title: 4-O-carboxymethyl ascochlorin causes ER stress and induced autophagy in human hepatocellular carcinoma cells.
abstract: The synthetic derivative of ascochlorin, 4-O-carboxymethyl ascochlorin (AS-6) is an agonist of the nuclear hormone receptor PPARγ and has been shown to induce differentiation in mouse pre-adipocytes and to ameliorate type II diabetes in a murine model. AS-6 was cytotoxic when added at micromolar concentrations to cultures of three different human cancer cell lines. We used gel electrophoresis and mass spectrometry to identify proteins with altered expression in human hepatocarcinoma cells (HepG2) cells after 12 h in the presence of AS-6 and found 58 proteins that were differentially expressed. Many of the proteins showing increased expression in cells treated with AS-6 are involved in protein quality control, including glucose-regulated protein 78 (GRP78/BiP), a regulator of ER stress responses, and the transcriptional regulator CHOP, which mediates ER stress-induced apoptosis. Cells treated with AS-6 undergo an autophagic response accompanied by increased expression of beclin1, ATG5, and LC3-II and autophagosome formation marked by the appearance of large vesicles containing LC3-II. Grp78 induction was inhibited when the PPARγ antagonist, GW9662, was added together with AS-6, and autophagy and cell death were partially blocked. 3-methyl-adenine (3-MA), an inhibitor of phosphatidyl inositol 3-kinase (PI3-kinase) prevented induction of ATG5 and activation of LC3-II and blocked autophagosome formation. 3-MA also blocked induction of GRP78 and CHOP, suggesting that PI3-kinase, which is known to mediate ER stress-induced autophagy, also plays a role in initiating apoptosis in response to ER stress. Together these data establish that the cytotoxicity of AS-6 operates by a mechanism dependent on ER stress-induced autophagy and apoptosis.
authors.affiliation: Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Jeong Han
authors.initials: JH
authors.lastname: Kang
authors.forename: Young-Chae
authors.initials: YC
authors.lastname: Chang
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D006016:Glycolates / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: C502778:ATG5 protein, human; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; D000091342:Endoplasmic Reticulum Chaperone BiP; D006016:Glycolates; C000717247:HSPA5 protein, human; C000717248:Hspa5 protein, mouse; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C043832:4-O-carboxymethylascochlorin