Apoptotic and anti-proliferative effects of 17beta-estradiol and 17beta-estradiol-like compounds in the Hep3B cell line.

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.

Roles of insulin-like growth factor II in cardiomyoblast apoptosis and in hypertensive rat heart with abdominal aorta ligation.

Although IGF-II activating the IGF-II receptor signaling pathway has been found to stimulate cardiomyocyte hypertrophy, the role of IGF-II in cardiac cell apoptosis remains unclear. This study aimed to identify the roles of IGF-II and/or IGF-II receptors (IGF-II/IIR) in cardiomyoblast apoptosis and in hypertensive rat hearts with abdominal aorta ligation. Cultured rat heart-derived H9c2 cardiomyoblasts and excised hearts from Sprague-Dawley rats with 0- to 20-day complete abdominal aorta ligation, a model of ANG II elevation and hypertension, were used. IGF-II/IIR expression, caspase activity, DNA fragmentation, and apoptotic cells were measured by RT-PCR, Western blot, agarose gel electrophoresis, and TUNEL assay following various combinations of ANG II, IGF-II/IIR antibody, CsA (calcineurin inhibitor), SP-600125 (JNK inhibitor), SB-203580 (p38 inhibitor), U-0126 (MEK inhibitor), or Staurosporine (PKC inhibitor) in H9c2 cells. ANG II-induced DNA fragmentation and TUNEL-positive cells were blocked by IGF-II/IIR antibodies and antisense IGF-II, but not by IGF-II sense. IGF-II-induced apoptosis was blocked by IGF-IIR antibody and CsA. The increased gene expressions of IGF-II and -IIR induced by ANG II were reversed by U-0126 and Sp600125, respectively. Caspase 8 activities induced by ANG II were attenuated by U-0126, SP-600125, and CsA. DNA fragmentation induced by ANG II was totally blocked by SP-600125, and CsA and was attenuated by U-0126. In rats with 0- to 20-day complete abdominal aorta ligation, the increases in IGF-II/IIR levels in the left ventricle were accompanied by hypertension as well as increases in caspase 9 activities and TUNEL-positive cardiac myocytes. ANG II-induced apoptosis was reversed by IGF-II/IIR blockade and coexisted with increased transactivation of IGF-II and -IIR, which are mediated by ERK and JNK pathways, respectively, both of which further contributed to cardiomyoblast apoptosis via calcineurin signaling. The increased cardiac IGF-II, IGF-IIR, caspase 9, and cellular apoptosis were also found in hypertensive rats with abdominal aorta ligation.

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.

Serum insulin-like growth factor-axis and matrix metalloproteinases in patients with rheumatic arthritis or rheumatic heart disease.

BACKGROUND: Insulin-like growth factor (IGF)-I plays an important role for maintaining cardiac functions. We clarified the unknown role of IGF-axis in rheumatic heart disease (RHD). METHOD: Interleukin (IL)-10, growth hormone (GH), IGF, IGF binding protein (IGFBP)-3 and matrix metalloproteinase (MMP) were measured by ELISA and zymography in 30 age range-matched normal subjects (control), 36 patients with acute phase of rheumatoid arthritis (RA) with positive rheumatoid factor (RF) and C-reactive protein (CRP), and in 43 patients with RHD with negative RF and CRP. RESULT: Compared with normal subjects, increased IL-10 level and decreased GH were found in RA group whereas unchanged IL-10 and decreased GH were found in RHD group. Compared with age range-matched normal subjects, decreased IGFBP-3, MMP-9 levels, unchanged IGF-I were found in RA group whereas decreased IGF-I levels, unchanged IGFBP3 and increased MMP-9 at age>30 years were found in RHD group. IGF-II was not changed in RA and RHD groups. CONCLUSION: These findings may imply that during inflammatory phase, the levels of anti-inflammation was high and total IGF-I and IGF bioavailability were maintained in patients with RA. Our findings in RHD may speculate that the long-term reduction of GH and IGF-I as well as the compensating effects of upregulated MMP-9 activity may be partially involved in the long-term pathogenesis from RHD to heart failure. Decreased GH, decreased IGF-I and increased MMP-9 activities may be possible diagnostic markers in RHD for developing heart failure.

Homocysteine and other biochemical parameters in Type 2 diabetes mellitus with different diabetic duration or diabetic retinopathy.

BACKGROUND: Cardiovascular risk-related markers in type 2 diabetes mellitus (DM) have not been well understood. METHODS: Serum and urine samples for biochemical and immunologic analysis were collected from 204 normal subjects and 257 type 2 DM patients, the latter of which were further classified by different diabetic duration with or without retinopathy. RESULTS: Glycosylated hemoglobin A1c, triglyceride, lipase, free fatty acid, albumin creatinine ratio (ACR), lactate dehydrogenase (LDH) and homocysteine were significantly increased in DM patients, whereas high density lipoprotein cholesterol and bilirubin were significantly decreased in DM patients, compared with normal subjects. Lipid profiles, ACR, bilirubin, uric acid, creatine kinase, and hsCRP were not changed in DM patients with different diabetic duration or diabetic retinopathy. Lactate dehydrogenase in DM patients with duration >20 years and homocysteine in patients with duration >10 years was significantly higher than those with duration <5 years. Homocysteine was significantly increased in DM patients with retinopathy, compared with DM patients without retinopathy. CONCLUSION: The increased triglyceride, lipase, free fatty acid, albumin creatinine ratio, lactate dehydrogenase and homocysteine as well as decreased high density lipoprotein cholesterol and anti-oxidative bilirubin in DM patients should be cautious and considered as risks for increasing DM complication. Homocysteine might be associated with longer diabetic duration and microvascular complication of retinopathy in diabetes.