Ji-Woong Son

Relationship between cyclooxygenase 8473T>C polymorphism and the risk of lung cancer: a case-control study

BackgroundCyclooxygenase-2 (COX-2) plays an important role in the development of lung cancer. DNA sequence variations in the COX-2 gene may lead to altered COX-2 production and/or activity, and so they cause inter-individual differences in the susceptibility to lung cancer. To test this hypothesis, we investigated the association between the 8473T>C polymorphism in the 3-untranslated region of the COX-2 gene and the risk of lung cancer in a Korean population.MethodsThe COX-2 genotypes were determined using PCR-based primer-introduced restriction analysis in 582 lung cancer patients and in 582 healthy controls that were frequency-matched for age and gender.ResultsThe distribution of the COX-2 8473T>C genotypes was not significantly different between the overall lung cancer cases and the controls. However, when the cases were categorized by the tumor histology, the combined 8473 TC + CC genotype was associated with a significantly decreased risk of adenocarcinoma as compared with the 8473 TT genotype (adjusted OR = 0.64; 95% CI = 0.46–0.90, P = 0.01). On the stratification analysis, the protective effect of the combined 8473 TC + CC genotype against adenocarcinoma was statistically significant in the males, older individuals and ever-smokers (adjusted OR = 0.59; 95% CI = 0.39–0.91, P = 0.02; adjusted OR = 0.55; 95% CI = 0.33–0.93, P = 0.03; and adjusted OR = 0.57; 95% CI = 0.37–0.87, P = 0.01, respectively).ConclusionThese findings suggest that the COX-2 8473T>C polymorphism could be used as a marker for the genetic susceptibility to adenocarcinoma of the lung.

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin-1–Syk and p62/SQSTM1

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin‐1/Syk‐dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin‐1 and Toll‐like receptor 2 (TLR2) were required for Mabc‐induced mRNA expression of pro‐interleukin (IL)‐1β, cathelicidin human cationic antimicrobial protein‐18/LL‐37 and β‐defensin 4 (DEFB4). Dectin‐1‐dependent Syk signaling, but not that of MyD88, led to the activation of caspase‐1 and secretion of IL‐1β through the activation of an NLRP3/apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc‐induced NLRP3/ASC inflammasome activation. Furthermore, Mabc‐induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.

Association of a common AGO1 variant with lung cancer risk: a two-stage case-control study.

Based on the important role of microRNA (miRNA) biosynthesis genes in carcinogenesis, we hypothesized that polymorphisms in the miRNA biosynthesis genes may modulate susceptibility to lung cancer. To test this hypothesis, we conducted a two‐stage study to evaluate the associations between single nucleotide polymorphisms (SNPs) in the miRNA biosynthesis genes and the risk of lung cancer. In stage 1 of the study, 24 SNPs in the 11 miRNA biosynthesis genes (DROSHA, DGCR8, RAN, XPO5, DICER, AGO1, AGO2, HIWI, GEMIN3, GEMIN4, and TRBP) were genotyped in 100 lung cancer patients and 100 healthy controls using a sequenome mass spectrometry‐based genotyping assay. One promising SNP (AGO1 rs636832Au2009>u2009G) was selected for stage 2 of the study, and genotyped by a melting‐curve analysis using fluorescence‐labeled hybridization probes in an independent set of 552 cases and 552 controls. The AGO1 rs636832Au2009>u2009G exhibited highly consistent results between the two stages of the study. In combined analysis, the 636832Au2009>u2009G was associated with a significantly decreased risk of lung cancer in a dose‐dependent manner (Ptrendu2009=u20096.0u2009×u200910−4). Individuals with at least one rs636832G allele were at a significantly decreased risk of lung cancer compared with those with the AA genotype (adjusted odds ratiou2009=u20090.67, 95% confidence intervalu2009=u20090.53–0.84, Pu2009=u20094.0u2009×u200910−4). This finding suggests that the AGO1 rs636832Au2009>u2009G might be a useful marker for determining the susceptibility to lung cancer and that the AGO1 gene might be involved in the development of lung cancer.

O6‐alkylguanine‐DNA alkyltransferase gene polymorphisms and the risk of primary lung cancer

O6‐alkylguanine‐DNA alkyltransferase (AGT) plays an important role in the repair of O6‐alkylguanine adducts, which are major mutagenic lesions produced by environmental carcinogens. Polymorphisms in the AGT gene may affect the capacity to repair DNA damage and thereby have influence on individuals susceptibility to smoking‐related cancer. To test this hypothesis, we investigated the potential association of AGT polymorphisms (485Cu2009>u2009A, Leu53Leu (Cu2009>u2009T) and Leu84Phe] with the risk of lung cancer in a Korean population. The AGT genotypes were determined in 432 lung cancer patients and in 432 healthy controls who were frequency‐matched for age and gender. The 485 AA genotype was associated with a significantly increased risk for overall lung cancer as compared with the 485 CC genotype and the combined 485 CCu2009+u2009CA genotype, respectively (adjusted odds ratio (OR)u2009=u20091.83, 95% confidence interval (CI)u2009=u20091.12–2.99, Pu2009=u20090.02, and Bonferroni corrected P‐value (Pc)u2009=u20090.04; and adjusted ORu2009=u20091.67, 95% CIu2009=u20091.05–2.66, Pu2009=u20090.03, respectively). When the lung cancer cases were categorized by the tumor histology, the 485 AA genotype was associated with a significantly increased risk of adenocarcinoma (AC) and small cell carcinoma (SmCC), respectively, as compared with the combined 485 CCu2009+u2009CA genotype (adjusted ORu2009=u20092.54, 95% CIu2009=u20091.39–4.66, Pu2009=u20090.003; and adjusted ORu2009=u20092.19, 95% CIu2009=u20091.06–4.55, Pu2009=u20090.04, respectively). However, the genotype distributions of the Leu53Leu and Leu84Phe polymorphisms were not significantly different between the lung cancer cases and the controls. On a promoter assay, the 485Cu2009>u2009A polymorphism did not have an effect on the promoter activity of the AGT gene. These results suggest that the effect of the AGT 485Cu2009>u2009A polymorphism on the risk of lung cancer may be secondary to linkage disequilibrium (LD) with either another AGT variant or with a true susceptibility gene, and that the AGT 485Cu2009>u2009A polymorphism could be used as a marker for the genetic susceptibility to lung cancer.

Expression and Regulation of the CC-Chemokine Ligand 20 During Human Tuberculosis

CC‐chemokine ligand 20 (CCL20), a unique chemokine ligand of CC‐chemokine receptor 6 (CCR6), play roles in various pathologic conditions. However, the characteristic expression profiles of CCL20 during human tuberculosis (TB) have been largely unknown. The present study analyzed the production and regulatory mechanisms of CCL20 in peripheral blood mononuclear cells (PBMC) and monocyte‐derived macrophages (MDM) from active pulmonary TB patients and healthy controls (HC). The 30‐kDa antigen (Ag) of Mycobacterium tuberculosis actively induced the production of CCL20 by human PBMC and MDM. A comparative analysis revealed that the expression of CCL20 protein was prominently up‐regulated in PBMC, MDM, bronchoalveolar lavage fluids (not in sera) from TB patients compared with the corresponding cells or body fluids from HC. Blockade of either tumour necrosis factor‐α or interferon‐γ, but not interleukin‐10, significantly attenuated the CCL20 production. In addition, recombinant CCL20 induced CCR6 expression by CD45RO+ T lymphocytes in a dose‐dependent manner. Furthermore, the expression of CCR6 was significantly increased in CD45RO+ T lymphocytes from TB patients, as compared with those from HC. Pharmacological inhibition studies showed that the 30‐kDa Ag‐induced CCL20 mRNA expression involves mitogen‐activated protein kinases (MAPK; extracellular signal‐regulated kinase 1/2 and p38)‐ and NF‐κB‐dependent signalling. Collectively, the present study demonstrated that TB patients show the up‐regulated expression of CCL20, which is modulated by proinflammatory cytokines, and through MAPK/NF‐κB‐mediated transcriptional mechanisms. The findings suggest important implications of potential roles of CCL20–CCR6 in immunopathogenesis of TB.

TP53 Mutations in Korean Patients with Non-small Cell Lung Cancer

Although TP53 mutations have been widely studied in lung cancer, the majority of studies have focused on exons 5-8 of the gene. In addition, TP53 mutations in Korean patients with lung cancers have not been investigated. We searched for mutations in the entire coding exons, including splice sites of the gene, in Korean patients with non-small cell lung cancer (NSCLC). Mutations of the gene were determined by direct sequencing in 176 NSCLCs. Sixty-nine mutations (62 different mutations) were identified in 65 tumors. Of the 62 mutations, 12 were novel mutations. TP53 mutations were more frequent in males, ever-smokers and squamous cell carcinomas than in females, never-smokers and adenocarcinomas, respectively (all comparisons, P<0.001). Missense mutations were most common (52.2%), but frameshift, nonsense, and splice-site mutations were frequently observed at frequencies of 18.8%, 15.9% and 10.1%, respectively. Of the 69 mutations, 9 (13.0%) were found in the oligomerization domain. In addition, the proportion of mutations in the oligomerization domain was significantly higher in adenocarcinomas than in squamous cell carcinomas (23.5% vs. 2.9%, P=0.01). Our study provides clinical and molecular characteristics of TP53 mutations in Korean patients with NSCLCs.

Nitric Oxide Synthesis is Modulated by 1,25-Dihydroxyvitamin D3 and Interferon-γ in Human Macrophages after Mycobacterial Infection

Background Little information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages. Methods Both iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors. Results Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-γ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-κB and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-γ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3. Conclusion These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

MIR144* inhibits antimicrobial responses against Mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2

ABSTRACT Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3′-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.

Ex Vivo Responses for Interferon‐gamma and Proinflammatory Cytokine Secretion to Low‐Molecular‐Weight Antigen MTB12 of Mycobacterium tuberculosis during Human Tuberculosis

MTB12 protein, also called CFP‐2, is a major and early secreted component of Mycobacterium tuberculosis. However, its role during mycobacterial infection has been poorly characterized. In this study, we purified the native MTB12 protein and investigated the profile of MTB12‐induced cytokines [interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6], in early tuberculosis (TB) patients (nu2003=u200320) and healthy controls (nu2003=u200335). The cytokine profiles were compared with those induced by the 30‐kDa antigen (Ag). In healthy controls, MTB12‐induced IFN‐γ production was markedly decreased in peripheral blood mononuclear cells compared with 30‐kDa Ag‐induced IFN‐γ. In TB patients, the mean IFN‐γ level induced by MTB12 was lower than that induced by the 30‐kDa Ag, albeit the difference was not significant. After 2u2003months of anti‐TB therapy, both the MTB12‐ and 30‐kDa‐induced IFN‐γ levels were significantly increased in TB patients. MTB12‐induced TNF‐α and IL‐6 levels were prominently upregulated in monocyte‐derived macrophages from TB patients, but they were not significantly different from those induced by the 30‐kDa Ag. Further, the activation of p38 mitogen‐activated protein kinase and extracellular signal‐regulated kinase was required for the induction of TNF‐α and IL‐6 by MTB12, as well as by the 30‐kDa Ag. Collectively, these data suggest that the MTB12 protein plays an essential role for proinflammatory responses through the MAPK pathway during the early stages of human TB, even though its T‐cell immunoreactivity is weaker than that of the 30‐kDa Ag.

A genetic variation in microRNA target site of KRT81 gene is associated with survival in early stage non-small cell lung cancer

BACKGROUNDnMicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC).nnnMETHODSnUsing public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs.nnnRESULTSnOf the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues.nnnCONCLUSIONnThe rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.BACKGROUNDnMicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC).nnnMETHODSnUsing public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs.nnnRESULTSnOf the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues.nnnCONCLUSIONnThe rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.