Permissiveness of human hepatocellular carcinoma cell lines for hepatitis C virus entry and replication.

Hepatitis C virus (HCV) is a globally prevalent pathogen and is associated with high death rates and morbidity. Since its discovery in 1989, HCV research has been impeded by the lack of a robust infectious cell culture system and thus in vitro studies on diverse genetic backgrounds are hampered because of the limited number of hepatoma cell lines which are able to support different aspects of the HCV life cycle. In the current study, we sought to expand the limited number of permissive cells capable of supporting the diverse phases of the HCV life cycle. Initially, we screened a panel of new hepatoma-derived cell lines, designated BCLC-1, -2, -3, -4, -5, -6, -9 and -10 cells, for their ability to express essential HCV receptors and subsequently to support HCV entry by using the well-characterized HCV pseudoparticle system (HCVpp). Apart from BCLC-9, all BCLC cell lines were permissive for HCVpp infection. Next, BCLC cells were subjected to short- and long-term HCV RNA replication studies using HCV subgenomic replicons. Interestingly, only BCLC-1, -5 and -9 cells, supported short-term HCV RNA replication, but the latter were excluded from further studies since they were refractory for HCV entry. BCLC-1, -5 were able to support long-term HCV replication too; yet BCLC-5 cells supported the highest long-term HCV RNA replication levels. Furthermore, cured BCLC-5 clones from HCV subgenomic replicon, showed increased permissiveness for HCV RNA replication. Strikingly, we were unable to detect endogenous BCLC-5 miR122 expression - an important HCV host factor- and as expected, the exogenous expression of miR122 in BCLC-5 cells increased their permissiveness for HCV RNA replication. However, this cell line was unable to produce HCV infectious particles despite ectopic expression of apolipoprotein E, which in other hepatoma cell lines has been shown to be sufficient to enable the HCV secretion process, suggesting a lack of other host cellular factor(s) and/or the presence of inhibitory factor(s). In conclusion, the establishment of these new permissive cell lines for HCV entry and replication, which possess a different genetic background compared to the well-established models, expands the current repertoire of hepatoma cell lines susceptible to the study of the HCV life cycle and also will aid to further elucidate the cellular determinants that modulate HCV replication, assembly and egress.

Restoring miR122 in human stem-like hepatocarcinoma cells, prompts tumor dormancy through Smad-independent TGF-ß pathway.

miR122 is the prevalent miRNA in adult healthy liver and it is responsible for liver stem cell differentiation towards hepatocyte lineage. Its expression is frequently lost in hepatocellular carcinoma (HCC). We studied the effects of restoring miR122 expression in a distinctive cell line derived from human HCC-BCLC9 cells-with a solid stem-like cell profile, high tumor initiating ability and undetectable miR122 expression. We generated a stable BCLC9 cell line that expresses miR122 (BCLC9-miR122). Restitution of miR122 in BCLC9 cells, decreases cell proliferation rate and reduces significantly tumor size in vivo. BCLC9-miR122 cells down-regulate expression of MYC, KLF4, FOXM1, AKT2 and AKT3 genes and up-regulate FOXO1 and FOXO3A gene expression. In addition, miR122 transfected cells decreased AKT2 kinase activation while decreased FOXO1 and FOXO3A protein inactivation. Reduction in tumor size in BCLC9-miR122 associated with an increase in p38MAPK protein expression and activation leading to a low phospho-ERK1/2 to phospho-p38 ratio. Treatment of miR122 positive cells with an inhibitor of TGFBR1 activation, abolished tumor dormancy program and recovered cell proliferation rate through a Smad-independent TGF-ß response.HCC stem-like cells can be directed towards cell differentiation and tumor dormancy by restoring miR122 expression. We demonstrate, for the first time, that dormancy program is achieved through a Smad-independent TGF-ß pathway. Restablishing miR122 expression is a promising therapeutic strategy that would work concurrently reducing tumor aggressiveness and decreasing disease recurrence.

Selective inhibition of hepatitis C virus infection by hydroxyzine and benztropine.

Hepatitis C virus (HCV) infection is a major biomedical problem worldwide as it causes severe liver disease in millions of humans around the world. Despite the recent approval of specific drugs targeting HCV replication to be used in combination with alpha interferon (IFN-α) and ribavirin, there is still an urgent need for pangenotypic, interferon-free therapies to fight this genetically diverse group of viruses. In this study, we used an unbiased screening cell culture assay to interrogate a chemical library of compounds approved for clinical use in humans. This system enables identifying nontoxic antiviral compounds targeting every aspect of the viral life cycle, be the target viral or cellular. The aim of this study was to identify drugs approved for other therapeutic applications in humans that could be effective components of combination therapies against HCV. As a result of this analysis, we identified 12 compounds with antiviral activity in cell culture, some of which had previously been identified as HCV inhibitors with antiviral activity in cell culture and had been shown to be effective in patients. We selected two novel HCV antivirals, hydroxyzine and benztropine, to characterize them by determining their specificity and genotype spectrum as well as by defining the step of the replication cycle targeted by these compounds. We found that both compounds effectively inhibited viral entry at a postbinding step of genotypes 1, 2, 3, and 4 without affecting entry of other viruses.

PGE2 induces angiogenesis via MT1-MMP-mediated activation of the TGFbeta/Alk5 signaling pathway.

The development of a new vascular network is essential for the onset and progression of many pathophysiologic processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite prostaglandin E(2) (PGE(2)). Here, we provide evidence for a novel signaling route through which PGE(2) activates the Alk5-Smad3 pathway in endothelial cells. PGE(2) induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGFbeta from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE(2). MT1-MMP-dependent transforming growth factor beta (TGFbeta) signaling through Alk5 is also required for PGE(2)-induced endothelial cord formation in vitro, and Alk5 kinase activity is required for PGE(2)-induced neovascularization in vivo. These findings identify a novel signaling pathway linking PGE(2) and TGFbeta, 2 effectors involved in tumor growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders.