title: Overexpression of regucalcin suppresses apoptotic cell death in the cloned rat hepatoma H4-II-E cells induced by a naturally occurring isothiocyanate sulforaphane.
abstract: The effect of regucalcin, a regulatory protein in intracellular signaling system, on cell death and apoptosis was investigated. Sulforaphane, a naturally occurring isothiocyanate, is known to induce cell cycle arrest and apoptosis in cancer cells, although its effect has not been clarified in the cloned rat hepatoma H4-II-E cells. Hepatoma H4-II-E cells (wild-type) and stable regucalcin/pCXN2-transfected cells were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS). Cells with subconfluency were changed to a medium containing either vehicle or sulforaphane (10(-7) or 10(-6) M) in the absence of FBS. After medium change, cells were cultured for 24, 48, or 72 h. The number of wild-type cells was significantly decreased in the presence of sulforaphane (10(-7) or 10(-6) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with sulforaphane (10(-7) or 10(-6) M) for 24 h. Sulforaphane (10(-7) or 10(-6) M)-induced cell number and DNA fragmentation was significantly suppressed in transfectants. The effect of sulforaphane (10(-6) M) in decreasing the number of wild-type cells was significantly prevented in the presence of caspase-3 inhibitor (10(-9) M), while the presence of Nomega-nitro-L-arginine methylester (NAME), an inhibitor of nitric oxide synthase, did not prevent sulforaphane-induced death of wild-type cells. Sulforaphane (10(-6) M) did not have a significant effect on cell number of transfectants in the presence of caspase-3 inhibitor or NAME. This study demonstrates that sulforaphane induces cell death and apoptosis in the cloned rat hepatoma H4-II-E cells, and that overexpression of regucalcin suppresses sulforaphane-induced apoptotic cell death which is partly mediated through caspase-3..
authors.affiliation: Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukaya
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Yamaguchi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases; D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D015238:Sulfotransferases; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D002135:Calcium-Binding Proteins; D061945:Caspase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D019323:omega-N-Methylarginine; D019001:Nitric Oxide Synthase; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C016766:sulforaphane