(The) study on anti-retroviral drug resistance in Korean patients-derived human immunodeficiency virus type-1 isolates

최주연 School of Life Sciences and Biotechnology, Korea U 2011 국내박사

The use of antiretroviral drugs has reduced the mortality and morbidity of patients with HIV/AIDS since zidovudine was introduced for the first time in 1987. Especially, the quality of life and the health condition of patients infected with HIV were improved with the aid of highly active antiretroviral therapy since the half of 1990. However, many drug-resistant variants have been reported after initiating HAART and the efficiency of HAART is limited by these variants despite of the rapid development of various antiretroviral drugs. Several methods to predict in patients’ condition were reported. The genotypic drug resistance assay is generally used and is a very useful method to investigate and predict antiretroviral drug resistance. Genotypic drug resistance assay has been the only method available to provide information related to drug-resistance in South Korea since 1999. Phenotypic assay is also useful method to predict a patients’ state related to antiretroviral drug resistance. The phenotypic drug susceptibility assay is used for optimal treatment in some developed country. However, commercial system and methods for phenotyping have not been introduced into South Korea. And, the use of commercial phenotypic assay kit is generally restricted to few countries. Preferentially, drug-resistant variants were analyzed using the Stanford database with sequences and mutation data of the HIV-1 genes for protease (codons 1-99) and reverse transcriptase (codons 1-250). And then, drug susceptibility was analyzed to antiretroviral drug resistance-related mutations. Of this drug-naïve patients’ group, 199 isolates (66.3%) showed at least 1 or more sites related to drug resistance. In particular, 13 isolates had the following mutation sites: PI-associated sites L10I, M36L, L63P, V77I and I93L and RTI-associated mutation sites such as M41L, D67N, T69S, K103N, V179D, T215Y and K219E. These antiretroviral drug resistance-related mutation sites also lead to severe drug resistance in treatment-experienced HIV-1 infected patients. To investigate and analyze difference between genotype and phenotype, I established the modified phenotypic assay to analyze infectivity and drug susceptibility using single cell round assay. In this study, phenotypic drug susceptibility was investigated in several patients’ derived pseudovirus compared to wild type, especially focused on multi-nRTI resistance. And, the genotypic drug resistance and phenotypic drug susceptibility of two different methods, Stanford HIV Drug Resistance Database and modified phenotypic drug susceptibility assay, were compared especially focused on the HIV-1 protease and reverse transcriptase gag-pol sequences. The IC50 for nine antiretroviral drugs was analyzed using WT-derived pseudovirus for establishing a standard level using XLfit. Percent inhibition shows a sigmoidal dose-response curve between serially diluted drugs and infectivity. For each antiretroviral drug, the IC50 was following as: ddI (6.4 M), d4T (5.0 M), ABC (2.3 M), 3TC (1.1 M), ddC (0.9 M) and AZT (22.3 nM) in NRTIs; APV (52.3 nM) and IDV (10.4 nM) in PIs; EFV (0.18 nM) in NNRTI. There was some discordance in comparing drug susceptibility results with the predicted genotypic drug resistance. Phenotypic drug resistance showed the following order for pseudoviruses from treatment-experienced patients with HIV/AIDS: Efavirenz (EFV, 21 to 1,319-fold change), Lamivudine (3TC, 31 to >189-fold change), Indinavir sulfate (IDV, 26 to 63-fold change), Amprenavir (APV, 4 to 35-fold change) and Zidovudine (AZT, 20 to 634-fold change). For patient KRC3221, the AZT-related phenotypic drug resistance was the greatest with 634-fold change compared with the wild type. And, phenotypic fold change was generally lower in ABC, d4T, ddI and ddC contrary to 3TC, AZT, EFV, APV and IDV. 3TC-related resistance shows most similar pattern among genotype and phenotypes except KRC3221. Especially, AZT-related drug resistance showed big differences among genotypic drug resistance, treatment history and phenotypic drug resistance in pseudoviruses derived from all treatment-experienced patients. For AZT-related resistance, all genotypic drug resistance levels were lower than those of phenotyping except for patient KRB5018. In particular, the pseudoviruses from patients KRB8067 and KRC2065 were predicted to be susceptible against AZT by genotyping though these patients had been undergoing an AZT-including regimen for more than two years. However, samples from these patients showed 27-fold and 95-fold increased phenotypic resistance levels compared with the WT virus, respectively. Results using phenotypic assays except AZT showed similar trends in contrast to the genotypic drug resistance based on the Stanford DB. In pseudoviruses derived from treatment-experienced patients, the fold change-based drug resistance level using ‘In house Phenotype’ was generally similar or higher than FC according to mutation patterns. Application of this modified phenotypic drug susceptibility assay is expected to help in predicting drug resistance as a guideline for clinicians to obtain a combined interpretation among genotyping, phenotyping and effective clinical treatments.

Molecular characteristics of second-line drug resistance in multidrug-resistant Mycobacterium tuberculosis

김창기 연세대학교 대학원 2011 국내박사

Tuberculosis (TB) is an infectious disease that usually attacks the respiratory system. Almost 10 million people worldwide contract TB each year. In Korea, TB incidence is 97 cases per 100,000 populations, which is highest among the member countries of the Organisation for Economic Cooperation and Development. Although there have been many efforts and resources contributed to control TB worldwide, an increase in drug-resistant TB cases is one of the major obstacles. Multidrug-resistant TB (MDR-TB) is defined as TB that is resistant to both isoniazid and rifampicin, which are the most important first-line drugs for TB. It takes approximately two years to cure MDR-TB with second-line drugs. Moreover, some MDR-TB cases are resistant to second-line drugs, which are called extensively drug-resistant TB (XDR-TB). It is extremely difficult to select drugs to treat XDR-TB; thus, treatment outcomes are poor. For successful control of drug-resistant TB, assessment of drug resistance rates and rapid and accurate drug susceptibility testing (DST) are necessary. Since conventional DST has many drawbacks, molecular DST has been introduced for detecting MDR-TB. However, there is little evidence of feasibility for molecular DST to detect XDR-TB. Therefore, the present study aims to analyze drug resistance in Korea and to evaluate rapid molecular detection of second-line drug resistance. DST results in 2009 were collected and divided into six groups: smear-negative new cases of health centers (HCs), smear-positive new cases of HCs, previously treated cases of HCs, health examination, referred cases from hospitals, and patients of the Korean National TB Association clinics. Also, we collected MDR-TB isolates whose mutations in seven loci associated with second-line drug resistance were identified and compared with conventional DST results. The resistance rate to isoniazid was highest, and the MDR rate of smear-positive new cases was 3.5%. XDR-TB accounted for 1% of the total cases and 12% of the MDR-TB cases. Resistance rates in MDR-TB varied greatly based on the drug and group. First-line drugs other than isoniazid and rifampicin showed very high resistance rates, with injectable agents demonstrating the lowest resistance rates among the drugs tested. The rates of resistance to fluoroquinolones were 2-4-fold higher in the private sector than in the public sector. Mutations in rpsL, 16S rRNA gene, and gyrA and gyrB were common in TB isolates with resistance to streptomycin, aminoglycosides, and fluoroquinolones, respectively. On the other hand, mutations in eis and gidB were not only present in resistant isolates, but also in susceptible isolates. This study demonstrates an increasing trend in MDR-TB rate among new cases of Korea. Also, we found that the proportion of XDR in MDR-TB cases was lower than the previously reported rate of 15%. A higher resistance rate of fluoroquinolones in the private sector underlines cautious use of these drugs and need for improvement in the treatment success rate for MDR-TB cases. Detecting major mutations which confer resistance to second-line drugs would be a reliable and specific method of identifying these strains. In summary, these results will be useful in helping to establish a treatment strategy for MDR-TB, and the data suggest that molecular DST can be used as a surrogate for conventional DST.

Rapid Molecular Detection and Characterization of Multidrug- and Extensively Drug-Resistance in Mycobacterium tuberculosis

조은진 경상대학교 대학원 2013 국내박사

The emergence and spread of drug-resistant strains of Mycobacterium tuberculosis (M. tuberculosis) are a serious threat to the control of tuberculosis. Treating patients infected with drug-resistant tuberculosis requires performing conventional drug-susceptibility testings (DST) on isolated bacteria, of which process takes upto 2 months. The delay in diagnosis drug-resistant can be resulted in worsening of the disease and the development of resistances to companion drugs. To avoid this delay, several rapid diagnostic methods have been developed that rely on the amplification of specific alleles known to be associated with resistance to specific drugs. The Line Probe Assay (LPA) has been officially endorsed by WHO for the detection of multidrug-resistant tuberculosis (MDR-TB) from cultured isolates and even directly from smear-positive sputum samples. Fortunately, in case of rifampicin (RIF), the diagnostic accuracy of detection is quite robust and up to 97% of isolates can be correctly identified as resistant. In the majority of RIF-resistant isolates, mutations occurred within an 81-bp region known as rifampicin resistance determining region (RRDR) in rpoB gene. Furthermore, RIF resistance generally occurs in combination with Isoniazid (INH) resistance; therefore, RIF resistance has been proposed as a surrogate marker for MDR-TB. This study analyzed the correlations between RIF resistance and mutations in rpoB as well as the predictive ability of RIF resistance for MDR-TB in Korea using the LPA. The results show that 92% of RIF-resistant strains were also resistant to INH, indicating that RIF resistance is a good surrogate marker for MDR in Korea, too. Direct molecular inspection of the rpoB gene using the LPA revealed mutations in 211 (98.14%) of the 215 RIF-resistant isolates and in 96 (100%) of the 96 RIF-resistant smear-positive sputum samples. The results showed that the LPA could be used to predict RIF- resistant phenotypes directly, at least in smear-positive sputum samples in Korea. Extensively drug-resistant tuberculosis (XDR-TB) has emerged worldwidely as a threat to public health and TB control. Improper treatment of patients with drug-resistant TB considered as the major cause of increasing XDR-TB cases. Despite of increasing XDR-TB cases, second-line TB drug testings are remained challenging beacuae of international standard methods are not well established and the reproducibility are not good, either. Therefore, for the rapid detection of extensively drug-resistant tuberculosis (XDR-TB), sloppy molecular beacons (SMBs) were designed to detect mutants with resistance to anti-TB drugs such as ofloxacin (OFX) and kanamycin (KM). Resistances to OFX or KM are caused by existence of mutations in a number of genes, including gyrA, gyrB, rrs, and the eis promoter. In this study, mutations were detected by melting temperature (Tm) shifts that occurred when SMBs bind to mismatched sequences. The resulting set of Tm values uniquely identifies the DNA sequences present in the sample. SMB assays were developed for the detection of mutations in gyrA and rrs, which closely related to the OFX and KM resistance in M. tuberculosis. The performance of this assay was evaluated by using DNA samples from clinical isolates. There were nearly 100% concordance between the SMB assay and DNA sequencing for gyrA and rrs genes analyzed in this study. SMB assay could detect gyrA gene mutations in 131 of 160 (81.88%) OFX-resistant isolates, and rrs gene mutations in 71 of 120 (59.20%) KM-resistant isolates. The gyrA-SMB assay could detect the presence of a mixed population, too. Despite the low sensitivity of the SMB assay, it will be a useful tool for identifying XDR-TB by detecting OFX and KM mutations, and also for detecting the early emergence of drug resistances in TB patients. The low sensitivity originated from the fact that several genes might involved in developing resistances of certain drug and can be overcome by adding additional targets relevant to drug resistances in the further. There are similar technologies such as TaqMan probe, and high-resolution Tm analysis which have limitations on targeting only a limited range of DNA sequences, and produce overlapping Tm peaks which are potentially difficult to analyze. However, the SMB assay could overcome such problems by taking longer probes. Through this study, a new diagnostic flatform, SMB assay to detect XDR-TB, was established and will be applied to GeneXpert MTB/RIF assay system to develop a better diagnostic system with simple, rapid, and less safety concerns.

Within-Host Ecology, Drug Pressure and the Evolution of Drug Resistance

Acosta, Monica Mercedes ProQuest Dissertations & Theses The Pennsylvania S 2020 해외박사(DDOD)

Pathogen evolution can impede the progress of human medicine. This is made no clearer than in the case of the evolution of antimicrobial drug resistance. Responsible for two million infections a year in the United States alone, drug resistance has triggered a modern health crisis; and heralds visions of a post-antibiotic era. While multiple factors have contributed to the problem, perhaps the biggest offender has been the misuse and overuse of antimicrobials. At the level of the individual, treatment regimens can vary in dose, duration, timing, etc. and are often optimized outside of the context of drug resistance. Evidence suggests that these details profoundly affect the ease by which resistance emerges and spreads within individuals. However, these details have to be contextualized. The evolution of drug resistance occurs within a dynamic host environment, variable in resources and defenses. Studying resistance evolution in its relevant ecological context is essential as drug treatment can interact with the within-host environment, producing non-straightforward effects that can be difficult to predict from simplified in vitro assays or from theoretical models. Despite this, there exist few realistic in vivo models of resistance emergence. Given that the evolution of resistance is a rare event, de-novo emergence models (i.e. from pathogen populations harboring no known resistant mutations) are even rarer. Understanding these biological details, however, has been identified as a priority by the World Health Organization and the medical community at large. We therefore use a unique in vivo model to study the effects of drug treatment and within host ecology on the de novo emergence of resistance. Drug treatment with the antimalarial drug atovaquone results in rapid selection for drug-resistance. We use Plasmodium chabaudi, a rodent malaria species and a workhorse in the field of pathogen ecology and evolution, for our de novo evolutionary experiments. We do this in two parts: first by independently varying drug treatment pressure (either drug concentration or treatment duration), and second by changing within-host parameters (such as parasite competition or the timing of drug treatment). We find that treatment failure (here defined as parasite relapse following drug treatment) and resistance evolution (relapse due to resistance) were differentially affected by dose, duration and population size, implicating complex effects of treatment on pathogen evolution and host health. In general, increases in dose and duration decreased competition between drug-resistant and sensitive parasites which increased the probability that treatment failure would occur due to resistance. Importantly, we show that the same drug regimens that optimize resistance prevention will not always result in parallel optimizations in preventing treatment failure (clinical effects). The second part of our work examines the effect of variation in host ecological parameters (here timing of drug treatment and within-host parasite competition) on resistance emergence. We show that differences in the timing of drug treatment, and associated differences in parasite population size, affected the evolution of drug resistance. As changes in population size are not independent from changes in other within-host aspects, we interpret these results conservatively, suggesting that increased population sizes at the time of drug treatment engender the evolution of drug resistance, but that these effects cannot be wholly separated from other within-host effects. Finally, we show that increasing within-host competition, by manually introducing drug-sensitive parasites into an existing infection during resistance emergence, changes infection dynamics in non-intuitive ways that require further investigation. Our work shows the importance of within-host ecology in the study of resistance emergence and supports further investigation of interactions between drug treatment and the host environment to better design chemotherapy protocols to minimize resistance.

Molecular epidemiology of antimalarial drug resistance genes in Plasmodium falciparum and Plasmodium vivax in Myanmar

하웅나우 경상국립대학교 대학원 2021 국내석사

말라리아는 열원충의 감염에 의해 야기되는 급성 열성감염질환으로서 전 세계적으로 매년 약 2억 명이 감염되며 약 420만 명이 말라리아로 사망한다. 미얀마는 동남아시아에 위치한 국가로서 동남아시아 국가들 중에서도 말라리아 유병률과 사망률이 높아 말라리아는 미얀마의 공중보건에 매우 중요한 위험 요소이다. 말라리아의 효율적 관리와 퇴치를 위협하는 항말라리아제 내성 열원충의 출현과 확산은 말라리아의 퇴치에 또 다른 중요한 걸림돌로 작용하고 있다. 열원충의 항말라리아제 내성은 표적 유전자의 돌연변이로 인해 획득되며 미얀마 열원충의 항말라리아제 내성 유전자에 대한 연구가 부족한 상태이다. 이 연구에서는 미얀마 만달레이지역에서 분리한 열대열원충과 삼일열원충에서 항말라리아제 내성 유전자의 분자역학적인 특성을 분석하였다. 항말라리아제 내성 유전자의 돌연변이는 미얀마 열대열원충과 삼일열원충 모두에서 확인되었으며 열대열원충의 pfdhfr과 pfdhps 그리고 삼일열원충의 pvdhps와 pvdhfr에서 sulfadoxin-pyrimethamine 내성과 관련된 변이가 매우 높은 비율로 관찰되었다. 열대열원충의pfmdr-1와 pfcrt, 삼일열원충의 pvmdr-1을 포함하는 chloroquine 내성 유전자에서는 중간 수준의 돌연변이 비율이 관찰되었다. 반면, 열대열원충의 pfk13, pfubp-1과 pfcytb 및 삼일열원충의 pvk12에서는 매우 낮은 수준의 변이가 관찰되거나 변이가 관찰되지 않았다. 따라서 만달레이지역에서 유행하고 있는 열대열원충과 삼일열원충에서 artemisinin과 atovaquone에 대한 내성은 높게 나타나지 않음을 알 수 있었다. 또한 만달레이지역의 열원충은 미얀마 북부나 국경지역(미얀마-태국, 미얀마-중국)의 열원충에 비교해서 낮은 수준의 항말라리아제 내성을 나타내는 것으로 생각된다. 이상의 연구 결과는 미얀마 항말라리아제 내성에 대한 이해를 확대함으로써 미얀마 말라리아 치료 및 퇴치를 위한 중요한 정보를 제공한다. Malaria is one of the most serious public health concerns in Myanmar. Different antimalarial drug regimens have periodically applied due to the emergence and spreading of drug resistance parasites, which threats malaria elimination program of the country. Antimalarial drug resistances of malaria parasites are acquired by mutations in target genes. Few approaches to determine antimalarial drug resistance in Myanmar malaria parasites have been done, but more expanded study is necessary to understand nationwide aspect of antimalarial drug resistance. In this study, molecular epidemiological study for antimalarial drug resistance genes in Plasmodium falciparum and P. vivax collected in Mandalay areas of Myanmar was conducted to gain in-depth information for antimalarial drug resistances in the areas. Substantial levels of mutations in antimalarial drug resistance genes were detected in both Myanmar P. falciparum and P. vivax isolates analyzed. Extremely high levels of mutations related with sulfadoxin-pyrimethamine resistance were found in pfdhfr and pfdhps of P. falciparum and pvdhps and pvdhfr of P. vivax. Meanwhile, moderate levels of mutations were detected in genes related with chloroquine resistance including pfmdr-1 and pfcrt of P. falciparum and pvmdr-1 of P. vivax. Low frequency or none of mutations were found in pfk13, pfubp-1, and pfcytb of P. falciparum and pvk12 of P. vivax, suggesting resistances to artemisinin and atovaquone are not critical concerns in the Mandalay areas, Myanmar. Overall patterns of molecular evidences for antimalarial drug resistance in malaria parasites in the Mandalay areas suggest that the parasite populations in the studied areas have lower levels of antimalarial drug resistance 2 compared to the parasites in boarder areas (Myanmar-Thailand and Myanmar-China) and Northern parts of Myanmar. Continuous monitoring of antimalarial drug resistances is necessary to provide useful information for the policy makers to plan proper antimalarial drug regime to control and eliminate malaria in Myanmar.

Role of Interleukin-8 in chemoresistance and progressive growth of hepatocellular carcinoma

박서영 Graduate School, Korea University 2014 국내박사

Interleukin-8 (IL-8) has been suggested as a prognostic biomarker for human hepatocellular carcinoma (HCC), but its roles in HCC progression and drug resistance have not been studied. This study investigates the role and underlying mechanism of IL-8 in the chemoresistance and progressive growth of HCC. HCC is one of the most drug-resistant cancers, and cancer stem cells are related to drug resistance. The side population (SP) of cells can be used as a model to study cancer stem cells in liver cancer. C-Jun N-terminal kinase (JNK) signaling maintains proliferation and migration in normal stem cells and also is involved in drug resistance. To date, the direct relationship between drug resistance and JNK signaling in liver cancer is unclear. In Chapter 1, the change of chemosensitivity and proportion of side population in hepatoma cells was examined by cell growth and flow cytometric analyses after anticancer treatments or knockdown of IL-8. Treatment of hepatoma cells with anticancer drugs increased the production of IL-8 and its receptor, as well as the proportion of SP cells in the cultures. In addition, recombinant IL-8 increased the SP fraction and the growth of drug-treated hepatoma cells. Silencing of IL-8 decreased both the proportion of SP cells and drug resistance properties in vitro. In Chapter 2, the correlation between the expression of IL-8 and ABC transporters was examined in both an in vivo xenograft tumor model, as well as in human HCC tissues. The drug-resistance genes breast cancer resistance protein (BCRP), multidrug resistance (MDR), and multidrug resistance-associated protein (MRP) were increased or decreased, dependent on IL-8 in HCC cells. In addition, knockdown of IL-8 in hepatoma cells resulted in significantly decreased tumor growth, and this correlated well with decreased serum IL-8 levels in vivo in xenograft models. In Chapter 3, the direct relationship between SP cells and JNK signaling was evaluated in HCC cells and tissue. The SP cell fraction was increased by anticancer drug administration and suppressed by co-administration of JNK inhibitor. Combined use of anticancer drug and JNK inhibitor was effective in suppressing anticancer drug resistance. These findings suggest that drug resistance in liver cancer might involve the increased number of SP cells resulting from JNK activation. In conclusion, these data suggest that IL-8 and the JNK signaling pathway play a critical role in HCC progression and acquisition of chemoresistance properties, and they may be potential therapeutic targets in the treatment of HCC.

Study of Tumor Microenvironment Using a 3D Culture Model of Canine Mammary Gland Tumor Cells

임가현 서울대학교 대학원 2024 국내박사

Tumor microenvironment refers to the complex ecosystem that forms within a tumor. It consists of cancer cells, immune cells, stromal cells, blood vessels, extracellular matrix, and more. In the tumor microenvironment, interactions between cancer cells, immune cells, and other components influence tumor growth, metastasis, immune evasion, angiogenesis, and drug resistance. Hence, understanding about the tumor microenvironment has become crucial in cancer research, with efforts to apply this knowledge to cancer diagnosis and treatment. Traditional cancer research predominantly has relied on 2D cell culture methods, which fails to accurately mimic the features of solid tumors. Notably, hypoxia, a hallmark of the tumor microenvironment, is challenging to induce in 2D cultures, and in vivo experiments face variability in oxygen tension across different tumors. Recognizing these limitations, 3D culture methods have gained attention. These methods allow researchers to better mimic the tumor microenvironment, modulate its components, and investigate tumor biology. Multicellular spheroid models, in particular, reflect the heterogeneity of tumor cells and create a hypoxic and acidic environment akin to solid tumors. They also mimic tumor growth, gene expression, and interactions between various components, similar to solid tumors. Multicellular spheroid models offer advantages such as precise control over tumor cells, high reproducibility, rapid model generation, and minimized influence of non-tumor cell types, making them suitable for cancer research. In this study, canine mammary gland tumor cells were used to establish multicellular spheroid models, enabling research into the tumor microenvironment. First, I established a multicellular tumor spheroid model using canine mammary gland tumor cells to mimic the tumor microenvironment in three-dimensional culture. To create spheroids from canine mammary gland tumor cells, I utilized ultra-low adhesion plates. I analyzed changes in gene expression related to hypoxia, tumor proliferation, and drug resistance using polymerase chain reaction (PCR) and Western blot analysis. When spheroids were formed from canine mammary gland tumor cells, I observed an increase in the expression of genes related to hypoxia, tumor proliferation, and drug resistance compared to conventional 2D cell culture methods (CIPp, COX2, 2.19 fold, p<0.05; HIF-1α, 4.49 fold, p<0.05; TGFβ, 3.90 fold, p<0.005; TSG6, 3.53 fold, p<0.05; VEGF, 3.80 fold, p<0.05; MRP1, 4.03 fold, p<0.001; P-gp, 2.17 fold, p<0.05), (CIPm, COX2, 4.72 fold, p<0.005; HIF-1α, 3.76 fold, p<0.05; TGFβ, 2.71 fold, p<0.01; TSG6, 4.88 fold, p<0.005; VEGF, 2.67 fold, p<0.005; MRP1, 1.78 fold, p<0.01; P-gp, 1.88 fold, p<0.01). Additionally, the expression of Procaspase3, related to cell death, and Cyclin D1, related to cell cycle arrest, decreased in spheroid formation compared to 2D cell culture methods (CIPp, Procaspase3, 0.28 fold, p<0.005; Cyclin D1, 0.27 fold, p<0.005), (CIPm, Procaspase3, 0.39 fold, p<0.01; Cyclin D1, 0.44 fold, p<0.01). Therefore, the multicellular tumor spheroids formed using canine mammary gland tumor cells can be evaluated as mimicking the tumor microenvironment. Secondly, I established a hybrid spheroid model by incorporating canine macrophages (DH82) and investigated the impact of macrophages on anti-cancer drug resistance within the tumor microenvironment. When forming the hybrid spheroid model with canine mammary gland tumor cells, I observed that hypoxic area increased compared to spheroids formed from canine mammary gland tumor cells alone (CIP, 5.23 fold, p<0.01; CIPm, 3.91 fold, p<0.05). Upon treatment with doxorubicin, there was a significant increase in the expression of hypoxia, tumor proliferation, and drug resistance-related genes in canine mammary gland tumor cells co-cultured with DH82. Additionally, the cell survival rate of canine mammary gland tumor cells co-cultured with DH82 was less reduced following doxorubicin treatment (CIPp, control group versus(vs) DH82 co-cultured group; 0.18 μM, 79.82% vs 90.01%, p<0.01; 0.37 μM, 66.67% vs 76.63%, p<0.05), (CIPm, control group vs DH82 co-cultured group; 0.18 μM, 73.07% vs 87.77%, p<0.01; 0.37 μM, 65.33% vs 81.03%, p<0.05). Moreover, the increase in cell death in canine mammary gland tumor cells co-cultured with DH82 was less pronounced after doxorubicin treatment (CIPp, control group vs DH82 co-cultured group; 0.18 μM, 16.89% vs 11.01%, p<0.05; 0.37 μM, 22.13% vs 16.15%, p<0.05; 0.92 μM, 23.70% vs 18.06%, p<0.05), (CIPm, control group vs DH82 co-cultured group; 0.18 μM, 30.82% vs 26.46%, p<0.05). The G2/M phase cell cycle arrest in canine mammary gland tumor cells co-cultured with DH82 was less pronounced after doxorubicin treatment (CIPp, ratio of G2/M to G0/G1 phases, control group vs DH82 co-cultured group; 0.37 μM, 1.51 vs 1.26, p<0.01; 0.92 μM, 1.37 vs 0.98, p<0.001), (CIPm, ratio of G2/M to G0/G1 phases, control group vs DH82 co-cultured group; 0.37 μM, 0.60 vs 0.47, p<0.05; 0.92 μM, 1.12 vs 0.84, p<0.001). Therefore, in the hybrid spheroid model with added canine macrophages, macrophages induced hypoxia, affecting gene expression in canine mammary gland tumor cells and promoting anti-cancer drug resistance. In this regard, the hybrid spheroid model with canine macrophages can be evaluated as mimicking the changes in the tumor microenvironment induced by tumor-associated macrophages. Thirdly, I established a spheroid model using canine mammary gland tumor cells with TSG6 gene knockdown to investigate the impact of TSG6 on drug resistance and angiogenesis in the tumor microenvironment. In the TSG6 knockdown spheroid model, a significant reduction in hypoxia was observed (CIPp, 0.37 fold, p<0.05; CIPm, 0.08 fold, p<0.005). When treating the TSG6 knockdown spheroid model with doxorubicin, a significant reduction in cell survival of canine mammary gland tumor cells was observed (CIPp, control group vs TSG6 knockdown group; 2.3 μM, 104.58% vs 85.29%, p<0.05; 4.6 μM, 95.43% vs 71.74%, p<0.05; 9.2 μM, 89.18% vs 70.37%, p<0.05), (CIPm, control group vs TSG6 knockdown group; 2.3 μM, 94.53% vs 72.24%, p<0.01; 4.6 μM, 93.91% vs 67.98%, p<0.01; 9.2 μM, 94.20% vs 65.82%, p<0.005). Similarly, in the TSG6 knockdown spheroid model, the expression of caspase3/7 in canine mammary gland tumor cells significantly increased upon doxorubicin treatment (CIPp, mean fluorescence intensity, control group vs TSG6 knockdown group; 2.3 μM, 41.74 vs 104.23, p<0.005; 4.6 μM, 72.84 vs 134.03, p<0.005; 9.2 μM, 70.43 vs 182.81, p<0.001), (CIPm, mean fluorescence intensity, control group vs TSG6 knockdown group; 2.3 μM, 34.72 vs 195.92, p<0.001; 4.6 μM, 35.73 vs 197.53, p<0.001; 9.2 μM, 35.81 vs 192.19, p<0.001). When assessing angiogenesis, the expression of CD31 in canine endothelial cells co-cultured with TSG6 knockdown model was significantly reduced (CIPp, 0.35 fold, p<0.05; CIPm, 0.32 fold, p<0.05). Moreover, the vascularization markers in canine endothelial cells treated with conditioned medium from TSG6 knockdown canine mammary gland tumor cells were significantly reduced. Therefore, TSG6 is evaluated as a factor that induces drug resistance and angiogenesis in the tumor microenvironment, and the knockdown spheroid model used for evaluation is considered suitable for studying the effects of a specific target gene within the tumor microenvironment. In conclusion, the spheroid model of canine mammary gland tumor cells established in this study provides a suitable method for tumor research, closely mimicking the tumor microenvironment when compared to conventional 2D cell culture. By introducing components of the tumor microenvironment, such as canine macrophages and endothelial cells, the roles of these components within the tumor microenvironment can be elucidated. Additionally, by regulating the expression of specific genes like TSG6 within tumor cells, spheroids can be formed to investigate the roles of specific genes within the tumor microenvironment. This spheroid model can be applied to study various types of tumors, including canine mammary tumors, and potentially aid in the development of anticancer strategies. Therefore, this study emphasizes that the spheroid model using canine mammary gland tumor cells is a valuable research method that closely mimics the tumor microenvironment and can serve as a foundation for tumor microenvironment studies in veterinary medicine. 종양 미세환경은 종양 내에서 형성되는 복합적인 생태계를 의미한다. 이는 종양세포와 면역세포, 연조직세포, 혈관, 세포외기질 등으로 이루어져 있다. 종양 미세환경 내에서 암세포와 세포 및 세포외 구성요소들은 서로 상호작용하여 종양의 증식, 전이, 면역회피, 혈관신생 그리고 항암제 내성에 영향을 미친다. 따라서 종양 연구에 있어서 종양미세환경에 대한 이해의 중요도가 높아졌으며 이를 연구하고 종양의 진단 및 치료에 적용하는 시도가 활발하게 진행되고 있다. 기존의 종양 연구에 있어 2차원 평면 배양 방법이 가장 전형적인 방법이었으나 실제 고형암의 특징을 반영하지 못한다. 특히, 종양의 hallmark에 해당하는 hypoxia는 종양미세환경의 주요한 요소인데 2D 평면 배양 방법으로는 이를 유도하기 어려우며 in vivo 실험 또한 종양 별 산소 압력이 다양하여 실험의 획일성을 조절하기 어렵다. 이러한 점에서 3차원 배양 방법은 종양 미세환경을 모방할 수 있고 연구자가 종양 미세환경의 구성 요소를 조절하여 종양 연구를 진행할 수 있다는 점에서 장점을 가진다. 특히나 multicellular spheroid model은 종양 미세환경 내 종양세포와 다른 세포의 heterogeneity를 반영할 수 있고 multiple cell layer를 가지기에 oxygen과 nutrient의 확산 저하로 고형암 내부의 hypoxic and acidic 환경을 반영한다. 더불어 종양세포와 다른 구성 요소들간의 상호작용으로 인해 종양의 성장 및 유전자 발현 등이 고형암과 유사하다 평가된다. 이러한 점에서 multicellular spheroid model은 종양 미세환경을 잘 모방하는 방법으로 고려되며 기존 2차원 평면 배양 방법과 비교할 시 종양 세포의 정밀한 제어와 재현성, 빠른 모델 생성 및 비종양 세포 유형의 영향을 최소화할 수 있는 능력과 같은 장점도 가지기에 종양 연구에 적합하다 평가된다. 따라서, 본 연구에서는 개 유선종양 세포를 사용하여 multicellular spheroid model을 확립하였고 이를 활용하여 종양 미세환경 연구를 진행하였다. 첫번째, 개 유선종양 세포의 multicellular tumor spheroid 모델을 확립하고 3차원 배양에 따른 저산소 유발 및 유전자 발현 변화를 확인하여 종양 미세환경을 모방하는 것을 확인하였다. 개 유선 종양 세포의 spheroid를 형성하기 위해 ultra-low adhesion plate를 사용하였고 중합효소 연쇄반응 기법과 western blot 분석 기법을 이용하여 저산소, 종양 증식 및 항암제 내성 관련 인자의 발현 변화를 분석하였다. 개 유선 종양 세포의 spheroid를 형성하였을 시, 기존 2차원 세포 배양 방법에 비해 저산소, 종양 증식 및 항암제 내성 관련 인자의 발현이 증가하였다 (CIPp, COX2, 2.19배, p<0.05; HIF-1α, 4.49배, p<0.05; TGFβ, 3.90배, p<0.005; TSG6, 3.53배, p<0.05; VEGF, 3.80배, p<0.05; MRP1, 4.03배, p<0.001; P-gp, 2.17배, p<0.05), (CIPm, COX2, 4.72배, p<0.005; HIF-1α, 3.76배, p<0.05; TGFβ, 2.71배, p<0.01; TSG6, 4.88배, p<0.005; VEGF, 2.67배, p<0.005; MRP1, 1.78배, p<0.01; P-gp, 1.88배, p<0.01). 더불어 세포사멸과 관련된 Procaspase3과 세포 주기 정지와 관련된 Cyclin D1의 발현도 spheroid 형성시 2 차원 세포 배양 대비 감소하였다 (CIPp, Procaspase3, 0.28배, p<0.005; Cyclin D1, 0.27배, p<0.005), (CIPm, Procaspase3, 0.39배, p<0.01, Cyclin D1 0.44배, p<0.01). 따라서 개 유선종양 세포로 형성한 multicellular tumor spheroid는 종양 미세환경을 모방한다고 생각할 수 있다. 두번째, 개 대식세포(DH82)를 추가한 hybrid spheroid model을 확립하고 종양 미세환경 내 대식세포에 의한 항암제 내성 변화를 확인하였다. 대식세포 hybrid spheroid model 형성시 개 유선종양 세포 단독 spheroid 대비 hypoxia 면적이 더 증가하였다 (CIP, 5.23 배, p<0.01; CIPm, 3.91 배, p<0.05). Doxorubicin 처치시 DH82 공배양한 개 유선종양 세포에서 저산소, 종양 증식 및 항암제 내성 관련 인자의 발현이 유의적으로 증가하였다. Doxorubicin 처치시 DH82를 공배양한 개 유선종양 세포의 세포 생존률이 덜 감소하였다 (CIPp, 대조군 vs DH82 공배양군; 0.18 μM, 79.82 % vs 90.01 %, p<0.01; 0.37 μM, 66.67% vs 76.63%, p<0.05), (CIPm, 대조군 vs DH82 공배양군; 0.18 μM, 73.07 % vs 87.77 %, p<0.01; 0.37 μM, 65.33% vs 81.03%, p<0.05). Doxorubicin 처치시 DH82를 공배양한 개 유선종양 세포의 세포사멸이 덜 증가하였다 (CIPp, 대조군 vs DH82 공배양군; 0.18 μM, 16.89 % vs 11.01 %, p<0.05; 0.37 μM, 22.13% vs 16.15%, p<0.05; 0.92 μM, 23.70% vs 18.06%, p<0.05), (CIPm, 대조군 vs DH82 공배양군; 0.18 μM, 30.82 % vs 26.46 %, p<0.05). Doxorubicin 처치시 DH82를 공배양한 개 유선종양 세포의 G2/M phase cell cycle arrest가 덜 증가하였다 (CIPp, G2/M과 G0/G1 주기의 비율, 대조군 vs DH82 공배양군; 0.37 μM, 1.51 vs 1.26, p<0.01; 0.92 μM, 1.37 vs 0.98, p<0.001), (CIPm, G2/M과 G0/G1 주기의 비율, 대조군 vs DH82 공배양군; 0.37 μM, 0.60 vs 0.47 p<0.05; 0.92 μM, 1.12 vs 0.84, p<0.001). 따라서 개 대식세포를 추가한 hybrid spheroid model에서 대식세포는 저산소를 유발하여 개 유선 종양 세포의 유전자 발현에 영향을 주어 항암제 내성을 유발하였다. 이러한 점에서 개 대식세포를 추가한 hybrid spheroid model은 대식세포에 의한 종양 미세환경의 변화를 모방한다고 생각할 수 있다. 세번째, TSG6 유전자 knockdown을 유발한 개 유선 종양 세포를 사용한 spheroid model를 형성하여 TSG6가 종양 미세환경내에서 항암제 내성과 혈관생성에 미치는 영향을 확인하였다. TSG6 knockdown spheroid model에서 유의적으로 hypoxia가 감소하였다 (CIPp, 0.37 배, p<0.05; CIPm, 0.08 배, p<0.005). TSG6 knockdown spheroid model에서 Doxorubicin 처치시 개 유선종양세포의 세포 생존률이 유의적으로 감소하였다 (CIPp, 대조군 vs TSG6 knockdown 군; 2.3 μM, 104.58 % vs 85.29 %, p<0.05; 4.6 μM, 95.43% vs 71.74%, p<0.05; 9.2 μM, 89.18 % vs 70.37 %, p<0.05), (CIPm, 대조군 vs TSG6 knockdown 군; 2.3 μM, 94.53 % vs 72.24 %, p<0.01; 4.6 μM, 93.91% vs 67.98%, p<0.01; 9.2 μM, 94.20 % vs 65.82 %, p<0.005). 마찬가지로 TSG6 knockdown spheroid model에서 Doxorubicin 처치시 개 유선종양세포의 caspase3/7의 발현이 유의적으로 증가하였다 (CIPp, 평균 형광강도, 대조군 vs TSG6 knockdown 군; 2.3 μM, 41.74 vs 104.23, p<0.005; 4.6 μM, 72.84 vs 134.03, p<0.005; 9.2 μM, 70.43 vs 182.81, p<0.001), (CIPm, 평균 형광강도, 대조군 vs TSG6 knockdown 군; 2.3 μM, 34.72 vs 195.92, p<0.001; 4.6 μM, 35.73 vs 197.53, p<0.001; 9.2 μM, 35.81 vs 192.19, p<0.001). 혈관신생을 평가하였을 시, 개 혈관내피세포를 추가한 TSG6 knockdown model에서 혈관내피세포의 CD31 발현이 유의적으로 감소하였다 (CIPp, 0.35 배, p<0.05; CIPm, 0.32 배, p<0.05). 또한 TSG6 knockdown 시킨 개 유선종양 세포의 세포 배양 상층액을 처치한 개 혈관내피세포에서 혈관생성 지표가 유의적으로 감소하였다. 따라서, TSG6가 종양 미세환경에서 항암제 내성과 혈관 신생에 유발하는 인자로 작용한다 평가되며 이를 평가하기 위해 사용한 knockdown spheroid model이 원하는 목표 유전자의 제거를 통해 그 유전자가 종양 미세환경 내에서 미치는 영향을 연구하는데 적합한 model이라 평가할 수 있다. 결론적으로, 이번 연구에서 확립한 개 유선종양 세포의 spheroid model은 종양 미세환경을 모방하여 기존 2차원 세포 배양에 비해 종양 연구에 적합한 방법이다. 개 대식세포 및 개 혈관내피 세포 등의 종양 미세환경 내 구성요소를 추가하여 각 구성요소들이 종양 미세환경에 미치는 역할을 규명할 수 있다. 또한, 종양 세포에서 TSG6와 같이 특정 유전자의 발현을 조절한 뒤 spheroid를 형성하여 종양 미세환경 내 특정 유전자의 역할을 파악할 수 있다. 이러한 spheroid model을 활용하여 개 유선 종양을 포함한 다양한 종양의 종양 미세환경에 대한 연구가 가능하며 항암제 개발 방법 사용될 수 있다. 따라서 이 연구는 개 유선종양 세포를 개발한 spheroid model은 종양 미세환경을 모방하는 유용한 연구 방법으로, 수의학에서 종양 미세환경의 연구에 토대가 될 수 있다고 생각한다.

Interpenetrating Polymer Network Hydrogels as An Engineered 3D Tumor Microenvironment for Drug Screening of Lung Carcinoma

이동신 인천대학교 일반대학원 2020 국내석사

암은 전세계 인류의 주요 사망 원인 중 하나로, 암의 치료를 위해 화학 요법, 방사선 요법 등의 다양한 임상 전략이 개발되고 발전하였지만 여전히 암 치료에 있어 높은 실패율을 보인다. 종양 미세 환경에 대한 불충분한 이해 또한 항암 치료와 신약 개발에 있어 심각한 어려움을 야기한다. 수십 년 동안 암세포의 진행 억제 또는 사멸을 유발하는 많은 화학요법 기반의 항암제들이 개발되었다. 그 중에서도, 게피니팁 등의 티로신키나아제 억제제는 높은 빈도로 EGFR의 비정상적인 활성화를 보이는 것으로 알려진 비소세포폐암 (NSCLS)를 치료하는 데에 효과를 보여 왔다. 비소세포폐암은 게피니팁에 의해 유발되는 EGFR 신호전달의 저해를 통해 효과적으로 치료될 수 있었으나, 치료 과정에서 돌연변이에 의한 수용체의 구조 변형 또는 EGFR의 신호전달을 대신할 수 있는 우회 경로를 통해 궁극적으로 항암제에 내성을 획득하게 된다. 따라서, 효과적인 암 치료 및 기초 연구를 위해서는 암세포가 항암제에 내성을 획득하는 메커니즘의 이해와 연구가 필요한 실정이다. 2D 배양, 동물 모델 등의 전임상 모델은 새로 개발되는 항암제의 임상 유효 성을 예측하고 기초적인 암 연구를 연구하는 데 이용되어 왔지만, 이러한 전임상 모델은 체내 종양 미세 환경을 모방하는 데 한계점을 지닌다. 하이드로 젤을 활용한 3차원 인공 종양 모델은 체내 종양 환경의 다양한 매개 변수를 모방할 수 있어, 기존 전임상 모델의 한계를 극복할 수 있는 새로운 플랫폼이라고 할 수 있다. 따라서, 하이드로젤을 기반으로 디자인된 3차원 종양 미세 환경을 이용하여 종양 미세 환경내 다양한 변수들이 암세포의 활동에 미치는 영향을 연구할 수 있다. 또한, 기능성 하이드로겔 물질의 공학적 응용을 통해 체내 종양 미세 환경의 특정한 특징들을 정확하게 재현함으로써 보다 진보한 암세포 연구 및 항암 효과의 예측을 할 수 있다. 그러나 체내 종양 미세 환경에 있어 중요한 요소들을 정확히 모방하기에 적절한 특성을 갖는 하이드로 겔 재료를 개발하는 것은 여전히 큰 과제로 남아 있다. 본 연구에서는 젤라틴과 폴리에틸렌 글리콜을 활용하여 새로운 유형의 상호침투 고분자 하이드로젤을 개발하고, 이를 활용하여 항암 물질의 스크리닝 및 폐암 세포의 내성 기전을 연구하기 위한 3차원 종양 미세 환경을 구현하고자 하였다. HRP를 이용한 산화적 가교 반응을 통해 하이드로젤을 제작하였으며, 젤 형성 과정에서 하이드로젤 구성 물질의 도입 정도를 조절하여 상전이 시간을 조절할 수 있었다. 특별히, 과산화수소의 농도를 조절하여 고분자 구성의 변화 없이도 IPN 하이드로젤의 기계적 특성을 조절할 수 있었으며, 단백질 분해 효소에 대한 구조적 안정성 또한 관찰할 수 있었다. 이러한 연구결과를 바탕으로 하이드로젤 내부에 폐암 세포인 HCC827과 HCC827GR을 캡슐화하여 3차원 폐암 미세 환경을 구현하였으며, 이를 활용해 상용화된 항암제인 티로신키나아제 억제제 게피티닙에 대한 폐암 세포의 약물 반응과 약물 내성을 유발하는 생물학적 기전을 연구할 수 있었다. 따라서, 이러한 결과들을 종합하였을 때 본 연구에서 개발한 IPN 하이드로 겔이 암세포의 기초 연구, 임상 결과를 개선하기 위해 항암 물질의 스크리닝 등 다양한 임상적 활용에 적용할 수 있는 종양 미세 환경을 조성하는 데 이용될 수 있을 것으로 판단한다. 핵심어: 고분자 하이드로젤, 인공 종양 미세환경, 폐암 세포, 약물 저항성 Cancer is a major one of the leading causes of death worldwide. Cancer treatment has limitations by high failure, despite the advances in clinical strategies such as chemotherapy, radiation therapy, and so on. Poor understanding of the cancer microenvironment also causes challenges in developing novel therapeutic agents. For decades, numerous chemotherapeutic anti-cancer drugs have developed that effectively inhibit the progression or survival of cancer cells. In particular, tyrosine kinase inhibitor (TKI) such as gefitinib has been widely used to treat non-small cell lung cancers (NSCLSs) baring mutations in the continuous activation of epidermal growth factor receptor (EGFR). EGFR mutant NSCLCs were effectively inhibited by gefitinib through the inhibition of the EGFR pathway for the survival of cancer cells. However, these cancers finally get resistance to the anti-cancer drug through the mutation of the drug binding site or development of the bypass pathway that can alter the role of EGFR. Therefore, understanding and studying the mechanism of how the cancer cells get resistance to anti-cancer drugs are indispensable for effective cancer treatment and basic cancer research. Although traditional pre-clinical models such as 2D culture and in vivo animal models have been utilized to predict the efficacy of newly developed anti-cancer drugs and studying basic cancer research, these pre-clinical models have limitations in simulating the efficacy of clinical outcomes due to poor recapitulation the native tumor microenvironments. Engineered hydrogels are assumed as advanced platforms to overcome these limitations through the mimicry of various parameters of the native tumor microenvironment. Hydrogel-based engineered 3D tumor microenvironments can be utilized to study the effects of independent or multiple cues within the tumor microenvironment on the cancer cell behavior and response against chemotherapeutic agents. Furthermore, emerging techniques with advanced hydrogel materials enable us to accurately recapitulate the specific features of the native tumor microenvironment that can contribute to advanced cancer research and the prediction of therapeutic agents. However, it is still challenging to develop hydrogel materials with appropriate characteristics to recapitulate the critical parameters in the native tumor microenvironment. Herein, we develop an interpenetrating polymer network (IPN) hydrogels composed of gelatin and poly (ethylene glycol), which can be utilized as an engineered 3D tumor microenvironment for the screening of the anti-cancer drugs and for exploring the drug resistance of lung carcinoma cells. Hydrogels are formed via horseradish peroxidase (HRP)-mediated oxidative crosslinking reaction, resulting in the IPN hydrogels with controllable phase transition time. The physicochemical properties of the IPN hydrogels such as mechanical property can be independently adjusted by different contents of H2O2. The IPN hydrogels exhibit structural stability against proteolytic enzymes. Based on this finding, we created engineered 3D lung carcinoma constructs to study the effect of the matrix stiffness on the drug resistance of human lung carcinoma cell line, HCC827, and HCC827GR. Moreover, we investigated the molecular mechanism that drives the response and the resistance of lung carcinoma cells against the EGFR TKI drugs. We propose that our IPN hydrogel is assumed as a promising material for engineered tumor microenvironment for various clinical applications, such as basic cancer research and screening of anticancer drugs for better clinical outcomes. Keywords: polymeric hydrogels, engineered 3D tumor microenvironment, lung cancer, drug resistance

The role of RhoGDI2 in drug resistance : 항암제에 대한 내성을 촉진시키는 RhoGDI2의 기능규명

백경은 경상대학교 대학원 2009 국내박사

Gastric cancer is still a serious health problem and remains the second most common type of fatal cancer worldwide. Despite the performance of extensive diagnostic and therapeutic investigations of gastric cancer, the prognosis for patients with advanced gastric cancer remains dismal, and little improvement in survival has been achieved in recent years. One reason is that gastric cancer is still too often diagnosed at a very advanced stage. For patients with advanced gastric cancer, chemotherapy has been the mainstream of treatment. However, the efficacy of chemotherapeutic agents in cancer treatment is limited by the development of resistance. RhoGDI2 promotes tumor growth and malignant progression in gastric cancer. In this study, the role of RhoGDI2 in drug-induced apoptosis is verified. Forced expression of RhoGDI2 in human gastric cancer cells, SNU-484, increased resistance against the induction of apoptosis by chemotherapeutic agents (cisplatin, etoposide, and staurosporin) and cisplatin-induced inhibition of tumor growth in nude mice model. Conversely, RhoGDI2 depletion by RNA interference sensitized MKN-28 cells, human gastric cancer cells which express the endogenous RhoGDI2, to drug-induced apoptosis. Next, VEGF-C was identified as a downstream target molecule for antiapoptotic ability of RhoGDI2 by using Microarray, RT-PCR, and Western blot analysis. Overexpression of RhoGDI2 increased the expression of VEGF-C and Bcl-2. Bcl-2 is one of the antiapoptotic Bcl-2 family member proteins and its expression has been known to be regulated by VEGF-C. Conversely, RhoGDI2 depletion inhibited their expression, respectively. Silencing of VEGF-C expression by RNAi in RhoGDI2 overexpressing cells suppressed Bcl-2 expression and augmented drug-induced apoptosis. Silencing of Bcl-2 expression by RNAi in RhoGDI2 overexpressing cells also increased drug-induced apoptosis. Finally, RhoGDI2-mediated drug resistance may be derived from its ability to sequentially activate PLC??/Rac1/PAK/NF-?eB. Overexpression of RhoGDI2 induced the activation of PLC??, Rac1, PAK, and NF-?eB, while RhoGDI2 depletion repressed their activities. Treatment of pharmacological inhibitors against these molecules in RhoGDI2 overexpressing cells suppressed RhoGDI2-mediated VEGF-C expression and drug resistance. It has not been reported about the role of RhoGDI2 in drug-induced apoptosis. All of these results suggest that RhoGDI2 is an antiapoptotic molecule that mediates cellular resistance to chemotherapeutic agents. Thus, RhoGDI2 may be a potent target for anticancer treatment.

Cisplatin resistance in human gastric cancer cell lines exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

오민영 고려대학교 보건대학원 2010 국내석사

Objectives : This study was performed to find the Cisplatin resistance and the mechanisms in gatric cancer cells exposed to 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Methods : Among gastric cancer cell lines, we selected three cell lines including SNU 601, MKN 45, SNU 668 according to Cisplatin resistance including weak, moderate and strong resistance cells. We compared drug resistance genes including breast cancer resistance protein (BCRP/ABCG2), major vault protein (MVP/lung resistance protein, LRP), P-glycoprotein (P-gp/multidrug resistance1, MDR1/ABCB1), multidrug resistance protein2 (MRP2/ABCC2), clusterin among cell lines after exposure of TCDD. We also compared cell viability of cells by exposure of TCDD with Cisplatin treatment. Results : Among drug resistance genes, only ABCG2 expression was significantly increased in three gastric cancer cell lines exposed to TCDD by PCR and Western blotting. Cell viability increased even after Cisplatin treatment in all three cell lines after exposure of TCDD. This result shows TCDD can increase the drug resistance of gastric cancer cells by increasing drug resistance protein, ABCG2. Conclusion : TCDD can increase of Cisplatin resistance through increase of ABCG2 in gastric cancer cells. Further study is needed to evaluate drug resistance of other cells lines related with exposure of environemental toxicants.