title: ERO1α-dependent endoplasmic reticulum-mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1).
abstract: Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.
authors.affiliation: Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
authors.forename: M
authors.initials: M
authors.lastname: Seervi
authors.affiliation: Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
authors.forename: P K
authors.initials: PK
authors.lastname: Sobhan
authors.affiliation: Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
authors.forename: J
authors.initials: J
authors.lastname: Joseph
authors.affiliation: Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
authors.forename: K
authors.initials: K
authors.lastname: Ann Mathew
authors.affiliation: Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
authors.forename: T R
authors.initials: TR
authors.lastname: Santhoshkumar
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D045325:HCT116 Cells; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000235:genetics; D000071717:X-Box Binding Protein 1
chemical_list: C514579:(4-benzylpiperazin-1-yl)acetic acid (3-allyl-2-hydroxybenzylidene)hydrazine; D004268:DNA-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D006835:Hydrazones; D008562:Membrane Glycoproteins; D010879:Piperazines; D034741:RNA, Small Interfering; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C405327:ERO1A protein, human; D010088:Oxidoreductases; D002118:Calcium