Junya Azumi

Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor β1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation

Objective and designTo clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs).MaterialUsing human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor β1 (TGFβ1).ResultsPPC suppressed ROS which are induced by TGFβ1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels.ConclusionThese results suggest that ROS and Nox4 induced by TGFβ1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.

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. (Cancer Sci 2011; 102: 934–941)

miR‐181a induces sorafenib resistance of hepatocellular carinoma cells through downregulation of RASSF1 expression

Sorafenib, a multi‐kinase inhibitor, is the only standard clinical drug for patients with advanced hepatocellular carcinoma (HCC); however, development of sorafenib resistance in HCC often prevents its long‐term efficacy. Therefore, novel targets and strategies are urgently needed to improve the antitumor effect of sorafenib. In the present study, we examined the novel mechanisms of sorafenib resistance of HCC cells by investigating the difference in sorafenib sensitivity between two HCC cell lines. Sorafenib induced more apoptosis of HepG2 cells compared to Hep3B cells. Sorafenib exposure to HepG2 cells but not Hep3B cells increased the expression of proapoptotic factor PUMA, and activated PARP and caspase‐3. Notably, microRNA‐181a (miR‐181a) expression levels were lower in HepG2 cells than in Hep3B cells. Exogenous miR‐181a expression in HepG2 cells reduced apoptosis, whereas inhibition of miR‐181a in Hpe3B cells increased apoptosis. In addition, we demonstrated that miR‐181a directly targets RASSF1, a MAPK signaling factor, and knockdown of RASSF1 increased sorafenib resistance. Taken together, these results suggest that miR‐181a provokes sorafenib resistance through suppression of RASSF1. Our data provide important insight into the novel therapeutic strategy against sorafenib resistance of HCC cells by targeting of miR‐181a pathway.

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-β 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.

CD117 expression is a predictive marker for poor prognosis in patients with non‑small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for >85% of incidences of lung cancer, for which the predicted 5-year survival rates are low and recurrence rates remain high. Although it has been reported that the patients with SCLC cells that possess the cluster of differentiation (CD) 117 marker exhibited poor prognosis and poor response to chemotherapy, no studies concerning the association of CD117 expression with prognosis of the patients with NSCLC have been reported. An in vitro study reportedly revealed that CD117-positive cell populations in NSCLC cell lines exhibited cancer stem cell (CSC) phenotypes including self-renewal and chemoresistance. Therefore, the present study hypothesized that if CD117-positive cells are CSC-like cells, CD117 positivity may be associated with the prognosis of patients with NSCLC. To confirm this hypothesis, the association between CD117 expression in patients with NSCLC and clinicopathological characteristics was investigated. CD177 expression was examined by immunohistochemistry in 99 patients with NSCLC who underwent curative surgical resection. Tumor samples in the present study included 73 samples of adenocarcinoma and 26 of squamous carcinoma. The associations of CD177 expression with clinicopathological features and prognosis were examined. The lymph node metastasis and rates of recurrence were significantly associated with overall survival rates through multivariate analysis (P<0.001 and P<0.001), respectively. A Kaplan-Meier analysis for relapse-free survival and the log-rank test revealed that the patients with CD117-positive cell populations exhibited shorter relapse-free survival rates compared with patients whose cells were CD117-negative (P=0.014). The multivariate analysis demonstrated that venous invasion, pathological stage, and CD117 expression were independent prognostic parameters for relapse-free survival in patients with NSCLC (P=0.001, P=0.001 and P=0.002), respectively. In conclusion, these data suggest that CD117 expression in NSCLC may serve as a useful marker for predicting the prognosis of patients with NSCLC.

Prognostic relevance of miR-137 in patients with hepatocellular carcinoma.

Cancer stem cells (CSCs) play a pivotal role in progression, metastasis and recurrence of cancer. Therefore, it is clinically useful to identify the relevant CSC marker that is associated with prognosis of hepatocellular carcinoma (HCC) and clarify its genetic and biological characteristics.

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/β-catenin signal enhances hepatic differentiation of human MSCs, we synthesized twenty-three derivatives of small molecule compounds originally reported to suppress the Wnt/β-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.

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.