title: Mitochondrial and endoplasmic reticulum stress pathways cooperate in zearalenone-induced apoptosis of human leukemic cells.
abstract: BACKGROUND
  Zearalenone (ZEA) is a phytoestrogen from Fusarium species. The aims of the study was to identify mode of human leukemic cell death induced by ZEA and the mechanisms involved.
  
  
  METHODS
  Cell cytotoxicity of ZEA on human leukemic HL-60, U937 and peripheral blood mononuclear cells (PBMCs) was performed by using 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Reactive oxygen species production, cell cycle analysis and mitochondrial transmembrane potential reduction was determined by employing 2',7'-dichlorofluorescein diacetate, propidium iodide and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. Caspase-3 and -8 activities were detected by using fluorogenic Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) and Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (IETD-AMC) substrates, respectively. Protein expression of cytochrome c, Bax, Bcl-2 and Bcl-xL was performed by Western blot. The expression of proteins was assessed by two-dimensional polyacrylamide gel-electrophoresis (PAGE) coupled with LC-MS2 analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) approach.
  
  
  RESULTS
  ZEA was cytotoxic to U937 > HL-60 > PBMCs and caused subdiploid peaks and G1 arrest in both cell lines. Apoptosis of human leukemic HL-60 and U937 cell apoptosis induced by ZEA was via an activation of mitochondrial release of cytochrome c through mitochondrial transmembrane potential reduction, activation of caspase-3 and -8, production of reactive oxygen species and induction of endoplasmic reticulum stress. Bax was up regulated in a time-dependent manner and there was down regulation of Bcl-xL expression. Two-dimensional PAGE coupled with LC-MS2 analysis showed that ZEA treatment of HL-60 cells produced differences in the levels of 22 membrane proteins such as apoptosis inducing factor and the ER stress proteins including endoplasmic reticulum protein 29 (ERp29), 78 kDa glucose-regulated protein, heat shock protein 90 and calreticulin, whereas only ERp29 mRNA transcript increased.
  
  
  CONCLUSIONS
  ZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. ratana@chiangmai.ac.th
authors.forename: Ratana
authors.initials: R
authors.lastname: Banjerdpongchai
authors.forename: Prachya
authors.initials: P
authors.lastname: Kongtawelert
authors.forename: Orawan
authors.initials: O
authors.lastname: Khantamat
authors.forename: Chantragan
authors.initials: C
authors.lastname: Srisomsap
authors.forename: Daranee
authors.initials: D
authors.lastname: Chokchaichamnankit
authors.forename: Pantipa
authors.initials: P
authors.lastname: Subhasitanont
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000502:physiology; D020298:U937 Cells; D015025:Zearalenone / Q000494:pharmacology*
chemical_list: D017382:Reactive Oxygen Species; D015025:Zearalenone