Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed in vitro to carbon ions and argon ions at the HIRFL

Human hepatoma (SMMC-7721) and normal liver (L02) cells were irradiated with c-rays, 12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou (HIRFL). By using the Calyculin-A induced premature chromosome condensation technique, chromatid-type breaks and isochromatid-type breaks were scored separately. Tumor cells irradiated with heavy ions produced a majority of isochromatid break, while chromatid breaks were dominant when cells were exposed to c-rays. The relative biological effectiveness (RBE) for irradiation-induced chromatid breaks were 3.6 for L02 and 3.5 for SMMC-7721 cell lines at the LET peak of 96 keVlm 1 12C6+ ions, and 2.9 for both of the two cell lines of 512 keVlm 1 36Ar18+ ions. It suggested that the RBE of isochromatid-type breaks was pretty high when high-LET radiations were induced. Thus we concluded that the high production of isochromatid-type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high-LET radiation exposure.

Effect of 80.55 MeV/u(12)C(6+) ions on radiosensitivity and cell cycle of human hepatoma cell lines

In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u(12)C(6+) ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 (survival fraction at 2 gray) of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to C-12(6+) ions.

beta-Glucan extracted from the medicinal mushroom Agaricus blazei prevents the genotoxic effects of benzo[a]pyrene in the human hepatoma cell line HepG2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

(-)-Cubebin is a lignan extracted from the seeds of the pepper Piper cubeba, a commonly eaten spice with beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated the effects of (-)-cubebin on the cytotoxicity, cell proliferation kinetics, mutagenicity and expression of p38 MAP kinase and glutathione S-transferase a2 (GSTa2) using real-time RT-PCR in Rattus norvegicus hepatoma cells. We found that 280 μM (-)-cubebin was cytotoxic after 24, 48 and 72. h of exposure, but not mutagenic at 0.28 μM, 2.8 μM and 28 μM after 26. h. Similarly, exposure to 0.28 μM, 2.8 μM and 28 μM (-)-cubebin for 24, 48, 72 and 96. h did not alter the cell proliferation kinetics. Cells exposed to 28 μM (-)-cubebin for 24. h did not exhibit changes in p38 MAP kinase and GSTa2 expression, indicating that cellular changes were not induced by extracellular stimuli and that (-)-cubebin is likely not metabolized via this pathway. Our results suggest that high levels of (-)-cubebin should be consumed with caution due to the cytotoxic effect observed at the highest concentration. However, at lower concentrations, no cytotoxic, mutagenic or proliferative effects were observed, providing further evidence of the safety of consuming (-)-cubebin. © 2013 Elsevier Inc.

Regulation of human GH receptor gene transcription by 20 and 22 kDa GH in a human hepatoma cell line.

The human GH gene is 1.7 kilobase pairs (kb) in length and is composed of five exons and four introns. This gene is expressed in the pituitary gland and encodes a 22 kDa protein. In addition to this predominant (75%) form, 5-10% of pituitary GH is present as a 20 kDa protein that has an amino acid (aa) sequence identical to the 22 kDa form except for a 15 aa internal deletion of residues 32-46 as a result of an alternative splicing event. Because it has been reported that non-22-kDa GH isoforms might be partly responsible for short stature and growth retardation in children, the aim of this study was to compare the impact of both 22 kDa and 20 kDa GH on GH receptor gene (GH receptor/GH binding protein (GHR/GHBP)) expression. Various concentrations of 20 kDa and 22 kDa GH (0, 2, 5, 12.5, 25, 50 and 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was measured by quantitative PCR. Addition of either 20 kDa or 22 kDa GH, at low or normal physiological concentrations (0, 2, 5, 12.5, 25 or 50 ng/ml) induced a dose-dependent increase in GHR/GHBP expression. However, a supraphysiological concentration of 20 kDa GH (150 ng/ml) resulted in a significantly lower (P<0.05) downregulation of GHR/GHBP gene transcription compared with the downregulation achieved by this concentration of 22 kDa GH. This difference might be explained by a decreased ability to form a 1 : 1 complex with GHR and/or GHBP, which normally occurs at high concentrations of GH. Nuclear run-on experiments and GHBP determinations confirmed the changes in GHR/GHBP mRNA levels. In conclusion, we report that both 20 kDa and 22 kDa GH, in low and normal physiological concentrations, have the same effect on regulation of GHR/GHBP gene transcription in a human hepatoma cell line. At a supraphysiological concentration of 150 ng/ml, however, 20 kDa GH has a less self-inhibitory effect than the 22 kDa form.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Piper and Vismia Species from Colombian Amazonia Differentially Affect Cell Proliferation of Hepatocarcinoma Cells

There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 mu g/mL) or even protected cells from basal death (P. putumayoense) in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS). These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.

EROD activities in a primary cell culture of grass carp (Ctenopharyngodon idellus) hepatocytes exposed to polychlorinated aromatic hydrocarbonas

Aryl hydrocarbon (Ah) receptor (Ah-agonist) effects of environmental samples containing polychlorinated aromatic hydrocarbons were evaluated using a 7-ethoxyresorufin-O-deethylase (FROD) assay of a primary hepatocyte culture from grass carp (Ctenopharyngodon idellus). The results were compared with those obtained from the assay using the rat hepatoma cell line H4IIE and chemical analysis using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). A dose-response relationship was observed between the EROD activities, either from primary hepatocyte culture assay or from H4IIE assay, and concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that the assay based on the H4IIE cell line (EC50 = 0.83 mug/mL) is more sensitive to TCDD than the assay based on primary hepatocyte Culture (EC50 = 9.7 pg/mL). In tests of environmental samples, the results from the assay using primary hepatocyte culture were comparable to those from the assay using the H4IIE cell line and chemical analysis of concentrations of mixtures of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDF). The lack of a change in the activities of glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) in cell culture upon exposure to TCDD indirectly indicates that the compound is persistent to biodegradation in the cell culture system. (C) 2004 Elsevier Inc. All rights reserved.

Mammalian cell cultures as a model system for the determination of cytotoxicity induced by cholesterol oxidation products

Oxysterols are products of cholesterol oxidation, which may be produced endogenously or may be absorbed from the diet where they are commonly found in foods of animal origin. Oxysterols are known to be cyctotoxic to cells in culture and mode of toxicity has been identified as apoptosis in certain cell lines. The cytotoxicity of the oxysterols 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) was examined in two human cell lines, HepG2, a hepatoma cell line, and U937, a monocytic cell line. Both 25-OH and 7β-OH were cytotoxic to the HepG2 cell line but apoptotic cells were not detected and it was concluded that cells underwent necrosis. 25-OH was not cytotoxic to the U937 cell line but it was found to have a cytostatic effect. 7β-OH was shown to induce apoptosis in the U937 line. The mechanism of oxysterol-induced apoptosis has not yet been fully elucidated, however the generation of an oxidative stress and the depletion of glutathione have been associated with the initial stages of the apoptotic process. The concentration of cellular antioxidant enzyme, superoxide dismutase (SOD) was increased in association with 7β-OH induced apoptosis in the U937 cell line. There was no change in the glutathione concentration or the SOD activity of HepG2 cells, which underwent necrosis in the presence of 7β-OH. Many apoptotic pathways center on the activation of caspase-3, which is the key executioner protease of apoptosis. Caspase-3 activity was also shown to increase in association with 7β-OH-induced apoptosis in U937 cells but there was no significant increase in caspase-3 activity in HepG2 cells. DNA fragmentation is regarded as the biochemical hallmark of apoptosis, therefore the comet assay as a measure of DNA fragmentation was assessed as a measure of apoptosis. The level of DNA fragmentation induced by 7β-OH, as measured using the comet assay, was similar for both cell lines. Therefore, it was concluded that the comet assay could not be used to distinguish between 7β-OH-induced apoptosis in U937 cells and 7β-OH-induced necrosis in HepG2 cells. The cytotoxicity and apoptotic potency of oxysterols 25-OH, 7β-OH, cholesterol- 5a,6a-epoxide (a-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 19-hydroxy-cholesterol (19-OH), and 7-ketocholesterol (7-keto) was compared in the U937 cell line. 7 β-OH, β-epoxide and 7-keto were found to induce apoptosis in U937 cells. 7β-OH-induced apoptosis was associated with a decrease in the cellular glutathione concentration and an increase in SOD activity, 7-keto and β-epoxide did not affect the glutathione concentration or the SOD activity of the cells.a-Epoxide, 19-OH and 25-OH were not cytotoxic to the U937 cell line.

Radiation-induced intercellular signaling mediated by cytochrome-c via a p53-dependent pathway in hepatoma cells

The tumor suppressor p53 has a crucial role in cellular response to DNA damage caused by ionizing radiation, but it is still unclear whether p53 can modulate radiation-induced bystander effects (RIBE). In the present work, three different hepatoma cell lines, namely HepG2 (wild p53), PLC/PRF/5 (mutation p53) and Hep3B (p53 null), were irradiated with c-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that the radiosensitivity of HepG2 cells was higher than that of PLC/PRF/5 and Hep3B cells. Only irradiated HepG2 cells, rather than irradiated PLC/PRF/5 or Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20 mu M pifithrin-alpha, an inhibitor of p53 function, or 5 lM cyclosporin A (CsA), an inhibitor of cytochrome- c release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation, cytochrome- c was released from mitochondria into the cytoplasm only in HepG2 cells in a p53- dependent manner, but not in PLC/PRF/5 and Hep3B cells. Interestingly, when 50 lg/ml exogenous cytochrome- c was added into cell co- culture medium, RIBE was significantly triggered by irradiated PLC/PRF/5 and Hep3B cells, which previously failed to provoke a bystander effect. In addition, this exogenous cytochrome- c also partly recovered the RIBE induced by irradiated HepG2 cells even with CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and that a p53- dependent release of cytochrome- c may be involved in the RIBE. Oncogene (2011) 30, 1947- 1955; doi: 10.1038/onc. 2010.567; published online 6 December 2010

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

Genotoxic, mutagenic and cytotoxic effects of the commercial dye CI Disperse Blue 291 in the human hepatic cell line HepG2

Textile dyes are discarded into the aquatic ecosystem via industrial effluents and potentially expose humans and local biota to adverse effects. The commercial dye CI Disperse Blue 291 which contains the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2), was tested for genotoxicity and cytotoxicity in the human hepatoma cell line HepG2, using the comet assay, micronucleus (MN) test and a cell viability test. Five different concentrations of the test compound were examined: 200 mu g/ml, 400 mu g/ml, 600 mu g/ml, 800 mu g/ml and 1000 mu g/ml. An increase in comet tail length and in the frequency of MN was detected with exposure of cells to concentrations of the commercial dye from 400 pg/ml. Furthermore, the dye was found to decrease cell viability. The results of this study demonstrate for the first time the genotoxic and mutagenic effects of the dye CI Disperse Blue 291 in mammalian cells, thus stressing the need to develop non-mutagenic dyes and to invest in improving the treatment of effluents. These measures will help to prevent harmful effects that these compounds can have on humans and aquatic organisms that come in contact with them. (C) 2007 Elsevier Ltd. All rights reserved.

α-Tocopherol transfer protein stimulates the secretion of α-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway

Vitamin E (α-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. α-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). α-Tocopherol transfer protein (αTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of αTTP-mediated α-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of α-tocopherol was more efficient in cells expressing αTTP than in matched cells lacking αTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on α-tocopherol secretion, indicating that αTTP-mediated α-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited α-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that αTTP present in the liver cytosol functions to stimulate secretion of cellular α-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.

Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.