Unveiling molecular mechanisms of action of anticancer arsenic- and gold-based drugs by metallomic and metalloproteomic approaches

Abstract

The introduction of metal/metalloid compounds to medicine has opened a new horizon for cancer therapy. Up to now, various metal-based compounds have been designed and utilized as anticancer agents. However, their modes of action and molecular targets still remain unclear. Further understanding the mechanisms underlying their antiproliferative activities can in-turn extend therapeutic potentials of anticancer metallodrugs and provide new insights into the exploitation of the mechanisms of other drugs. S-dimethylarsino-glutathione (ZIO-101, darinaparsin®) exhibits broader antitumor activity and less toxicity than arsenic trioxide (ATO, Trisenox®), a clinically used drug for the treatment of acute promyelocytic leukemia (APL). However, its molecular mechanisms

remain largely concealed. By utilizing on-line coupling of column-type gel electrophoresis with inductively coupled plasma mass spectrometry (GE-ICP-MS), histone H3.3 was identified for the first time as a ZIO-101 but not ATO binding protein in the nuclei of leukemia cells. Such binding was subsequently validated both in vitro and in cellulo by various biophysical techniques. Mechanistic studies reveal that the binding of ZIO-101 to histone H3.3 induces the downregulation of HDAC1 and consequently dramatic upregulation of CDKN1A, resulting in TRAIL-induced apoptosis. This study proposes an unprecedented mechanism underlying the anticancer activity of ZIO-101, providing significant insights into the distinct molecular mechanisms and therapeutic effects of organic and inorganic arsenic drugs. By integrating dynamic transcriptomic analysis and biochemical assays, ZIO-101 is delineated to exert antiproliferative effects against leukemia cells via activating ferroptosis pathway, a newly discovered iron-dependent programmed cell death, at the early stage as evidenced by abnormally elevated intracellular iron contents and lipid peroxidation. Silencing heme oxygenase 1 (HMOX1), an important iron homeostasis related gene, can effectively attenuate ZIO-101 induced ferroptosis, with iron accumulation and lipid peroxidation being significantly alleviated. Significantly, ZIO-101 and kinase inhibitors can synergistically kill leukemia cells, with combination index of less than 1.0 under all the tested drug concentrations. These findings on ferroptosis-mediated anti-leukemia activity of ZIO-101 based on the dynamic and temporal transcriptomic analysis provide promising approaches to combat drug-resistant leukemia by combination of ZIO-101 with kinase inhibitors, and the methodology might be further exploited for uncovering the modes of action of other drugs. Due to its superiority of matching metals/metalloids to their bound proteins simultaneously, GE-ICP-MS was further utilized to identify the putative protein targets of Auranofin, a gold-based anti-rheumatic drug which is now being investigated for cancer treatment. Four gold-binding proteins were identified in A2780 cells treated with Auranofin. Particularly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important enzyme involved in glycose metabolism, was preliminarily validated to be a potential target of Auranofin by various biochemical techniques. Auranofin is discovered to dose-dependently inhibit the enzymatic activity of GAPDH and suppress the glycolytic metabolism in A2780 cells. Silencing GAPDH can effectively elevate the viability of Auranofin-treated A2780 cells, indicating that reducing GAPDH expression level confers resistance to Auranofin in A2780 cells.

The study in this thesis provides an effective and general approach to explore the molecular mechanisms of action underlying the anticancer effects of metallodrugs in various cells and further facilitates the development of novel therapeutic strategies.