Didox, a ribonucleotide reductase inhibitor with iron chelator properties, counteracts the in vitro and in vivo growth of rhabdomyosarcoma cells

Abstract

Ribonucleotide reductase (RR) is the rate-limiting enzyme for NTPs conversion into dNTPs, playing a central role in genome replication and maintenance. It is composed by two catalytic (RRM1) and two regulatory (alternatively RRM2 and p53R2) subunits, of which RRM2′s functionality depends on a diferric center in the active site and is one of the most expressed genes in many tumors, among which Rhabdomyosarcoma (RMS), a rare and aggressive pediatric tumor. Didox (3,4-dihydroxy-benzohydroxamic acid) is a highly effective RRM2 inhibitor with iron chelating properties which shows fewer in vivo side effects than classical RR inhibitors. In the present work, we analyzed the impact of didox on RMS cells. Our data clearly showed that didox effectively reduces cell viability, clonogenic capability and motility of both RD and RH30 cells (representative of embryonal named ERMS and alveolar subtype named ARMS), with higher potency in ARMS cells. Interestingly, didox is effective in inhibiting the cell viability of RMS radioresistant. Mechanistically, didox modulates the main iron-related proteins (TfR1 and H-ferritin), confirming its iron chelating properties; it induces mitochondrial ROS formation and caused an increase in double positive annexin-V/PI cells, confirming apoptosis as mechanism of cell death. Moreover, didox also potently reduces in vivo tumor proliferation of RH30 cells, without significant side effects on animals. Finally, the combination of sublethal doses of Actinomycin-D and didox is effective in decreasing cell viability and clonogenicity of RMS cells. Therefore, our data suggests the effectiveness of the RR inhibitor didox on both in vitro and in vivo RMS proliferation.