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Background & Aims: Our previous studies showed that CD133, EpCAM, and aldehyde dehydrogenase (ALDH) are useful markers to identify cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) tissues. The present study aims to evaluate chemosensitivity and invasion capability of HCC based on CSC marker profiles, and to explore the underlying molecular mechanisms., Methods: Hepatoma cell lines were separated into subpopulations according to CD133, EpCAM, and ALDH expression profiles. Epithelial mesenchymal transition (EMT) and hedgehog (Hh) signaling were examined to identify their links with chemoresistance and aggressive invasion., Results: Well-differentiated cell lines were positive for CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) at 1.5-15% and 2.3-8.3%; whereas, poorly-differentiated cells were almost all negative for these markers. FACS-enriched CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) Hep3B and Huh-7 cells formed more spheroids in vitro. CD133(-)/ALDH(low) HLE cells were more resistant to cisplatin, doxorubicin or sorafenib than their positive counterparts. CD133(-)/EpCAM(-) Huh-7 cells or CD133(-)/ALDH(-) HLE cells exhibited a higher invasion rate than their positive counterparts. HLE and HLF cells acquired EMT in double negative subpopulations. Hh activity in Huh-7 CD133(-)/EpCAM(-) cells was higher than in their positive counterparts, and the inhibition of Hh activity by cyclopamine resulted in reduced cell proliferation., Conclusions: Well-differentiated CD133(+)/ALDH(high) or CD133(+)/EpCAM(+) cells appear to be a CSC/initiating subpopulation; whereas, in poorly-differentiated hepatoma cells, EMT and enhanced hedgehog signaling activity may be responsible for their chemoresistance and invasion. These findings underscore the significance of EMT and enhanced Hh signaling in liver cancer stem or initiating cells., (Copyright © 2011 European Association for the Study of the Liver. All rights reserved.)

The capacity of stem cells to differentiate into specific cell types makes them very promising in tissue regeneration and repair. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, hepatocyte growth factor (HGF), an important morphogen in liver development, was co-printed with collagen I (Col) to create arrays of protein spots on glass. Human adipose stem cells (ASCs) were cultured on top of the HGF/Col spots for 2weeks. The effects of surface-immobilized HGF on hepatic differentiation of ASCs were analyzed using RT-PCR, ELISA and immunocytochemistry. Stimulation of stem cells with HGF from the bottom-up caused an upregulation in synthesis of α-fetoprotein and albumin, as determined by immunocytochemistry and ELISA. RT-PCR results showed that the mRNA levels for albumin, α-fetoprotein and α1-antitrypsin were 10- to 20-fold higher in stem cells cultured on the HGF/Col arrays compared to stem cells on Col only spots. Our results show that surfaces containing HGF co-printed with ECM proteins may be used to differentiate mesenchymal stem cells such as ASCs into hepatocyte-like cells. These results underscore the utility of growth factor-containing culture surfaces for stem cell differentiation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Cancer stem cells (CSCs) are thought to be a critical subpopulation in tumor development, progression, metastasis and recurrence, and the identification of these cells is an initial step in understanding their role in oncogenesis and in seeking valuable markers for diagnosis or development of targeting therapeutics., Aims: To identify CSCs in hepatocellular carcinoma (HCC) specimens and define their tissue specificity., Methods: Immunohistochemical staining of CSC markers: CD44, CD90, CD133 and aldehyde dehydrogenase (ALDH) was performed in 25 HCC specimens, 4 hepatoblastomas, 8 peri-malignant tissues, and 19 cases of viral hepatitis., Results: The positivity of CD44 staining in HCC specimens was significantly lower than in viral hepatitis specimens. The positive rate of CD133 in HCC was similar to viral hepatitis specimens. CD133(+) cells were largely localized to ALDH-positive cells in HCC as revealed by confocal microscopy. In contrast, the co-expression of both markers was visualized within vessels or in the portal areas in viral hepatitis. Moreover, among 7 liver specimens adjacent to HCC tissue, 3-6 samples were positive for CD44, CD90, CD133 and ALDH, especially in dysplastic cells. One of 4 hepatoblastoma cases was positive for all these markers; whereas, the other three specimens were negative for all these CSC markers., Conclusions: In HCC and dysplastic tissues, clusters of CD133(+)/ALDH(high) cells were identified. The use of cancer stem cell markers to screen tissues with chronic liver diseases provides limited guidance in the identification of malignant cells., (Copyright 2010 Elsevier Inc. All rights reserved.)

One of the pathways by which alcohol induces hepatocyte apoptosis is via oxidative stress. We screened several chemically-synthesized small molecules and found LAS-0811, which inhibits oxidative stress. In this study, we elucidated its role in inhibiting alcohol-induced apoptosis in hepatocyte-like VL-17A cells. VL-17A cells were pre-incubated with LAS-0811, followed by ethanol incubation. Ethanol-induced reactive oxygen species and apoptosis were significantly inhibited in LAS-0811 pre-treated cells. VL-17A cells were transfected with a reporter (ARE/TK-GFP) plasmid containing green fluorescent protein (GFP) as a reporter gene and the anti-oxidant response element as the promoter. LAS-0811 pre-treatment significantly induced the GFP expression compared to the cells treated with ethanol alone. LAS-0811 induced the activation of nrf2 and enhanced the expression and activity of glutathione peroxidase, one of the downstream targets of nrf2. The results indicate that LAS-0811 protects VL-17A cells against ethanol-induced oxidative stress and apoptosis at least in part via nrf2 activation.

Small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in plant and animal cells. RNA dependent DNA methylation (RdDM) accounts for TGS in plants, but it is unclear whether siRNA induces RdDM in mammalian cells. To determine whether stable expression of short hairpin siRNA (shRNA) induces DNA methylation in mammalian cells, we transduced rat hepatic stellate SBC10 cells with lentiviral vectors which encode an U6 promoter-driven shRNA expression cassette homologous to the transforming growth factor-beta receptor (TGFbetaRII) promoter region. Sequencing analysis of bisulfite-modified genomic DNA showed the methylation of cytosine residues both in CpG dinucleotides and non-CpG sites around the target region of the TGFbetaRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFbetaRII mRNA levels which were reversed by treatment with 5-aza-2-deoxycytidine. Our results demonstrate that recombinant lentivirus-mediated shRNA delivery resulted in the methylation of the homologous promoter area in mammalian cells, and this approach may be used as a tool for transcriptional gene silencing by epigenetic modification of mammalian cell promoters.

Background/aims: Some of the mutant forms of cellular proteins not only lose their function, but also cause diseases by their toxic effects. One of the challenging tasks in the field of gene therapy will be "gene replacement" accomplished by inhibiting mutant gene expression and providing normal function of the same gene, simultaneously. Although lung involvement in alpha1-antrypsin (alpha1-AT) deficiency is caused by the lack of alpha1-AT function, the liver involvement is due to the accumulation of the mutated alpha1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of alpha1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning alpha1-AT protein in the liver., Methods: For the inhibition of alpha1-AT gene expression, panels of alpha1-AT-specific hammerhead ribozymes designed to target different GUC sites in the alpha1-AT mRNA were evaluated in a human hepatoma cell-line, transduced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which contained a functional alpha1-AT ribozyme and was combined with a modified alpha1-AT gene, whose product was engineered to be resistant to the specific alpha1-AT ribozyme. This construct was transduced into target hepatoma cells., Results: The transduced hepatoma cells showed the effective expression of modified alpha1-AT, under the conditions where the endogenous alpha1-AT gene expression was inhibited., Conclusion: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of alpha1-AT deficiency.

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In the present study, we investigated the association of transforming growth factor-beta 1 (TFG-beta 1) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro alpha 1 (I) collagen, and TGF-beta 1. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. When compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-beta 1 mRNA levels. Moreover, steady-state levels of TGF-beta 1 and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-beta 1 transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-beta 1 gene expression is significantly enhanced in man during active liver disease.