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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. CONCLUSION: ZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.

SUBMITTER: Banjerdpongchai R

PROVIDER: S-EPMC3018374 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Mitochondrial and endoplasmic reticulum stress pathways cooperate in zearalenone-induced apoptosis of human leukemic cells.

Banjerdpongchai Ratana R Kongtawelert Prachya P Khantamat Orawan O Srisomsap Chantragan C Chokchaichamnankit Daranee D Subhasitanont Pantipa P Svasti Jisnuson J

Journal of hematology & oncology 20101230

<h4>Background</h4>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.<h4>Methods</h4>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 redu ...[more]

PMID: 21190589

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Project description:BackgroundHuman head and neck squamous carcinoma is the 6th most prevalent carcinoma worldwide. Although many novel therapies have been developed, the clinical treatment for patients remains non-ideal. Chloride intracellular channel 4 (CLIC4), one of the seven members of the CLIC family, is a newly found Cl(-) channel that participates in various biological processes, including cellular apoptosis and differentiation. Accumulating evidence has revealed the significant role of CLIC4 in regulating the apoptosis of different cancer cells. Here, we investigated the functional role of CLIC4 in the apoptosis of HN4 cells, a human head and neck squamous carcinoma cell line.ResultsIn the present study, we used immunohistochemical staining to demonstrate that the expression level of CLIC4 is elevated in the tissue of human oral squamous carcinoma compared with healthy human gingival tissue. Specific CLIC4 small interfering RNA was used to knockdown the expression of CLIC4. The results showed that knockdown of CLIC4 with or without 100 μM adenosine triphosphate (ATP) treatment significantly increased the expression of Bax, active caspase 3, active caspase 4 and CHOP but suppressed Bcl-2 expression in HN4 cells. Moreover, the results from the TdT-mediated dUTP nick end labeling assay indicated that CLIC4 knockdown induced a higher apoptotic rate in HN4 cells under the induction of ATP. In addition, knockdown of CLIC4 dramatically enhanced ATP-induced mitochondrial membrane depolarization in HN4 cells. Moreover, intracellular Ca(2+) measurement revealed that Ca(2+) release induced by ATP and thapsigargin, a Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, was significantly enhanced by the suppression of CLIC4 in HN4 cells.ConclusionsKnockdown of CLIC4 enhanced ATP-induced apoptosis in HN4 cells. Both the pathways of mitochondria and endoplasmic reticulum stress were involved in CLIC4-mediated cell apoptosis. Based on our finding, CLIC4 may be a potential and valuable target for the clinical treatment of head and neck squamous carcinoma.

Project description:Isoniazid (INH) is a first-line antituberculosis drug. The incidence of adverse reactions accompanied by inflammation in the liver during drug administration to tuberculosis patients is high and severely affects clinical treatment. To better understand the mechanism of hepatotoxicity induced by INH under the inflammatory state, we compared the differences in levels of hepatotoxicity from INH between normal zebrafish and zebrafish in an inflammatory state to elucidate the hepatotoxic mechanism using different endpoints such as mortality, malformation, inflammatory effects, liver morphology, histological changes, transaminase analysis, and expression levels of certain genes. The results showed that the toxic effect of INH in zebrafish in an inflammatory state was more obvious than that in normal zebrafish, that liver size was significantly decreased as measured by liver fatty acid binding protein (LFABP) reporter fluorescence and intensity, and that alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly increased. Hematoxylin and eosin (HE) staining and electron microscopy showed that hepatocyte injury was more obvious in the inflammatory state. In the inflammatory state, INH significantly increased the expression levels of endoplasmic reticulum stress (ERS)-related factors (GRP78, ATF6, PERK, IRE1, XBP1s, GRP94, and CHOP), autophagy-related factors (beclin 1, LC3, Atg3, and Atg12), and apoptosis-related factors (caspase-3, caspase-8, caspase-9, Bax, p53, and Cyt) in larvae. Correlational analyses indicated that the transcription levels of the inflammatory factors interleukin-1b (IL-1b), tumor necrosis factor beta (TNF-β), cyclooxygenase 2 (COX-2), and TNF-ɑ were strongly positively correlated with ALT and AST. Furthermore, the ERS inhibitor sodium 4-phenylbutyrate (4-PBA) could ameliorate the hepatotoxicity of INH-lipopolysaccharide (LPS) in zebrafish larvae. These results indicated that INH hepatotoxicity was enhanced in the inflammatory state. ERS and its mediated autophagy and apoptosis pathways might be involved in INH-induced liver injury promoted by inflammation.

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Mitochondrial and endoplasmic reticulum stress pathways cooperate in zearalenone-induced apoptosis of human leukemic cells.

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Mitochondrial and endoplasmic reticulum stress pathways cooperate in zearalenone-induced apoptosis of human leukemic cells.

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