Human Tumors Immunobiology Unit, Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Medical Oncology Unit 3, Department of Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori and University of Milan, Italy.
Clin Cancer Res. 2012 Jun 15;18(12):3316-27. doi: 10.1158/1078-0432.CCR-11-2232. Epub 2012 May 2.
To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression.
Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal-regulated kinase (MEK)-, BRAF(V600E)-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents.
Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAF(V600E)-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAF(V600E)-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAF(V600E)-specific inhibitors.
Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways.
评估 Apollon 在黑色素瘤抵抗内在和外在凋亡途径中的作用，并确定降低其表达的策略。
评估 Apollon 在体外和体内黑色素瘤细胞中的表达。通过 Western blot 和/或流式细胞术评估 Apollon 调节和黑色素瘤凋亡，以响应细胞毒性药物、丝裂原活化蛋白/细胞外信号调节激酶（MEK）-、BRAF(V600E)-和 mTOR 特异性抑制剂、TRAIL 和抗 HLA 类 II 单克隆抗体（mAb）。使用线粒体去极化、半胱氨酸蛋白酶激活、凋亡测定和基因表达谱来测试 Apollon 沉默通过 siRNA 对黑色素瘤对抗肿瘤药物的反应的影响。
Apollon 在黑色素瘤细胞中，无论是在体外还是在体内，都持续表达，并且表达水平高于良性黑素细胞病变。黑色素瘤凋亡与 Apollon 蛋白在响应细胞毒性药物、MEK 或 BRAF(V600E)-特异性抑制剂时的下调显著相关。MEK 和 mTOR 抑制剂联合治疗以及通过特异性 mAb 连接 HLA 类 II，可促进 Apollon 下调并增强黑色素瘤凋亡。抗肿瘤药物诱导的 Apollon 下调不依赖于半胱氨酸蛋白酶，但依赖于蛋白酶体。通过 siRNA 敲低 Apollon 可触发凋亡并/或显著增强黑色素瘤细胞对细胞毒性药物、MEK 和 BRAF(V600E)-特异性抑制剂以及可溶性或膜结合 TRAIL 的敏感性。Apollon 沉默可促进不同抗肿瘤药物和改变 MEK 或 BRAF(V600E)-特异性抑制剂调节的基因谱的线粒体去极化和半胱氨酸蛋白酶-2、半胱氨酸蛋白酶-8、半胱氨酸蛋白酶-9 和半胱氨酸蛋白酶-3 的激活。
针对 Apollon 可能会显著改善黑色素瘤细胞对触发内在或外在凋亡途径的抗肿瘤药物的细胞死亡。
