Park Young-Shin, Kwon Yeo-Jung, Chun Young-Jin
College of Pharmacy, Chung-Ang University, Seoul, Korea.
Toxicol Res. 2017 Jul;33(3):211-218. doi: 10.5487/TR.2017.33.3.211. Epub 2017 Jul 15.
Cytochrome P450 1B1 (CYP1B1) acts as a hydroxylase for estrogen and activates potential carcinogens. Moreover, its expression in tumor tissues is much higher than that in normal tissues. Despite this association between CYP1B1 and cancer, the detailed molecular mechanism of CYP1B1 on cancer progression in HeLa cells remains unknown. Previous reports indicated that the mRNA expression level of , an E3 ligase for ISGylation, is promoted by CYP1B1 suppression using specific small interfering RNA, and that ISGylation may be involved in ubiquitination related to β-catenin degradation. With this background, we investigated the relationships among CYP1B1, Herc5, and β-catenin. RT-PCR and western blot analyses showed that CYP1B1 overexpression induced and CYP1B1 inhibition reduced, respectively, the expression of Wnt/β-catenin signaling target genes including β-catenin and cyclin D1. Moreover, HeLa cells were treated with the CYP1B1 inducer 7,12-dimethylbenz[α]anthracene (DMBA) or the CYP1B1 specific inhibitor, tetramethoxystilbene (TMS) and consequently DMBA increased and TMS decreased β-catenin and cyclin D1 expression, respectively. To determine the correlation between CYP1B1 expression and ISGylation, the expression of ISG15, a ubiquitin-like protein, was detected following CYP1B1 regulation, which revealed that CYP1B1 may inhibit ISGylation through suppression of ISG15 expression. In addition, the mRNA and protein expression levels of Herc5 were strongly suppressed by CYP1B1. Finally, an immunoprecipitation assay revealed a direct physical interaction between Herc5 and β-catenin in HeLa cells. In conclusion, these data suggest that CYP1B1 may activate Wnt/β-catenin signaling through stabilization of β-catenin protein from Herc5-mediated ISGylation for proteosomal degradation.
细胞色素P450 1B1（CYP1B1）作为雌激素的羟化酶，可激活潜在致癌物。此外，其在肿瘤组织中的表达远高于正常组织。尽管CYP1B1与癌症存在这种关联，但CYP1B1在HeLa细胞中促进癌症进展的详细分子机制仍不清楚。先前的报道表明，使用特异性小干扰RNA抑制CYP1B1可促进ISGylation的E3连接酶的mRNA表达水平，并且ISGylation可能参与与β-连环蛋白降解相关的泛素化过程。在此背景下，我们研究了CYP1B1、Herc5和β-连环蛋白之间的关系。逆转录聚合酶链反应（RT-PCR）和蛋白质印迹分析表明，CYP1B1的过表达分别诱导，而CYP1B1的抑制分别降低了包括β-连环蛋白和细胞周期蛋白D1在内的Wnt/β-连环蛋白信号靶基因的表达。此外，用CYP1B1诱导剂7,12-二甲基苯并[α]蒽（DMBA）或CYP1B1特异性抑制剂四甲氧基芪（TMS）处理HeLa细胞，结果DMBA分别增加和TMS降低了β-连环蛋白和细胞周期蛋白D1的表达。为了确定CYP1B1表达与ISGylation之间的相关性，在CYP1B1调节后检测了泛素样蛋白ISG15的表达，结果显示CYP1B1可能通过抑制ISG15的表达来抑制ISGylation。此外，CYP1B1强烈抑制Herc5的mRNA和蛋白质表达水平。最后，免疫沉淀试验揭示了HeLa细胞中Herc5与β-连环蛋白之间存在直接的物理相互作用。总之，这些数据表明，CYP1B1可能通过稳定β-连环蛋白蛋白，使其免受Herc介导的用于蛋白酶体降解的ISGylation作用，从而激活Wnt/β-连环蛋白信号通路。
