Nonalcoholic steatohepatitis induced by a high‐fat diet promotes diethylnitrosamine‐initiated early hepatocarcinogenesis in rats

It has been suggested that patients with nonalcoholic steatohepatitis (NASH) may have high risk for liver cancer. However, it is unknown whether high‐fat diet (HFD) induced NASH promotes hepatocarcinogenesis. In this study, Sprague‐Dawley rats were injected with a low dose of hepatic carcinogen diethylnitrosamine (DEN) and then fed either Lieber‐DeCarli control diet (CD) or HFD for 6 weeks. Liver histology and the hepatic placental form of glutathione S‐transferase (P‐GST) positive foci were examined. Expression levels of proliferating cell nuclear antigen (PCNA), cyclinD1, phosphorylated mitogen‐activated protein kinase (MAPK) including extracellular signal‐regulated kinase (ERK) and p38, as well as tumor necrosis factor‐alpha (TNF‐α), and nuclear factor‐kappaB (NF‐κB) were measured in the liver. Induction of lipid peroxidation end products (malondialdehyde plus 4‐hydroxynonenal) in liver and apoptotic hepatocytes were also assessed. Results showed that HFD‐fed rats developed significantly higher incidence and multiplicity of P‐GST positive foci along with more fat accumulation, infiltration of inflammatory cells and higher lipid peroxidation in the liver, when compared with rats fed the CD. This high prevalence of hepatic lesions in the liver was accompanied by greater PCNA expression and cyclinD1 protein concentration but little change in hepatocyte apoptosis. HFD feeding elevated hepatic phosphorylated ERK but reduced phosphorylated p38 when compared with the CD feeding. In addition, a significantly higher expression of TNF‐α mRNA and nuclear NF‐κB p65 protein were observed in HFD group than in CD group. These data clearly demonstrate that NASH induced by HFD promoted DEN‐initiated early hepatocarcinogenesis, which was associated with elevated TNF‐α/NF‐κB signaling and MAPK related hepatocyte proliferation. © 2008 Wiley‐Liss, Inc.     

Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice

The farnesoid X receptor (FXR) controls the synthesis and transport of bile acids (BAs). Mice lacking expression of FXR, designated Fxr-null, have elevated levels of serum and hepatic BAs and an increase in BA pool size. Surprisingly, at 12 months of age, male and female Fxr-null mice had a high incidence of degenerative hepatic lesions, altered cell foci and liver tumors including hepatocellular adenoma, carcinoma and hepatocholangiocellular carcinoma, the latter of which is rarely observed in mice. At 3 months, Fxr-null mice had increased expression of the proinflammatory cytokine IL-1beta mRNA and elevated beta-catenin and its target gene c-myc. They also had increased cell proliferation as revealed by increased PCNA mRNA and BrdU incorporation. These studies reveal a potential role for FXR and BAs in hepatocarcinogenesis.     

Dietary lycopene and tomato extract supplementations inhibit nonalcoholic steatohepatitis‐promoted hepatocarcinogenesis in rats

Epidemiological and experimental studies provide supportive evidence that lycopene (LY), a major carotenoid from tomatoes and tomato products, may act as a chemopreventive agent against certain types of cancers. We recently showed that high‐fat diet (HFD)‐induced nonalcoholic steatohepatitis (NASH) promoted diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis in a rat model. Using this model, we investigated the efficacy of an equivalent dosage of dietary LY from either a pure compound or a tomato extract (TE) against NASH‐promoted hepatocarcinogenesis. Six groups of rats were injected with DEN and then fed either Lieber‐DeCarli control diet or HFD with or without LY or TE for 6 weeks. Results showed that both LY and TE supplementations significantly decreased the number of altered hepatic foci expressing the placental form of glutathione S‐transferase in the livers of HFD‐fed rats. This was associated with significantly lower proliferating cell nuclear antigen positive hepatocytes and cyclinD1 protein, as well as decreased activation of extracellular signal‐regulated kinase and nuclear NF‐κB. Although both LY and TE supplementations reduced HFD‐induced lipid peroxidation in the livers, we observed significantly decreased cytochrome P450 2E1, inflammatory foci and mRNA expression of proinflammatory cytokines (TNF‐α, IL‐1β and IL‐12) in the HFD+TE fed group but increased nuclear NF‐E2‐related factor‐2 and heme oxygenase‐1 proteins in the HFD+LY fed group, relative to HFD feeding alone. These data indicate that LY and TE can inhibit NASH‐promoted hepatocarcinogenesis mainly as a result of reduced oxidative stress, which could be fulfilled through different mechanisms.     

Gut-liver axis-mediated mechanism of liver cancer: A special focus on the role of gut microbiota

Gut microbiota and the mammalian host share a symbiotic relationship, in which the host provides a suitable ecosystem for the gut bacteria to digest indigestible nutrients and produce useful metabolites. Although gut microbiota primarily reside in and influence the intestine, they also regulate liver function via absorption and subsequent transfer of microbial components and metabolites through the portal vein to the liver. Due to this transfer, the liver may be continuously exposed to gut-derived metabolites and components. For example, short-chain fatty acids (SCFA) produced by gut microbiota, through the fermentation of dietary fiber, can suppress inflammation via regulatory T cell induction through SCFA-induced epigenetic mechanisms. Additionally, secondary bile acids (BA), such as deoxycholic acid, produced by gut bacteria through the 7α-dehydroxylation of primary BAs, are thought to induce DNA damage and contribute to the remodeling of tumor microenvironments. Other substances that are also thought to influence liver function include lipopolysaccharides (components of the outer membrane of gram-negative bacteria) and lipoteichoic acid (cell wall component of Gram-positive bacteria), which are ligands of innate immune receptors, Toll-like receptor-4, and Toll-like receptor-2, respectively, through which inflammatory signaling is elicited. In this review, we focus on the role of gut microbiota in the liver microenvironment, describing the anatomy of the gut-liver axis, the role of gut microbial metabolites, and the relationships that exist between gut microbiota and liver diseases, including liver cancer.     

Silencing of microRNA‐122 is an early event during hepatocarcinogenesis from non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus‐independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH‐HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18 weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR‐122 in non‐tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks. Expression of miR‐122 was further decreased in HCCs relative to non‐tumor LC at the age of 18 weeks. Expression of miR‐122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR‐122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR‐122 is an early event during hepatocarcinogenesis from NASH, and that miR‐122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.     

Interplay between bile acids and the gut microbiota promotes intestinal carcinogenesis

The gut microbiota and the bile acid pool play pivotal roles in maintaining intestinal homeostasis. Bile acids are produced in the liver from cholesterol and metabolized in the intestine by the gut microbiota. Gut dysbiosis has been reported to be associated with colorectal cancer. However, the interplay between bile acid metabolism and the gut microbiota during intestinal carcinogenesis remains unclear. In the present study, we investigated the potential roles of bile acids and the gut microbiota in the cholic acid (CA; a primary bile acid)‐induced intestinal adenoma‐adenocarcinoma sequence. Apc ^min/+ mice, which spontaneously develop intestinal adenomas, were fed a diet supplemented with 0.4% CA for 12 weeks. Mice that were fed a normal diet were regarded as untreated controls. In CA‐treated Apc ^min/+ mice, the composition of the gut microbiota was significantly altered, and CA was efficiently transformed into deoxycholic acid (a secondary bile acid) by the bacterial 7α‐dehydroxylation reaction. The intestinal adenoma‐adenocarcinoma sequence was observed in CA‐treated Apc ^min/+ mice and was accompanied by an impaired intestinal barrier function and IL‐6/STAT3‐related low‐grade inflammation. More importantly, microbiota depletion using an antibiotic cocktail globally compromised CA‐induced intestinal carcinogenesis, suggesting a leading role for the microbiota during this process. Overall, our data suggested that the crosstalk between bile acids and the gut microbiota mediated intestinal carcinogenesis, which might provide novel therapeutic strategies against intestinal tumor development.     

Ezetimibe suppresses development of liver tumors by inhibiting angiogenesis in mice fed a high‐fat diet

Nonalcoholic steatohepatitis (NASH) is a common cause of liver cirrhosis and hepatocellular carcinoma (HCC). However, effective therapeutic strategies for preventing and treating NASH‐mediated liver cirrhosis and HCC are lacking. Cholesterol is closely associated with vascular endothelial growth factor (VEGF), a key factor that promotes HCC. Recent reports have demonstrated that statins could prevent HCC development. In contrast, we have little information on ezetimibe, an inhibitor of cholesterol absorption, in regards to the prevention of NASH‐related liver cirrhosis and HCC. In the present study, a steatohepatitis‐related HCC model, hepatocyte‐specific phosphatase and tensin homolog (Pten)‐deficient (Pten^Δhep) mice were fed a high‐fat (HF) diet with/without ezetimibe. In the standard‐diet group, ezetimibe did not reduce the development of liver tumors in Pten^Δhep mice, in which the increase of serum cholesterol levels was mild. Feeding of a HF diet increased serum cholesterol levels markedly and subsequently increased serum levels of VEGF, a crucial component of angiogenesis. The HF diet increased the number of VEGF‐positive cells and vascular endothelial cells in the tumors of Pten^Δhep mice. Kupffer cells, macrophages in the liver, increased VEGF expression in response to fat overload. Ezetimibe treatment lowered cholesterol levels and these angiogenetic processes. As a result, ezetimibe also suppressed inflammation, liver fibrosis and tumor growth in Pten^Δhep mice on the HF diet. Tumor cells were highly proliferative with HF‐diet feeding, which was inhibited by ezetimibe. In conclusion, ezetimibe suppressed development of liver tumors by inhibiting angiogenesis in Pten^Δhep mice with hypercholesterolemia.     

Inhibition of microRNA‐214 ameliorates hepatic fibrosis and tumor incidence in platelet‐derived growth factor C transgenic mice

Differentially regulated microRNA (miRNA) are associated with hepatic fibrosis; however, their potential usefulness for blocking hepatic fibrosis has not been exploited fully. We examined the expression of miRNA in the liver of a transgenic mouse model in which platelet‐derived growth factor C (PDGF‐C) is overexpressed (Pdgf‐c Tg), resulting in hepatic fibrosis and steatosis and the eventual development of hepatocellular carcinoma (HCC). Robust induction of miR‐214 correlated with fibrogenesis in the liver of Pdgf‐c Tg mice, atherogenic high‐fat diet‐induced NASH mice, and patients with chronic hepatitis B or C. Pdgf‐c Tg mice were injected with locked nucleic acid (LNA)‐antimiR‐214 via the tail vein using Invivofectamine 2.0 and the degree of hepatic fibrosis and tumor incidence were evaluated. Pdgf‐c Tg mice treated with LNA‐antimiR‐214 showed a marked reduction in fibrosis and tumor incidence compared with saline or LNA‐miR‐control‐injected control mice. In vitro, LNA‐antimiR‐214 significantly ameliorated TGF‐β1‐induced pro‐fibrotic gene expression in Lx‐2 cells. MiR‐214 targets a negative regulator of EGFR signaling, Mig‐6. Mimic‐miR‐214 decreased the expression of Mig‐6 and increased the levels of EGF‐mediated p‐EGFR (Y1173 and Y845) and p‐Met (Tyr1234/1235) in Huh‐7 cells. Conversely, LNA‐antimiR‐214 repressed the expression of these genes. In conclusion, miR‐214 appears to participate in the development of hepatic fibrosis by modulating the EGFR and TGF‐β signaling pathways. LNA‐antimiR‐214 is a potential therapy for the prevention of hepatic fibrosis.     

NAFLD exacerbates cholangitis and promotes cholangiocellular carcinoma in mice

Nonalcoholic fatty liver disease (NAFLD) is an increasingly common condition, affecting up to 25% of the population worldwide. NAFLD has been linked to several conditions, including hepatic inflammation, fibrosis, and hepatocellular carcinoma (HCC), however the role of NAFLD in cholangitis and the development of cholangiocellular carcinoma (CCC) remains poorly understood. This study investigated whether a high-fat diet (HFD) promotes cholangitis and the development of CCC in mice. We used liver-specific E-cadherin gene (CDH1) knockout mice, CDH1^∆Liv, which develop spontaneous inflammation in the portal areas along with periductal onion skin-like fibrosis, similar to that of primary sclerosing cholangitis (PSC). An HFD or normal diet (ND) was fed to CDH1^∆Liv mice for 7 mo. In addition, CDH1^∆Liv mice were crossed with LSL-Kras^G12D mice, fed an HFD, and assessed in terms of liver tumor development. The extent of cholangitis and number of bile ductules significantly increased in mice fed an HFD compared with ND-administered CDH1^∆Liv mice. The numbers of Sox9 and CD44-positive stem cell-like cells were significantly increased in HFD mice. LSL-Kras^G12D /CDH1^∆Liv HFD mice exhibited increased aggressiveness along with the development of numerous HCC and CCC, whereas LSL-Kras^G12D/CDH1^∆Liv ND mice showed several macroscopic tumors with both HCC and CCC components. In conclusion, NAFLD exacerbates cholangitis and promotes the development of both HCC and CCC in mice.     

B lymphocytes repress hepatic tumorigenesis but not development in Hras12V transgenic mice

Increasing reports show noninflammation underlying HCC, challenging our understanding of the roles of the immune system in hepatocarcinogenesis. By exploring a mouse model of hepatic tumor induced by hepatocyte‐specific expression of the Hras12V oncogene without obvious inflammation, we found that the proportion of B cells, but not T cells, progressively and significantly decreased in 3, 5‐month‐old transgenic mice (Tg) compared with non‐transgenic mice. Notably, the proportions of total and activated B and T cells all significantly decreased in 9‐month‐old Tg with multiple massive hepatic tumors. Together with the decreased B cell proportion, serum IgG1/2 also significantly decreased in 5, 9‐month‐old Tg. Interestingly, homozygous Tg showed significantly higher B cell proportion and IgG2 levels, accompanied by significantly lower incidences of liver nodules but not adenomas and carcinomas compared with heterozygous Tg. Treatment of Tg with PCI‐32765, a potent Bruton's tyrosine kinase (BTK) inhibitor for suppressing B cell proliferation and activation, significantly decreased the B cell proportion and IgG2 levels, accompanied by a significantly higher incidence of liver nodules, but had no effects on adenoma and carcinoma. Treatment of Tg with insulin‐like growth factor 1 (IGF‐1) significantly increased the B cell proportion and IgG2 levels, accompanied by a significantly lower incidence of liver nodules and carcinoma, but had no effects on adenoma. Conclusively, B cells and IgG2 may play important roles in suppressing hepatic tumorigenesis, but not development. In addition, hepatocyte‐specific expression of the ras oncogene may play roles in suppressing B cells, while developed hepatic tumors suppress both B and T cells.     

Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4

Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.     

Liver specific overexpression of platelet‐derived growth factor‐B accelerates liver cancer development in chemically induced liver carcinogenesis

A genetic basis of hepatocellular carcinoma (HCC) has been well‐established and major signaling pathways, such as p53, Wnt‐signaling, transforming growth factor‐β (TGF‐β) and Ras pathways, have been identified to be essential to HCC development. Lately, the family of platelet‐derived growth factors (PDGFs) has shifted to the center of interest. We have reported on spontaneously developing liver fibrosis in PDGF‐B transgenic mice. Since HCC rarely occurs in healthy liver, but dramatically increases at the cirrhosis stage of which liver fibrosis is a preliminary stage, we investigated liver cancer development in chemically induced liver carcinogenesis in these mice. HCC induction was performed by treatment of the mice with diethylnitrosamine and phenobarbital. At an age of 6 months, the tumor development of these animals was analyzed. Not only the development of dysplastic lesions in PDGF‐B transgenic mice was significantly increased but also their malignant transformation to HCC. Furthermore, we were able to establish a key role of PDGF‐B signaling at diverse stages of liver cancer development. Here, we show that development of liver fibrosis is likely through upregulation of TGF‐β receptors by PDGF‐B. Additionally, overexpression of PDGF‐B also leads to an increased expression of β‐catenin as well as vascular endothelial growth factor and platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31), all factors with established roles in carcinogenesis. We were able to extend the understanding of key genetic regulators in HCC development by PDGF‐B and decode essential downstream signals.     

Secondary bile acid‐induced dysbiosis promotes intestinal carcinogenesis

The gut microbiota plays an important role in maintaining intestinal homeostasis. Dysbiosis is associated with intestinal tumorigenesis. Deoxycholic acid (DCA), a secondary bile acid increased by a western diet, correlates with intestinal carcinogenesis. However, evidence relating bile acids, intestinal microbiota and tumorigenesis are limited. In our study, we investigated the effect of DCA on induction of intestinal dysbiosis and its roles in intestinal carcinogenesis. Alteration of the composition of the intestinal microbiota was induced in DCA‐treated APC^min/+ mice, which was accompanied by impaired intestinal barrier, gut low grade inflammation and tumor progression. The transfer of fecal microbiota from DCA‐treated mice to another group of Apc^min/+ mice increased tumor multiplicity, induced inflammation and recruited M2 phenotype tumor‐associated macrophages. Importantly, the fecal microbiota transplantation activated the tumor‐associated Wnt/β‐catenin signaling pathway. Moreover, microbiota depletion by a cocktail of antibiotics was sufficient to block DCA‐induced intestinal carcinogenesis, further suggesting the role of dysbiosis in tumor development. Our study demonstrated that alteration of the microbial community induced by DCA promoted intestinal carcinogenesis.     

PTPRO-mediated autophagy prevents hepatosteatosis and tumorigenesis

Autophagy plays a critical role in the progression of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Protein tyrosine phosphatase receptor type O (PTPRO) was recently identified as a tumor suppressor, but little is known about its role in NASH. Here, we investigated the role of PTPRO-dependent autophagy in insulin resistance, lipid metabolism, and hepatocarcinogenesis. Wild-type (WT) and ptpro^−/− mice were fed a high-fat diet (HFD) for another 16 weeks after diethylnitrosamine (DEN) injection to induce NASH. Ptpro^−/− mice exhibited severe liver injury, insulin resistance, hepatosteatosis and autophagy deficiency compared with WT littermates. PTPRO deletion also promoted the induction of lipogenic target genes and decreases in β-oxidation-related genes. Increased activation of AKT and accumulation of cytoplasmic p53 was detected in ptpro^−/− mice, which in combination repressed autophagy. Intriguingly, hyperinsulinemia involving AKT activation was also exacerbated in HFD-fed mice due to PTPRO deletion. Activation of AKT induced stabilization of the MDMX/MDM2 heterocomplex, thus promoting p53 accumulation in the cytoplasm. Inhibition of AKT restored autophagy and p53 accumulation in hepatocytes, indicating that AKT acts upstream of p53. Due to hyperinsulinemia and autophagy deficiency, a HFD could aggravate steatohepatitis in ptpro^−/− mice. Importantly, the expression of PTPRO was much decreased in human steatohepatitis, which was associated with increased p62 accumulation. Together, these data indicate that PTPRO regulates insulin and lipid metabolism via the PI3K/Akt/MDM4/MDM2/P53 axis by affecting autophagy.     

Gut microbiota and immune system in liver cancer: Promising therapeutic implication from development to treatment

Liver cancer is a leading cause of death worldwide, and hepatocellular carcinoma (HCC) is the most frequent primary liver tumour, followed by cholangiocarcinoma. Notably, secondary tumours represent up to 90% of liver tumours. Chronic liver disease is a recognised risk factor for liver cancer development. Up to 90% of the patients with HCC and about 20% of those with cholangiocarcinoma have an underlying liver alteration. The gut microbiota-liver axis represents the bidirectional relationship between gut microbiota, its metabolites and the liver through the portal flow. The interplay between the immune system and gut microbiota is also well-known. Although primarily resulting from experiments in animal models and on HCC, growing evidence suggests a causal role for the gut microbiota in the development and progression of chronic liver pathologies and liver tumours. Despite the curative intent of “traditional” treatments, tumour recurrence remains high. Therefore, microbiota modulation is an appealing therapeutic target for liver cancer prevention and treatment. Furthermore, microbiota could represent a non-invasive biomarker for early liver cancer diagnosis. This review summarises the potential role of the microbiota and immune system in primary and secondary liver cancer development, focusing on the potential therapeutic implications.     

Therapeutic and analysis model of intrahepatic metastasis reflects clinical behavior of hepatocellular carcinoma.

This study was designed to establish an intrahepatic metastasis model to investigate the biology and therapy of hepatocellular carcinoma (HCC) in mice. A fragment of mouse HCC tumor CBO140C12 was orthotopically implanted into the mouse liver. The number of intrahepatic metastatic colonies and the volume of the implanted tumor increased in a time-dependent manner. At 28 days after fragment implantation, all mice showed intrahepatic metastasis. Intravenous administrations of cisplatin and doxorubicin at 7 and 21 days after the implantation significantly suppressed the growth of the primary tumor nodule, but tended to inhibit intrahepatic metastasis. However, a marked decrease of body weight was observed during the experiment. On the other hand, an inhibitor of matrix metalloproteinases (MMPs), ONO-4817, decreased the gelatinase activity of MMP-9 secreted by CBO140C12 cells, and significantly reduced the number of colonies of intrahepatic metastasis when administered orally. Our established model, which is focused on intrahepatic metastasis, is suitable for evaluating the therapeutic effect of HCC and for analyzing intrahepatic metastasis, because this model reflects the clinical features of HCC and all the steps of tumor metastasis.     

Therapeutic and Analysis Model of Intrahepatic Metastasis Reflects Clinical Behavior of Hepatocellular Carcinoma

This study was designed to establish an intrahepatic metastasis model to investigate the biology and therapy of hepatocellular carcinoma (HCC) in mice. A fragment of mouse HCC tumor CBO140C12 was orthotopically implanted into the mouse liver. The number of intrahepatic metastatic colonies and the volume of the implanted tumor increased in a time-dependent manner. At 28 days after fragment implantation, all mice showed intrahepatic metastasis. Intravenous administrations of cisplatin and doxorubicin at 7 and 21 days after the implantation significantly sup- pressed the growth of the primary tumor nodule, but tended to inhibit intrahepatic metastasis. However, a marked decrease of body weight was observed during the experiment. On the other hand, an inhibitor of matrix metalloproteinases (MMPs), ONO–4817, decreased the gelatinase activity of MMP–9 secreted by CBO140C12 cells, and significantly reduced the number of colonies of intrahepatic metastasis when administered orally. Our established model, which is focused on intrahepatic metastasis, is suitable for evaluating the therapeutic effect of HCC and for analyzing intrahepatic metastasis, because this model reflects the clinical features of HCC and all the steps of tumor metastasis.     

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway

Hepatitis B virus (HBV)‐X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c‐myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (?)/HBV(?) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c‐myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L‐02 cell line or CHL cell lines upon transfection with MCV‐HBx vectors. Stem‐like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming‐related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1‐afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1‐afp vectors. AFP‐siRNA vectors were able to inhibit tumour colony formation and reprogramming‐related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV‐induced hepatocarcinogenesis.     

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin,a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticarcinogenic agent. However, its short half‐life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of “resistant hepatocyte” model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (p < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB‐induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways. © 2008 Wiley‐Liss, Inc.