Abstract
BackgroundZearalenone (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.MethodsCell 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.ResultsZEA 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.ConclusionZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.
Highlights
The phytoestrogen zearalenone (ZEA) is one of the most active naturally occurring estrogenic compounds [1,2]
Activation of caspase-9 leads to subsequent activation of executioner caspases, such as caspase-3, -6, -7, which in turn stimulates a series of apoptotic events, eventually leading to cell death [9,12,13]
Using two human leukemic HL-60 and U937 cell lines we found that human leukemic cell apoptosis induced by ZEA was related to caspase-3 and -8 activation, mitochondrial transmembrane potential (MTP) reduction and cytochrome c release
Summary
The phytoestrogen zearalenone (ZEA) is one of the most active naturally occurring estrogenic compounds [1,2]. ZEA has been shown to induce apoptosis in human hepatocytes (HepG2) via p53-dependent mitochondrial signaling pathway with the up regulation of ATM and GADD45 involved in DNA repair [8]. There are two major pathways involved in apoptosis: mitochondria-initiated intrinsic pathway and death receptor-stimulated extrinsic pathway [9,10,11]. In the former pathway, proapoptotic signals provoke release from mitochondrial inter-membranous space into cytosol of cytochrome c, which forms a complex with Apaf-1 and dATP, known as apoptosome, and triggers caspase-9 activation. The aims of the study was to identify mode of human leukemic cell death induced by ZEA and the mechanisms involved
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