Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W Campbell Rd., Richardson, TX-75080, USA.
Metallomics. 2022 Sep 15;14(9). doi: 10.1093/mtomcs/mfac061.
Cis-diamminedichloroplatinum(II) (cisplatin) is a widely used metal-based chemotherapeutic drug for the treatment of cancers. However, intrinsic and acquired drug resistance limit the efficacy of cisplatin-based treatments. Increased production of intracellular thiol-rich molecules, in particular metallothioneins (MTs), which form stable coordination complexes with the electrophilic cisplatin, results in cisplatin sequestration leading to pre-target resistance. MT-1/-2 are overexpressed in cancer cells, and their expression is controlled by the metal response element (MRE)-binding transcription factor-1 (MTF-1), featuring six Cys2His2-type zinc fingers which, upon zinc metalation, recognize specific MRE sequences in the promoter region of MT genes triggering their expression. Cisplatin can efficiently react with protein metal binding sites featuring nucleophilic cysteine and/or histidine residues, including MTs and zinc fingers proteins, but the preferential reactivity towards specific targets with competing binding sites cannot be easily predicted. In this work, by in vitro competition reactions, we investigated the thermodynamic and kinetic preferential reactivity of cisplatin towards human Zn7MT-2, each of the six MTF-1 zinc fingers, and the entire human MTF-1 zinc finger domain. By spectroscopic, spectrometric, and electrophoretic mobility shift assays (EMSA), we demonstrated that cisplatin preferentially reacts with Zn7MT-2 to form Cys4-Pt(II) complexes, resulting in zinc release from MT-2. Zinc transfer from MT-2 to the MTF-1 triggers MTF-1 metalation, activation, and binding to target MRE sequences, as demonstrated by EMSA with DNA oligonucleotides. The cisplatin-dependent MT-mediated MTF-1 activation leading to apo-MT overexpression potentially establishes one of the molecular mechanisms underlying the development and potentiation of MT-mediated pre-target resistance.
顺式-二氨二氯铂(II)（顺铂）是一种广泛用于治疗癌症的金属基化疗药物。然而，内在和获得性药物耐药性限制了基于顺铂的治疗效果。细胞内富含巯基的分子（特别是金属硫蛋白（MTs））产量增加，这些分子与亲电顺铂形成稳定的配位复合物，导致顺铂被隔离，从而导致前靶向耐药。MT-1/-2 在癌细胞中过度表达，其表达受金属反应元件（MRE）结合转录因子-1（MTF-1）控制，MTF-1 具有六个 Cys2His2 型锌指，在锌金属化后，识别 MT 基因启动子区域中的特定 MRE 序列，触发其表达。顺铂可以有效地与含有亲核半胱氨酸和/或组氨酸残基的蛋白质金属结合位点反应，包括 MTs 和锌指蛋白，但对于具有竞争结合位点的特定靶标的优先反应性不能轻易预测。在这项工作中，我们通过体外竞争反应，研究了顺铂对人 Zn7MT-2、MTF-1 的每个锌指以及整个人类 MTF-1 锌指结构域的热力学和动力学优先反应性。通过光谱、光谱和电泳迁移率变动分析（EMSA），我们证明顺铂优先与 Zn7MT-2 反应形成 Cys4-Pt(II) 复合物，导致 MT-2 中的锌释放。MT-2 中的锌从 MT-2 转移到 MTF-1 触发 MTF-1 的金属化、激活和与靶 MRE 序列结合，这通过与 DNA 寡核苷酸的 EMSA 得到证明。顺铂依赖性 MT 介导的 MTF-1 激活导致 apo-MT 过表达，这可能是 MT 介导的前靶向耐药发展和增强的分子机制之一。
