title: Mechanism of the induction of endoplasmic reticulum stress by the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT): Activation of PERK/eIF2α, IRE1α, ATF6 and calmodulin kinase.
abstract: The endoplasmic reticulum (ER) plays a major role in the synthesis, maturation and folding of proteins and is a critical calcium (Ca(2+)) reservoir. Cellular stresses lead to an overwhelming accumulation of misfolded proteins in the ER, leading to ER stress and the activation of the unfolded protein response (UPR). In the stressful tumor microenvironment, the UPR maintains ER homeostasis and enables tumor survival. Thus, a novel strategy for cancer therapeutics is to overcome chronically activated ER stress by triggering pro-apoptotic pathways of the UPR. Considering this, the mechanisms by which the novel anti-cancer agent, Dp44mT, can target the ER stress response pathways were investigated in multiple cell-types. Our results demonstrate that the cytotoxic chelator, Dp44mT, which forms redox-active metal complexes, significantly: (1) increased ER stress-associated pro-apoptotic signaling molecules (i.e., p-eIF2α, ATF4, CHOP); (2) increased IRE1α phosphorylation (p-IRE1α) and XBP1 mRNA splicing; (3) reduced expression of ER stress-associated cell survival signaling molecules (e.g., XBP1s and p58(IPK)); (4) increased cleavage of the transcription factor, ATF6, which enhances expression of its downstream targets (i.e., CHOP and BiP); and (5) increased phosphorylation of CaMKII that induces apoptosis. In contrast to Dp44mT, the iron chelator, DFO, which forms redox-inactive iron complexes, did not affect BiP, p-IRE1α, XBP1 or p58(IPK) levels. This study highlights the ability of a novel cancer therapeutic (i.e., Dp44mT) to target the pro-apoptotic functions of the UPR via cellular metal sequestration and redox stress. Assessment of ER stress-mediated apoptosis is fundamental to the understanding of the pharmacology of chelation for cancer treatment.
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Angelica M
authors.initials: AM
authors.lastname: Merlot
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia; Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Nurul H
authors.initials: NH
authors.lastname: Shafie
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Yu
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Vera
authors.initials: V
authors.lastname: Richardson
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Patric J
authors.initials: PJ
authors.lastname: Jansson
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Sumit
authors.initials: S
authors.lastname: Sahni
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Darius J R
authors.initials: DJR
authors.lastname: Lane
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Zaklina
authors.initials: Z
authors.lastname: Kovacevic
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: danutak@med.usyd.edu.au.
authors.forename: Danuta S
authors.initials: DS
authors.lastname: Kalinowski
authors.affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: d.richardson@med.usyd.edu.au.
authors.forename: Des R
authors.initials: DR
authors.lastname: Richardson
mesh_terms: D051702:Activating Transcription Factor 6 / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects* / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007502:Iron Chelating Agents / Q000138:chemical synthesis / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013882:Thiosemicarbazones / Q000138:chemical synthesis / Q000494:pharmacology*; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D000071717:X-Box Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics* / Q000378:metabolism
chemical_list: C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D000970:Antineoplastic Agents; C497249:DDIT3 protein, human; C494651:DNAJC3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007502:Iron Chelating Agents; D013882:Thiosemicarbazones; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C539263:di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; D051742:Transcription Factor CHOP; C460640:EIF2AK3 protein, human; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004722:Endoribonucleases; D003676:Deferoxamine
keywords: Anti-cancer drug; Dp44mT; Endoplasmic reticulum; Endoplasmic reticulum stress; Unfolded protein response