Idoxifene and estradiol enhance antiapoptotic activity through estrogen receptor-beta in cultured rat hepatocytes

Oxidative stress plays a causative role in the development of hepatic fibrosis and apoptosis. Estradiol (E2) is an antioxidant, and idoxifene is a tissue-specific selective estrogen-receptor modulator. We have previously demonstrated that E2 inhibits hepatic fibrosis in rat models of hepatic fibrosis and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on the antiapoptotic role of idoxifene and E2, and the functions of ER subtypes ER- and ER- in hepatocytes undergoing oxidative stress. Lipid peroxidation was induced in cultured rat hepatocytes with ferric nitrilotriacetate solution with idoxifene or E2. Oxidative stress-induced early apoptosis was linked to its ability to inhibit not only the expression of Bcl-2 and Bcl-X_L but the production of antioxidant enzymes as well and to stimulate Bad expression. Hepatocytes possessed functional ER-, but not ER-, to respond directly to idoxifene and E2. Idoxifene and E2 suppressed oxidative stress-induced reactive oxygen species generation and lipid peroxidation, and their antiapoptotic effects on the activation of activator protein-1 and nuclear factor-B, the loss of antioxidant enzyme activity, and Bcl-2 family protein expression in early apoptotic hepatocytes were blocked by the pure ER antagonist ICI 182,780. Our results indicate that idoxifene and E2 could enhance antiapoptotic activity through ER- during oxidative damage in hepatocytes.     

Gankyrin-mediated dedifferentiation facilitates the tumorigenicity of rat hepatocytes and hepatoma cells

Gankyrin is a critical oncoprotein overexpressed in human hepatocellular carcinoma (HCC). However, the mechanism underlying gankyrin-mediated hepatocarcinogenesis remains elusive. Herein, we provide evidence that gankyrin expression was progressively elevated in liver fibrosis, cirrhosis, and HCC. Levels of gankyrin expression were closely associated with the dedifferentiation status of hepatoma in patients. Decrease of hepatocyte characteristic markers and increase of cholangiocyte-specific markers were observed in rat primary hepatocytes with enforced gankyrin expression and diethylnitrosamine (DEN)-triggered rat hepatocarcinogenesis. Overexpression of gankyrin also attenuated the hepatic function of primary hepatocytes, which further suggests that gankyrin promotes the dedifferentiation of hepatocytes. Moreover, elevated expression of gankyrin closely correlated with the expression of HCC stem/progenitor cell markers in DEN-triggered hepatocarcinogenesis and human HCCs. Hepatoma cells derived from suspension-cultured spheroids exhibited a higher gankyrin level, and enforced gankyrin expression in hepatoma cells remarkably enhanced cluster of differentiation (CD)133, CD90, and epithelial cellular adhesion molecule expression, indicating a role of gankyrin in hepatoma cell dedifferentiation and the generation of hepatoma stem/progenitor cells. In contrast, down-regulation of gankyrin in hepatoma cells by lentivirus-mediated microRNA delivery significantly improved their differentiation status and attenuated malignancy. Interference of gankyrin expression in hepatoma cells also diminished the proportion of cancer stem/progenitor cells and their self-renewal capacity. Furthermore, gankyrin was found to bind hepatocyte nuclear factor 4α (HNF4α), which determines hepatocyte differentiation status and enhances proteasome-dependent HNF4α degradation in hepatoma cells. The inverse correlation of gankyrin and HNF4α was further confirmed in primary hepatocytes, DEN-induced hepatocarcinogenesis, and human HCCs. CONCLUSION: Gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells via, at least partially, down-regulation of HNF4α facilitates HCC development, and interference of gankyrin expression could be a novel strategy for HCC prevention and differentiation therapy.     

Metformin enhances certain insulin actions in cultured rat hepatoma cells

Summary The effect of the oral antidiabetic agent metformin on insulin regulation of glycogen metabolism, tyrosine-aminotransferase activity, and [1-¹⁴C]aminoisobutyric acid uptake was studied in H4IIE cultured rat hepatoma cells. Metformin enhanced both basal (from 0.213±0.016 to 0.262±0.024 nmol/mg protein,p<0.01) and insulin stimulated [³H] glucose incorporation into glycogen in a time-dependent and dose-dependent manner. A small effect of metformin was seen at 1 μmol/l, and its greatest effects were obtained at 10 μmol/l. At the same concentrations, metformin did not influence basal tyrosine-aminotransferase activity but it potentiated insulin stimulated tyrosine-aminotransferase activity (+ 29.2±1.4%,p<0.01) and prevented the loss of tyrosine-aminotransferase responsiveness to insulin in H4IIE cells desensitised by a previous exposure to insulin. In contrast, metformin had no effect on basal or insulin-stimulated [1-¹⁴C]aminoisobutyric acid uptake. Over the concentrations of metformin that enhanced insulin action in H4IIE cells, the drug had no significant effect on insulin binding to its receptor. These studies suggest, therefore, that metformin may influence cellular metabolism by potentiating certain insulin actions through mechanisms that may be beyond insulin receptor binding.     

Increase in insulin binding affinity by chloroquine in cultured rat hepatoma cells

Chloroquine increased the binding of 125I-labeled insulin to rat hepatoma cells (R-Y121B) at 4 degrees C. The effect of chloroquine on insulin binding was amplified at 23 degrees C, and a large increase in cell-associated radioactivity was observed. Scatchard analysis showed that chloroquine increased the affinity of insulin for cells at both 4 degrees C and 23 degrees C, and that this increase eventually caused the accumulation of insulin internalized by cells at 23 degrees C.     

Cholesteryl ester cycle in cultured hepatoma cells

The existence of a cholesteryl ester cycle in cultured Fu5AH hepatoma cells was documented and factors affecting the rate of turnover of the cholesteryl ester cycle in this cell line were explored. The influence of the physical state of the lipid inclusion in which the cholesteryl esters are stored could be addressed in this cell line because these cells can be induced to store cholesteryl esters in anisotropic (liquid-crystalline) cytoplasmic inclusions by exposure to free cholesterol-rich phospholipid dispersions or in isotropic (liquid) inclusions by addition of oleic acid to the phospholipid dispersions. To examine the relative rates of turnover of the cholesteryl ester cycle in the cells with the two types of inclusions, the fraction of cholesteryl linolenate, a cholesteryl ester present in low amounts in these inclusions, was examined after cells were exposed to medium containing linolenate. After 12 h, cells with anisotropic inclusions contained 17.5% cholesteryl linolenate and cells with isotropic inclusions contained 29.8% cholesteryl linolenate, suggesting an approximately 2-fold difference in turnover of the cholesteryl ester pool. To determine whether this difference was due to a differential rate of cholesteryl ester hydrolysis, the acyl CoA: cholesterol acyl transferase arm of the cholesteryl ester cycle was blocked using a specific inhibitor, Sandoz 58-035. In the presence of this compound, cholesteryl ester was hydrolysed twice as fast in cells with isotropic inclusions as compared to that in cells with anisotropic inclusions. The difference in rate of turnover of the cholesteryl ester cycle was shown to be related to the rate of hydrolysis of cholesteryl ester which, in turn, is related to the physical state of the stored cholesteryl ester.     

Glutathione S-transferases in primary cultured rat hepatocytes

The present study is aimed to elucidate the changes in glutathione S-transferase (GST) activity and GST subunit components in primary cultured rat hepatocytes. Enzyme activity was measured with l-chloro-2,4-dinitrobenzene as cosubstrate. The activity decreased at 48 hr, and subsequently increased and returned to levels initially observed at 12 hr by 120 hr. Phenobarbital caused an induction of GST activity in culture at 72 and 168 hr. Immunocytochemical studies were performed using a peroxidase-anti-peroxidase technique with three polyclonal antibodies: anti-Ya, Yb₁ and Yp. With anti-Ya, hepatocytes were persistently positive up to 144 hr in cell culture. With anti-Yb₁, hepatocytes were positive at 24 hr, though positivity then gradually decreased. On the other hand, with anti-Yp, cells were almost negative at 48 hr and became obviously positive at 96 hr. Immunoelectron microscopy with anti-Ybj using the avidin-biotin ferritin method revealed ferritin particles in the ribosomes on endoplasmic reticulum as well as in the free cytoplasmic space. In conclusion, the GST subunit components are in a state of dynamic change in cultured rat hepatocytes, and overall time-dependent increase in the total activity of the enzyme can be accounted for by increased expression of the Yp subunit. Finally, the intracellular localization of Yb₁ subunit was clarified in the present report.     

Deficiency of methylthioadenosine phosphorylase in human leukemic cells in vivo

Cells from 20 patients with leukemia and 9 with solid tumors were assayed for the enzyme methylthioadenosine phosphorylase, which function in both purine and polyamine metabolism in rapidly dividing cells. As determined by autoradiography of viable cells, and by direct enzymatic analysis, samples from one individual with pre-T-cell acute lymphoblastic leukemia and one with common acute lymphoblastic leukemia were methylthioadenosine phosphorylase deficient. In contrast, other leukemias of similar antigenic phenotype, as well as normal peripheral blood lymphocytes, thymic lymphocytes, and normal bone marrow cells, had substantial methylthioadenosine phosphorylase activity. This evidence suggests that the complete absence of methylthioadenosine phosphorylase distinguishes some leukemic cells in vivo from their nonmalignant counterparts.     

Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes

The objective of this study was to investigate the mechanisms by which tamoxifen modifies cholesterol metabolism in cellular models of liver metabolism, HepG2 cells and rat hepatocytes. The effect of tamoxifen on cholesterol and triglyceride-palmitate synthesis was measured using isotopomer spectral analysis (ISA) and gas chromatography—mass spectrometry (GC-MS) and compared with the effects of progesterone, estradiol, the antiestrogen ICI 182,780, and an oxysterol, 25-hydroxycholesterol (25OHC). Cholesterol synthesis in cells incubated in the presence of either [1-¹³C]acetate, [U-¹³C]glucose, or [4,5-¹³C]mevalonate for 48 hours was reduced in the presence of 10 μmol/L tamoxifen and 12.4 μmol/L 25OHC in both HepG2 cells and rat hepatocytes. The ISA methodology allowed a clear distinction between effects on synthesis and effects on precursor enrichment, and indicated that these compounds did not affect enrichment of the precursors of squalene. Progesterone was effective in both cell types at 30 μmol/L and only in HepG2 cells at 10 μmol/L. Estradiol and ICI 182,780 at 10 μmol/L did not inhibit cholesterol synthesis. None of the compounds altered the synthesis of triglyceride-palmitate in either cell type. Treatment of cells with tamoxifen produced accumulation of three sterol precursors of cholesterol, zymosterol, desmosterol, and Δ⁸ cholesterol. This pattern of precursors indicates inhibition of Δ^24,25 reduction in addition to the previously described inhibition of Δ⁸ isomerase. We conclude that tamoxifen is an effective inhibitor of the conversion of lanosterol to cholesterol in cellular models at concentrations comparable to those present in the plasma of tamoxifen-treated individuals. Our findings indicate that this mechanism may contribute to the effect of tamoxifen in reducing plasma cholesterol in humans.     

肝细胞与肝癌细胞中磷脂酰乙醇胺N-甲基转移酶2表达及活性的比较

目的 比较研究原代培养的大鼠肝细胞与大鼠肝癌细胞内磷脂酰乙醇胺Ｎ－甲基转移酶２（ｐｈｏｓｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅＮ－ｍｅｔｈｙｌｔｒａｎｓｆｅｒｓａｅ２．ＰＥＭＴ２）表达水平和活性,了解ＰＥＭＴ２与细胞生长、增殖的关系。方法 利用免疫细胞化学及蛋白质免疫印迹方法观察ＰＥＭＴ２蛋白的表达,以「＾３Ｈ－Ｃ 同位素掺入法测定了ＰＥＭＴ２的活性,流式细胞仪分析了两种细胞的细胞周期中ＤＮＡ的     

Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357

BACKGROUND/AIMS: Due to a particular resistance against conventional chemotherapeutics, palliative treatment of hepatocellular carcinomas (HCC) is highly ineffective. Recent demonstration of both proliferation-inhibition and apoptosis of hepatoma cells by a histone deacetylase inhibitor (HDAC-I) treatment opens up a promising new approach. However, little is known about tumor cell death mechanisms and HDAC-I influences on healthy hepatocytes. METHODS: HDAC-I substances with favourable in vivo profiles, valproate (VPA) and ITF2357, were investigated on HCC cell lines and primary human hepatocytes (PHH). Histone acetylation and apoptosis-modulating proteins were investigated by western-blotting, proliferation by sulforhodamin B binding, toxicity by enzyme release, apoptosis by FACS analysis. RESULTS: VPA and ITF2357 inhibited proliferation in HCC cell lines. Both substances induced considerable cellular damage in HCC-derived cells, but PHH tolerated these substances well. A downregulation of anti- and upregulation of proapoptotic factors was found. Moreover, Bcl-X(L) transfection into HCC cells abrogated apoptosis induced by both substances, indicating that modulation of intracellular pro- and anti-apoptotic proteins is a key event in VPA or ITF2357 induced tumor-cell death. CONCLUSIONS: Preferential induction of cell death in HCC-derived cell lines, without toxicity in PHH, demonstrates the potential of VPA and ITF2357 to become promising new tools in the fight against HCC.     

Acquisition, storage and release of iron by cultured human hepatoma cells.

BACKGROUND/AIMS: The recovery from iron overload is hampered by the limited number of pathways and therapeutic agents available for the augmentation of iron secretion/excretion. The present study was aimed to investigate the process of iron storage and release by cultured human hepatoma cells, the role of transferrin receptors and ferritin in this process as well as the effect of iron chelators. METHODS: We followed the acquisition, storage and release of iron by cultured cells HepG2 and Hep3B by biochemical means and electron microscopy. RESULTS: The uptake of iron from diferric transferrin (Trf) was extremely low, while iron as ferric-ammonium-citrate (FAC) was taken up readily, especially by Hep3B cells. Up to 80% of the iron taken up by hepatoma cells was released to the medium. The rate of spontaneous iron release depended on the extent of iron loading. ApoTrf and deferoxamine facilitated release after 1- and 7-day iron-exposure. Up to a third of the radio-iron released from the cells was associated with ferritin. The release of ferritin-iron was not enhanced by either deferoxamine or Trf. CONCLUSIONS: Ferritin-iron release appeared to be an important mechanism of iron discarding in cultured human hepatoma cells, independent of the activity of chelating agents.     

Probucol enhances cholesterol transport in cultured rat hepatocytes

Probucol has been investigated extensively in vivo and ex vivo. However, little information is available on direct treatment of cultured cells. Treatment of cultured hepatocytes by 1 to 10 microM probucol for 16 to 20 hours had no apparent deleterious effect on the cell, and in fact decreased lactic dehydrogenase leakage. Also, total cellular cholesterol was increased, cholesterol synthesis was decreased, and cholesterol esterification was increased. The increase in cellular cholesterol was not the result of increased lipoprotein uptake but appeared to be the result of an interaction between cell and lipoprotein in which the cell became enriched and the lipoprotein depleted in cholesterol. This type of change in lipoprotein composition after treatment is also observed clinically.     

Growth factors and gene expression in cultured rat hepatocytes.

BACKGROUND/AIM: The aim of this study was to evaluate the effect of replication on function variables in cultured hepatocytes. METHODS: Isolated rat hepatocytes were cultured in HCM medium and plated on collagen-coated dishes at cell densities from 0.2 x 10(5) (subconfluent) to 1.0 x 10(5) x cm(-2) (confluent) with and without addition of hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-I. The synthesis rate was measured for DNA, albumin, urea, and glucose together with mRNA levels (Northern blots) for albumin, urea cycle enzymes, and acute phase and "house-keeping" proteins. RESULTS: In subconfluent culture the synthesis of DNA and urea was higher (118% and 112%, respectively), and of albumin and glucose lower (40% and 67%, respectively) than in confluent culture. The mRNA levels of carbamoylphosphate synthase, argininosuccinate synthetase, argininosuccinate lyase, arginase, a2-macroglobulin, beta-fibrinogen, and albumin were lower (23%, 58%, 77%, 33%, 12%, 50%, and 51%, respectively) in subconfluent culture compared with confluent culture. Relatively increased levels were found for beta-actin (109%) and alpha-tubulin (136%). In subconfluent culture hepatocyte growth factor increased the DNA synthesis rate 6-fold, epidermal growth factor 3-fold, and insulin-like growth factor-I 2-fold; that of albumin, urea and glucose was not increased significantly. In confluent culture the effect of growth factors on synthesis rates was not significant, and the growth factors had little influence on mRNA levels. CONCLUSIONS: Hepatocytes produce urea at the same rate in subconfluent as in confluent culture in spite of a lower mRNA level of urea cycle enzymes. Hepatocyte growth factor and epidermal growth factor increase DNA synthesis markedly in subconfluent culture only, without significantly changing the ratio between subconfluent and confluent culture of other variables. This suggests that active replication is not an important cause of the relatively low liver-specific function of hepatocytes in subconfluent culture.     

Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes

Aim:   Glycogen synthesis, and glucose and lactate production were examined in cultured rat hepatocytes preincubated with metformin (0–500 µ m ) for 24 h.
Methods:   Cells incubated with[1-¹³C]-glucose and [1-¹³C]-lactate allowed us to study the effect of metformin on glucose production from glycogenolysis and gluconeogenesis in a detailed manner using NMR spectroscopy. ¹H and ¹³C-filtered ¹H-NMR spectra were recorded by using flow-injection technique.
Results:   Metformin decreased glycogen synthesis in a dose-dependent manner with an IC₅₀ value of 196.5 µ m . This effect could not be reversed by the presence of the glycogen phosphorylase inhibitor DAB, suggesting that glycogenolysis was not affected. A clear correlation between glucose production and glycogen content (0.97 < R < 0.99; p < 0.001) and lactate production and glycogen content (0.97 < R < 0.99; p < 0.001) was observed. Moreover, a strong inhibition (62%, p < 0.001) of glucose produced from lactate/pyruvate (3 m m /0.3 m m ) was observed in cells treated with 350 µ m metformin.
Conclusion:   Hepatocytes preincubated for 24 h in the presence of metformin at clinically relevant concentrations showed impaired glycogenesis as well as gluconeogenesis.     

培养人胎肝细胞的形态与功能研究

目的探讨人胎肝细胞分离与培养的简易方法．方法采用体外两步灌流法分离人胎肝细胞，选择条件培养液加以培养，并进行形态学和蛋白分泌功能的动态观察．结果用该法分离的肝细胞经台盼蓝拒染试验证实存活率在９５％以上，培养时间可达２周，并保持正常形态和良好的蛋白合成分泌功能．结论用酶灌流分离和条件培养的胎肝细胞性能良好，可用作肝细胞移植的供体．     

The histone-deacetylase inhibitor SAHA potentiates proapoptotic effects of 5-fluorouracil and irinotecan in hepatoma cells

Treatment for advanced stages of hepatocellular carcinoma (HCC) remains unsatisfactory. While 5-fluorouracil (5-FU) and irinotecan are first-line treatment options for other gastrointestinal tumors, their effect on HCCs is low. Histone-deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) have shown antitumoral activity at micromolar concentrations in a variety of human cancers in vitro and in vivo. Here, we investigated the effects of a combination of 5-FU, irinotecan and SAHA on growth inhibition and apoptosis induction in HCC cell lines. HepG2, Hep1B and MH-7777A hepatoma cell lines and human foreskin fibroblasts as non-transformed controls were incubated with 5-FU, irinotecan and SAHA either alone or in combination. While the single agents did not show any effects on growth of the cell lines, the combination of 5-FU and irinotecan (both 10 M) led to a moderate increase in apoptosis and proliferation inhibition. Adding 1 M SAHA increased the apoptosis rate in hepatoma cell lines up to 92% after 72 h, while fibroblasts showed no response (5.5% apoptosis). Induction of apoptosis was paralleled by loss of the mitochondrial transmembrane potential, downregulation of bcl-2 expression and activation of caspase 3 but not caspase 8. In summary, SAHA sensitized HCC cell lines for treatment with an otherwise ineffective combination of 5-FU and irinotecan and led to mitochondrial apoptosis induction. The use of the triple combination could optimize treatment results in vivo and needs further evaluation.M. Ocker and A. Alajati contributed equally to this work.     

Insulin action in cultured HTC and H35 rat hepatoma cells: receptor binding and hormone sensitivity

Insulin receptors and several biological effects of insulin were measured in H35 and HTC cells, two rat hepatoma lines in permanent cell culture that have been employed previously to study glucocorticoid action. In H35 cells, insulin at concentrations as low as 1 pM both enhanced tyrosine amino-transferase activity and stimulated [3H]uridine incorporation into RNA. In this cell line, the binding of approximately 10 molecules of insulin/cell was sufficient to elicit these two biological responses. In contrast, in HTC cells, much higher concentrations of insulin (3 nM or greater) were needed to stimulate both tyrosine aminotransferase activity and other biological functions. When insulin receptors were measured, both cell types had a major insulin-binding site with a Kd of approximately 20 nM. H35 cells, however, had approximately 6 times more receptors per cell than HTC cells. In both cell types, insulin degradation was minimal. These findings indicate that several biological functions in H35 cells are extremely sensitive to insulin in vitro. In contrast, HTC cells are much less sensitive to the hormone. This lack of sensitivity in HTC cells most likely reflects a decrease in the number of insulin receptors present. In addition, the very large differences in responsiveness suggest that postreceptor alterations are also operative.