Liver Cell Proliferation Research Articles

Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment.

Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening.

Antioxidant and Anticancer Properties of the Extracts from Lepisorus thunbergianus (Kaulf.) Ching

Lepisorus thunbergianus (Kaulf.) Ching has been used in folk medicine in Korea. In this study, a L. thunbergianus methanol extract and its fractions were investigated for their antioxidant properties. The results showed that the ethyl acetate and butanol fractions of L. thunbergianus possess potent DPPH radical scavenging activities. Both fractions also possessed reducing power and inhibited reactive oxygen species formation. In addition, the cytotoxic activity of the L. thunbergianus n-hexane fraction (HF) was investigated. The results suggested that the HF remarkably suppressed proliferation of human breast, liver and colon cancer cells. These results demonstrate, for the first time, that L. thunbergianus extract induces apoptosis in SW620 cells, suggesting that L. thunbergianus may have potential as a therapeutic agent for colon cancer.

HBx triggers either cellular senescence or cell proliferation depending on cellular phenotype.

Replicative senescence is a hallmark of chronic liver diseases including chronic hepatitis B virus (HBV) infection, whereas HBV-encoded oncoproteins HBx and preS2 have been found to overcome senescence. HBx possesses a C-terminal truncation mainly in hepatocellular carcinomas but also in noncancerous liver tissues. Here, by cell counting, BrdU incorporation, MTT proliferation assay, cell cycle analysis, SA-βgal staining and Western blotting in primary and malignant cells, we investigated the effect of HBx C-terminal mutants on cellular senescence. HBx C-terminal mutants were found to trigger cellular senescence in primary MRC5 cells, and malignant liver cells Huh7, and SK-Hep1. In contrast, these mutants promoted the proliferation of HepG2 malignant liver cells. The pro-senescent effect of HBx relied on an increased p16(INK4a) and p21(Waf1/Cip1) expression, and a decreased phosphorylation of Rb. Together, these results suggest that the two main variants of HBx present in HBV-infected liver possess opposite effects on cellular senescence that depend on the phenotype of infected cells.

Hepatic regeneration in a rat model is impaired by chemotherapy agents used in metastatic colorectal cancer

PurposeAdministering Oxaliplatin prior to resection of colorectal liver metastases often induces a Sinusoidal Obstruction Syndrome (SOS), which can affect postoperative patient outcome. Bevacizumab (Anti-VEGF-A) can decrease the severity of SOS and the associated risk of postoperative liver failure. We investigated the impact of both Oxaliplatin (Oxali) and Bevacizumab on liver regeneration in a rat model. Material and methodsMale Wistar rats underwent a 70% partial hepatectomy (PH) 3 days after a 2 ml intraperitoneal injection of either saline (controls, n = 17), or Oxaliplatin 10, 20 or 50 mg/kg, 5-Fluorouracil 100 mg/kg (5-FU) and Bevacizumab 5 or 10 mg/kg in various combinations (total 98 rats, 11 groups, n = 5–18/group). Liver regeneration was assessed by remnant liver weight recovery and cell proliferation by immunodetection of BrDU incorporation (days 1, 2, 3, 7). Hepatic mRNA expression levels of VEGF-A and of its 2 receptors (Flt-1 and KDR) were quantified by PCR technique. ResultsLiver regeneration was impaired for 3 days post PH by Oxali 20 alone and Oxali 10 + 5-FU, without any rescue effect by neither Bevacizumab 5 nor 10 mg/kg. Unlike in humans, there were no sinusoidal changes. VEGF-A mRNA expression and receptor 2 (KDR) expressions decreased 24 h post PH in a similar fashion in controls, Oxali 20 and Oxali 10 + 5-FU groups. All groups had recovered over 60% of their liver weight by day 7. ConclusionOxaliplatin causes early hepatocyte proliferation impairment post PH, unaffected by Bevacizumab and unexplained by changes in VEGF-A signalling in a Wistar rat model.

Comparative gene expression analysis in the liver, kidney and blood vessels during renal injury after repeated exposure to tacrolimus in Sprague-Dawley rats

To elucidate the molecular mechanisms associated with renal injury based on multi-organ interactions, we simultaneously examined the changes in expression profiles of the liver, kidneys, and blood vessels after treatment with the nephrotoxic drug tacrolimus. Sprague-Dawley rats were treated daily with tacrolimus and sacrificed 28 d after oral administration. Serum biochemistry analysis of the major injury markers was performed. Histopathological characteristics were also observed. Total RNA was extracted from the liver, kidneys, and blood vessels from the thoracic aorta, followed by microarray analysis. Differentially expressed genes were selected based on 1.5-fold changes and statistical significance (P<0.05). The effects of three dosages of tacrolimus on transcription levels were analyzed within and among the three organs. Gene functions, as well as the biological and toxicological functions of the differentially regulated genes, were analyzed using Ingenuity Pathways Analysis (IPA). IPA identified genes involved in metabolic activation, including lipid metabolism, renal tubule injury, and cell proliferation in tacrolimus-treated livers, kidneys, and blood vessels, respectively. In response to tacrolimus treatment, genes related to lipid metabolic responses were regulated in the three organs similarly. Genes associated with inflammatory response were regulated in the liver and kidneys similarly. Based on the results from this study, we suggest the molecular pathways involved in the response to tacrolimus in multiple organs. We also provide information about candidate genes, to evaluate the toxicity induced by tacrolimus. These results might be helpful to elucidate the underlying mechanisms of nephrotoxicity by interaction among multiple organs.

Abstract 5388: Methionine S-adenosyltransferase 2A (MAT2A) inhibitors for cancer treatment

Abstract Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. In normal liver, methionine S-adenosyltransferase 1 (MAT1), but not MAT2A is expressed. MAT2A is induced in liver cancer. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells and liver cells in vitro and in vivo. Using a biotinylated FIDAS analog, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. FIDAS agents do not bind MAT1A. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC and liver cancer cell growth. FIDAS agents also inhibited specific histone methylation and repressed transcription of c-myc and other oncogenes. FIDAS agents delivered orally repressed CRC xenografts in athymic nude mice. Based on SAR and docking studies, new generations of FIDAS agents were synthesized and tested. An active in vivo metabolite of FIDAS was identified. PK/PD studies were performed. Our findings suggest that FIDAS analogs targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment. Citation Format: Wen Zhang, Vitaliy Sviripa, David Watt, Chunming Liu. Methionine S-adenosyltransferase 2A (MAT2A) inhibitors for cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5388. doi:10.1158/1538-7445.AM2015-5388

Abstract 2812: The chemo-prevention effect of statin on hepatocellular carcinoma cell lines

Abstract Hepatocellular carcinoma (HCC) is rated as the fifth most common malignancy and third in cancer related deaths worldwide. Statins, HMG-CoA reductase inhibitors, are potent cholesterol lowering drugs. Recent epidemiology suggests that statins prevent aggressive HCC development (J Clin. Oncol. 2012; 30:623-630). In previous cell experiments we have shown that statins down-regulate nuclear Akt responses to insulin (J Biol Chem 2010; 285, 27900). We showed that atorvastatin decreases nuclear phosphorylated Akt (pAkt) levels in pancreatic and lung cancer cell lines within minutes. This rapid effect was apparently not mediated by HMG-CoA reductase inhibition; instead there were indications that it was mediated by the P2×7 receptor. Akt has oncogenic activity in HCC and many other cancer types, and we have now tested the possibility that the P2×7-Akt pathway is of importance for the anticipated anticancer effects of statins in HCC. We performed western blotting, immunocytochemistry and transfections assays and found that statins decrease insulin induced pAkt levels in the nucleus. Further, statin decrease the proliferation of liver cells significantly. Hepatocytes lacking P2×7 receptor was insensitive to these effects of statins. Overexpression of P2×7 restituted the statin effect on pAkt levels. Overexpression of Hepatit B X protein vector has been shown to increase pAkt levels (FEBS J 279, 1220) and we found that statin counteract the Hepatit B vector-induced pAkt. These data provide evidence for a signaling pathway by which statins decrease pAkt and its downstream effect on pGSK3β Ser9 in hepatocytes via the P2×7 receptor. As the Akt pathway is deregulated and may act as a driver in HCC development this signaling pathway may have a role in the indicated chemo-preventive effect of statins. Citation Format: Aram Ghalali, Javier Martin-Renedo, Johan Högberg, Ulla Stenius. The chemo-prevention effect of statin on hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2812. doi:10.1158/1538-7445.AM2015-2812

Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production

Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA – hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia.

Synergistic Induction of Potential Warburg Effect in Zebrafish Hepatocellular Carcinoma by Co-Transgenic Expression of Myc and xmrk Oncogenes

Previously we have generated inducible liver tumor models by transgenic expression of Myc or xmrk (activated EGFR homolog) oncogenes in zebrafish. To investigate the interaction of the two oncogenes, we crossed the two transgenic lines and observed more severe and faster hepatocarcinogenesis in Myc/xmrk double transgenic zebrafish than either single transgenic fish. RNA-Seq analyses revealed distinct changes in many molecular pathways among the three types of liver tumors. In particular, we found dramatic alteration of cancer metabolism based on the uniquely enriched pathways in the Myc/xmrk tumors. Critical glycolytic genes including hk2, pkm and ldha were significantly up-regulated in Myc/xmrk tumors but not in either single oncogene-induced tumors, suggesting a potential Warburg effect. In RT-qPCR analyses, the specific pkm2 isoformin Warburg effect was found to be highly enriched in the Myc/xmrk tumors but not in Myc or xmrk tumors, consistent with the observations in many human cancers with Warburg effect. Moreover, the splicing factor genes (hnrnpa1, ptbp1a, ptbp1b and sfrs3b) responsible for generating the pkm isoform were also greatly up-regulated in the Myc/xmrk tumors. As Pkm2 isoform is generally inactive and causes incomplete glycolysis to favor anabolism and tumor growth, by treatment with a Pkm2-specific activator, TEPP-46, we further demonstrated that activation of Pkm2 suppressed the growth of oncogenic liver as well as proliferation of liver cells. Collectively, our Myc/xmrk zebrafish model suggests synergetic effect of EGFR and MYC in triggering Warburg effect in the HCC formation and may provide a promising in vivo model for Warburg effect.

MicroRNA-17-5p activates hepatic stellate cells through targeting of Smad7

A considerable amount of research has focused on the roles of microRNAs (miRNA) in the pathophysiology of liver fibrosis in view of their regulatory effects on hepatic stellate cell (HSC) functions, including proliferation, differentiation, and apoptosis. Recently, miR-17-5p was shown to promote cell proliferation and migration in liver. Transforming growth factor-β1 (TGF-β1) has been characterized as the master fibrogenic cytokine that stimulates HSC activation and promotes progression of liver fibrosis. The issue of whether miR-17-5p plays a role in TGF-β1-induced hepatic fibrogenesis remains to be established. In this study, we demonstrated a dose-/time-dependent increase in miR-17-5p expression in TGF-β1-treated HSCs. Enhanced miR-17-5p expression was additionally observed in CCl4-induced rat liver fibrosis. Inhibition of miR-17-5p led to suppression of HSC proliferation induced by TGF-β1 without affecting cellular apoptosis. Notably, miR-17-5p was significantly associated with TGF-β1-induced expression of type I collagen and α-SMA in HSC. Furthermore, Smad7, a negative regulator of the TGF-β/Smad pathway, was confirmed as a direct target of miR-17-5p. Serum miR-17-5p levels were significantly higher in patients with cirrhosis, compared to healthy controls. Our results collectively indicate that miR-17-5p promotes HSC proliferation and activation, at least in part, via reduction of Smad7, supporting its potential utility as a novel therapeutic target for liver fibrosis.

Proteomic analysis of cell proliferation in a human hepatic cell line (HL-7702) induced by perfluorooctane sulfonate using iTRAQ

Perfluorooctane sulfonate (PFOS) is a commonly used and widely distributed perfluorinated compound proven to cause adverse health outcomes. However, how PFOS affects liver cell proliferation is not well understood. In this experiment, we exposed a human liver cell line (HL-7702) to 50μM PFOS for 48h and 96h. We identified 52 differentially expressed proteins using a quantitative proteomic approach. Among them, 27 were associated with cell proliferation, including hepatoma-derived growth factor (Hdgf) and proliferation biomarkers Mk167 (Ki67) and Top2α. Results from MTT, cell counting, and cell cycle analysis showed low-dose PFOS (<200μM) stimulated HL-7702 cell viability at 48h and 96h, reduced the G0/G1 percentage, and increased the S+G2/M percentage. Moreover, levels of Cyclin D1, Cyclin E2, Cyclin A2, Cyclin B1 and their partner Cdks were elevated, and the expression of regulating proteins like c-Myc, p53, p21 waf/cip1 and Myt1, as well as the phosphorylation levels of p-Wee1(S642), p-Chk1(S345) and p-Chk2(T68), were disturbed. We hypothesized that low-dose PFOS stimulated HL-7702 proliferation by driving cells into G1 through elevating cyclins/cdks expression, and by promoting cell cycle progression through altering other regulating proteins. This research will shed light on the mechanisms behind PFOS-mediated human hepatotoxicity.

β-Arrestin1 enhances hepatocellular carcinogenesis through inflammation-mediated Akt signalling.

G-protein-coupled receptors (GPCR) constitute the largest known superfamily for signal transduction and transmission, and they control a variety of physiological and pathological processes. GPCR adaptor β-arrestins (ARRBs) play a role in cancerous proliferation. However, the effect of ARRBs in inflammation-mediated hepatocellular carcinogenesis is unknown. Here we show that ARRB1, but not ARRB2, is upregulated in inflammation-associated hepatocellular carcinoma (HCC) and paracancerous tissues in humans. A genotoxic carcinogen, diethylnitrosamine (DEN), significantly induces hepatic inflammation, TNF-α production and ARRB1 expression. Although ARRB1 deficiency does not affect hepatic inflammation and TNF-α production, it markedly represses hepatocellular carcinogenesis by suppressing malignant proliferation in DEN-treated mice. Furthermore, TNF-α directly induces hepatic ARRB1 expression and enhances ARRB1 interaction with Akt by binding to boost Akt phosphorylation, resulting in malignant proliferation of liver cells. Our data suggest that ARRB1 enhances hepatocellular carcinogenesis by inflammation-mediated Akt signalling and that ARRB1 may be a potential therapeutic target for HCC.

Immunomodulator 'mushroom beta glucan' induces Wnt/β catenin signalling and improves wound recovery in tilapia and rat skin: a histopathological study.

The present study aims to investigate the effects of mushroom beta glucan (MBG) on wound recovery in partial hepatectomy (PH) in Nile tilapia (Oreochromis niloticus) and in rat skin wound healing examination. Following PH, we focussed on the effects on liver repair ability using in vitro and in vivo tests. In vitro, we examined whether the MBG has an impact on liver cell proliferation, mainly through 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) cell proliferation assay detection method. Results showed that MBG treatment was remarkable in enhancing cell proliferation of hepatocytes and in maintaining the cellular viability. Immunohistochemical staining to analyse Wnt/β-catenin signalling also showed that MBG has the effect of promoting cell proliferation of liver tissues after PH surgery.

Inhibition of hepatitis B virus gene expression and replication by hepatocyte nuclear factor 6.

Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanistically, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a cis element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms. HBV is a major human pathogen whose replication is regulated by host factors. Liver-enriched transcription factors are critical for many liver functions, including metabolism, development, and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this study, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.

Pre-stimulated Mice with Carbon Tetrachloride Accelerate Early Liver Regeneration After Partial Hepatectomy.

The liver has a high capacity of its regeneration. Most hepatic cells are quiescent unless otherwise stimulated such as their injury or ablation. A previous study suggest that pre-activated hepatic cells have a positive effect on their regeneration. In this study, we examined whether the pre-activated hepatic cells for regeneration accelerate the subsequent liver regeneration. We administered a single injection of carbon tetrachloride (CCl4) to mice 7 days before partial hepatectomy (PHx). Liver weight/body weight ratio and several parameters for cell proliferation such as mitotic index and the number of Ki67 positive cells in the liver were examined after PHx as indexes of liver regeneration. Compared to control mice, those pre-stimulated with CCl4 showed earlier liver regeneration 48 h after PHx. Regardless of their accelerated regeneration, pre-stimulated mice showed less cell proliferation than did control mice during liver regeneration. Hepatic fibrosis was not observed in both control and CCl4-pretreated mice after PHx. Mice pre-treated with CCl4 showed the higher matrix metalloproteinase 9 (MMP9) expression than those pre-treated with olive oil. When matrix metalloproteinase 9 (MMP9) activity was inhibited, the pre-stimulated mice did not demonstrate accelerated liver regeneration and they returned to the original state for cell proliferations after PHx. Pre-activated liver by CCl4 promoted its subsequent regeneration after PHx. This was not a cause of fibrosis and partly dependent on MMP9 pre-activity rather than cell proliferation in liver. Our findings would not only provide a novel strategy for liver regeneration without cell proliferation as much as possible and also propose a new method for liver transplantation.

Human health implications from co-exposure to aflatoxins and fumonisins in maize-based foods in Latin America: Guatemala as a case study

Co-occurrence of fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in maize has been demonstrated in many surveys. Combined-exposure to FB1 and AFB1 was of concern to the Joint FAO/WHO Expert Committee on Food Additives because of the known genotoxicity of AFB1 and the ability of FB1 to induce regenerative proliferation in target tissues. Humans living where maize is a dietary staple are at high risk for exposure to both mycotoxins. Our work has focused on Guatemala, a country in Central America where maize is consumed in large amounts every day and where intake of FB1 has been shown to be potentially quite high using biomarker-based studies. In 2012 a survey was conducted which analysed maize samples for FB1 and AFB1 from all 22 departments of Guatemala. The results show that the levels of AFB1 exposure are also potentially quite high in Guatemala, and likely throughout Central America and Mexico. The implications of co-exposure for human health are numerous, but one area of particular concern is the potential of FB1 to modulate AFB1 hepatoxicity and/or hepatocarcinogenicity. Both the mechanism of action of FB1 and its ability to promote liver carcinogenicity in rats and rainbow trout is consistent with this concern. In farm and laboratory animals FB1 inhibits ceramide synthases, key enzymes inde novo ceramide biosynthesis. The inhibition of sphingolipid signalling pathways mediating programmed cell death and activation of pathways stimulating cell proliferation in livers of individuals exposed to AFB1 could contribute to the tumorigenicity of AFB1. Studies investigating the health effects of either toxin should consider the potential for co-exposure to both toxins. Also, in countries where maize-based food are prepared by alkaline treatment of the maize kernels, the effect of traditional processing on AFB1 levels and toxicity needs to be determined, especially for maize highly contaminated with AFB1.

Induction of cell proliferation in the rat liver by the short-term administration of ethyl tertiary-butyl ether.

In the present study, in continuation of our previous experiment in order to investigate the mode of action (MOA) of ethyl tertiary-butyl ether (ETBE) hepatotumorigenicity in rats, we aimed to examine alterations in cell proliferation, that are induced by short-term administration of ETBE. F344 rats were administered ETBE at doses of 0, and 1,000 mg/kg body weight twice a day by gavage for 3, 10, 17 and 28 days. It was found that the previously observed significant increase of P450 total content and hydroxyl radical levels after 7 days of ETBE administration, and 8-OHdG formation at day 14, accompanied by accumulation of CYP2B1/2B2, CYP3A1/3A2, CYP2C6, CYP2E1 and CYP1A1 and downregulation of DNA oxoguanine glycosylase 1, was preceded by induction of cell proliferation at day 3. Furthermore, we observed an increase in regenerative cell proliferation as a result of ETBE treatment at day 28, followed by induction of cell cycle arrest and apoptosis by day 14. These results indicated that short-term administration of ETBE led to a significant early increase in cell proliferation activity associated with induction of oxidative stress, and to a regenerative cell proliferation as an adaptive response, which could contribute to the hepatotumorigenicity of ETBE in rats.

Molecular signalling in hepatocellular carcinoma: Role of and crosstalk among WNT/ß-catenin, Sonic Hedgehog, Notch and Dickkopf-1.

Hepatocellular carcinoma is the sixth most common cancer worldwide. In the majority of cases, there is evidence of existing chronic liver disease from a variety of causes including viral hepatitis B and C, alcoholic liver disease and nonalcoholic steatohepatitis. Identification of the signalling pathways used by hepatocellular carcinoma cells to proliferate, invade or metastasize is of paramount importance in the discovery and implementation of successfully targeted therapies. Activation of Wnt/β-catenin, Notch and Hedgehog pathways play a critical role in regulating liver cell proliferation during development and in controlling crucial functions of the adult liver in the initiation and progression of human cancers. β-catenin was identified as a protein interacting with the cell adhesion molecule E-cadherin at the cell-cell junction, and has been shown to be one of the most important mediators of the Wnt signalling pathway in tumourigenesis. Investigations into the role of Dikkopf-1 in hepatocellular carcinoma have demonstrated controversial results, with a decreased expression of Dickkopf-1 and soluble frizzled-related protein in various cancers on one hand, and as a possible negative prognostic indicator of hepatocellular carcinoma on the other. In the present review, the authors focus on the Wnt⁄β-catenin, Notch and Sonic Hedgehog pathways, and their interaction with Dikkopf-1 in hepatocellular carcinoma.

State of the art: hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, mainly occurring in cirrhotic livers (in 80–90% of cases) after years of chronic inflammation [1,2]. The pathogenesis of HCC is a multistep process associated with changes in the host gene expression of multiple redundant negative-growth regulatory pathways that protect cells against transformation. An imbalance between the proliferation and apoptosis of liver cells is thought to promote tumor development [3]. Annually, 3–6% of cirrhotic patients will develop HCC, mainly men with advanced liver disease. Recent trends in the USA show that men are affected three-times more frequently than women, Asians are affected twice as often as blacks and Hispanics, and blacks are affected twice as often as whites [4]. One explanation for the disproportionate effects of HCC in men may be due to the higher estrogen concentrations present in women, which suppress IL-6 production and inhibit chemically induced liver carcinogenesis [1]. Epidemiology & etiology HCC is the principal cause of death in patients with compensated cirrhosis worldwide. It is largely a problem of less developed regions, where 83% (50% in China alone) of the estimated 782,000 new cases of liver cell cancer worldwide occur (based on 2012 data) [5]. HCC is the fifth most common cancer in men (554,000 cases; 7.5% of the total) and the ninth in women (228,000 cases; 3.4% of the total) [5]. Regions of high incidence (age adjusted rates per 100,000 inhabitants per year in men) are eastern and south-eastern Asia (31.9 and 22.2, respectively). Intermediate rates occur in southern Europe (9.5) and North America (9.3), and the lowest rates are in northern Europe (4.6) and south-central Asia (3.7) [5]. The global age distribution of HCC cases is related to the dominant viral hepatitis in the underlying population and the age at which it was acquired. For example, the disease burden is highest in areas with endemic hepatitis B virus (HBV) infection, such as in sub-Saharan Africa, where the virus is mainly transmitted at birth (with >90% of these cases becoming chronic HBV carriers). However, the diagnosis of HCC often occurs later in North America and southern Europe, where the most common etiology is HBV or hepatitis C virus (HCV) acquired later in life [2]. Several factors have been reported to increase HCC risk among HBV carriers, including male sex, older age, Asian or African ethnicity, family history of HCC, viral (higher levels of HBV repli cation, HBV genotype, longer duration of infection or coinfection with HCV, HIV or hepatitis D virus), clinical (cirrhosis) and environmental or lifestyle factors (exposure to aflatoxin, chronic excessive alcohol consumption or tobacco smoking). Other risk factors that increase the risk in HCV-infected patients include coinfection with HIV, HBV, HCV genotype 1b, old age, the presence of diabetes and obesity and a high level of chronic alcohol consumption. However, both HBV and HCV are highly amenable to preventive measures with antivirals or vaccination (for HBV). In Italy, for example, the Italian Liver Cancer (ITA.LI.CA) database reveals a decline in HCV infection with a concomitant increase in alcohol consumption as the primary etiology for HCC [6].

Effects of cadmium on cell proliferation, apoptosis, and proto-oncogene expression in zebrafish liver cells

Cadmium (Cd) is one of the major transitional metal that has toxic effects in aquatic organisms and their associated ecosystem; however, its hepatic toxicity and carcinogenicity are not very well characterized. We used a zebrafish liver (ZFL) cell line as a model to investigate the mechanism of Cd-induced toxicity on hepatocytes. Our results showed that Cd can be effectively accumulated in ZFL cells in our exposure experiments. Cell cytotoxicity assays and flow cytometer measurements revealed that Cd2+ stimulated ZFL cell proliferation with decreasing apoptotic cell numbers indicating potentially tumorigenic effects of Cd in ZFL cells. Gene expression profiles also indicated that Cd downregulated oncogenes p53 and rad51 and upregulated immediate response oncogenes, growth arrest and DNA damage-inducible (gadd45) genes, and growth factors. We also found dramatic changes in the gene expression of c-jun and igf1rb at different exposure time points, supporting the notion that potentially tumorigenic of Cd-is involved in the activation of immediate early genes or genes related to apoptosis in cancer promotion.