Abstract
The role of regucalcin, a regulatory protein in intracellular signaling pathway, in cell death was investigated by using the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin. The hepatoma cells (wild‐type) and stable regucalcin/pCXN2 transfectants were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24–72 h in a medium containing either vehicle, insulin (10^−8^ or 10^−7^M) or insulin‐like growth factor‐I (IGF‐I; 10^−9^ or 10^−8^M) in the absence of FBS. The number of wild‐type cells was significantly decreased by culture for 24, 48, or 72 h in the presence of insulin (10^−8^ or 10^−7^M) or IGF‐I (10^−9^ or 10^−8^M). Agarose gel electrophoresis showed the presence of low‐molecular‐weight deoxyribonucleic acid (DNA) fragments of adherent wild‐type cells cultured with insulin or IGF‐I. The effect of insulin or IGF‐I in stimulating cell death and DNA fragmentation in hepatoma cells (wild‐type) was significantly prevented in transfectants overexpressing regucalcin. Meanwhile, epinephrine (10^−6^ or 10^−5^M) or transforming growth factor‐β1 (10^−13^ or 10^−12^M) did not cause cell death of hepatoma cells. Insulin‐induced decrease in the number of wild‐type cells was significantly prevented by culture with caspase‐3 inhibitor (10^−8^M), although the effect of IGF‐I was not inhibited. The effect of insulin or IGF‐I in inducing the death of hepatoma cells (wild‐type) was significantly prevented in the presence of N ω‐nitro‐l‐arginine methylester (NAME), an inhibitor of nitric oxide synthase. Genistein (10^−6^M), an inhibitor of protein tyrosine kinase, or vanadate (10^−5^M), an inhibitor of protein tyrosine phosphatase, caused a significant decrease in the number of hepatoma cells (wild‐type). The effect of insulin in inducing the death of wild‐type cells was not seen in the presence of genistein or vanadate. The effect of IGF‐I on the death of wild‐type cells was observed in the presence of genistein or vanadate. The effect of genistein on cell death was significantly prevented in transfectants. Such effect was not seen with vanadate. This study demonstrates that insulin or IGF‐I stimulates cell death and apoptosis in the hepatoma cells, and that overexpression of regucalcin has a suppressive effect on cell death induced by insulin or IGF‐I that is mediated through different signaling pathway. © 2005 Wiley‐Liss, Inc.