ERO1α-dependent endoplasmic reticulum-mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1)

Cell Death Dis. 2013 Dec 19;4(12):e968. doi: 10.1038/cddis.2013.502.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport / drug effects
  • Calcium / metabolism*
  • Cell Line, Tumor
  • DNA-Binding Proteins / genetics
  • Endoplasmic Reticulum / drug effects*
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum Stress / drug effects*
  • HCT116 Cells
  • HeLa Cells
  • Heat-Shock Proteins / genetics
  • Humans
  • Hydrazones / pharmacology*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Microscopy, Fluorescence
  • Mitochondria / drug effects*
  • Mitochondria / metabolism*
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Piperazines / pharmacology*
  • RNA Splicing / genetics
  • RNA Splicing / physiology
  • RNA, Small Interfering / genetics
  • Regulatory Factor X Transcription Factors
  • Transcription Factors / genetics
  • X-Box Binding Protein 1

Substances

  • (4-benzylpiperazin-1-yl)acetic acid (3-allyl-2-hydroxybenzylidene)hydrazine
  • DNA-Binding Proteins
  • Heat-Shock Proteins
  • Hydrazones
  • Membrane Glycoproteins
  • Piperazines
  • RNA, Small Interfering
  • Regulatory Factor X Transcription Factors
  • Transcription Factors
  • X-Box Binding Protein 1
  • XBP1 protein, human
  • ERO1A protein, human
  • Oxidoreductases
  • Calcium
  • molecular chaperone GRP78