Mi-Jin Lee

Involvement of GADD153 and cardiac ankyrin repeat protein in hypoxia-induced apoptosis of H9c2 cells.

Oxidative stress is the main cause of cardiac injury during ischemia/reperfusion but the molecular mechanism for this process is unclear. In this study, it was found that hypoxia induces apoptosis in rat embryonic heart-derived H9c2 cells leading to the induction of GADD153, which is an apoptosis-related gene. Therefore, this study addressed the molecular role of GADD153 in hypoxia-induced apoptosis. The stable or inducible overexpression of GADD153 sensitized the H9c2 cells to apoptotic cell death. The results suggest that the transactivation domain of the GADD153 might be responsible for this cell execution and play a role in the nucleoplasmic localization of GADD153. The cells transiently transfected with the antisense GADD153 were more resistant to hypoxia-induced apoptosis than the vector control cells. Furthermore, GADD153 transcriptionally down-regulated the expression of the cardiac ankyrin repeat protein gene (CARP), which is a nuclear transcriptional co-factor that negatively regulates the expression of the cardiac gene. The ectopic expression of CARP in H9c2 cells increased the resistance to hypoxia-induced apoptosis. These results suggest that GADD153 overexpression and the concomitant down-regulation of CARP might have a causative role in the apoptotic cell injury of hypoxic H9c2 cells.

EFNA1 ligand and its receptor EphA2: potential biomarkers for hepatocellular carcinoma.

Novel biomarkers are needed for early detection and progression evaluation of hepatocellular carcinoma (HCC). The purpose of this study was to identify useful biomolecular markers for HCC. The 26 genes that encode membrane or secretory proteins were identified from cDNA microarray data. We further examined the expression of EFNA1 and its receptor EphA2 and determined their biological implications during the development and progression of HCC. The EFNA1 mRNA was overexpressed in most HCCs as compared with its expression in corresponding nontumor tissues (36 out of 40 cases, 90%), but EphA2 expression was noted in only half of the HCC tissues (20 of 40 cases, 50%). In most of the hepatoma cell lines, the EFNA1 protein expression was positively associated with alpha‐fetoprotien (AFP) expression but inversely associated with EphA2 expression. Furthermore, EFNA1 levels were detectable in the supernatant of the cultured hepatoma cells and in the serum of patients with HCC. In contrast, EphA2 expression was prominent in highly invasive hepatoma cells, and its overexpression was significantly correlated with decreased differentiation (r = 0.0248, p < 0.010) and poor survival (p = 0.0453) for HCC patients. EFNA1 and EphA2 may be useful serum markers for the detection of HCC development and progression, respectively.

Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells

Cholangiocarcinoma (CC) is an intrahepatic bile duct carcinoma with a high mortality rate and a poor prognosis. Sarcomatous change/epithelial mesenchymal transition (EMT) of CC frequently leads to aggressive intrahepatic spread and metastasis. The aim of this study was to identify the genetic alterations and gene expression pattern that might be associated with the sarcomatous change in CC. Previously, we established 4 human CC cell lines (SCK, JCK1, Cho-CK, and Choi-CK). In the present study, we characterized a typical sarcomatoid phenotype of SCK, and classified the other cell lines according to tumor cell differentiation (a poorly differentiated JCK, a moderately differentiated Cho-CK, and a well differentiated Choi-CK cells), both morphologically and immunocytologically. We further analyzed the genetic alterations of two tumor suppressor genes (p53 and FHIT) and the expression of Fas/FasL gene, well known CC-related receptor and its ligand, in these four CC cell lines. The deletion mutation of p53 was found in the sarcomatoid SCK cells. These cells expressed much less Fas/FasL mRNAs than did the other ordinary CC cells. We further characterize the gene expression pattern that is involved in the sarcomatous progression of CC, using cDNA microarrays that contained 18,688 genes. Comparison of the expression patterns between the sarcomatoid SCK cells and the differentiated Choi-CK cells enabled us to identify 260 genes and 247 genes that were significantly over-expressed and under-expressed, respectively. Northern blotting of the 14 randomly selected genes verified the microarray data, including the differential expressions of the LGALS1, TGFBI, CES1, LDHB, UCHL1, ASPH, VDAC1, VIL2, CCND2, S100P, CALB1, MAL2, GPX1, and ANXA8 mRNAs. Immunohistochemistry also revealed in part the differential expressions of these gene proteins. These results suggest that those genetic and gene expression alterations may be relevant to the sarcomatous change/EMT in CC cells.

Identification of Cystatin B as a Potential Serum Marker in Hepatocellular Carcinoma

Purpose: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. Experimental Design: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry. The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). Results: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001). The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. Conclusion: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and α-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.

Lipocalin‐2 negatively modulates the epithelial‐to‐mesenchymal transition in hepatocellular carcinoma through the epidermal growth factor (TGF‐beta1)/Lcn2/Twist1 pathway

Lipocalin‐2 (Lcn2) is preferentially expressed in hepatocellular carcinoma (HCC). However, the functional role of Lcn2 in HCC progression is still poorly understood, particularly with respect to its involvement in invasion and metastasis. The purpose of this study was to investigate whether Lcn2 is associated with the epithelial‐mesenchymal transition (EMT) in HCC and to elucidate the underlying signaling pathway(s). Lcn2 was preferentially expressed in well‐differentiated HCC versus liver cirrhosis tissues, and its expression was positively correlated with the stage of HCC. The characteristics of EMT were reversed by adenoviral transduction of Lcn2 into SH‐J1 cells, including the down‐regulation of N‐cadherin, vimentin, alpha‐smooth muscle actin, and fibronectin, and the concomitant up‐regulation of CK8, CK18, and desmoplakin I/II. Knockdown of Lcn2 by short hairpin RNA (shRNA) in HKK‐2 cells expressing high levels of Lcn2 was associated with EMT. Epidermal growth factor (EGF) or transforming growth factor beta1 (TGF‐β1) treatment resulted in down‐regulation of Lcn2, accompanied by an increase in Twist1 expression and EMT in HCC cells. Stable Lcn2 expression in SH‐J1 cells reduced Twist1 expression, inhibited cell proliferation and invasion in vitro, and suppressed tumor growth and metastasis in a mouse model. Furthermore, EGF or TGF‐β1 treatment barely changed EMT marker expression in SH‐J1 cells ectopically expressing Lcn2. Ectopic expression of Twist1 induced EMT marker expression even in cells expressing Lcn2, indicating that Lcn2 functions downstream of growth factors and upstream of Twist1. Conclusion: Together, our findings indicate that Lcn2 can negatively modulate the EMT in HCC cells through an EGF (or TGF‐β1)/Lcn2/Twist1 pathway. Thus, Lcn2 may be a candidate metastasis suppressor and a potential therapeutic target in HCC. (Hepatology 2013;58:1349–1361)

Genome-wide expression patterns associated with oncogenesis and sarcomatous transdifferentation of cholangiocarcinoma

BackgroundThe molecular mechanisms of CC (cholangiocarcinoma) oncogenesis and progression are poorly understood. This study aimed to determine the genome-wide expression of genes related to CC oncogenesis and sarcomatous transdifferentiation.MethodsGenes that were differentially expressed between CC cell lines or tissues and cultured normal biliary epithelial (NBE) cells were identified using DNA microarray technology. Expressions were validated in human CC tissues and cells.ResultsUsing unsupervised hierarchical clustering analysis of the cell line and tissue samples, we identified a set of 342 commonly regulated (>2-fold change) genes. Of these, 53, including tumor-related genes, were upregulated, and 289, including tumor suppressor genes, were downregulated (<0.5 fold change). Expression of SPP1, EFNB2, E2F2, IRX3, PTTG1, PPARγ, KRT17, UCHL1, IGFBP7 and SPARC proteins was immunohistochemically verified in human and hamster CC tissues. Additional unsupervised hierarchical clustering analysis of sarcomatoid CC cells compared to three adenocarcinomatous CC cell lines revealed 292 differentially upregulated genes (>4-fold change), and 267 differentially downregulated genes (<0.25 fold change). The expression of 12 proteins was validated in the CC cell lines by immunoblot analysis and immunohistochemical staining. Of the proteins analyzed, we found upregulation of the expression of the epithelial-mesenchymal transition (EMT)-related proteins VIM and TWIST1, and restoration of the methylation-silenced proteins LDHB, BNIP3, UCHL1, and NPTX2 during sarcomatoid transdifferentiation of CC.ConclusionThe deregulation of oncogenes, tumor suppressor genes, and methylation-related genes may be useful in identifying molecular targets for CC diagnosis and prognosis.

Activation of mammalian target of rapamycin complex 1 (mTORC1) and Raf/Pyk2 by growth factor-mediated Eph receptor 2 (EphA2) is required for cholangiocarcinoma growth and metastasis†‡

Eph receptor 2 (EphA2) overexpression is frequently accompanied by the loss of its cognate ligand during tumor progression. However, the molecular mechanism of this ligand‐independent promotion of tumor by EphA2 remains unclear in highly malignant and fatal cholangiocarcinoma (CC). We examined the biological role of EphA2 in tumor growth and metastasis in CC tissues and cells according to the degree of differentiation and we explored the downstream signaling pathways of EphA2. Growth factor‐mediated EphA2 overexpression itself leads to the activation of the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal‐regulated kinase (ERK) pathways through ligand‐independent activation of EphA2 (phosphorylation of S897). An in vitro soft agar assay and in vivo orthotopic or subcutaneous tumor model showed that EphA2 enhanced colony formation and accelerated tumor growth, and which seemed to be mainly associated with Akt (T308)/mTORC1 activation. Aberrant expression and activation of EphA2 was also associated with poorer differentiation and higher metastatic ability. Enhanced metastatic ability was also observed in an orthotopic tumor model or lung metastasis model, correlating with Pyk2(Y402)/c‐Src/ERK activation in addition to activation of the canonical Raf/MEK/ERK pathway. The mTORC1 and Raf/Pyk2 pathways also appeared to affect each other. These results suggest that growth factor‐mediated EphA2 might be involved in tumor growth and metastasis through activation of the mTORC1 and Raf/Pyk2 pathways. Therapeutic strategies that target EphA2 and its downstream effectors may be useful to control CC. (HEPATOLOGY 2013;57:2248–2260)

Enhancement of parthenolide-induced apoptosis by a PKC-alpha inhibition through heme oxygenase-1 blockage in cholangiocarcinoma cells

Cholangiocarcinoma (CC) is a chemoresistant intrahepatic bile duct carcinoma with a poor prognosis. The aims of this study were to identify molecular pathways that enhance sesquiterpene lactone parthenolide (PTL)-induced anticancer effects on CC cells. The effects of PTL on apoptosis and hemoxygenase-1 (HO-1) induction were examined in CC cell lines. The enhancement of PTL-mediated apoptosis by modulation of HO-1 expression and the mechanisms involved were also examined in an in vitro cell system. Low PTL concentrations (5 to 10 µM) led to Nrf2-dependent HO-1 induction, which attenuated the apoptogenic effect of PTL in Choi-CK and SCK cells. PTL-mediated apoptosis was enhanced by the protein kinase C-alpha inhibitor Ro317549 (Ro) through inhibition of expression and nuclear translocation of Nrf2, resulting in blockage of HO-1 expression. Finally, HO-1 silencing resulted in enhancement of apoptotic cell death in CC cells. The combination of PTL and Ro efficiently improved tumor growth inhibition compared to treatment with either agent alone in an in vivo subcutaneous tumor model. In conclusion, the modulation of HO-1 expression substantially improved the anticancer effect of PTL. The combination of PTL and Ro could prove to be a valuable chemotherapeutic strategy for CC.

Involvement of GADD153 and cardiac ankyrin repeat protein in cardiac ischemia-reperfusion injury

Oxidative stress is critical for causing cardiac injuries during ischemia-reperfusion (IR), yet the molecular mechanism for this remains unclear. In the present study, we observe that hypoxia and reoxygenation, a component of ischemia, effectively induces apoptosis in the cardiac myocytes from neonatal rats and it concomitantly leads to induction of GADD153, an apoptosis-related gene. Furthermore, IR injury of rat heart showed a GADD153 overexpression in the ischemic area where the TUNEL reaction was positive. A downregulation of cardiac ankyrin repeat protein (CARP) was also observed in this ischemic area. Promoter deletion and reporter analysis revealed that hypoxia transcriptionally activates a GADD153 promoter through the AP-1 element in neonatal cardiomyocytes. Ectopic overexpression of GADD153 resulted in the downregulation of CARP expression. Accordingly, the induction of GADD153 mRNA were followed by the CARP down-regulation in an in vivo rat coronary ischemia/reperfusion injury model. These results suggest that GADD153 over-expression and the resulting downregulation of CARP may have causative roles in apoptotic cell death during cardiac IR injury.

Disruption of a regulatory loop between DUSP1 and p53 contributes to hepatocellular carcinoma development and progression.

BACKGROUND & AIMSnAltered expression of dual specificity phosphatase 1 (DUSP1) is common in tumors including hepatocellular carcinoma (HCC), and is predictive of tumor progression and poor prognosis. However, the tumor suppressive role of DUSP1 has yet to be clearly elucidated.nnnMETHODSnThe molecular mechanisms of tumor suppression that were investigated were induction of apoptosis, cell cycle inhibition, and regulation of p53. Additionally, the antitumor effect of DUSP1 was assessed using a mouse model. Associated signaling pathways in HCC cells and tissues were examined.nnnRESULTSnDownregulation of DUSP1 expression was significantly correlated with poor differentiation (p<0.001) and advanced HCC stage (p=0.023). DUSP1 expression resulted in HCC suppression and longer survival (p=0.0002) in a xenoplant mice model. DUSP1 inhibited p38 MAPK phosphorylation and subsequently suppressed HSP27 activation, resulting in enhanced p53 phosphorylation at sites S15, S20, and S46 in HCC cells. Enhanced p53 activation induced the expression of target genes p21 and p27, which are linked to cell cycle arrest and apoptosis. Thus, DUSP1 was potentially linked to p53 activation via the p38 MAPK/HSP27 pathway. Wild-type but not mutant p53 transcriptionally upregulated DUSP1 via its DNA-binding domain. DUSP1 and p53 might collaborate to suppress tumors in hepatocarcinogenesis via a positive regulatory loop.nnnCONCLUSIONSnOur results revealed that disruption of a positive regulatory loop between DUSP1 and p53 promoted HCC development and progression, providing a rationale for a therapeutic agent that restores DUSP1 in HCC.