Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4‐II‐E cells induced by tumor necrosis factor‐α or thapsigargin

Abstract

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of 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. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619-626). After culture for 72 h, cells were further cultured for 24-72 h in medium without FBS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The number of wild-type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (0.1 or 1 ng/ml) or thapsigargin (10(-7) or 10(-6) M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF-alpha (10 ng/ml) or thapsigargin (10(-5) M) caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity in wild-type cells was significantly increased by culture with TNF-alpha (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10(-5) M) caused a significant increase in Ca(2+)/calmodulin-dependent NO synthase activity in wild-type cells or transfectants. TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-nitro-L-arginine (10(-4) M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with thapsigargin (10(-6) M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin-induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF-alpha or thapsigargin.

Increased regucalcin gene expression extends survival in breast cancer patients: Overexpression of regucalcin suppresses the proliferation and metastatic bone activity in MDA-MB-231 human breast cancer cells in vitro

“…MDA-MB-231 wild-type cells (1x10 5 /ml/well) and MDA-MB-231 cells (1x10 5 /ml/well) transfected with either full length, deleted exon 4 or deleted exons 4 and 5 regucalcin cDNAs were cultured using a 24-well plate in DMEM containing 10% FBS and 1% penicillin and streptomycin for 5 days. Subconfluent cells were cultured for additional 3 days in the presence or absence of LPS (0.1 or 1 µg/ml), TNF-α (0.1 or 1 ng/ml) (30). In separate experiments, wild-type MDA-MB-231 cells (1x10 5 /ml/well) or transfectants were cultured for 5 days to confluent, and then for an additional 24 h in the presence or absence of LPS (1 ng/ml) or Bay K 8644 (10 µM) with or without caspase-3 inhibitor (10 µM) (29).…”

Combination of alendronate and genistein synergistically suppresses osteoclastic differentiation of RAW267.4 cells in vitro

“…RAW267.4 cells (1x10 5 /ml cells/well; 2 ml medium added per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days when confluence was reached (24). The cells were then cultured for an additional 2 days in the presence or absence of alendronate (0.1, 1, 10 or 100 µM), genistein (0.1, 1, 10 or 100 µM), or alendronate (0.1, 1, 10 or 100 µM) plus genistein (0.1, 1, 10 or 100 µM).…”

Potential suppressive effects of gentian violet on human breast cancer MDA-MB-231 cells in vitro: Comparison with gemcitabine

“…Subsequent to the culture process, the cells were detached from each culture dish and counted (16,17). In addition, to determine the effects of GV on MDA-MB-231 cells that reached confluence, the cells (1x10 5 cells/ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S in the absence of GV for 7 days until they reached confluence, and then the cells were cultured in the presence of GV (1, 10, 50, 100 or 200 nM) for 3 days (18). Following this, the cells were detached from each culture dish and counted.…”

“…Following trypsinization of each culture dish using 0.2% trypsin plus 0.02% EDTA in Ca 2+ /Mg 2+ -free PBS for 2 min at 37˚C, detached cells from the dishes were collected after centrifugation at 150 x g for 5 min (16)(17)(18). The cells were resuspended in PBS solution and stained with eosin.…”