Cisplatin (cis-[Pt(NH3)2Cl2]) is one of the most successful and widely utilized Pt(II)-based chemotherapeutic drugs. Cisplatin demonstrated efficacy in the treatment of various human cancers, including testicular, ovarian, bladder, and lung tumors. Cisplatin's antineoplastic activity is caused by the formation of Pt(II)-DNA adducts that introduce lesions and distortion in the DNA structure that cannot be repaired, eventually leading to cancer cell apoptosis. Despite cisplatin’s therapeutic benefits, intrinsic and acquired resistance limit its efficacy. In pre-target resistance, molecular processes upstream of Pt(II) binding to DNA, prevent its reaction with its pharmacological target. One of predominant mechanism in pre-target resistance is cisplatin sequestration via covalent binding by intracellular thiol-rich molecules like metallothioneins (MTs). High MTs expression levels correlate with cisplatin drug resistance in preclinical and clinical studies, but the molecular mechanism of MT-mediated resistance remains still elusive. The transcriptional induction of the MT gene is mediated by Metal-Responsive Transcription Factor 1 (MTF-1) which bind with DNA motifs present in MT gene promoters. By biochemical and spectroscopic analysis we investigated the relative reactivity of cisplatin towards Zn(II)7MT-2, each of MTF-1 Cys2/His2 zinc fingers, and the entire MTF-1 hexa-zinc finger domain. By performing cis-[Pt(NH3)2Cl2] binding competition experiments between MT-2 and individual and apo-/holo- zinc fingers, we demonstrate that cisplatin preferentially binds to Zn7MT-2 over MTF-1. Preferential Pt(II) binding to MT cysteine thiolates result in Zn(II) displacement, release and subsequent Zn(II) binding to MTF-1 zinc fingers, thereby potentially activating MTF-1. Analysis of reaction kinetics reveal indeed that the rate of cisplatin binding to Zn7MT-2 is faster than its binding to each of the individual MTF-1 zinc fingers. Initial experiments confirm that similar processes occur with the entire MTF-1 hexa- zinc finger domain. The work substantiates that zinc release upon cisplatin binding to MTs leads to MTF-1 activation, which in turn induces apoMT expression. The consequent increased apoMT levels would lead to cellular cisplatin sequestration and promote MT-mediated drug inactivation. Overall, the work provides a molecular mechanism for metallothionein-mediated cisplatin pre-target resistance.

Molecular mechanism of Pt(II)-drug resistance via metallothionein-dependent MTF-1 activation

ACS Fall 2021

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