑制导致Bcl-(xL)表达下调，并使前列腺癌细胞对TRAIL诱导的凋亡致敏。通过化学抑制剂2-甲氧基抗霉素A(3)抑制Bcl-(xL)功能，证实了Bcl-(xL)在TRAIL凋亡调控中的作用，这导致细胞对TRAIL凋亡致敏。研究了DETANONOate和TRAIL诱导凋亡的信号传导。DETANONOate通过诱导膜去极化改变线粒体，并在半胱天冬酶9和3无下游激活的情况下释放适量的细胞色素c和Smac/DIABLO。然而，DETANONOate和TRAIL的联合作用导致线粒体途径激活以及半胱天冬酶9和3激活，并诱导凋亡。这些发现表明，DETANONOate介导的前列腺癌细胞对TRAIL诱导凋亡的致敏作用是通过抑制组成型NF-κB活性和Bcl-(xL)表达实现的。
