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 Nω-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..

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