Abstract
Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.
Highlights
Pharmacological induction of proteotoxic stress is a promising strategy for anti-melanoma intervention
Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention
Inspired by structural features displayed by established small molecule heat shock response inducers including the benzoquinone ansamycin antibiotic geldanamycin (Gel) and the quinone-methide celastrol (Cel; Fig. 1A) (54 –57, 58 – 63), we examined the activity of synthetic triphenylmethane derivatives with phenolic and/or quinone-methide functional groups including aurin-tricarboxylic acid (ATA; 5,5Ј-(3-carboxy-4-oxocyclohexa-2,5-dien-1-ylidenemethylene)bis(2-hydroxybenzoic acid), CAS #4431-00-9; Fig. 1A), an experimental therapeutic tested previously for suppression of thrombocyte aggregation and cardioprotection [51,52,53]
Summary
Pharmacological induction of proteotoxic stress is a promising strategy for anti-melanoma intervention. We describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Strategies that aim at increasing proteotoxic stress through pharmacological modulation of proteasomal, autophagic-lysosomal, or heat shock response functions are pursued for experimental and investigational chemotherapeutic intervention targeting malignant melanoma [27,28,29,30,31,32,33, 37,38,39,40]. Further analysis revealed that aurin displays inhibitory activity targeting Hsp90␣, causing early heat shock factor (HSF) transcriptional activation, up-regulation of heat shock response gene expression, and unfolded protein response (UPR) signaling followed by induction of Noxa-dependent apoptotic cell death
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