Since there is evidence for estrogen and estrogen-like compounds to have beneficial effect on the pathogenesis of hepatocellular carcinoma (HCC), this study was designed to investigative the apoptotic and anti-proliferative effects of these compounds on the human hepatoma Hep3B cell line. The Hep3B cells were treated with 17beta-estradiol (E2), diethylstilbestrol (DES), tamoxifen, and genistein. After treatments of these compounds at the concentration of 10(-6) or 10(-8) M, the Hep3B cells were demonstrated to have significant DNA fragmentation, nucleus condensation, cytochrome-c leaking from the mitochondria and caspase-3 activation by DAPI and Western blotting. The cells were also observed to have declined proliferative potential by MTT assay, arrested cell cycle by flow-cytometry measurements. However, the cytochrome-c leaking from the mitochondria induced by E2 and E2-like compounds was blocked totally by ICI 182,780 treatment. These finding suggest that estrogen and the estrogen-like compounds may induce anti-proliferative and apoptotic effects in Hep3B cells, and the E2 and the E2-like compounds mediated apoptotic effect was estrogen receptor dependent. Among the drugs tested, E2, E2 agonists (DES and genistein) and partial antagonist (tamoxifen), all showed the stronger anti-tumor potential.
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.
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