Liver-enriched activator protein 1 as an isoform of CCAAT/enhancer-binding protein beta suppresses stem cell features of hepatocellular carcinoma.

PURPOSE: Liver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs. MATERIALS AND METHODS: Differences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence. RESULTS: LAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell-associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo. CONCLUSION: LAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.

The potential role of liver stem cells in initiation of primary liver cancer.

Identification of the cellular origin of primary liver cancer remains challenging. Some data point toward liver stem cells (LSCs) or liver progenitor cells (LPCs) not only as propagators of liver regeneration, but also as initiators of liver cancer. LSCs exhibit a long lifespan and strong duplicative potential upon activation and are inclined to accumulate more mutations that can be passed down to the next generations. Recent evidence shows that dysregulation of signaling pathways associated with self-renewal of LSCs can drive their aberrant proliferation and even malignant transformation. If LSCs could be proved to be an initiator of liver carcinogenesis, they would be promising for ultra-early diagnosis and targeting therapy of liver cancer. This review mainly summarizes the potential role of LSCs in the carcinogenesis of primary liver cancer.

Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.

Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1ß and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.

Cuprous oxide nanoparticles selectively induce apoptosis of tumor cells.

In the rapid development of nanoscience and nanotechnology, many researchers have discovered that metal oxide nanoparticles have very useful pharmacological effects. Cuprous oxide nanoparticles (CONPs) can selectively induce apoptosis and suppress the proliferation of tumor cells, showing great potential as a clinical cancer therapy. Treatment with CONPs caused a G1/G0 cell cycle arrest in tumor cells. Furthermore, CONPs enclosed in vesicles entered, or were taken up by mitochondria, which damaged their membranes, thereby inducing apoptosis. CONPs can also produce reactive oxygen species (ROS) and initiate lipid peroxidation of the liposomal membrane, thereby regulating many signaling pathways and influencing the vital movements of cells. Our results demonstrate that CONPs have selective cytotoxicity towards tumor cells, and indicate that CONPs might be a potential nanomedicine for cancer therapy.

Murine embryonic stem cell-derived hepatocytes correct metabolic liver disease after serial liver repopulation.

Although embryonic stem (ES) cell-derived hepatocytes have the capacity for liver engraftment and repopulation, their in vivo hepatic function has not been analyzed yet. We aimed to determine the metabolic function and therapeutic action of ES cell-derived hepatocytes after serial liver repopulations in fumaryl acetoacetate hydrolase knockout (Fah(-/-)) mice. Albumin expressing (Alb(+)) cells were obtained by hepatic differentiation of ES cells using two frequently reported methods. After transplantation, variable levels of liver repopulation were found in Fah(-/-) mice recipients. FAH expressing (FAH(+)) hepatocytes were found either as single cells or as nodules with multiple hepatocytes. After serial transplantation, the proportion of the liver that was repopulated by the re-transplanted FAH(+) hepatocytes increased significantly. ES cell-derived FAH(+) hepatocytes were found in homogenous nodules and corrected the liver metabolic disorder of Fah(-/-) recipients and rescued them from death. ES cell-derived hepatocytes had normal karyotype, hepatocytic morphology and metabolic function both in vitro and in vivo. In conclusion, ES cell-derived hepatocytes were capable of liver repopulation and correction of metabolic defects after serial transplantation. Our results are an important piece of evidence to support future clinical applications of ES cell-derived hepatocytes in treating liver diseases.

A potential role for the homeoprotein Hhex in hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC), the most common primary malignant tumor of the liver, often associated with the dysregulation of transcriptional pathways involved in cell growth and differentiation. The hematopoietically expressed homeobox protein (Hhex) is an important transcription factor throughout liver development and is essential to liver bud formation and hepatoblast differentiation. Here, we report a relationship between Hhex expression and HCC. First, adenovirus-mediated Hhex delivery into the hepatoma cell line, Hepa1-6, resulted in decreased expression of several proto-oncogenes (c-Jun and Bcl2), increased expression of some tumor suppressor genes (P53 and Rb), and enhanced expression of a cluster of hepatocytic and bile ductular markers. Second, Hhex expression significantly attenuated Hepa1-6 tumorigenicity in nude mice. Third, we report a correlation between Hhex expression and the differentiation state of human HCC. In 24 cases of clinical specimens, there was a significant difference in Hhex expression between poorly differentiated HCC and well-differentiated HCC (P < 0.001). Taken together, these results indicate that Hhex is a potential candidate molecular marker for HCC pathological evaluation, suggesting a need to evaluate Hhex as a potential target for therapeutic intervention.

Long-term persistence of hepatitis B surface antigen and antibody induced by DNA-mediated immunization results in liver and kidney lesions in mice.

DNA-mediated immunization has been recognized as a new approach for prevention and treatment of hepatitis B virus (HBV) infection. However, the side effects of this approach have not been well described. Here we report that DNA-mediated immunization by intramuscular injection of plasmid DNA encoding HBV surface antigen (HBsAg) induced long-term persistence of HBsAg and HBsAg-specific antibody (anti-HBs) in the sera of the immunized BALB/c mice and resulted in liver and kidney lesions. The lesions persisted for 6 months after injection. Lesions were also found in normal mice injected with the sera from immunized mice, and in HBV-transgenic mice injected with anti-HBs antibody, or sera from immunized mice. Furthermore, lesions were accompanied by deposition of circulating immune complex (CIC) of HBsAg and anti-HBs antibody in the damaged organs. These results indicate that long-term persistence of HBsAg and anti-HBs in the immunized mice can result in deposited CIC in liver and kidney, and in development of lesions. The use of DNA containing mammalian replication origins, such as the plasmids used in this study, is not appropriate for human vaccines due to safety concerns relating to persistence of DNA; nevertheless, the safety of DNA-mediated immunization protocols still needs to be carefully evaluated before practical application.

[Immunopathology of hepatitis B virus transgenic mouse strain C57-TgN (adr2.0) SMMU].

AIM: To make immunopathological study of hepatitis B virus transgenic mice line C57-TgN (HBV adr2.0) SMMU. METHODS: Twenty transgenic C57-TgN mice of SPF grade and corresponding control mice C57BL/6 were used for the study. The expressions of CD3, CD4 and CD8 on lymphocytes in peripheral blood from transgenic and normal C57BL/6 mice were detected by flow cytometry. T lymphocyte subsets in the liver tissues from transgenic mice and hepatitis B patients were analyzed by EnVision staining. RESULTS: The expression of CD3, CD4 and CD8 on lymphocytes in peripheral blood from transgenic mice was lower than those from normal mice. The infiltrative mononuclear cells in the liver tissues of transgenic mice were mainly CD3+ CD4+ cells, and no CD8+ CD57+ cells was found. The infiltrative mononuclear cells in the liver tissues of human hepatitis B patients were mainly CD3+ CD4+ or CD3+ CD8+ cells. CONCLUSION: T cell subsets in the peripheral blood and liver tissues of transgenic mice C57-TgN (adr2.0) SMMU are different from those in human chronic hepatitis B patients.

Expression of hepatitis B virus X protein in transgenic mice.

AIM: To establish a mice model harboring hepatitis B virus x gene (adr subtype) for studying the function of hepatitis B virus X protein, a transactivator of viral and cellular promoter/enhancer elements. METHODS: Expression vector pcDNA3-HBx, containing CMV promoter and hepatitis B virus x gene open reading fragment, was constructed by recombination DNA technique. Hela cells were cultured in DMEM and transfected with pcDNA3-HBx or control pcDNA3 plasmids using FuGENE6 Transfection Reagent. Expression of pcDNA3-HBx vectors in the transfected Hela cells was confirmed by Western blotting. After restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes. The pups were evaluated by multiplex polymerase chain reaction (PCR) at genomic DNA level. The x gene transgenic mice founders were confirmed at protein level by Western blotting, immunohistochemistry and immunogold transmission electron microscopy. RESULTS: Expression vector pcDNA3-HBx was constructed by recombination DNA technique and identified right by restriction endonuclease digestion and DNA direct sequencing. With Western blotting, hepatitis X protein was detected in Hela cells transfected with pcDNA3-HBx plasmids, suggesting pcDNA3-HBx plasmids could express in eukaryotic cells. Following microinjection of coding sequence of pcDNA3-HBx, the embryos were transferred to oviducts of pseudopregnant females. Four pups were born and survived. Two of them were verified to have the HBx gene integrated in their genomic DNA by multiplex PCR assay, and named C57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3 respectively. They expressed 17KD X protein in liver tissue by Western blotting assay. With the immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of transgenic mice, which was furthermore confirmed by immunogold transmission electon microscopy. CONCLUSION: We have constructed the expression vector pcDNA3-HBx that can be used to study the function of HBx gene in eukaryotic cells in vitro. We also established HBx gene (adr subtype) transgenic mice named C57-TgN (HBx)SMMU harboring HBx gene in their genome and express X protein in hepatocytes, Which might be a valuable animal system for studying the roles of HBx gene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.