Glycolytic inhibition by resveratrol prevents myoblast cell death caused by glucose deprivation and hypoxia; a possible application to the three-dimensional tissue construction.

Decreased cell viability resulting from a severe condition of nutrients deprivation and hypoxia has been the major obstacle in three-dimensional (3D) tissue construction. Therefore, technical improvement which prevents cell death caused by starvation and low oxygen is desired for the development of large, thick tissues. We focused on the anti-glycolytic effect of resveratrol (RSV), a naturally-occurring polyphenol known as a caloric restriction mimetic, and investigated its cytoprotective effect in two-dimensional (2D) and 3D-cell culture using H9c2 rat myoblast cells. Glucose deprivation by culturing with low glucose media caused time- and dose-dependent cell death in H9c2 cells. In contrast, RSV treatment at 100 µM significantly increased the cell viability by preventing cell death. RSV showed anti-glycolytic effect associated with a down-regulation of glycolytic genes (GLUT1, PKM2) and glucose uptake activity, and increased the activation of AMP-activated protein kinase (AMPK), an essential cellular energy sensor activated in the condition of energy deprivation. RSV treatment markedly improved the viability of myoblast cells cultured in a hypoxic, low glucose condition and attenuated the up-regulation of glycolytic genes by hypoxic response. In 3D-cultured model, spheroids constructed with RSV-treated cells showed improved cell viability and intact histological appearance compared with control. These results suggest that glycolytic inhibition by RSV decreases the glucose usage of myoblast cells, therefore, prevents cell death caused by nutrient deprivation and hypoxic condition. Our finding provides useful information to improve cell viability in a condition that nutrients and oxygen are low in supply, and be a possible application to the 3D-tissue construction.

Liver-specific dysregulation of clock-controlled output signal impairs energy metabolism in liver and muscle.

The liver is the major organ maintaining metabolic homeostasis in animals during shifts between fed and fasted states. Circadian oscillations in peripheral tissues including the liver are connected with feeding-fasting cycles. We generated transgenic mice with hepatocyte specific E4BP4, D-box negative regulator, overexpression. Liver-specific E4BP4 overexpression was also achieved by adenoviral gene transfer. Interestingly, hepatic E4BP4 overexpression induced marked insulin resistance, that was rescued by DBP, a competing D-box positive regulator, overexpression. At basal conditions hepatocyte E4BP4 transgenic mice exhibited increased gluconeogenesis with reduced AKT phosphorylation in liver. In muscle, AKT phosphorylation was impaired after insulin stimulation. Such muscle insulin resistance was associated with elevated free fatty acid flux from the liver and reduced fatty acid utilization as an energy source during the inactive phase. E4BP4, one of the clock-controlled output genes, are key metabolic regulators in liver adjusting liver and muscle metabolism and insulin sensitivity in the feeding-fasting cycles. Its tuning is critical for preventing metabolic disorders.

HDAC9 Is Preferentially Expressed in Dedifferentiated Hepatocellular Carcinoma Cells and Is Involved in an Anchorage-Independent Growth.

Aberrant activation of histone deacetylases (HDACs) is one of the causes of tumor cell transformation in many types of cancer, however, the critical HDAC responsible for the malignant transformation remain unclear. To identify the HDAC related to the dedifferentiation of hepatocellular carcinoma (HCC) cells, we investigated the expression profile of HDACs in differentiated and undifferentiated hepatoma cells. We found that HDAC9, a member of the class II HDAC, is preferentially expressed in undifferentiated HCC cells. Analysis of 373 HCC patients in The Cancer Genome Atlas (TCGA) database revealed that the expression of HDAC9 mRNA positively correlated with the markers of mesenchymal phenotype and stemness, and conversely, negatively correlated with hepatic differentiation markers. HDAC9 was transcriptionally upregulated in epithelial-mesenchymal transition (EMT)-induced HCC cells treated with TGF-ß. Genetic and pharmacological inhibition of HDAC9 in undifferentiated HCC cells showed decreased sphere-forming activity, which indicates an ability of anchorage-independent cell growth and self-renewal. We also showed that aldehyde dehydrogenase 1A3 (ALDH1A3) was downregulated in HDAC9-suppressing cells, and ALDH inhibitor disulfiram significantly decreased the sphere formation of undifferentiated HCC cells. Together, our data provide useful information for the development of HDAC9-specific inhibitors for the treatment of HCC progression.

Hedgehog Signal Inhibitor GANT61 Inhibits the Malignant Behavior of Undifferentiated Hepatocellular Carcinoma Cells by Targeting Non-Canonical GLI Signaling.

The Hedgehog (HH)-GLI pathway plays an important role in cell dedifferentiation and is therefore pivotally involved in the malignant transformation of cancer cells. GANT61, a selective inhibitor of GLI1 and GLI2, was reported as a promising treatment for cancer in various tissues; however, the biological impact of GANT61 in hepatocellular carcinoma (HCC), especially in undifferentiated HCC cells, remains unclear. In this study, we investigated the antitumor effect of GANT61 using two undifferentiated hepatoma cell lines: HLE and HLF. Quantitative PCR and RT-PCR analyses revealed that these cells express GLI transcripts, showing mesenchymal phenotypes characterized by the loss of epithelial and hepatic markers and specific expression of epithelial-mesenchymal transition (EMT)-related genes. GANT61 significantly reduced the proliferation and cell viability after drug treatment using 5-FU and Mitomycin C. We showed that GLI transcript levels were down-regulated by the MEK inhibitor U0126 and the Raf inhibitor sorafenib, suggesting that non-canonical signaling including the Ras-Raf-MEK-ERK pathway is involved. Sphere formation and migration were significantly decreased by GANT61 treatment, and it is suggested that the underlying molecular mechanisms are the down-regulation of stemness-related genes (Oct4, Bmi1, CD44, and ALDH) and the EMT-related gene Snail1. The data presented here showed that direct inhibition of GLI might be beneficial for the treatment of dedifferentiated HCC.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

All-trans retinoic acid ameliorates hepatic stellate cell activation via suppression of thioredoxin interacting protein expression.

Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome-wide screening of RA-responsive genes by in silico analysis of RA-response elements, and identified 26 RA-responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all-trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP-mediated HSC activation was suppressed by antioxidant N-acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.

Impact of Preferentially Expressed Antigen of Melanoma on the Prognosis of Hepatocellular Carcinoma.

BACKGROUND: Retinoids, vitamin A and its derivatives, have an antitumor effect on hepatocellular carcinoma (HCC). The function of retinoids is exerted by the complex of retinoic acid (RA) with the heterodimer of retinoid X receptor and the RA receptor. The preferentially expressed antigen of melanoma (PRAME) acts as a dominant repressor of RA signaling by binding to the complex. The significance of PRAME on the prognosis of HCC remains to be clarified. METHODS: PRAME mRNA expression was examined by quantitative real-time polymerase chain reaction in both tumor and non-tumor tissues of 100 HCC patients who received surgical resection. The effect of PRAME knockdown on DR5-mediated RA transcriptional activity was examined. RESULTS: In tumor tissues, there were significant associations among PRAME expression, clinical stage, tumor markers, and tumor numbers. In non-tumor tissues, there were significant associations among PRAME expression, overall survival, and disease-free survival. The knockdown of PRAME caused no reduction in DR5-mediated transcriptional activity of RA, suggesting that PRAME acts via other mechanisms than the DR5 RA-responsive elements. CONCLUSION: Our findings indicate that PRAME expression is a novel prognostic marker in HCC patients.

Wnt/Beta-Catenin Signal Inhibitor HC-1 Sensitizes Oral Squamous Cell Carcinoma Cells to 5-Fluorouracil through Reduction of CD44-Positive Population.

BACKGROUND: Oral squamous cell carcinoma is a prevalent and frequently lethal malignancy worldwide. Existence of treatment-resistant cancer stem cells is considered to be associated with tumor formation, recurrence and metastasis. Wnt/beta-catenin signal is one of the crucial signaling pathways for cancer stem cells. Wnt/beta-catenin signal inhibitor may reduce the population of cancer stem cells and improve therapeutic effects on the cancers. METHODS: The effects of three derivatives of Wnt/beta-catenin signal inhibitors, HC-1, IC-2 and PN3-13, which we recently developed, on oral squamous cell carcinoma cell line HSC2, were examined by luciferase reporter assay, WST assay, cell sorting assay and apoptosis assay. RESULTS: The reporter assay showed that these small molecule compounds reduced Wnt/beta-catenin transcriptional activity in HSC2 cells. Of these compounds, IC-2 and PN3-13 inhibited cell viability in a dose-dependent manner, whereas HC-1 did not at even higher concentrations. Notably, however, the cell-sorting assay revealed that HC-1 significantly reduces the CD44-positive population of oral squamous cell carcinoma cells, compared to other compounds without affecting cell viability. In addition, HC-1 increases the cytotoxicity of HSC2 cells to 5-fluorouracil. The combination treatment of HC-1 with 5-fluorouracil significantly increased the apoptotic cells whereas treatment by either compound did not. CONCLUSION: These data suggest that HC-1 is an effective compound to target cancer stem cells, and the combination treatment of HC-1 and 5-fluorouracil can stimulate the tumor suppressive effect on oral squamous cell carcinoma cells.

Involvement of glucocorticoid in induction of lingual T1R3 in rodents.

We previously reported that in rats, chronic exposure to stress inhibits the induction of the common receptor (T1R3) for sweet and umami tastes. Here, we investigated whether endogenous glucocorticoids (GCs) might be responsible for this inhibition. In addition, we used mouse taste-bud cells (TB cells) expressing T1R3 to examine the effect of exogenous GC on T1R3 induction. Both adrenal glands were removed from rats [adrenalectomized (ADX) rats] and T1R3 mRNA expression in fungiform papillae was examined by real-time RT-PCR. T1R3 mRNA expression was significantly reduced in the ADX rats (versus sham-ADX rats). The reduced mRNA expression was restored to the level seen in the sham-ADX rats by administration of dexamethasone (DEX) at the smallest dose tested (0.1ng/kg, i.p.). However, with larger doses of DEX (10 and 1000ng/kg, i.p.) there was no such restoration (i.e., the expression level did not differ from that seen in ADX rats). Expression of the mRNA for the GC receptor-α was detected in mouse TB cells by RT-PCR. Significantly reduced T1R3 mRNA expression, as measured by real-time RT-PCR, was observed in TB cells at 24h after application of DEX (0.1, 1.0, or 10µM). These results suggest that in rodents: (a) a low concentration of endogenous GC is necessary and sufficient for induction of T1R3 expression, and that higher concentrations may actually inhibit such induction, and (b) this inhibitory effect may be due, at least in part, to a direct action of GC on taste cells.

Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib.

To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all-trans retinoic acid (ATRA), NIK-333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 µM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK-333 and Am80. We found that ATRA induced AMP-activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT-1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase-3, was observed. Suppression of AMP-activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells.

Identification of the small molecule compound which induces hepatic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) are expected to have utility as a cell source in regenerative medicine. Because we previously reported that suppression of the Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we synthesized twenty-three derivatives of small molecule compounds originally reported to suppress the Wnt/ß-catenin signal in human colorectal cancer cells. We then screened these compounds for their ability to induce hepatic differentiation of human UE7T-13 MSCs. After screening using WST assay, TCF reporter assay, and albumin mRNA expression, IC-2, a derivative of ICG-001, was identified as a potent inducer of hepatic differentiation of human MSCs. IC-2 potently induced the expression of albumin, complement C3, tryptophan 2,3-dioxygenase (TDO2), EpCAM, C/EBPα, glycogen storage, and urea production. Furthermore, we examined the effects of IC-2 on human bone marrow mononuclear cell fractions sorted according to CD90 and CD271 expression. Consequently, CD90+ CD271+ cells were found to induce the highest production of urea and glycogen, important hepatocyte functions, in response to IC-2 treatment. CD90+ CD271+ cells also highly expressed albumin mRNA. As the CD90+ CD271+ population has been reported to contain a rich fraction of MSCs, IC-2 apparently represents a potent inducer of hepatic differentiation of human MSCs.

Biological and clinical implications of retinoic acid-responsive genes in human hepatocellular carcinoma cells.

BACKGROUND & AIMS: Accumulating data from epidemiological and experimental studies have suggested that retinoids, which are vitamin A derivatives, exert antitumor activity in various organs. We performed a gene screening based on in silico analysis of retinoic acid response elements (RAREs) to identify the genes facilitating the antitumor activity of retinoic acid (RA) and investigated their clinical significance in hepatocellular carcinoma (HCC). METHODS: In silico analysis of RAREs was performed in the 5-kb upstream region of EST clusters. Chromatin immunoprecipitation analysis of the retinoic acid receptors and gene expression analysis were performed in HuH7, HepG2, and MCF7 cells treated with all-trans RA (ATRA). mRNA expression of RA-responsive genes was investigated using tumor and non-tumor tissues of clinical HCC samples from 171 patients. The association between gene expression and survival of patients was examined by Cox regression analysis. RESULTS: We identified 201 candidate genes with promoter regions containing consensus RARE and finally selected 26 RA-responsive genes. Of these, downregulation of OTU domain-containing 7B (OTUD7B) gene, which was upregulated by ATRA, in tumor tissue was associated with a low cancer-specific survival of HCC patients. Functional analyses revealed that OTUD7B negatively regulates nuclear factor κB (NF-κB) signaling and decreases the survival of HCC cells. CONCLUSIONS: We identified RA-responsive genes which are regulated by retinoid signal and found that low-OTUD7B mRNA expression is associated with a poor prognosis for HCC patients. OTUD7B-mediated inhibition of NF-κB signaling may be an effective target for antitumor therapy for HCC.

Retinoids and their target genes in liver functions and diseases.

Retinoids have been reported to prevent several kinds of cancers, including hepatocellular carcinoma (HCC). Retinoic acid (RA) coupled with retinoic acid receptor/retinoid X receptor heterodimer exerts its functions by regulating its target genes. We previously reported that transgenic mice, in which RA signaling is suppressed in a hepatocyte-specific manner, developed liver cancer at a high rate, and that disruption of RA functions led to the increased oxidative stress via aberrant metabolisms of lipid and iron, indicating that retinoids play an important role in liver pathophysiology. These data suggest that exploring the metabolism of retinoids in liver diseases and their target genes provides us with useful information to understand the liver functions and diseases. Consequently, the altered metabolism of retinoids was observed in liver diseases, including non-alcoholic fatty liver disease. In this review, we summarize the metabolism of retinoids in the liver, highlight the functions of retinoids in HCC, non-alcoholic fatty liver disease, and alcoholic liver disease, and discuss the target genes of RA. Investigation of retinoids in the liver will likely help us identify novel therapies and diagnostic modalities for HCC.

Identification of the genes chemosensitizing hepatocellular carcinoma cells to interferon-α/5-fluorouracil and their clinical significance.

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-ß signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.

The nucleocapsid protein of measles virus blocks host interferon response.

Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-α/ß and γ-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.

c-Jun N-terminal kinase activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of retinoic acid receptor α protein in hepatic cells.

We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis.

Disrupted plasma membrane localization of equilibrative nucleoside transporter 2 in the chemoresistance of human pancreatic cells to gemcitabine (dFdCyd).

Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.

Iron state in association with retinoid metabolism in non-alcoholic fatty liver disease.

AIM: We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non-alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. METHODS: Thirty-six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism-related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. RESULTS: In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. CONCLUSION: The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.

Preparation of an artificial cell cycle progressor with a novel mechanism.

Control of cell cycle progression in somatic cells or terminally differentiated cells is a key technology for cell-based therapies such as regenerative therapy. We have prepared an artificial cell cycle progression peptide composed of a human immunodeficiency virus-derived Tat protein transduction domain (PTD) and a p53 genetic suppressor element (GSE123). The peptide significantly promoted hepatocyte growth factor-induced DNA synthesis and the proliferation of primary mouse hepatocytes, which are highly differentiated somatic cells. The addition of a nuclear localization signal (NLS) sequence to the peptide increased the internalization of the peptide to the nuclear fraction. Distribution analysis using a fluoresein isothiocyanate-labeled peptide indicated that the NLS enabled the peptide to escape from the lysosomes to the cytosol. As a result, the NLS-Tat-GSE123 peptide induced potent cell proliferation of primary mouse hepatocytes in vitro. The use of this peptide as an artificial cell growth enhancer, bypassing a specific receptor, is a useful tool for the study of regenerative therapy and cell signaling.

Control of ER stress by a chemical chaperone counteracts apoptotic signals in IFN-gamma-treated murine hepatocytes.

Apoptosis of hepatocytes plays a key role in the pathogenesis of immune-mediated hepatitis. However, the detailed mechanisms of apoptotic signaling remain unclear. In this study, we investigated the involvement of ER stress in a model of IFN-gamma-induced apoptosis of hepatocytes in vitro, using a chemical chaperone reagent, glycerol. IFN-gamma-induced apoptotic events (mitochondrial release of cytochrome c, enzymatic activation of caspase-3 and -9) were markedly inhibited by glycerol. Glycerol induced partial inhibition of cytotoxicity indicated by lactate dehydrogenase release from the cytosol but had no inhibitory effect on the induction of IRF-1 gene expression and reactive oxygen species, required for hepatocyte apoptosis by IFN-gamma. Induction of caspase-4 and -12 gene expression, positively correlated with ER stress, was attenuated by glycerol. Gene analysis revealed that induction of ER stress-related genes, C/EBP homologue protein (CHOP/GADD153) and TRB3, was suppressed completely by glycerol treatment. These results suggest that ER stress plays a crucial role in mediating apoptosis of hepatocytes induced by IFN-gamma, and a chemical chaperone is an effective inhibitor of the ER stress.