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Hepatic perivascular epithelioid cell tumors (PEComas) are very rare. We report a primary hepatic PEComa with a review of the literature. A 56-year-old women presented with a nodular mass detected during the management of chronic renal failure and chronic hepatitis C. Diagnostic imaging studies suggested a nodular hepatocellular carcinoma in segment 5 of the liver. The patient underwent partial hepatectomy. A brown-colored expansile mass measuring 3.2×3.0 cm was relatively demarcated from the surrounding liver parenchyma. The tumor was mainly composed of epithelioid cells that were arranged in a trabecular growth pattern. Adipose tissue and thick-walled blood vessels were minimally identified. A small amount of extramedullary hematopoiesis was observed in the sinusoidal spaces between tumor cells. Tumor cells were diffusely immunoreactive for human melanoma black 45 (HMB45) and Melan A, focally immunoreactive for smooth muscle actin, but not for hepatocyte specific antigen (HSA).

Streptozotocin (STZ) is widely used to induce diabetic rodent models. It is specifically toxic to pancreatic beta cells and causes severe destruction and dysfunction. We investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on an STZ-induced diabetic mouse model. PLAG attenuated the glucose increase and maintained serum insulin at levels similar to those seen with control mice. In pancreatic beta cell line INS-1, STZ-induced cell apoptosis and intracellular reactive oxygen species (ROS) generation were significantly reduced to nearly normal levels after PLAG treatment. Glucose transporter 2 (GLUT2) localization analyses and glucose uptake assays showed that PLAG accelerated GLUT2 internalization, which ameliorated excessive entry of glucose, as well as STZ. STZ-induced cytotoxic effects were significantly reduced in PLAG-treated groups. The biological activity of PLAG was further confirmed in GLUT2-silenced cells, and the specificity of PLAG was verified using its derivative 1-palmitoyl-2-linoleoyl-3-hydroxyl-rac-glycerol (PLH). Our results suggest that PLAG may be a useful agent for protecting beta cells in the setting of excessive glucose influx.

The expression and function of caldesmon (CAD) in urothelial bladder carcinoma (BC) have not been reported. Here, we investigated the expression, prognostic value, and potential functional mechanism of CAD in primary non-muscle-invasive bladder cancer (NMIBC). Protein profiling of tissue samples using antibody microarrays showed significantly higher CAD expression in muscle-invasive BC tissues compared with NMIBC tissues. We then validated the CAD expression in BC cells by immunohistochemistry analysis using paraffin-embedded tissue blocks and western blots using BC cell lines. In addition, we examined the expression of CAD variants by reverse transcription-polymerase chain reaction, and confirmed the expression of low-molecular-weight isoforms (L-CAD), specifically encoded by WI-38 L-CAD II (transcript variant 2), in BC cells. Survival analysis in an independent primary NMIBC cohort comprising 132 patients showed that positive CAD expression was significantly associated with poorer prognosis than no CAD expression with regard to recurrence- and progression-free survival (p = 0.001 and 0.014, respectively). Multivariate analyses further indicated that positive CAD expression was an independent predictor of progression-free survival (p = 0.032; HR = 5.983). Data obtained from in vitro silencing and overexpression studies indicated that L-CAD promotes migration and invasiveness of BC cells. Immunofluorescence assays showed dramatic structural changes in the actin cytoskeleton of BC cells after L-CAD overexpression. Our findings collectively suggest that L-CAD overexpression in primary NMIBC is significantly associated with tumor progression and that a possible mechanism for L-CAD's activity is implicated in increased cell motility and invasive characteristics through morphological changes in BC cells.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), a chemically synthesized monoacetyldiaglyceride, is one of the constituents in Sika deer antlers and has been known traditionally as having immunomodulatory effects. However, the mechanism by which PLAG controls neutrophil migration, which evokes liver injury in the hepatitis animal model, remains largely unknown. This study was designed to evaluate the immunomodulatory effects of PLAG on cytokine secretion and neutrophil migration in vivo and in vitro. The injection of mice with Concanavalin A (Con A) induced leukocyte infiltration in the liver and increased plasma cytokine levels. Pretreatment with PLAG reduced the levels of interleukin (IL)-4, IL-6, IL-10, and CXCL2, but maintained interferon (IFN)-γ levels and modulated neutrophil recruitment towards the liver. Furthermore, the mRNA and protein levels of IL-4 and CXCL2 in liver tissue were also decreased in the Con A-treated mice. Liver histology analyses showed that PLAG reduced Con A-induced hepatic necrosis, which was accompanied by leukocyte infiltration. The in vitro studies revealed that PLAG reduced IL-4 secretion in Con A stimulated T cell and blocked signal transducer and activator of transcription 6 (STAT6) Con A induced hepatocyte. PLAG attenuated IL-4 induced activation of atypical protein kinase C (PKC)/ STAT6 in hepatocytes and inhibited neutrophil migration towards the liver tissue through suppression of IL-8/vascular cell adhesion molecule (VCAM) expression. These results suggest that PLAG could mitigate excess neutrophil migration into liver tissue and potentially have a therapeutic effect on immune-mediated liver injury. This article is protected by copyright. All rights reserved

BACKGROUND: Kallikrein-related peptidase 6 (KLK6) encodes a trypsin-like serine protease that is up-regulated in several cancers, although the putative functions of KLK6 in cancer have not been elucidated. In the current study, overexpression of KLK6 was identified in colon cancer, and the possibility that KLK6 may be a suitable candidate as a tumor marker was examined. METHODS: Messenger RNA (mRNA) transcript levels and protein up-regulation of KLK6 in colon cancer tissues was examined using reverse transcriptase-polymerase chain reaction, immunohistochemistry, and clinicopathologic analyses. Cell proliferation, invasiveness, and antiapoptotic activity were determined in colon cancer cells that were transfected with small-interfering RNA (siRNA) of KLK6. RESULTS: KLK6 mRNA was up-regulated significantly in tumor tissues compared with nontumor regions. KLK6 protein was strongly expressed in adenocarcinomas but was not expressed in normal mucosa or in premalignant dysplastic lesions. Sera from patients with colon cancer revealed an increase in KLK6 secretion (0.25 μg/mL; P = .031) compared with noncancer cells (0.19 μg/mL). Clinicopathologic and immunohistochemical studies of 143 patients with colon cancer revealed a significant correlation between KLK6 expression and Dukes disease stage (P = .005). High KLK6 expression was associated significantly with shorter overall (P = .001) and recurrence-free survival (P = .001). The rates of proliferation and invasiveness were decreased by 50% in cells that were transfected with KLK6 siRNA. The overexpression of KLK6 led to decreased activity of the E-cadherin promoter. CONCLUSIONS: KLK6 was up-regulated significantly in tissues and sera from patients with colon cancer and was associated closely with a poor prognosis, suggesting that KLK6 may be used as a potential biomarker and a therapeutic target for colon cancer. Cancer 2011;. © 2010 American Cancer Society.

5-Lipoxygenase (5-LOX) is important enzyme in the biosynthesis of leukotrienes, and is a potential target in the treatment of asthma and allergy. We designed and synthesized a series of benzoxazoles and benzothiazoles as 5-LOX inhibitors. Fourteen compounds prepared showed the inhibition of LTC4 formation with IC50 value of 0.12–23.88 μM. Also two compounds 2d and 2g showed improved airway hypersensitiveness.

Background Cysteine proteases like cathepsins are widely distributed proteolytic enzymes and form tight equimolar complexes with cystatins at their active sites. Among cystatins, CST1, encoding cystatin SN, is a member of the type 2 salivary cystatin family found in a variety of fluids and secretions, including plasma, tears, and saliva. CST1 was identified as an upregulated gene in gastric cancer tissues compared to noncancerous regions using our Affymetrix GeneChip microarray. Methods The upregulation of CST1 in gastric cancer was analyzed using RT-PCR (n = 15), immnohistochemistry, and clinicopathological (n = 77) analysis. CST1-siRNA was used for the suppression of CST1 gene expression and cathepsin proteolytic activity was assayed. Results CST1 was upregulated in cancerous lesions of gastric cancer tissues compared to noncancerous regions and clinicopathological analysis showed a significant correlation between high expression of CST1 and pTNM stage (p = 0.044). In CST1-siRNA transfected cells, cell proliferation was reduced and the proteolytic activity of cathepsins was increased. Conclusions CST1 might be highly involved in gastric tumorigenesis and regulate the proteolytic activity of cysteine proteases.

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the bile duct epithelium, is one of the leading causes of death from cancer, worldwide. However, the mechanisms related to it remain largely unknown. In this study, an analysis of the gene expression profiles for ICC was done using the frequency of the ESTs obtained from nine cDNA libraries that constructed from 4 ICC cell lines and 4 normal liver tissues. One hundred and thirty-seven genes were identified as being either up- or down-regulated in human ICC cells. Thirty genes were randomly selected to confirm their differential expression in 4 human ICC cell lines and 5 ICC tissues compared to normal liver tissues by semi-quantitative RT-PCR. Among these genes, ANXA1, ANXA2, AMBP, and SERPINC1 were further verified by immunohistochemical analyses. In conclusion, these identified genes represent potential biomarkers for ICC and represent potential targets for elucidating the molecular mechanisms that are associated with ICC.

Many fundamental processes, including oncogenesis, have implicated HECT domain proteins with ubiquitin ligase activity. The protein human upstream regulatory element binding protein 1 (hUREB1) is a HECT domain protein whose function is not defined yet. Here, we investigate the function of hUREB1 as a ubiquitin–protein ligase in human colorectal cells. Ectopic expression of the HECT domain of hUREB1 reduces the protein level and transcriptional activity of the p53 tumor suppressor, which is abrogated by the deletion in the HECT domain or point mutations in the essential residues of the HECT domain. The ubiquitination and destabilization of p53 is observed in cells treated with the protease inhibitor MG132, implying that the HECT domain of hUREB1 suppresses the transcriptional activity of p53 through a ubiquitin-dependent degradation pathway. Based on the results of Northern blot analysis, RT-PCR, and immunohistochemical analyses, the over-expression of hUREB1 is associated with colorectal carcinoma. Moreover, protein levels of hUREB1 and p53 were inversely correlated. These findings suggest that hUREB1 can function, at least in part, as a negative regulator of p53 during the colorectal carcinoma progression through the ubiquitination pathway mediated by the HECT domain.

S100A6 (calcyclin) is an acidic calcium binding protein with two EF-hand motifs and overexpressed in several tumors including intrahepatic carcinoma. TNFalpha, a strong NF-kappaB activator required for hepatocyte proliferation during liver regeneration, triggered the expression of S100A6 mRNA in human hepatoblastoma cell line HepG2. Transient expression of NF-kappaB (p65) increased S100A6 promoter activity and expression of inhibitor of NF-kappaB (IkappaBalpha) decreased TNFalpha-induced S100A6 promoter activity. To confirm the involvement of NF-kappaB in S100A6 promoter activation, we analyzed serially deleted promoter constructs of the S100A6 gene by luciferase reporter assay and found a NF-kappaB-responsive DNA fragment at the position between -584 and -361. Electrophoretic mobility shift assays showed that TNFalpha induced p65 binding to a potential NF-kappaB binding site at -460/-451. Furthermore, treatment of cells with CAPE (caffeic acid phenethyl ester), a specific NF-kappaB (p65) inhibitor, decreased NF-kappaB binding and promoter activity. These results suggest that NF-kappaB transcription factor contributes to the activation of S100A6 gene expression in response to TNFalpha in HepG2 cells.

To discover the molecular mechanism of N-Myc downstream-regulated gene 2 (NDRG2), a newly found differentiation-related tumor suppressor, the relationships between NDRG2 and E-cadherin were investigated in tumor cells and tissues. Positive correlations between the expression of E-cadherin and NDRG2 were shown in several colon cancer cell lines as well as in colon cancer tissues. According to the transcription assays using a reporter plasmid containing E-cadherin promoter region (-368~+51), NDRG2 introduction into colon cancer cell lines induced upregulation of E-cadherin promoter activity and its transcription. On the contrary, inhibition of NDRG2 expression by siRNA treatment caused the decrease of E-cadherin transcription. Snail, a zinc-finger transcriptional repressor, was shown to be a mediator of NDRG2-regulated E-cadherin expression. The enhancement of glycogen synthase kinase 3β (GSK-3β) activity by NDRG2 overexpression caused proteasomal degradation of Snail transcription factor followed by transcriptional de-repression of E-cadherin. We also found that NDRG2 could mediate cell density-regulated E-cadherin expression. The increase of NDRG2 expression with cell density preceded E-cadherin expression, and the regulation of Snail activity by GSK-3β was also related to this process.

NAD glycohydrolase (NADase) is present in many organisms from bacteria to mammals. In any given organism, this enzyme is ubiquitous in many tissues. However, its precise localization and its physiological significance have not been defined. We have determined the distribution of NADase in normal human and rabbit tissues by immunoblotting and immunohistochemistry, using a polyclonal antibody raised in goats. Immunoblot analyses revealed that NADase was highly expressed in the heart, lung, stomach, and liver tissues of the rabbit. From immunohistochemical studies of NADase, high concentrations in both human and rabbit tissues were found in hepatocytes and sinusoidal lining cells, sinus histiocytes of the lymph node, spleen and thymus, glomerular capillary endothelial cells of the kidney, cardiac muscle, endothelium of blood vessels, and erythrocytes.

Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.

Many biological systems are regulated by an intricate set of feedback loops that oscillate with a circadian rhythmicity of roughly 24 hr. For example, increases in body temperature, heart rate, blood pressure, and cortisol secretion occur early in the day. Histone proteins exist in a delicate balance between acetylated and deacetylated states. Acetylation leads to nucleosomal relaxation and increased expression of numerous genes. Recent work implicates this chromatin remodeling response in hypertrophic growth of the myocardium. trichostatin A(TSA), a potent inhibitor of histone deactylase(HDAC) activity, imposes a dose-dependent blockade to hypertrophy and fetal gene activation in neonatal cardiac cells. To explore the role of HDAC activation in an in vitro, we studied the effects of TSA, NaB and melatonin in neonatal and H9c2 cells. Chemicals such as TSA and NaB treated neonatal cardiac cells had an increase of global histone H3 and H4(P

Hypoxia-inducible factors (HIFs) are transcription factors that activate the transcription of target genes involved in crucial aspects of cancer development. This study investigated the expression of HIFs and their contribution to the regulation of target genes related to angiogenesis and glucose metabolism in gastric cancer. The data showed that HIFs were over-expressed in gastric cancer and that activation of the target genes was observed mainly in the early stages. Moreover, the results of the present study revealed that only HIF-1alpha, but not HIF-2alpha dimerizes with HIF-1beta and then regulates expression of target genes in response to hypoxia. The results of the present study demonstrate that HIF-1alpha and HIF-1beta enhances expression of VEGF and glucose metabolism-related genes in response to hypoxia in gastric cancer. These data offer important information regarding HIF pathways in the development of gastric cancer.

S100A6 (calcyclin) is a small calcium-binding protein which has been implicated in several cellular processes such as cell cycle progression, cytoskeleton rearrangement, and exocytosis. Also the upregulation of S100A6 has been reported in a variety of tumors and linked to metastasis. However, exact intracellular roles of S100A6 related with apoptosis have not been clarified yet. Here we demonstrated that the upregulation of S100A6 enhances the cell death rate compared to the control under the apoptotic conditions. In exogenously S100A6 induced Hep3B cells, cell viability was significantly decreased compared with mock and S100A6-knockdown cells under calcium ionophore A23187 treatment. The exogenously introduced S100A6 significantly affected the caspase-3-like activity in programmed cell death through the enhanced caspase-3 expression, which was verified by promoter assay in wild or mutant S100A6-transfected Hep3B cells. Next, the promoter activity of caspase-3 was increased by 2.5-folds in wild-type S100A6-transfected cells compared to mutant 2 (E67K, mutant of EF-hand motif) or control. Our results suggest that S100A6 might be involved in the processing of apoptosis by modulating the transcriptional regulation of caspase-3.

A time sequential study was performed to investigate the histological and ultrastructural findings of fumonisin B1-induced apoptosis in the male Sprague-Dawley rat liver. Six hours after administration of FB1, marked morphologic changes of hepatocytes included the appearance of small vacuoles along the margin of cell membrane. Twelve hours after injection of FB1, acidophilic degeneration of cells occurred, but no fragmented nucleus was evident around the centrilobular area, with few apoptotic cells. By electron microscope, the degenerated acidophilic cells revealed following changes: characteristic formation of cytoplasmic vacuoles, condensed cytoplasm, detachment from neighboring cells, and as well as margination of nuclear chromatin and swollen mitochondria with amorphous matrical deposit. The number of apoptotic cells or bodies was further enhanced at 24 hours in the vicinity of dense acidophilic cells, resulting in a marked increase over the values of control rats. Serum analysis revealed the elevation of cholesterol levels from the beginning to the end of this experiment. Morphologic data and serum findings in this study support the theory that FB1-induced alteration of membrane lipid constituents of the hepatocytes are likely to be early key events in explaining the FB1 apoptotic effect.

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah- 1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating ǰ -catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A mono- clonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis. (Immune Network 2004;4(1):23-30)

Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis. (Im m u ne N etw ork 2003; 3(1):16-22)