Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells

Glucose is known to affect mRNA levels of several genes. In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose. Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations. These data suggest that glucose affects insulin receptor gene expression.     

Iron accumulation and iron-regulatory protein activity in human hepatoma (HepG2) cells

Iron may populate distinct hepatocellular iron pools that differentially regulate expression of proteins such as ferritin and transferrin receptor (TfR) through iron-regulatory mRNA-binding proteins (IRPs), and may additionally regulate uptake and accumulation of non-transferrin-bound iron (NTBI). We examined iron-regulatory protein (IRP) binding activity and ferritin/TfR expression in human hepatoma (HepG2) cells exposed to iron at different levels for different periods. Several iron-dependent RNA-binding activities were identified, but only IRP increased with beta-mercaptoethanol. With exposures between 0 and 20 microg/ml iron, decreases in IRP binding accompanied large changes in TfR and ferritin expression, while chelation of residual iron with deferoxamine (DFO) caused a large increase in IRP binding with little additional effect on TfR or ferritin expression. Cellular iron content increased beyond 4 days of exposure to iron at 20 microg/ml, when IRP binding, TfR, and ferritin had all reached stable levels. However, iron content of the cells plateaued by 7 days, or decreased with 24 h exposure to very high concentrations (>50 microg/ml) of iron. These results indicate that iron-replete HepG2 cells exhibit a narrow range of maximal responsiveness of the IRP-regulatory mechanism, whose functional response is blunted both by excessive iron exposure and by removal of iron from a chelatable pool. HepG2 cells are able to limit iron accumulation upon higher or prolonged exposure to NTBI, apparently independent of the IRP mechanism.     

Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells.

Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.     

Oxidative stress decreases extracellular homocysteine concentration in human hepatoma (HepG2) cell cultures

BACKGROUND: Mild hyperhomocysteinemia is associated with premature vascular disease. The mechanism behind the vascular injuries is, however, still unknown. Homocysteine may be catabolized in the trans-sulfuration pathway to cysteine. Cystathionine beta-synthase, which catalyses the first step in the trans-sulfuration pathway is redox-sensitive. We have therefore investigated total extracellular homocysteine turnover in the presence of oxidative stress in human cell lines. METHODS: The turnover of total extracellular homocysteine in HeLa and hepatoma cell cultures has been investigated in the presence of hydrogen peroxide. Furthermore, the effect of hydrogen peroxide on the removal of high amounts of exogenously added homocysteine was also studied. RESULTS: Total extracellular homocysteine concentration in hepatoma cell cultures decreased in the presence of hydrogen peroxide, whereas the extracellular homocysteine concentration in HeLa cell cultures was not influenced. There was no significant change of intracellular homocysteine in any type of cell cultures. Furthermore, the presence of hydrogen peroxide did not increase the removal of exogenously added homocysteine. CONCLUSION: The presence of hydrogen peroxide probably increases the activity of the trans-sulfuration pathway in hepatoma cell cultures, which increases the intracellular use of homocysteine and lowers its extracellular release. Consequently this mechanism might tend to lower total plasma homocysteine concentration in oxidative stress.     

The apoptotic effect of sarsasapogenin from Anemarrhena asphodeloides on HepG2 human hepatoma cells

Sarsasapogenin, a kind of mainly effective components of Anemarrhena asphodeloides Bunge (Liliaceae) has the effects of being anti-diabetes and improving memory. However, there are few reports focusing on its anti-tumor effects. In this study, the sarsasapogenin was extracted from rhizomes of A. asphodeloides Bunge and applied to inhibit HepG2 human hepatoma cells. MTT assay showed that sarsasapogenin induced a distinct dose- and time-dependent diminution of cell viability with IC(50) of 42.4+/-1.0microg/ml for 48h. Furthermore, sarsasapogenin-induced apoptosis of HepG2 cells was verified by Hoechst 33258 staining, electron microscopy, DNA fragmentation and PI staining. Flow cytometry analysis showed that sarsasapogenin-induced cell apoptosis was through arrest of cell cycle in G(2)/M phase. Hence we proposed that sarsasapogenin could be used as an anti-liver cancer drug for future studies.     

Catabolic properties of aglycofibrinogen synthesized by tunicamycin-treated human hepatoma (HepG2) cells and rabbit hepatocytes

Human hepatoma cell (HepG2) or rabbit hepatocyte monolayers were incubated with [35S]methionine in presence or absence of tunicamycin, a potent inhibitor of asparagine-linked glycosylation. The 35S-labeled nonglycosylated and control fibrinogens purified from the media were used to evaluate the influence of the oligosaccharide on the catabolic properties of this glycoprotein. Plasmin, pronase, cathepsin D or cathepsin B each degraded the nonglycosylated and control fibrinogens similarly, as evidenced by the release of trichloroacetic acid-soluble radioactivity and by SDS-polyacrylamide gel electrophoresis and autoradiography of plasmic digests. Nonglycosylated and control fibrin clots also showed no differences in susceptibility to plasmic digestion. The two forms of fibrinogen demonstrated the same plasma half-life in rabbits. These data indicate that the oligosaccharide does not influence the proteolytic stability or the in vivo plasma survival of fibrinogen, and suggest that other biochemical determinants may influence the catabolic properties of this molecule.     

Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

The binding and metabolism of [3H]vitamin A-containing chylomicron (CM) remnants by the human hepatoma cell line HepG2 were studied. Mesenteric lymph chylomicrons were collected from [3H]retinol-fed rats and incubated with lipoprotein lipase to obtain CM remnants. At 4 degrees C, specific CM remnant binding was inhibited by an excess of unlabeled CM remnants. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 microgram triglyceride/ml). CM remnant uptake at 37 degrees C was greater than that of CM and at least 70 times more efficient than the pinocytosis of sucrose. CM remnant binding increased with the extent of lipolysis. Addition of human apolipoprotein E enhanced both CM remnant and CM binding. After internalization, HepG2 cells hydrolyzed CM remnant-[3H]retinyl esters, and radiolabeled metabolites accumulated. As a function of the concentration of [3H]retinoid initially bound to cells, retinol and retinyl esters accumulated as the major cell-associated metabolites. In contrast, retinol was the major metabolite in the medium only at low retinoid concentrations; other more polar metabolites accumulated at higher concentrations (greater than 110 pmol retinoid/mg cell protein). The accumulation in the medium of labeled metabolites derived from CM remnant-retinoid was reduced when cells were preincubated in unlabeled retinol-supplemented media. The specific activity of retinol in the medium indicated that CM remnant-vitamin A had mixed with the cellular store prior to its secretion as retinol. These results indicate that HepG2 cells internalize CM remnants in part by specific binding sites, and that the metabolism of CM remnant-retinoids by the HepG2 cell involves retinyl ester hydrolysis and the secretion of retinol and other more polar metabolites. These processes were regulated in part by the concentration of retinoid delivered by the CM remnant and by the initial retinoid content of the cell.     

Expression of calcium-binding protein regucalcin mRNA in the cloned human hepatoma cells (HegG2): Stimulation by insulin

We have isolated an endogenous positive inotropic factor (EPIF) from porcine left heart ventricular tissue, which demonstrated to have only weak digitalis-like properties including the inhibition of myocardial Na⁺,K⁺-ATPase. EPIF completely lacks digitalis-like toxicity such as after-contractions in larger doses. In our recent studies, we have demonstrated that EPIF produces a decrease in the amplitude of the post-rest rapid cooling contracture which indicated that EPIF may release Ca²⁺from the sarcoplasmic reticulum. In the present study, the effects of EPIF were investigated on the Ca²⁺uptake and release properties of SR enriched membrane vesicles from rat heart. At pH 6.8 and in the presence of oxalate, EPIF dose-dependently inhibited the ATPdependent uptake of Ca²⁺by SR vesicles. Concentrations as low as 25 ul (in 1 mL uptake medium) of EPIF caused a 45-47% reduction in the uptake of Ca²⁺within 3-4 min. Increases in EPIF concentration to 50 ul/mL caused additional reduction of only 15-20% in the uptake of Ca²⁺. Concentrations of 25 ul/mL of EPIF had little or no effects on passive release of actively loaded Ca²⁺in SR vesicles. On doubling the concentrations to 50 ul/mL EPIF, however, enhanced the release of Ca²⁺by 25-28% during 1-2 min. and 44-48% after 4 min of incubation of Ca²⁺loaded vesicles in the release medium. Relatively smaller effects of EPIF on Ca²⁺release implies that EPIF may mainly lower the uptake of Ca²⁺in SR. This reduced uptake of Ca²⁺may be explained by the EPIF-induced inhibition of Ca²⁺pump.     

Anti-proliferative effects of the novel squamosamide derivative (FLZ) on HepG2 human hepatoma cells by regulating the cell cycle-related proteins are associated with decreased Ca(2+)/ROS levels

FLZ is a synthetic novel squamosamide derivative and has previously been proved to be a potential drug for Parkinson's disease and Alzheimer's disease. FLZ has strong antioxidant activity, which implies that FLZ could eliminate excessive intracellular reactive oxygen species (ROS) in tumor cells and induce a pathway related to low cellular ROS levels, thereby inhibiting tumor cells proliferation. However, few reports have focused on the antitumor effects of FLZ. In this study, we investigated the antitumor efficacy of FLZ in HepG2 cells and the mechanism of cell growth inhibition. FLZ effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner; meanwhile, it was minimally toxic to normal cells. FLZ induced a significant decrease in oxidative stress through elimination of excessive intracellular ROS and strengthening of the glutathione antioxidant system. In addition, FLZ can effectively attenuate redundant [Ca(2+)](i), thereby avoiding uncontrolled amplification by Ca(2+)/ROS positive feedback. Furthermore, Western blot showed that FLZ inhibited phosphorylation of Akt and retinoblastoma protein (Rb), down-regulated the expressions of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), and enhanced the expression of CDK inhibitor p27(kip1), while did not affect CDK4 expression. These results suggest that FLZ has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the expression or activation of cell-cycle regulatory proteins, which are associated with decreased Ca(2+)/ROS levels, and indicate that FLZ is a potential liver cancer drug worthy of further research and development.     

Differential effects of some antibiotics on paraoxonase enzyme activity on human hepatoma cells (HepG2) in vitro

Serum paraoxonase (aryldialkylphosphatase, EC 3.1.8.1., PON1) is an esterase protein synthesised by the liver and released into the serum, where it is associated with HDL lipoproteins. In this study, we have determined the in vitro effects of the following antibiotics: sodium ampicillin, ciprofloxacin, Rifamycin SV and clindamiycin phosphate, on human hepatoma (HepG2) cells (liver hPON1). All the antibiotics caused a dose-dependent and time-dependent decrease in the paraoxonase activity while Rifamycin SV was the most effective antibiotic due to its low 50% inhibition concentration (IC₅₀) value. Liver hPON1 activity was determined using paraoxon as a substrate. The IC₅₀ values of the drugs were calculated from graphs of hydratase activity (%) by plotting concentration of the drugs that showed an inhibition effect.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Cadmium cation increases the production and mRNA levels of insulin-like growth factor-binding protein-1 in HepG2

The production of insulin-like growth factor-binding protein-1 (IGFBP-1) in HepG2 was increased by cadmium cation (Cd2+) at 3 microM, but not by other divalent cations. The mRNA level of IGFBP-1 was also increased by the administration of 3 microM of Cd(2+). These results suggest that Cd(2+) impacts the gene expression of IGFBP-1, which leads to production of IGFBP-1.     

Malondialdehyde modification of lipoprotein(a) produces avid uptake by human monocyte-macrophages.

Increased plasma levels of the apoB-100-containing lipoprotein(a) (Lp(a)) are associated with an increased risk for atherosclerosis and myocardial infarction, but the mechanisms by which lipoprotein(a) may accelerate these processes remain obscure. In this study we have investigated the impact of the association of apoprotein(a) with the low density lipoprotein (LDL)-like Lp(a) particle upon specificity of receptor recognition after lipoprotein modification by malondialdehyde or transition metal-induced oxidation. We have determined that radioiodination labels both apoprotein components of Lp(a), that malondialdehyde modification produces an anionic lipoprotein comparable to native Lp(a) in Stokes' radius, and that N,N'-disubstituted 1-amino-3-iminopropene derivatives preferentially cross-link apoprotein(a) to apoB-100 protein. Like LDL, native Lp(a) is recognized in human monocyte-macrophages by the LDL receptor. Like LDL, progressive modification of Lp(a) by malondialdehyde abolishes lipoprotein recognition by the LDL receptor and produces uptake and hydrolysis by the scavenger receptor of human monocyte-macrophages. We propose that intimal retention of Lp(a) by extracellular components of the atherosclerotic reaction places the lipoprotein in a microenvironment favoring subsequent peroxidative modification. The chronic production of lipid peroxide-modified Lp(a) together with unmitigated cellular clearance by scavenger receptors may contribute to the accumulation of lipoprotein-derived lipid in macrophage-derived foam cells of the atherosclerotic reaction.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2

We have demonstrated that physiological concentrations of 17 beta-estradiol increase nuclear estrogen-specific binding sites in the human hepatoma cell line HepG2 7- to 10-fold and the rate of accumulation of secreted apolipoprotein C-II (apo-C-II), 2.5-fold (Tam, S-P., Archer, T. K., and Deeley, R. G. (1985) J. Biol. Chem. 260, 1670-1675). Apo-C-II is the major activator of lipoprotein lipase, an enzyme which plays a key role in lipoprotein catabolism. In order to define more precisely the mechanism by which estrogen influences apo-C-II production, we have synthesized a triacontanucleotide DNA probe that is complementary to apo-C-II mRNA. We have used the probe both in Northern hybridization experiments and in DNA excess titrations to quantify apo-C-II mRNA in hormonally treated HepG2 cells and various primate tissues. These studies revealed that: 1) the concentration of apo-C-II mRNA in HepG2 cells is comparable with that present in human liver; 2) treatment of the cells with low levels of estrogen results in a doubling of the apo-C-II mRNA concentration; 3) the apo-C-II mRNA concentration in monkey liver is 60- to 70-fold greater than in the intestine and 2.5-fold higher than in human liver.