Jee Young Kwon

Lack of genotoxic potential of ZnO nanoparticles in in vitro and in vivo tests

The industrial application of nanotechnology, particularly using zinc oxide (ZnO), has grown rapidly, including products such as cosmetics, food, rubber, paints, and plastics. However, despite increasing population exposure to ZnO, its potential genotoxicity remains controversial. The biological effects of nanoparticles depend on their physicochemical properties. Preparations with well-defined physico-chemical properties and standardized test methods are required for assessing the genotoxicity of nanoparticles. In this study, we have evaluated the genotoxicity of four kinds of ZnO nanoparticles: 20nm and 70nm size, positively or negatively charged. Four different genotoxicity tests (bacterial mutagenicity assay, in vitro chromosomal aberration test, in vivo comet assay, and in vivo micronucleus test, were conducted, following Organization for Economic Cooperation and Development (OECD) test guidelines with good laboratory practice (GLP) procedures. No statistically significant differences from the solvent controls were observed. These results suggest that surface-modified ZnO nanoparticles do not induce genotoxicity in in vitro or in vivo test systems.

Microarray analysis of gene expression alteration in human middle ear epithelial cells induced by micro particle

OBJECTIVES The primary aim of this study is to reveal the effect of particulate matter (PM) on the human middle ear epithelial cell (HMEEC). METHODS The HMEEC was treated with PM (300 μg/ml) for 24 h. Total RNA was extracted and used for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed by using Pathway Studio 9.0 software. For selected genes, the changes in gene expression were confirmed by real-time PCR. RESULTS A total of 611 genes were regulated by PM. Among them, 366 genes were up-regulated, whereas 245 genes were down-regulated. Up-regulated genes were mainly involved in cellular processes, including reactive oxygen species generation, cell proliferation, apoptosis, cell differentiation, inflammatory response and immune response. Down-regulated genes affected several cellular processes, including cell differentiation, cell cycle, proliferation, apoptosis and cell migration. A total of 21 genes were discovered as crucial components in potential signaling networks containing 2-fold up regulated genes. Four genes, VEGFA, IL1B, CSF2 and HMOX1 were revealed as key mediator genes among the up-regulated genes. A total of 25 genes were revealed as key modulators in the signaling pathway associated with 2-fold down regulated genes. Four genes, including IGF1R, TIMP1, IL6 and FN1, were identified as the main modulator genes. CONCLUSIONS We identified the differentially expressed genes in PM-treated HMEEC, whose expression profile may provide a useful clue for the understanding of environmental pathophysiology of otitis media. Our work indicates that air pollution, like PM, plays an important role in the pathogenesis of otitis media.

Genome-wide profiling induced by ionizing radiation (IR) in non-small cell lung cancer (NSCLC) grown as three-dimensional spheroid

Lung cancer is one of the most common human malignancies. Approximately over 75% of lung cancer is non-small cell lung cancer (NSCLC). The p53 tumor suppressor gene is mutated in approximately 50% of NSCLC. Radiotherapy using γ-ray was conventionally used for NSCLC treatment. In tumors, radiation induced apoptosis is modulated by their microenvironmental conditions. The definite mechanism of ionizing radiation is required for effective remedial value to NSCLC. However, most studies were performed in monolayer cell culture system which is unable to reflect in vivo microenvironment. Therefore, there is a need for in vitro model which is able to mimic in vivo solid tumor. In this study, we investigated cellular responses, by analysis of gene expression profile against IR to NSCLC forming in vivo mimic spheroids. The results showed that apoptosis inducible radiation dose was determined by acridine orange (AO) staining on three-dimensional (3D) spheroid. Our microarray data exhibited the altered gene expression levels between monolayer cells and 3D cells against IR in p53 null cancer cells. Surprisingly, we found that several genes related with microenvironment such as focal adhesion, adhesion junction and hypoxia had higher expression level in 3D culture system than two-dimensional (2D) culture system. In conclusion, 3D culture system was able to describe and mimic the microenvironment on in vivo solid tumor. Therefore, we suggested that the in vivo mimic 3D culture system approach might provide important clues for the clarification of mechanism of radiation effect for p53 mutated or deleted cancer cells.

Genome-wide microarray investigation of molecular targets and signaling networks in response to high-LET neutron in in vivo-mimic spheroid of human carcinoma

Although conventional clinical treatment with low LET (linear energy transfer) including gamma-ray and X-ray has been widely used for radiotherapy in various cancers, however, ineffective outcomes occur due to radioresistance caused by p53 mutation. High LET has become alternative since it is able to induce apoptosis regardless of p53 status. Indeed, the molecular mechanisms toward high LET have been suggested. Nevertheless, most studies have been done in monolayer culture system which cannot promptly represent solid tumor microenvironment. Here we applied in vivo mimic 3D spheroid to conduct microarray-based genomic expression and molecular signaling pathway analyses under neutron irradiation. As a result, 3D spheroid system was achieved using thermorevesible gel system. An effective apoptosis-inducible dose of neutron was determined by Acridine Orange (AO) staining in 3D spheroid. Differentially expressed genes in both unique and common responses to neutron were identified in the 3D spheroid compared to the monolayer cells. Total 95 and 169 genes were notably altered at transcription level toward neutron in monolayer and 3D spheroid system, respectively. Based on microarray data, putative apoptosis signaling was depicted using Pathway Studio software. In 3D-in vivo mimic model, the molecular networks interacted with ITGB1, MAP4K4, PAPPA, and SGK1 might be suggested as plausible molecular pathways. In conclusion, we demonstrate novel molecular signaling and corresponding targets of in vitro solid tumor following high LET exposure. This result might provide critical clues for clarification of neutron-induced apoptosis mechanism.

Identification of Potential Novel Biomarkers and Signaling Pathways Related to Otitis Media Induced by Diesel Exhaust Particles Using Transcriptomic Analysis in an In Vivo System

Introduction Air pollutants are associated with inflammatory diseases such as otitis media (OM). Significantly higher incidence rates of OM are reported in regions with air pollution. Diesel exhaust particles (DEPs) comprise a major class of contaminants among numerous air pollutants, and they are characterized by a carbonic mixture of polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, and small amounts of sulfate, nitrate, metals and other trace elements. DEP exposure is a risk factor for inflammatory diseases. Our previous study identified potential biomarkers using gene expression microarray and pathway analyses in an in vitro system. Although in vitro investigations have been conducted to elucidate plausible biomarkers and molecular mechanisms related to DEP exposure, in vivo studies are necessary to identify the exact biological relevance regarding the incidence of OM caused by DEP exposure. In this study, we identified potential molecular biomarkers and pathways triggered by DEP exposure in a rodent model. Methods Transcriptomic analysis was employed to identify novel potential biomarkers in the middle ear of DEP-exposed mice. Results A total of 697 genes were differentially expressed in the DEP-exposed mice; 424 genes were upregulated and 273 downregulated. In addition, signaling pathways among the differentially expressed genes mediated by DEP exposure were predicted. Several key molecular biomarkers were identified including cholinergic receptor muscarinic 1 (CHRM1), erythropoietin (EPO), son of sevenless homolog 1 (SOS1), estrogen receptor 1 (ESR1), cluster of differentiation 4 (CD4) and interferon alpha-1 (IFNA1). Conclusions Our results shed light on the related cell processes and gene signaling pathways affected by DEP exposure. The identified biomarkers might be potential candidates for determining early diagnoses and effective treatment strategies for DEP-mediated disorders.

Recognition of potential predictive markers for diagnosis in Korean serous ovarian cancer patients at stage IIIc using array comparative genomic hybridization with high resolution

Ovarian cancer is the second most frequently diagnosed gynecologic malignancy, and causes higher mortality than any other cancer in the reproductive system. Recognizing predictive markers of development and progression of this cancer would facilitate to individualize therapy and improve survival of ovarian cancer patients. Detection of genetic aberrations using array based comparative genomic hybridization (array CGH) has been implicated as one of important tools for identifying diagnostic and prognostic markers in various cancers, leading to the interesting study that exploration of copy number alterations in serous ovarian cancer at IIIc stage with the ultimate goal to discover potential predictive markers. To gain the best opportunities for exploring genomic imbalance involved in modulation of cancer development and progression, homogenous samples were selected as concerning with histology and stage in which we applied the array CGH with high resolution of one million formats. Here, the array CGH analysis obviously revealed significant DNA copy number changes (gain and loss) in Korean ovarian cancer patients at serous histological IIIc stage. We discovered DNA copy number gain in chromosome 6p22.3 whereas DNA copy number loss in chromosome 8p21.1-p12 and chromosome 11p15.4, with relatively high frequency of genomic alteration (83.3%). Upon these chromosomal regions, we eventually identified 32 genes including DUSP4, ID4, NRG1, and RRM1. We also classified their functions by gene ontology (GO) analysis using DAVID program and consequently demonstrated various interesting GO terms including biological regulation and positive regulation of cell growth. Taken together, these identified genes might be considered as potential predictive markers for further target-based strategies for diagnosis in Korean ovarian cancer patients at serous histological IIIc stage. These studies might also help understanding of tumorigenesis and the progression of ovarian carcinomas, in the aspect of genetic variations.

Genomic approach to understand association between single nucleotide polymorphisms and risk of Korean serous ovarian cancer at stage IIIc

Globally, ovarian cancer is responsible for approximately 125,000 deaths each year. Identifying the genetic contributions to ovarian cancer will lead to advances in diagnosis and therapy. Here we analyzed SNPs through genotyping in Korean serous ovarian cancer patients at stage IIIc for the purpose of applying a pathway analysis-based approach using candidate loci. The results of statistical analysis based on the Korean HapMap showed that a total of 759 SNPs had significant differences in ovarian cancer. Our biological pathway analysis included the comparison of all SNPs with SNPs of serous ovarian cancer patients in The Cancer Genome Atlas (TCGA) to identify the molecular pathway in Korean serous ovarian cancer. The results suggest that genetic alterations associated with these signaling pathways might contribute to the discovery of unique biomarkers for diagnostic predictor of Korean serous ovarian cancer at stage IIIc.

Abstract 4861: Identification of novel potential targets for selenomethionine-mediated chemoprevention in colorectal carcinoma mouse model via proteomics analysis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite some controversy, selenomethionine (SeMet) mediated protection against colorectal carcinoma (CRC) might be a very promising non-cytotoxic option. However, responsive molecular targets and underlying mechanisms of SeMet mediated chemoprevention are still unclear. Our aim was to discover new targets of SeMet mediated chemoprevention in CRC using proteomics analysis. We found dietary SeMet supplementation before carcinoma initiation effectively suppressed polyp incidence and dysplastic lesions without any adverse effects. To determine chemopreventive targets of SeMet, we employed 2-dimensional gel electrophoresis based proteomics analysis in CRC mouse model. Pretreatment with SeMet apparently modulated the expression of 30 proteins with functions in major processes like chronic inflammation, oxidative-stress and apoptosis as discovered through pathway analysis with Pathway studio software.We validated four proteins selected from pathway analysis including prohibitin, purine nucleoside phosphorylase, annexin 2 and c-reactive protein by immuno-histochemistry. 8-hydroxy-2′-deoxyguanosine (8-OHdG), a known oxidative stress marker was decreased by SeMet treatment in CRC mice as seen by immunohistochemistry. Further network analysis was done among these new four validated proteins, 8-OHdG and colorectal cancer. These four proteins found by proteomics analysis might be considered as potential chemopreventive biomarkers of SeMet against colon cancer and can help develop and improve approaches in preventive, therapeutic and prognostic aspects. Citation Format: Md. Mujibur Rahman, Jee Young Kwon, Young Rok Seo. Identification of novel potential targets for selenomethionine-mediated chemoprevention in colorectal carcinoma mouse model via proteomics analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4861. doi:10.1158/1538-7445.AM2013-4861

Abstract LB-167: Identification of ionizing radiation (IR)-responsive mRNA in p53-deficient human non-small cell lung cancer (NSCLC) grown in three-dimensional cell culture system

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Non-small cell lung cancer (NSCLC) has been known as the most common human malignancy. The p53 tumor suppressor gene is mutated in approximately 50 % of NSCLC. For treatment of lung cancer, radiotherapy using -ray is general treatment. However, many patients suffer from recurred disease. Therefore, the definite mechanism of radiation based on in vivo mimic system is required for effective remedical value to NSCLC. Most of radiation biology studies were performed in conventional monolayer culture system which can not represent in vivo tumor environment. Hence, study applying in vivo mimic three-dimensional culture system is necessary to elucidate the molecular mechanism of solid tumor. Here, we analyzed gene expression profile induced by ionizing radiation (IR) in H1299 cells grown as spheroid in three-dimensional cell culture system. Our data showed that different gene expression profiles among monolayer culture system, three-dimensional small spheroid and three-dimensional large spheroid system. Particularly, genes related with microenvironment including adhesion junction and hypoxia had different expression level in three-dimensional spheroid system compare with monolayer culture system. This study suggested that in vivo mimic three-dimensional cell culture engineering approach might provide critical clues for understanding of cellular responses in p53 mutated or deleted lung cancer cells against IR. (* This work was supported by Nuclear Research Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST). (Grand code: 2007-2001431, 2008-2001694, 2009-0078295) from Korea Science and Engineering Foundation) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-167.

Abstract A40: The enhancement of p53 stability via the inhibition of JNK-modulated ubiquitination with the activation of redox factor 1 (Ref-1) in response to non-genotoxic antioxidant selenomethionine (semet)

A40 p53 has been known to play an important role as an tumor suppressor gene for the maintaining of genome stability. In the field of cancer prevention study, p53 has been issued to clarify the mechanism of activation under the non-genotoxic cancer preventive agents containing antioxidant selenium. In previous our study, p53 has been reported to be the function of transcriptional activation activated without the genotoxicity through the redox modulation in response to selenomethionine (SeMet). In this study, we investigated that the mechanism of p53 protein stabilization enhanced by SeMet. Our data showed that the ubiquitinated p53 was decreased in the cells exhibiting the downregulation of JNK which is one of E3-ubiquitin ligases compared with in mock-treated cells suggesting that the JNK-modulated p53 ubiquitination might be inhibited by SeMet. To define the mechanism of the enhanced p53 stability in the presence of SeMet, redox factor 1 (Ref-1) which has been documented as one of the factors modulating the redox status in response to antioxidants was downregulated using siRNA. Our data showed the first time that the interaction of JNK and p53 was increased in Ref-1 siRNA-treated cells in the presence of SeMet implicating that the Ref-1 activated by SeMet might be involved in the inhibition of JNK-mediated p53 ubiquitination. Our study suggested that the enhancement of p53 stabilization in response to SeMet might provide an important clue for the cancer prevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A40.