Growth Inhibition Of HepG2 Cells Research Articles

Ecotoxicological evaluation of 4-aminobiphenyl using a test battery

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control, and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective, and quantitative test battery should be developed. In this study, the effects of 4-aminobiphenyl (4-ABP) were investigated using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, development of zebrafish embryos, and inhibition of mammalian cell proliferation. The growth inhibition effects of 4-ABP were assessed on mouse connective tissue fibroblast cells (L929 cells) and human hepatocelluar carcinoma cells (HepG2 cells) by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-trazolium bromide reduction assay. The results reveal that 4-aminobiphenyl is toxic for aquatic organisms and mammalian cells. The system most sensitive to 4-aminobiphenyl is D. magna immobilization, followed by development of zebrafish embryos, and inhibition of cell proliferation. L929 and HepG2 cell growth inhibition bioassays show low sensitivity. These findings indicate that a single model for the possible harmful effect of 4-ABP has its limitations; only a test battery, composed of bioassays on different species, can provide an accurate assessment of the action of 4-ABP in the whole environment.

Transcription activation of FLRG and follistatin by activin A, through Smad proteins, participates in a negative feedback loop to modulate activin A function.

Signaling of TGFbeta family members such as activin is tightly regulated by soluble binding proteins. Follistatin binds to activin A with high affinity, and prevents activin binding to its own receptors, thereby blocking its signaling. We previously identified FLRG gene from a B-cell leukemia carrying a t(11;19)(q13;p13) translocation. We and others have already shown that FLRG, which is highly homologous to follistatin, may be involved in the regulation of the activin function through its binding to activin. In this study, we found that, like follistatin, FLRG protein inhibited activin A signaling as demonstrated by the use of a transcriptional reporter assay, and blocked the activin A-induced growth inhibition of HepG2 cells. We have recently shown that the TGFbeta-induced expression of FLRG occurs at a transcriptional level through the action of Smad proteins. Here we show that activin A increases FLRG and follistatin at both the mRNA and protein levels. We found that Smad proteins are involved in the activin A-induced transcription activation of FLRG and follistatin. Finally we demonstrate that FLRG protein regulates its own activin-induced expression. In conclusion, activin A induces FLRG and follistatin expression. This observation, in conjunction with the antagonistic effect of FLRG and follistatin on activin signaling, indicates that these two proteins participate in a negative feedback loop which regulates the activin function.

Activation of Erk1/Erk2 and transiently increased p53 levels together may account for p21 expression associated with phorbol ester-induced transient growth inhibition in HepG2 cells

In HepG2 cells grown in the presence of serum, enhanced Raf-activation correlated with transient growth inhibition. The activation of Raf was increased either by the phorbol ester-induced activation of protein kinase C (PKC) or by the addition of the PKC inhibitor bisindolylmaleimide I (BIM). Either of these treatments increased the cellular levels of p21 by an Erk1/Erk2 MAP kinase cascade-dependent way, since this increase was prevented by the MEK-inhibitor PD98059. Nevertheless, the growth inhibition correlated with the transient increase of p53 levels as well. Either the activation of PKC with phorbol ester or the addition of BIM to cells growing in serum induced a rapid but transient increase of p53 levels, which preceded growth inhibition. This increase of p53 levels was probably due to the transient stabilisation of p53 and did not require the activation of Erk1/Erk2.

Involvement of p-15INK4b and p-16INK4a Gene Expression in Saikosaponin a and TPA-Induced Growth Inhibition of HepG2 Cells

Saikosaponin a, a purified ingredients of Chinese herb with known antitumor activity can inhibit cell growth and DNA synthesis of hepatoma cell line HepG2. Both mRNA and protein of the CDK inhibitor p-16INK4a and p-15INK4b in HepG2 were greatly induced by saikosaponin a while that of p-21CIP, p-27KIP and other cell cycle related genes were not. In addition, reduced phosphorylation of RB protein is observed in saikosaponin a-treated HepG2. Staurosporin, one of the PKC inhibitors, significantly prevented the saikosaponin a induced growth inhibition suggesting PKC pathway be involved. On the other hand, the phorbol ester tumor promoter TPA (12-O-Tetredecanolyphorbol 13-acetate) also inhibited HepG2 growth and specifically induced p-16INK4a and p-15INK4b mRNA expression. The results suggest that both saikosaponin a and TPA-induced HepG2 growth inhibition are associated with p-15INK4a and p-16INK4b gene expression and might be mediated by PKC signaling pathway.

Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.

Possible involvement of p21/waf1 in the growth inhibition of HepG2 cells induced by hepatocyte growth factor

Possible involvement of p21/waf1 in the growth inhibition of HepG2 cells induced by hepatocyte growth factor

Induction of apoptosis in human hepatoma cells by alpha-fetoprotein.

We have investigated the effects of purified human alpha-fetoprotein (AFP) on the growth of the human hepatocarcinoma-cells HepG2 in culture. Cancer-derived AFP (cAFP), isolated from the culture medium of AFP-secreting HepG2 cells and embryonal AFP (eAFP), isolated from human cord serum, were used for these studies. Both AFP pre parations studied were shown to induce strong dose-dependent inhibition of HepG2 cell proliferation and complete growth arrest at high protein concentrations (more than 0.1 mg/ml). To test whether AFP may trigger an endogenous suicide program in hepatoma cells, we examined whether DNA fragmentation preceded cell death. After exposure of the cells of the high AFP dose (1.0 mg/ml), DNA fragmentation was detected as early as 2 h after treatment, and 70% of cells were apoptotic by 24 h. DNA fragmentation was shown to precede other signs of cell death for several hours. Typical morphological changes of apoptosis were observed after 4 h of exposure of cells to high AFP doses. Low concentrations of cAFP and eAFP (less than 0.1 mg/ml) failed to induce growth inhibition of HepG2 cells, rather showing a weak stimulative effect, demonstrating a biphasic AFP activity. Cell pretreatment with the transcriptional inhibitor actinomycin D had no measurable influence on AFP cytotoxicity. These findings demonstrate that protein synthesis is not required for this mechanism of cell death. The charcoal-treated ligand-free eAFP (eAFPp) had a dose-dependent growth-inhibitory activity, similar to intact protein, but slightly less intensive. The similar growth-inhibitory activities of cAFP, eAFP and eAFPp, which have a significant difference in bound-ligand content, provide evidence that the main role in cell growth regulation may be attributed to the protein moiety of the entire AFP molecule, but not to its ligands. These biologically active AFP ligands could, however, modulate AFP-growth-regulating activity. Growth factor deprivation distinctly enhanced the cytostatic activity of high AFP concentrations and also increased the mitogenic activity of low AFP levels, showing the interdependence of the growth-regulative activity of AFP and growth factors. The findings of this study demonstrated that AFP is directly introduced into the intracellular pathways of cell growth regulation and programmed cell death.

Partial down‐regulation of protein kinase C reverses the growth inhibitory effect of phorbol esters on HepG2 cells

Phorbol ester treatment of HepG2, a human tumorigenic cell line, caused rapid morphological changes characterized by a flattening and spreading of the cells that coincided with a rapid inhibition of thymidine incorporation. Within 24 h, cell division was completely inhibited, suggesting the cells had entered a quiescent state. Continued incubation in the presence of phorbol esters resulted in the resumption of thymidine incorporation and cell division, but this coincided with only a partial down-regulation of PKC activity. Seventy-two hours of treatment was required to obtain down-regulation of greater than 80% of the PKC activity, but reversal of the inhibitory effects occurred between 24 and 48 h after the addition of phorbol esters, when a large proportion of the PKC activity was still present. Northern blot analysis of a number of transcripts showed that the steady-state levels of c-myc and transforming growth factor beta 1 (TGF-beta 1) messages increased only after 3 h of phorbol ester treatment and returned to normal levels after 24 h. C-fos, albumin, and alphafetoprotein messages were not affected, suggesting the differentiation state of the cells was not altered. Therefore, phorbol ester activation of PKC causes an inhibition of HepG2 cell growth initially, but this is unlike the promotion of differentiation seen in other systems. Partial down-regulation of PKC activity causes a reversal of the growth inhibition and the cells return to a normal growth rate. This effect is also clearly different from systems in which phorbol esters have been shown to have a mitogenic effect on cells.