Jong Wan Kim

A novel adipokine CTRP1 stimulates aldosterone production

Comμlement‐Clq TNF‐related protein 1 (CTRP1), a member of the CTRP superfamily, is expressed at high levels in adipose tissues of obese Zucker diabetic fatty (fa/fa) rats, and CTRP1 expression is induced by proinflammatory cytokines, including TNF‐a and IL‐ip. In the present study, we investigated stimulation of aldosterone production by CTRP1, since it was observed that CTRP1 was specifically expressed in the zona glomerulosa of the adrenal cortex, where aldosterone is produced. Increased aldosterone production by CTRP1 in cells of the human adrenal cortical cell line H295R was dose‐dependent. Expression levels of aldosterone synthase CYP11B2 were examined to investigate the molecular mechanisms by which CTRP1 enhances the production of aldosterone. The expression of CYP11B2 was greatly increased by treatment with CTRP1, as was the expression of the transcription factors NGFIB and NURR1, which μlay critical roles in stimulation of CYP11B2 gene expression. It was also revealed that angiotensin II‐induced aldosterone production is, at least in part, mediated by the stimulation of CTRP1 secretion, not by the increase of CTRP1 mRNA transcription. In addition, the levels of CTRP1 were significantly up‐ regulated in hypertensive patients serum. As CTRP1 was highly expressed in obese subjects as well as up‐regulated in hypertensive patients, CTRP1 may be a newly identified molecular link between obesity and hypertension.—Jeon, J. H., Kim, K., Kim, J. H., Baek, A., Cho, H., Lee, Y. H., Kim, J. W., Kim, D., Han, S. H., Lim, J.‐S., Kim, K.‐I., Yoon, D. Y., Kim, S.‐Y., Oh, G. T., Kim, E., Yang, Y. A novel adipokine CTRP1 stimulates aldosterone production. FASEBJ. 22, 1502–1511 (2008)

Investigation of Toxin Gene Diversity, Molecular Epidemiology, and Antimicrobial Resistance of Clostridium difficile Isolated from 12 Hospitals in South Korea

BACKGROUNDnClostridium difficile is a major cause of antibiotic-associated diarrhea. The objective of this study was to characterize clinical isolates of C. difficile obtained from various regions in Korea with regard to their toxin status, molecular type, and antimicrobial susceptibility.nnnMETHODSnWe analyzed a total of 408 C. difficile isolates obtained between 2006 and 2008 from 408 patients with diarrhea in 12 South Korean teaching hospitals. C. difficile toxin genes tcdA, tcdB, cdtA, and cdtB were detected by PCR. Molecular genotyping was performed by PCR ribotyping. Antimicrobial susceptibilities of the 120 C. difficile isolates were assessed by agar dilution methods.nnnRESULTSnAmong 337 toxigenic isolates, 105 were toxin A-negative and toxin B-positive (A(-)B(+)) and 29 were binary toxin-producing strains. PCR ribotyping showed 50 different ribotype patterns. The 5 most frequently occurring ribotypes comprised 62.0% of all identified ribotypes. No isolate was susceptible to cefoxitin, and all except 1 were susceptible to piperacillin and piperacillin-tazobactam. The resistance rates of isolates to imipenem, cefotetan, moxifloxacin, ampicillin, and clindamycin were 25%, 34%, 42%, 51%, and 60%, respectively. The isolates showed no resistance to metronidazole or vancomycin.nnnCONCLUSIONSnThis is the first nationwide study on the toxin status, including PCR ribotyping and antimicrobial resistance, of C. difficile isolates in Korea. The prevalence of A-B+ strains was 25.7%, much higher than that reported from other countries. Binary toxin-producing strains accounted for 7.1% of all strains, which was not rare in Korea. The most prevalent ribotype was ribotype 017, and all A-B+ strains showed this pattern. We did not isolate strains with decreased susceptibility to metronidazole or vancomycin.

Up-regulation of Mac-2 binding protein by hTERT in gastric cancer

Mac‐2 binding protein (Mac‐2BP) is a secreted tumor antigen that is elevated in many cancers and implicated in tumor metastasis, as well as cell adhesion and immune functions. We focused on the human telomerase reverse transcriptase (hTERT) induced Mac‐2BP expression and the relationship between Mac‐2BP expression and the progression of gastric cancer. A cDNA expression array analysis was performed on the telomerase‐negative cell line, SW13, which was engineered to overexpress hTERT when compared with the parental SW13 cell. hTERT‐induced Mac‐2BP expression was confirmed via RT‐PCR and Northern blotting. ELISA and flow cytometric analyses revealed that Mac‐2BP protein was increased by 2‐ to 4‐fold in hTERT‐overexpressing cells compared with the mock control. Mac‐2BP expression was significantly reduced when the overexpressed hTERT was neutralized by the introduction of hTERT‐specific siRNA. These results suggest that Mac‐2BP expression is modulated by hTERT. Mac‐2BP levels in both gastric cancer cells and tumor tissues were determined via Northern blot analysis and immunohistochemistry. Mac‐2BP protein was highly expressed in most gastric cancer cell lines, and gastric tumor tissues were stained more densely than normal tissues. The intracellular and secreted Mac‐2BP levels were also evaluated via ELISA, indicating that Mac‐2BP was expressed and secreted more abundantly in gastric cancer patients than in healthy donors. The elevated serum Mac‐2BP level in gastric tumor patients was also significantly associated with distant metastasis (p = 0.05) and higher tumor stage (p = 0.04). Our findings suggest that Mac‐2BP is induced by hTERT, and that it may prove to be a useful prognostic marker for the detection of malignant progression of metastatic stomach cancers.

Dysregulation of overexpressed IL-32α in hepatocellular carcinoma suppresses cell growth and induces apoptosis through inactivation of NF-κB and Bcl-2

IL-32 is a newly discovered cytokine. Recently, various reports suggest that it plays a role as a proinflammatory mediator and may be involved in several cancer carcinogenesis. However, IL-32 expression in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated the expression and role of IL-32α in hepatocellular carcinoma, because IL-32 was identified as an upregulated gene in hepatocellular carcinoma tissues compared to nontumorous regions using DNA microarray. IL-32α was overexpressed in tissue and serum from patients with HCC and localized in the cytoplasm and nucleus of hepatocellular carcinoma tumor cells. Moreover, secreted IL-32α concentration in the serum of patients with hepatocellular carcinoma was elevated as compared with those in the normal serum using a developed sandwich ELISA. Furthermore, IL-32α suppression in hepatocellular carcinoma decreased expression of phospho-p38 MAPK, NF-κB, and antiapoptotic protein Bcl-2 and induced expression of proapoptotic proteins as well as p53 and PUMA resulting in the suppression of cell growth and induction of intrinsic apoptosis. Based on our results, we suggest that IL-32α is involved in the progression of hepatocellular carcinoma and may be a useful biomarker for diagnosis and therapeutic target of hepatocellular carcinoma.

Impaired responses of leukemic dendritic cells derived from a human myeloid cell line to LPS stimulation

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-α, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40-mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPSstimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-α and IL-1β, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.

ESM-1 silencing decreased cell survival, migration, and invasion and modulated cell cycle progression in hepatocellular carcinoma.

Endothelial cell-specific molecule-1 (ESM-1) is a secretory proteoglycan comprising a mature polypeptide of 165 amino acids and a single dermatan sulfate. The aim of this study was to evaluate endothelial cell-specific molecule-1 (ESM-1) as a hepatocellular carcinoma (HCC) marker and to analyze the effect of ESM-1 gene silencing in hepatocellular carcinoma cells. RT-PCR and Western Blot analysis revealed overexpression of ESM-1 in human HCC liver tissue and in serum from patients with HCC. Sandwich ELISA assay was used for quantitative analysis of ESM-1 in serum. Levels of ESM-1 were significantly elevated in the serum of patients with HCC (nxa0=xa040) as compared to serum from patients with hepatitis (AH, nxa0=xa040; CH, nxa0=xa039) or liver cirrhosis (nxa0=xa040) or from healthy subjects (nxa0=xa040). The accuracy of ESM-1 for HCC was higher than that of α-fetoprotein (AFP) according to ROC curve analysis. Expression of ESM-1 siRNA decreased cell survival through the inhibition of NF-κB pathway and induced cell cycle arrest by PTEN induction resulting in the inhibition of cyclin D1 in SK-Hep1 cells. Furthermore, ESM-1 silencing inhibited cell migration and invasion of SK-Hep1 cells. This study demonstrates that ESM-1 as a potential tumor marker is overexpressed in most tissues and serum in the presence of HCC and is involved with cell survival, cell cycle progression, migration, and invasion of hepatocellular carcinoma cells. Based on our results, we suggest that ESM-1 or a combination of ESM-1 and AFP is useful markers for diagnosis of HCC and ESM-1 may be useful therapeutic target of hepatocellular carcinoma.

[Dissemination of IMP-1 and OXA type beta-lactamase in carbapenem-resistant Acinetobacter baumannii].

BACKGROUNDnAcinetobacter baumannii is an aerobic, gram-negative, glucose-nonfermenting bacterium, which has emerged as a serious opportunistic pathogen. In recent years, the increasing instance of carbapenem-resistant A. baumannii producing metallo-beta-lactamases (MBLs) or OXAtype beta-lactamases is causing a serious clinical problem. In this study, we investigated the prevalence of Ambler class A, B, and D beta-lactamases and their extended-spectrum derivatives in carbapenem-resistant A. baumannii isolates.nnnMETHODSnA total of 31 consecutive, non-duplicate, carbapenem-resistant A. baumannii were isolated from three university hospitals in the Chungcheong province of Korea. The modified Hodge and inhibitor-potentiated disk diffusion tests were conducted for the screening of carbapenemase and MBL production, respectively. PCR and DNA sequencing were performed for the detection of beta-lactamase genes. We also employed the enterobacterial repetitive intergenic consensus (ERIC)-PCR method for the epidemiologic study.nnnRESULTSnTwenty-three of 31 isolates harbored bla(OXA-2)(51.6%), bla(OXA-23)(22.6%), bla(IMP-1)(48.4%),and bla(VIM-2)(3.2%). All of the OXA-2-producing strains also evidenced MBLs. The strains that harbored bla(OXA-23)were isolated only in hospital C, and only in a limited fashion. The ERIC-PCR pattern of the five OXA-23 strains indicated that the isolates were closely related in terms of clonality. The six strains producing IMP-1 isolated from hospital A were confirmed to be identical strains.nnnCONCLUSIONSnA. baumannii strains harboring IMP-1 or OXA-type beta-lactamases are currently widely distributed throughout the Chungcheong province of Korea. The most notable finding in this study was that a bla(OXA-2)-producing A. baumannii harboring MBL, which has not been previously reported, can also lead to outbreaks.

ESM-1 regulates cell growth and metastatic process through activation of NF-κB in colorectal cancer.

In our previous study, we reported that endothelial cell specific molecule-1 (ESM-1) was increased in tissue and serum from colorectal cancer patients and suggested that ESM-1 can be used as a potential serum marker for early detection of colorectal cancer. The aim of this study was to evaluate the role of ESM-1 as an intracellular molecule in colorectal cancer. ESM-1 expression was knocked down by small interfering RNA (siRNA) in colorectal cancer cells. Expression of ESM-1 siRNA decreased cell survival through the Akt-dependent inhibition of NF-κB/IκB pathway and an interconnected reduction in phospho-Akt, -p38, -ERK1, -RSK1, -GSK-3α/β and -HSP27, as determined by a phospho-MAPK array. ESM-1 silencing induced G(1) phase cell cycle arrest by induction of PTEN, resulting in the inhibition of cyclin D1 and inhibited cell migration and invasion of COLO205 cells. Consistently, ESM-1 overexpression in HCT-116 cells enhanced cell proliferation through the Akt-dependent activation of NF-κB pathway. In addition, ESM-1 interacted with NF-κB and activated NF-κB promoter. This study demonstrates that ESM-1 is involved in cell survival, cell cycle progression, migration, invasion and EMT during tumor invasion in colorectal cancer. Based on our results, ESM-1 may be a useful therapeutic target for colorectal cancer.

Identification of endothelial cell-specific molecule-1 as a potential serum marker for colorectal cancer.

No ideal serum markers for screening colorectal cancer (CRC) have been identified. The aim of this study was to determine the usefulness of endothelial cell‐specific molecule‐1 (ESM‐1) as a serum marker for CRC. Illumina microarray was carried out to search CRC‐related biomarkers. cDNA microarray detected that ESM‐1 was one of the overexpressed genes in CRC. Overexpression of ESM‐1 mRNA was confirmed in tissues of CRC by RT‐PCR and real‐time PCR. Immunohistochemical staining showed strong expression of ESM‐1 in the cytoplasm of tumor cells. Overexpression of ESM‐1 in human serum with CRC was found by Western blot analysis. For quantitative analysis of ESM‐1 in serum, we determined the ESM‐1 levels in serum specimens using an ELISA kit. We showed that the ESM‐1 levels in the serum of patients with CRC were significantly elevated (70.1u2003±u200329.7u2003pg/mL) compared to healthy subjects (29.7u2003±u200314.9u2003pg/mL). The accuracy, sensitivity, and specificity of ESM‐1 for CRC were 0.94, 99%, and 73%, respectively, by receiver operating characteristics curve analysis. The positive predictive value and negative predictive value were 63% and 95%, respectively. The likelihood ratios of a positive or negative test result were 73 and 0.27, respectively. When analyzed with a Cox regression model, a higher serum ESM‐1 level (≥76.0u2003pg/mL) was correlated with poor prognosis. This study suggests that expression of ESM‐1 is increased in tissue and serum of CRC patients and that ESM‐1 can be used as a potential serum marker for the early detection of CRC. (Cancer Sci 2010); 101: xxx–xxx

Overexpression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in colorectal cancer.

BACKGROUNDnCarcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer.nnnMETHODSnCEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses.nnnRESULTSnCEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes stage (p<0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p<0.001) and shorter recurrence-free survival (p<0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness.nnnCONCLUSIONSnCEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.