Yue-Xiang Chen

Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4α gene†

Previous studies have shown that hepatocyte nuclear factor‐4α (HNF4α) is a central regulator of differentiated hepatocyte phenotype and forced expression of HNF4α could promote reversion of tumors toward a less invasive phenotype. However, the effect of HNF4α on cancer stem cells (CSCs) and the treatment of hepatocellular carcinoma (HCC) with HNF4α have not been reported. In this study, an adenovirus‐mediated gene delivery system, which could efficiently transfer and express HNF4α, was generated to determine its effect on hepatoma cells (Hep3B and HepG2) in vitro and investigate the anti‐tumor effect of HNF4α in mice. Our results demonstrated that forced re‐expression of HNF4α induced the differentiation of hepatoma cells into hepatocytes, dramatically decreased “stemness” gene expression and the percentage of CD133+ and CD90+ cells, which are considered as cancer stem cells in HCC. Meanwhile, HNF4α reduced cell viability through inducing apparent apoptosis in Hep3B, while it induced cell cycle arrest and cellular senescence in HepG2. Moreover, infection of hepatoma cells by HNF4α abolished their tumorigenesis in mice. Most interestingly, systemic administration of adenovirus carrying the HNF4α gene protected mice from liver metastatic tumor formation, and intratumoral injection of HNF4α also displayed significant antitumor effects on transplanted tumor models. Conclusion: The striking suppression effect of HNF4α on tumorigenesis and tumor development is attained by inducing the differentiation of hepatoma cells—especially CSCs—into mature hepatocytes, suggesting that differentiation therapy with HNF4α may be an effective treatment for HCC patients. Our study also implies that differentiation therapy may present as one of the best strategies for cancer treatment through the induction of cell differentiation by key transcription factors. (HEPATOLOGY 2008.)

Hepatocyte Nuclear Factor 4α Suppresses the Development of Hepatocellular Carcinoma

Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that plays a key role in hepatocyte differentiation and the maintenance of hepatic function, but its role in hepatocarcinogenesis has yet to be examined. Here, we report evidence of a suppressor role for HNF4α in liver cancer. HNF4α expression was progressively decreased in the diethylinitrosamine-induced rat model of liver carcinogenesis. In human liver tissues, HNF4α expression was decreased in cirrhotic tissue and further decreased in hepatocarcinoma relative to healthy tissue. Notably, an inverse correlation existed with epithelial-mesenchymal transition (EMT). Enforced expression of HNF4α attenuated hepatocyte EMT during hepatocarcinogenesis, alleviated hepatic fibrosis, and blocked hepatocellular carcinoma (HCC) occurrence. In parallel, stem cell marker gene expression was inhibited along with cancer stem/progenitor cell generation. Further, enforced expression of HNF4α inhibited activation of β-catenin, which is closely associated with EMT and hepatocarcinogenesis. Taken together, our results suggest that the inhibitory effect of HNF4α on HCC development might be attributed to suppression of hepatocyte EMT and cancer stem cell generation through an inhibition of β-catenin signaling pathways. More generally, our findings broaden knowledge on the biological significance of HNF4α in HCC development, and they imply novel strategies for HCC prevention through the manipulation of differentiation-determining transcription factors in various types of carcinomas.

Long-Term Effects of Mid-Dose Ursodeoxycholic Acid in Primary Biliary Cirrhosis: A Meta-analysis of Randomized Controlled Trials

OBJECTIVES:The effect of ursodeoxycholic acid (UDCA) treatment on survival and liver histological progression of primary biliary cirrhosis (PBC) remains uncertain. The aim of this study is to assess the long-term efficacy of mid-dose UDCA treatment for PBC.METHODS:Electronic databases including Medline, Embase, Cochrane controlled trials register, Science Citation Index, and PUBMED (updated to Nov 2005), and manual bibliographical searches were conducted. A meta-analysis of all long-term randomized controlled trials (RCTs) comparing mid-dose UDCA with placebo or no treatment was performed.RESULTS:Seven RCTs and six reports of their extended follow-up including 1,038 patients were assessed. UDCA could significantly improve liver biochemistry, but had no effect on pruritus and fatigue. UDCA could delay the progression of PBC, especially for early-stage patients. Meta-analysis of the seven RCTs including their extended follow-up showed a significant reduction of the incidence of liver transplantation (OR 0.65, p = 0.01), and a marginally significant reduction of the rate of death or liver transplantation (fixed-effect model: OR 0.76, p = 0.05; random-effect model: OR 0.77, p = 0.3) in the UDCA group, except death (OR 1.01, p = 1). In the sensitivity analyses, which included studies administrating placebo as control, long-term studies (≥48 months), or large size studies (total number of patients ≥100), we all found long-term treatment with UDCA could significantly reduce the incidence of liver transplantation, and death or liver transplantation.CONCLUSIONS:Long-term treatment with mid-dose UDCA can improve liver biochemistry and survival free of liver transplantation in patients with PBC. In addition, UDCA therapy can delay the histological progression in the early-stage patients.

Gemcitabine in the chemoradiotherapy for locally advanced pancreatic cancer: A meta-analysis

AIMS Whether gemcitabine based chemoradiotherapy (GEM-based CRT) is superior to 5-fluorouracil based chemoradiotherapy (5-FU-based CRT) for locally advanced pancreatic cancer (LAPC) remains uncertain. The aim of the present study was to evaluate the effect of GEM-based CRT compared with 5-FU-based CRT. METHODS Electronic database including Medline, Embase, Cochrane controlled trials register, PubMed (update to December 2010) and manual bibliography searches were carried out. A meta-analysis of all randomized clinical trials (RCTs) or other comparative studies comparing GEM-based CRT and 5-FU-based CRT were performed. RESULTS Three RCTs and one retrospective comparative study including 229 patients were assessed. Meta-analysis showed survival advantage of GEM-based CRT compared with 5-FU-based CRT for 12-month (12-mo) survival rates (SRs) (RR=1.54, 95% CI 1.05-2.26, p=0.03). Moreover, there were also trends of benefit for SR after 6-months (RR 1.13, 95% CI 0.98-1.30, p=0.09) and 24-months (24-mo: RR 2.41, 95% CI 0.90-6.48, p=0.08), though the trends did not reach statistical significance. More frequent severe acute hematologic toxicities were found in the GEM-based CRT group. CONCLUSIONS The meta-analysis found that GEM-based CRT was better than 5-FU-based CRT in the treatment of LAPC, especially for 12-mo SRs. However, the acute toxicity should be carefully regarded.

Recombinant Adenovirus Carrying the Hepatocyte Nuclear Factor-1alpha Gene Inhibits Hepatocellular Carcinoma Xenograft Growth in Mice

Hepatocyte nuclear factor‐1alpha (HNF1α) is one of the key transcription factors of the HNF family, which plays a critical role in hepatocyte differentiation. Substantial evidence has suggested that down‐regulation of HNF1α may contribute to the development of hepatocellular carcinoma (HCC). Herein, human cancer cells and tumor‐associated fibroblasts (TAFs) were isolated from human HCC tissues, respectively. A recombinant adenovirus carrying the HNF1α gene (AdHNF1α) was constructed to determine its effect on HCC in vitro and in vivo. Our results demonstrated that HCC cells and HCC tissues revealed reduced expression of HNF1α. Forced reexpression of HNF1α significantly suppressed the proliferation of HCC cells and TAFs and inhibited the clonogenic growth of hepatoma cells in vitro. In parallel, HNF1α overexpression reestablished the expression of certain liver‐specific genes and microRNA 192 and 194 levels, with a resultant increase in p21 levels and induction of G2/M arrest. Additionally, AdHNF1α inhibited the expression of cluster of differentiation 133 and epithelial cell adhesion molecule and the signal pathways of the mammalian target of rapamycin and transforming growth factor beta/Smads. Furthermore, HNF1α abolished the tumorigenicity of hepatoma cells in vivo. Most interestingly, intratumoral injection of AdHNF1α significantly inhibited the growth of subcutaneous HCC xenografts in nude mice. Systemic delivery of AdHNF1α could eradicate the orthotopic liver HCC nodules in nonobese diabetic/severe combined immunodeficiency mice. Conclusion: These results suggest that the potent inhibitive effect of HNF1α on HCC is attained by inducing the differentiation of hepatoma cells into mature hepatocytes and G2/M arrest. HNF1α might represent a novel, promising therapeutic agent for human HCC treatment. Our findings also encourage the evaluation of differentiation therapy for tumors of organs other than liver using their corresponding differentiation‐determining transcription factor. (HEPATOLOGY 2011)

Adenovirus-mediated transfer of siRNA against PAI-1 mRNA ameliorates hepatic fibrosis in rats ☆

BACKGROUND/AIMS Plasminogen activator inhibitor-1 (PAI-1) is a potential profibrotic molecule. The aim of this study was to evaluate the therapeutic effect of PAI-1 small interfering RNA (siRNA) on experimental hepatic fibrosis and investigate the intrinsic mechanisms. METHODS Hepatic fibrosis in rats was induced by dimethylnitrosamine (DMN) administration or bile duct ligation (BDL). An adenovirus carrying PAI-1 shRNA (AdshPAI) was generated and administered via tail vein injection. The expression of PAI-1 was confirmed by real-time RT-PCR and immunohistochemistry. The effect of AdshPAI on fibrosis was evaluated by histological and immunohistochemical examination. RESULTS We found that PAI-1 was downregulated after AdshPAI administration. Liver fibrosis was significantly improved after AdshPAI administration in both DMN and BDL models. AdshPAI treatment facilitated matrix degradation by correcting the levels of matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) through upregulation of MMP9, MMP13 and downregulation of TIMP-1. Moreover, AdshPAI treatment stimulated hepatocellular proliferation and inhibited cellular apoptosis. CONCLUSIONS This study suggests that AdshPAI treatment has a protective effect on hepatocytes and ameliorates liver fibrogenesis. Inhibiting the upregulation of PAI-1 during liver fibrosis may be an antifibrotic pathway worth exploiting.

Treatment of experimental hepatic fibrosis by combinational delivery of urokinase‐type plasminogen activator and hepatocyte growth factor genes

Purpose: To investigate the effect of combinational delivery of urokinase‐type plasminogen activator (uPA) and hepatocyte growth factor (HGF) genes on hepatic fibrosis.

The Restorative Effect of Taurine on Experimental Nonalcoholic Steatohepatitis

Our objective was to explore the restorative effect of taurine on experimental nonalcoholic steatohepatitis (NASH). Thirty-six SD rats were randomly divided into three groups, 12 in each group: the normal group was fed standard rat diet; the model group and the treatment group were both fed a high-fat rat diet for 12 weeks, and the rats in the treatment group were simultaneously injected with taurine subcutaneously for 8 weeks. Hepatic histological change was observed; TNF-α and TGF-β1 protein expression was identified by immunohistochemistry; mRNA expression of TNF-α, TGF-β1, type I procollagen, and adiponectin was measured by RT-PCR; body weight, weight gain, liver weight, and liver index were measured; and biochemical parameters monitored included serum transaminases, serum lipids, fasting plasma glucose, and hepatic level of oxidative stress. Rats in the model group showed a significant increase in liver weight, liver index, serum transaminase activities, serum triglyceride, fasting plasma glucose, and oxidative stress; the mRNA expression of TNF-α, TGF-β1, and type I procollagen increased, whereas the expression of adiponectin decreased significantly, compared with that in the normal group. The typical hepatic lesions of NASH were observed histologically in the model group. Taurine treatment resulted in a significant decrease in liver weight, liver index, serum transaminase activities, serum triglyceride, fasting plasma glucose, and oxidative stress; the mRNA expression of TNF-α, TGF-β1, and type I procollagen decreased, but the expression of adiponectin increased significantly, compared with that in the model group. Histological improvement was observed in the treatment group. In conclusion, taurine could inhibit lipid peroxidation, improve lipid and glucose metabolism, decrease synthesis of TNF-α and TGF-β1, promote synthesis of adiponectin, and have a restorative effect on experimental NASH.

Inhibition of extracellular signal-regulated kinase 1 by adenovirus mediated small interfering RNA attenuates hepatic fibrosis in rats†

Extracellular signal‐regulated kinase 1 (ERK1) is a critical part of the mitogen‐activated protein kinase signal transduction pathway, which is involved in hepatic fibrosis. However, the effect of down‐regulation of ERK1 on hepatic fibrosis has not been reported. Here, we induced hepatic fibrosis in rats with dimethylnitrosamine administration or bile duct ligation. An adenovirus carrying small interfering RNA targeting ERK1 (AdshERK1) was constructed to determine its effect on hepatic fibrosis, as evaluated by histological and immunohistochemical examination. Our results demonstrated that AdshERK1 significantly reduced the expression of ERK1 and suppressed proliferation and levels of fibrosis‐related genes in hepatic stellate cells in vitro. More importantly, selective inhibition of ERK1 remarkably attenuated the deposition of the extracellular matrix in fibrotic liver in both fibrosis models. In addition, both hepatocytes and biliary epithelial cells were proven to exert the ability to generate the myofibroblasts depending on the insults of the liver, which were remarkably reduced by AdshERK1. Furthermore, up‐regulation of ERK1 paralleled the increased expression of transforming growth factor β1 (TGF‐β1), vimentin, snail, platelet‐derived growth factor‐BB (PDGF‐BB), bone morphogenetic protein 4 (BMP4), and small mothers against decapentaplegic‐1 (p‐Smad1), and was in reverse correlation with E‐cadherin in the fibrotic liver. Nevertheless, inhibition of ERK1 resulted in the increased level of E‐cadherin in parallel with suppression of TGF‐β1, vimentin, snail, PDGF‐BB, BMP4, and p‐Smad1. Interestingly, AdshERK1 treatment promoted hepatocellular proliferation. Conclusion: Our study provides the first evidence for AdshERK1 suppression of hepatic fibrosis through the reversal of epithelial‐mesenchymal transition of both hepatocytes and biliary epithelial cells without interference of hepatocellular proliferation. This suggests that ERK1 is implicated in hepatic fibrogenesis and selective inhibition of ERK1 by small interfering RNA may present a novel option for hepatic fibrosis treatment. (HEPATOLOGY 2009.)

Hepatic stellate cells modulate the differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells.

Differentiation of stem cells is tightly regulated by the microenvironment which is mainly composed of nonparenchymal cells. Herein, we investigated effect of hepatic stellate cells (HSCs) in different states on mesenchymal stem cells (MSCs) differentiation. Rat HSCs were isolated and stayed quiescent within 5 days. Primary HSCs were activated by being in vitro cultured for 7 days or cocultured with Kupffer cells for 5 days. MSCs were cocultured with HSCs of different states. Expression of hepatic lineage markers was analyzed by RT‐PCR and immunofluorescence. Glycogen deposition was detected by periodic acid‐schiff staining. MSCs cocultured with HSC‐T6 or Kupffer cell activated HSCs were morphologically transformed into hepatocyte‐like cells. Hepatic‐specific marker albumin was expressed in 78.3% of the differentiated MSCs 2 weeks after initiation of coculture. In addition, the differentiated MSCs also expressed α‐fetoprotein, cytokeratin‐18, glutamine synthetase and phosphoenolpyruvate carboxykinase. Glycogen deposition was detectable in 55.4% of the differentiated MSCs 6 weeks after initiation of coculture. However, the quiescent HSCs or culture activated HSCs did not exert the ability to modulate the differentiation of MSCs. Moreover, Kupffer cell activated HSCs rather than culture activated HSCs expressed hepatocyte growth factor mRNA. We draw the conclusion that fully activated HSCs could modulate MSCs differentiation into hepatocyte‐like cells. J. Cell. Physiol. 217: 138–144, 2008.