Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644.
The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 microg/ml). The effect of LPS (0.1 or 1.0 microg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 microg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 microg/ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 microg/ml)-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca(2+) entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.
Izumi
Takako
T
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
Yamaguchi
Masayoshi
M
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002999:Clone Cells; D003992:Dibucaine / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology; D015238:Sulfotransferases; D014407:Tumor Cells, Cultured
D002120:Calcium Channel Agonists; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; 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; D019311:Staurosporine; D003992:Dibucaine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium