Overexpression of regucalcin suppresses cell proliferation in cloned rat hepatoma H4‐II‐E cells: Involvement of intracellular signaling factors and cell cycle‐related genes

Abstract

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation 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 monolayters. The proliferation of cells was significantly suppressed in transfectants cultured for 24-72 h. The proliferation of wild-type cells was significantly inhibited when the cells were cultured for 72 h in a medium containing an inhibitor of transcriptional activity or protein synthesis. Such an effect was not seen in transfectants. The presence of various inhibitors of protein kinase including PD 98059 (10(-7) or 10(-6) M), dibucaine (10(-6) M), wortmannin (10(-8) or 10(-6) M), or genistein (10(-5) M) caused a significant inhibition of the proliferation of wild-type cells. These inhibitory effects were not seen in transfectants. Staurosporine (10(-8) - 10(-7) M) significantly inhibited the proliferation of wild-type cells and transfectants. Also, the effect of vanadate (10(-5) M), an inhibitor of protein tyrosine phosphatase, or Bay K 8644 (10(-6) M), an agonist of calcium entry into cells, in inhibiting the proliferation of wild-type cells was not observed in transfectants. Moreover, the proliferation of wild-type cells was significantly inhibited in the presence of roscovitine (10(-7) or 10(-6) M) or sulforaphane (10(-7) M), which induces cell-cycle arrest. Such effect was not seen in transfectants. The inhibitory effect of sodium butyrate (8.3 x 10(-4) M) on proliferation of wild-type cells was also induced in transfectants. Gene expression in hepatoma cells cultured for 72 h with 10% FBS was determined by using reverse transcription-polymerase chain reaction (RT-PCR). The expression of p21 mRNA was significantly enhanced in transfectants, while cdc2a and chk2 mRNA expression were not significantly changed. Insulin-like growth factor-I (IGF-I) mRNA expression was significantly suppressed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation that is partly mediated through various intracellular signaling-related factors, and that the effect may be partly involved in the change in p21 or IGF-I mRNA expression. The finding further supports that regucalcin plays an important role as a suppressor in the enhancement of cell proliferation.

Identification of biomarkers associated with the development of hepatocellular carcinoma in CuZn superoxide dismutase deficient mice

“…Over-expression of RGN in the rat hepatoma cell line H4-II-E was shown to suppress the expression of oncogenes c-myc, Haras, and c-src, and increase the expression of tumor suppressor genes p53 and Rb [23,24]. Recent studies in hepatoma cell lines also showed growth suppression effects by RGN [25,26]. Therefore, upregulation of RGN may provide the benefit of suppressing hepatocarcinogenesis in Sod1−/− mice, whereas reduced expression of RGN may have the opposite effect.…”

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

“…The breast cancer MDA-MB-231 cells (1x10 5 /ml per well) were cultured in a 24-well plate using DMEM containing 10% FBS and 1% P/S in the presence or absence of GV (1, 10, 50, 100 or 200 nM) for 1, 3, 7 or 14 days in a water-saturated atmosphere containing 5% CO 2 and 95% air at 37˚C (15)(16)(17). In separate experiments, the MDA-MB-231 cells (1x10 5 /ml per well) were cultured in DMEM containing 10% FBS and 1% P/S in the presence of either ethanol (0.1% final concentration; control), sodium butyrate (10 and 100 µM), roscovitine (10 and 100 nM), sulforaphane (1 and 10 nM), PD98059 (1 µM), staurosporin (0.1 µM), wortmannin (1 µM), DRB (1 µM) or gemcitabine (100 nM) for 3-7 days.…”

“…In separate experiments, the MDA-MB-231 cells (1x10 5 /ml per well) were cultured in DMEM containing 10% FBS and 1% P/S in the presence of either ethanol (0.1% final concentration; control), sodium butyrate (10 and 100 µM), roscovitine (10 and 100 nM), sulforaphane (1 and 10 nM), PD98059 (1 µM), staurosporin (0.1 µM), wortmannin (1 µM), DRB (1 µM) or gemcitabine (100 nM) for 3-7 days. 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 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.…”

Potential significance of the regucalcin gene in human carcinoma

“…Nuclear regucalcin suppressed Ca Overexpression of regucalcin was found to play a role as a suppressor protein in cell proliferation that is mediated through various signaling stimulations in the cloned normal rat kidney proximal tubular epithelial NRK52E cells and the cloned rat hepatoma H4-II-E cells [31,34,35]. Regucalcin caused G1 and G2/M phase cell cycle arrest in these cells [31,36]. The anti-cell proliferation effect of regucalcin was not dependent on apoptosis; regucalcin suppresses apoptosis induced through multisignaling pathway [reviewed in Ref.…”