Opposing action of estrogen receptors alpha and beta on tumor necrosis factor-alpha gene expression and caspase-8-mediated apoptotic effects in HA22T cells.

Hepatocellular carcinoma (HCC), the major manifestation of primary liver cancer, is one of the most frequent and malignant cancers worldwide, especially in Taiwan. Estrogen receptors (ERs) have been reported to play either a proliferation- or apoptosis-enhancing role in the differentiation of cancers, including HCC. In a previous experiment, we showed that transient overexpressed estrogen receptor-alpha induced early stage HCC cell line Hep 3B cell apoptosis by increasing the hTNF-alpha gene expression in a ligand-independent manner. To further clarify if the apoptotic effect occurs in poorly differentiated HCC cell line, HA22T, and elucidate the roles of ERs and TNF-alpha, DNA fragmentation and caspase activity were measured in late stage HCC cell line, HA22T, by measuring the expression of hER-alpha and hER-beta using a Tetracycline-inducible system (Tet-on). Increased DNA fragmentation and caspase-3 activity were found in hERbeta-overexpressed HA22T cells treated with estrogen (10(-8) M) but not in hERalpha-overexpressed HA22T cells. Using RT-PCR/PCR and western blotting in HA22T cells, overexpressed hER-beta was also found to increase the expression of hTNF-alpha mRNA and induce hTNF-alpha-dependent luciferase activity in a ligand-dependent manner. Additionally, LPS treatment and hER-beta overexpression both enhance caspase-8 activities, whereas neither hER-beta nor E2 treatment affected caspase-9 activities. In addition, the overexpressed hER-beta plus E2 enhanced DNA fragmentation and caspase-8 activities were only partially reduced by anti-hTNF-alpha (0.1 ng/ml), which was possibly due to the involvement of P53 and TGF-beta. Taken together, our data indicates that overexpressed hER-beta but not hER-alpha may induce caspase-8-mediated apoptosis by increasing the hTNF-alpha gene expression in a ligand-dependent manner in poorly differentiated HA22T cells.

Regulation of extracellular signal-regulated protein kinase signaling in human osteosarcoma cells stimulated with nicotine.

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Currently, there is limited information on the regulation of mitogen-activated protein kinases (MAPK) expression in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of MAPKs in human osteosarcoma cell line U2OS cells. Furthermore, various pharmacological agents were added to search the possible regulation mechanisms on nicotine-induced MAPKs expression. METHODS: Cytotoxicity and western blot assays were used to investigate the effects of U2OS cells exposed to nicotine. In addition, various pharmacological agents [NS-398, dexamethasome, 2-oxothiazolidine-4-carboxylic acid (OTZ), herbimycin A, and curcumin] were added to test how they modulated the effects of nicotine-induced MAPKs expression. RESULTS: Concentrations of nicotine higher than 5 mm demonstrated cytotoxicity to U2OS cells (p<0.05). A nicotine concentration of 5 mm was found to induce extracellular signal-regulated kinase (ERK) phosphorylation in a time-dependent manner (p<0.05). In addition, amounts of ERK protein were unaffected by nicotine during the same time interval. By contrast, nicotine has no effect on either c-Jun N-terminal kinase (JNK) or p38, respectively. In addition, NS-398, dexamethasone, OTZ, herbimycin A, and curcumin were found to inhibit the nicotine-induced ERK expression (p<0.05). CONCLUSIONS: The activation of ERK expression by nicotine suggests a potential role for nicotine in the pathogenesis of cigarette smoking-associated periodontal disease. In addition, nicotine-induced ERK expression was down-regulated by NS-398, dexamethasone, OTZ, herbimycin A, and curcumin.