Abstract
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.