Wang Ying-Jan, Jeng Jiiang-Huei, Chen Rong-Jane, Tseng How, Chen Li-Ching, Liang Yu-Chih, Lin Chien-Huang, Chen Chien-Ho, Chu Jan-Show, Ho Wei-Lu, Ho Yuan-Soon
Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan.
Mol Carcinog. 2002 Aug;34(4):199-210. doi: 10.1002/mc.10066.
Our previous studies demonstrated that the oral antifungal agent ketoconazole (KT) induces apoptosis and G0/G1 phase cell cycle arrest in human cancer cell lines. In this study, we first demonstrated that KT (1 microM) potentiated the apoptotic effects of nocodazole (ND, 1 nM) in COLO 205 cancer cells. We further demonstrated the therapeutic efficacy of a combined treatment of KT (50 mg/kg/three times per week) and ND (5 mg/kg/three times per week) in vivo by treating athymic mice bearing COLO 205 tumor xenografts. The antitumor effects of ND were significantly potentiated by KT in mice after 6 wk of treatment. No gross signs of toxicity were observed in mice receiving these treatment regimens. The apoptotic cells were detected in a microscopic view of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and by observation of DNA fragmentation in KT + ND-treated tumor tissues. The levels of cell cycle regulatory proteins were determined by Western blot analysis. Treatment with KT inhibits tumor growth through elevation of p53, p21/CIP1, and p27/KIP1 as well as inhibition of cyclin D3 and cyclin-dependent kinase 4 protein expression. Immunohistochemical staining analysis showed that p53, p21/CIP1, and p27/KIP1 immunoreactivity were induced in the tumor tissues. To clarify the roles of the p21/CIP1 and p27/KIP1 protein expression involved in G(0)/G(1) arrest and/or apoptosis induced by a combined treatment with KT and ND, antisense oligodeoxynucleotides (ODNs) specific to p21/CIP1 and p27/KIP1 were used. Our results demonstrated that apoptotic phenomena, including BAX induction and cytochrome C released from mitochondria induced by KT + ND, were significantly attenuated by pretreatment the cells with the p27/KIP1-specific antisense ODNs. These results indicate that p27/KIP1 protein does indeed play a critical role in the KT + ND-induced apoptosis. Our study revealed the molecular mechanism of KT + ND in regression of the tumor growth. The apoptotic effects of KT in a great variety of cancer cells make it a very attractive agent for cancer chemotherapy.
我们之前的研究表明，口服抗真菌药酮康唑（KT）可诱导人癌细胞系发生凋亡并使细胞周期停滞于G0/G1期。在本研究中，我们首先证明了KT（1微摩尔）可增强诺考达唑（ND，1纳摩尔）对COLO 205癌细胞的凋亡作用。我们通过对携带COLO 205肿瘤异种移植的无胸腺小鼠进行治疗，进一步证明了KT（50毫克/千克/每周三次）与ND（5毫克/千克/每周三次）联合治疗在体内的疗效。治疗6周后，KT显著增强了ND对小鼠的抗肿瘤作用。接受这些治疗方案的小鼠未观察到明显的毒性迹象。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色的显微镜观察以及对KT + ND处理的肿瘤组织中DNA片段化的观察，检测到了凋亡细胞。通过蛋白质印迹分析确定细胞周期调节蛋白的水平。KT治疗通过提高p53、p21/CIP1和p27/KIP1水平以及抑制细胞周期蛋白D3和细胞周期蛋白依赖性激酶4蛋白表达来抑制肿瘤生长。免疫组织化学染色分析表明，肿瘤组织中诱导了p53、p21/CIP1和p27/KIP1免疫反应性。为了阐明p21/CIP1和p27/KIP1蛋白表达在KT和ND联合治疗诱导的G(0)/G(1)期停滞和/或凋亡中的作用，使用了针对p21/CIP1和p27/KIP1的反义寡脱氧核苷酸（ODN）。我们的结果表明用p27/KIP1特异性反义ODN预处理细胞后，KT + ND诱导产生的包括BAX诱导和线粒体释放细胞色素C在内的凋亡现象显著减弱。这些结果表明p27/KIP1蛋白在KT + ND诱导的凋亡中确实起着关键作用。我们的研究揭示了KT + ND使肿瘤生长消退的分子机制。KT对多种癌细胞的凋亡作用使其成为一种极具吸引力的癌症化疗药物。
