Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes

Abstract

Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.

The Quinone Methide Aurin Is a Heat Shock Response Inducer That Causes Proteotoxic Stress and Noxa-dependent Apoptosis in Malignant Melanoma Cells

“…Interestingly, Noxa-induced apoptosis downstream of ER stress causing PERK/phospho-eIF2␣/ATF4-dependent PMAIP1 upregulation has been substantiated earlier in celastrol-exposed hepatocellular carcinoma cells (65). In this context, it will be interesting to examine how Hsp90-directed antagonistic activity synergizes with proteasomal impairment, UPR signaling (resulting in up-regulated expression of the pro-apoptotic transcription factor CHOP), and oxidative stress, all of which are potentially critical factors contributing to aurin-induced Noxadependent melanoma cell apoptosis (11,39,65,66,68).…”

“…5B) (39). Inhibitory phosphorylation of eIF2␣ (eukaryotic translation initiation factor) through activation of PERK (double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase) is an established hallmark of cytotoxic ER stress known to occur upstream of DDIT3/CHOP up-regulation.…”

“…7F). Due to the established causative involvement of PMAIP1 up-regulation in cancer cell mitochondrial apoptosis that occurs in response to various genotoxic, proteotoxic, and oxidative stress stimuli (39,41,44,65,66,68), a genetic target modulation approach employing siRNA-based antagonism of PMAIP1 expression was used to further substantiate the causative involvement of Noxa in aurin-induced melanoma cell apoptosis. First, immunoblot analysis confirmed that aurin-induced up-regulation of Noxa protein levels (detectable within 12 h of exposure time) was fully suppressed in siPMAIP1-cotreated A375 melanoma cells (Fig.…”

“…Specifically, Hsp90 serves as an essential factor stabilizing oncogenic V600E BRAF in malignant melanoma cells, and its inhibition has emerged as a promising strategy for antimelanoma intervention (34 -37). Therefore, strategies that aim at increasing proteotoxic stress through pharmacological modulation of proteasomal, autophagic-lysosomal, or heat shock response functions are now pursued for experimental and investigational chemotherapeutic intervention targeting malignant melanoma (27)(28)(29)(30)(31)(32)(33)(37)(38)(39)(40).…”

Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions

“…Upon thermal stress, HaCaT keratinocytes responded with induction of both HSPA1A and A6, although the latter to many more fold at the mRNA level. Given its detection in nonstressed conditions (Gomez-Sucerquia et al 2012 and our report), its capacity for significant fold induction (Chow et al 2010;Qiao et al 2012), and its likely contribution to post-stress cell survival (Noonan et al 2007b), we sought to better define the control of its basal and stress-inducible expression in keratinocytes, a cell type with wide dependence on chaperone function and likely to encounter diverse stress conditions. Nevertheless, even with this better-defined control of its expression, any HSPA6 contribution to chaperoning or stress recovery would be restricted to species carrying the gene such as human, goat, and swine (Banerjee et al 2014;Dezeure et al 1993, andParsian et al 2000).…”