대한응급의학회지제 19 권제 3 호 Volume 19, Number 3, June, 2008 원 저 Paraquat 로저항성을유발시킨인간간암세포주에서전사반응에대한 DNA microarray 분석 충북대학교의과대학응급의학교실, 충북대학교의과대학흉부외과교실 1 김 @ 훈ㆍ이석우ㆍ김시욱 DNA Microarray Analysis of Transcriptional Responses in HepG2 Cells with Induced Paraquat Resistance Hoon Kim, M.D., Suk-Woo Lee, M.D., Si-Wook Kim, M.D. 1 Purpose: To date, paraquat poisoning has almost universally resulted in unfavorable outcomes, and it has become a big issue in clinical toxicology. Current efforts to overcome its toxicity have focused on drugs with anti-oxidant capacity such as ascorbic acid in order to combat over-production of reactive oxygen species (ROS) by paraquat radicals, which are mainly induced by NADPH-cytochrome P450 reductase. Unfortunately, this strategy of treatment has not yielded satisfactory results. In search of a new approach to cope with PQ toxicity, we developed an in vitro culture model of cells resistant to lethal doses of PQ, and we then investigated resistance mechanisms using DNA microarray technology, a tool for simultaneously measuring a number of gene expression changes. Methods: This experiment was conducted in vitro using the hepatocelluar carcinoma cell line (HepG2) to assay xenobitotics metabolism. We induced resistant of these cells to up to 100 um PQ by treating with escalating doses of PQ for about 5 months. Cytotoxicity was studied using the MTT method, and optical density was measured at 540 nm using an ELISA reader. We examined morphological changes in cells after drug treatment using an inverted microscope, and we investigated gene expression profiles in control and resistant cells by use of DNA microarray. Results: Results of MTT assays indicated that resistant cells showed relatively high survivals against a 100 mm dose, but that the control group had zero percents of survival at a 1 mm dose. In the comparing gene expression levels between the control group and the resistant group, 6,717 genes found to be differentially expressed. In the analysis of anti-apoptosis genes in particular, the resistant group showed more expression of genes with anti-apoptotic functions than did the control group. In examining the expression of cytochrome P450 genes related to xenobiotic metabolism and PQ radical induction, expression of the cytochrome P450 1B1 gene was significantly higher in the resistant group than in the control group. Conclusion: Although cytochrome P450 is known to be responsible for redox cycling of PQ as an electron transferor, this study suggest that up-regulation of the cytochrome P450 1B1 gene can corelate with PQ resistance. Therefore, induction of cytochrome P450 1B1 can be a new therapeutic approach to reduce PQ toxicity through actual PQ degradation, rather than simply through neutralization of ROS. Key Words: Paraquat, DNA Microarray, Cytochrome P450 Department of Emergency Medicine, Department of Chest surgery 1, College of Medicine, Chungbuk National University, Cheongjoo, Korea 책임저자 : 김 @ 훈충청북도청주시흥덕구개신동충북대학교병원응급의학교실 Tel: 043) 269-6080, Fax: 043) 269-6954 E-mail: nichekh2000@chungbuk.ac.kr 접수일 : 2008년 3월 27일, 1차교정일 : 2008년 5월 19일게재승인일 : 2008년 6월 2일 서론 Paraquat (1,1 -dimethy-4,4 -bipyridinium dichloride) 는 1958년최초로개발된이후흔히사용되는비선택성접촉성광범위제초제로서국내에서는그람옥손, 속사포, 파라코등의이름으로시판되고있다. 강한독성으로소량에서도인체에흡수되면치명적이어서약물중독에 * 이논문은 2007학년도충북대학교학술연구지원사업의연구의한사망원인으로는최고를차지하며우리나라에서는연비지원에의하여연구되었음. 322
김 @ 훈외 : Paraquat 로저항성을유발시킨인간간암세포주에서전사반응에대한 DNA microarray 분석 / 323 간 500명이상의사망자가발생한다고알려져있다 1). Paraquat의독성작용은세포내에서 cytochrome P450 reductase, glutathione reductase 등의효소에의한 paraquat 이온의순환적환원으로활성산소종인 superoxide radicals의과다생성으로세포막지질과산화, 세포자멸 (apoptosis) 유도등이발생하여산화적세포독성이발생하게되어임상적으로소량의음독시에도 paraquat 는폐세포에서의지속적인독성작용으로인하여수주에서수개월에거쳐서폐섬유화를통한호흡부전으로사망하는사례를자주볼수있다 2). 임상적으로 paraquat 중독치료를위해다양한항산화제가중요한근간으로인식되고있다. 이에현재급성 paraquat 중독의치료시유해활성산소종제거에활용되고있는대표적인항산화제들로 tocopherol, ascorbic acid 등의비타민계항산화제, melatonin, glutathione, metallothionein, n-acetylcysteine, liposomal antioxidants 등이있다. 그외에 chelating agents로 desferoxamine hydroxypyridin-4-one, 그리고 superoxide radicals 제거와관련된효소인 superoxide dismutase( 이하 SOD) 흡인치료등이현재임상적으로활용되고있는약물들이다 3,4). 하지만현재까지외부에서투여하는항산화제중심의 paraquat 치료는만족스럽지않은임상결과를보이고있다. 이에 paraquat 치료에대한새로운접근이필요하며, 특히소량의중독과만성적중독에서 paraquat의대사촉진유도, 각장기의 paraquat 저항성증대방안을찾는노력이필요하다. 이에우리는 paraquat에대한내재적저항성과관련된특정유전자들을조사하여 paraquat중독치료에대한새로운접근을시도하기위하여인간간암세포주에비치명적인용량의 paraquat를장기간처리하여고농도의 paraquat에저항성인상태로유도한후, DNA microarray 기법을활용하여유도된저항성세포군에서발현하는유전자변화의차이를정상세포군과비교분석하였다. 대상과방법 1. 실험재료본실험에사용된 paraquat, dimethyl sulfoxide, tetrazolium bromide은 Sigma (St. Louis, MO, USA) 사에서, Dulbeco s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin 등은 Gibco-BRL (Rockville, MD, USA) 사에서구입하였고, ECL protein detection kit, nitrocellulose membrane은 Amersham (Arlington Heighs, IL, USA) 사에서구입하였다. RNA 추출을위하여 ultraspec kit (Biotecx Lab. Inc., USA) 를이용하였다. 본실험에서사 용된 DNA microchip은 Human whole 35K Oligo chip (Operon Co., Germany) 을사용하였다. 2. 세포의배양과저항성유도본실험은간암세포주 HepG2 세포를사용하여 10% heat-inactivated FBS를첨가한 DMEM을배양액으로 5% CO 2, 37 C 에서배양하였으며, 저항성의유도는계대배양시저농도 (1 nm paraquat) 에서부터점차적으로비치명적인용량으로농도를높여처리함으로서세포가죽지않도록유도하였다. 이러한유도과정에서생존하는세포군은점차서서히 paraquat의농도를높이면서계대배양을지속시켰으며고농도 (100 um paraquat) 에서도생존을유지하는상태까지배양을시행하였다. 3. 세포생존력검사 (MTT assay) 세포생존정도를평가하기위해 ELISA microreader 650 (Biorad Laboratories, CA, USA) 을이용하여 Microculture tetrazolium 법에의해 540 nm에서흡광도를측정하였다. 4. 세포형태변화관찰유도된 paraquat 저항성세포군은도립현미경 (Inverted Microscope; 대물렌즈가표본아래부착되어배양용기내세포나조직의관찰에용이한생물현미경 ) 이용하여정상세포군 (paraquat 미처리군 ) 과비교하여세포의형태변화양상을함께관찰하였다. 5. RNA 추출전체 RNA 조제는 Ultraspec kit (Biotecx Lab. Inc., USA) 을이용하여사용설명서에따라분리하였다. 최종적으로조제된 RNA 농도는분광광도계를이용하여 260 nm 에서측정하였다. 6. DNA microarray 분석 Operon Human Whole 35 K Oligo chip (Operon Co., Germany) 을이용하여대조군과비교하여유도된저항성군에서의유전자변화를분석하였다. 각샘플의 RNA을 Cy3와 Cy5의형광물질을이용하여역전사과정에 labeling 후에 DNA microarray slide glass에점적하여 hybridization을시킨후 microarray scanner를이용하여 scanning을진행하면서각 spot에서방사되는 Cy3와 Cy5의형광강도를수치화하였다. 이러한수치화과정은
324 / 대한응급의학회지 : 제 19 권제 3 호 2008 scanner의분석용 software (GenePix pro 4.1) 프로그램을이용하였고, 대조군과비교하여유도된저항성군에서의유전자변화가 2배이상증가또는감소가있을때의미있는유전자변화로서정의하였다 (Fig. 1). 저항성세포군과정상세포군에 paraquat를농도별로처리한후 48시간에서의생존율을비교한결과, 정상세포군에서 1 um 농도에서는 95% 이상의세포손상이관찰된반면에, 저항성세포군에서는 100 um 농도에서도약 80% 의세포생존을보였다 (Fig. 2). 결 과 2. 저항성세포군의형태학적변화 1. 저항성세포군과정상세포군의생존률비교 정상적인세포군은비교적다각형형태로여러세포들이 Fig. 1. Schematic DNA microarray process. Total mrna extracted from normal cells & resistant cells was labeled with Cyanine (CY3) or Cyanine (Cy5) by a reverse transcription reaction using reverse transcriptase. After this, two labeled cdnas were mixed and placed on Operon Human genome oligo 35 K micorarry. Hybridized slides were scanned with the Axon Instruments GenePix 4000 B scanner and the scanned images were analyzed with the software program GenePix Pro 5.1. Fig. 2. Comparison of cytotoxic activity of paraquat between resistant group and control group. HeG2 cells were treated with various concentrations of paraquat for the indicated times. Cytotoxicity was determined by MTT assay, and relative absorbance comparing with control group without paraquat treatment was calculated according to doses of paraquat. A B Fig. 3. The morphological change of normal HeG2 cells and induced HeG2 cells resistant up to 100 um PQ. The morphology of the cells was observed under phase contrast microscopy ( 300). (A) Normal cells (B) Induced resistant cells up to 100 um PQ treatment
김 @ 훈외 : Paraquat 로저항성을유발시킨인간간암세포주에서전사반응에대한 DNA microarray 분석 / 325 군집을이루면서분열성장하는양상인반면저항성세포군에서는돌기형태로정상세포보다외형이더가늘고긴모양을보이면서각세포는분리된상태로분열성장하는양상을보였다 (Fig. 3). 한유전자발현변이를확인한결과, 총 6,717개의유전자들이 DNA microarray 분석에서유의한수준의 2배이상 3. RNA 추출 정상세포군과저항성세포군에서전체 RNA를분광광도계로정량한결과, 각각 419.50 ug과 91.12 ug 이였으며각각 30 ug을 DNA microarray 분석에사용하였다. 추출의순도를확인하기위해전기영동을실시한결과비교적높은순도로분리됨을확인하였다 (Fig. 4). 4. DNA Microarray 분석결과 36,912개의유전자가부착된 Operon Human Whole 35K Oligo chip을활용한 DNA Microarray 분석을통한정상세포군과비교하여상대적인저항성세포군에서유전자발현정도와차이를조사한결과, 그림 5에서처럼녹색과적색이혼재된양상의소견이관찰되었고, 녹색과적색의강도도 hybridization 반응정도에따라다양하게관찰되었다 (Fig. 5). Hybridization 후저항성세포군의정상세포군에대비 Fig. 5. Hybridization result of oparray Human whole 35 K oligo chip. We investigated scan image and scatter plot of oparray Human whole 35K oligo chip. Total RNA extracted from normal HepG2 cells was labeled with Cy3 (green). Total RNA extracted from resistant HepG2 cells was labeled with Cy5 (red). The intensity ratio of Cy3 and Cy5 reflect the degree of expression of specific genes. Sample name 260 nm 280 nm Ratio (260 nm/280 nm) concentration (ug/ul) total RNA (ug) Normal cells 0.681 0.34 2.03 5.45 419.50 Resistant cells 0.242 0.11 2.15 1.94 091.12 Fig. 4. Total RNA concentration results and agarose gel image. Total genomic DNA was extracted and the concentrations of total RNA were determined using Shimadzu spectrophotometer. the extraction purity were identified by being electrophoresed on 1.6% agarose gel.
326 / 대한응급의학회지 : 제 19 권제 3 호 2008 유전자발현변이를나타내었다. 이중 2배이상유의한유전자발현의증가를보인경우가 3,385개 (50.4%), 2배이상유의한유전자발현의감소를보인경우가 3,328개 (49.6%) 로서비슷한양상을보였다 (Fig. 6). 또한 100배이상유전자발현의증가를보인경우가 47개 (0.7%), 100배이하유전자발현감소를보인경우가 11개 (0.2%) 였다. 이중세포자멸과관련된유전자발현발생을조사한결과, HIV-1 Tat interactive protein 2, interferon, lectin, clusterin, BCL2-associated athanogene, TNF receptor -associated factor 5, phosphatase 등 13개의반세포자멸 (anti-apoptosis) 유전자발현이증가하였으며, amyloid beta precursor protein binding protein 1, BCL2-related ovarian killer, microtubule-associated protein tau, BCL2-associated transcription factor 1 등 37개의세포자멸유전자발현이억제되었다. 반면에 PH domain and leucine rich repeat protein phosphatase, ring finger and FYVE-like domain containing 1, tumor necrosis factor receptor superfamily, member 21, BCL2-associated transcription factor 1 등반세포자멸과관련된 7개의유전자발현이억제되었고, cyclin-dependent kinase inhibitor 2A, pleiomorphic adenoma gene-like 1, p21/cdc42/ Rac1-activated kinase 1, adhesion molecule with Ig-like domain 2 등 27개의세포자멸과관련된유전자발현이증가한소견을보였다 (Table 1). 또한약물의대사와 paraquat의독성유발과연관이있는 cytochrome P450 효소계유전자발현과관련하여 cytochrome P450 family 1 subfamily B polypeptide 1, cytochrome P450 family 26 subfamily B polypeptide 1, cytochrome P450 family 11 subfamily A polypeptide 1 등의유전자발현이증가소견을보였다 (Table 2). 고찰세포내로유입된 paraquat는 cytochrome P450 효소등의전자전달계관련단백질에의하여 paraquat 이온이형성되어산소와반응하여유해한자유산소기를생산하여독성을유발하는것으로잘알려져있다 5). 세포내에서의방어기전은이러한자유산소기의제거를위한catalase, SOD 등이중요한역할을하는것으로알려져있다 2,5). 이에 SOD 흡입치료등이임상적으로시도되기도한다. 하지만 Fig. 6. Signal intensity ratio histogram. Signal intensity of Cy3/Cy5 show differential transcriptional expression of resistant HeG2 cells in comparisons with normal HeG2 cells. The number of spots with 2-fold changes were 6717(Log intensity >1.0 or <-1.0).
김 @ 훈외 : Paraquat 로저항성을유발시킨인간간암세포주에서전사반응에대한 DNA microarray 분석 / 327 Table 1. Up-and Down- regulated gene expressions related with apoptotic function GenBank Accession No. Updated gene title Signal intensity NM_006410 HIV-1 Tat interactive protein 2, 30kDa 221.30 AF208043 interferon, gamma-inducible protein 16 017.80 NM_002305 lectin, galactoside-binding, soluble, 1 (galectin 1) 009.86 NM_203339 clusterin 009.55 NM_004323 BCL2-associated athanogene 008.40 NM_004619 TNF receptor-associated factor 5 005.78 NM_000314 phosphatase and tensin homolog 003.66 NM_001065 tumor necrosis factor receptor superfamily, member 1A 003.10 NM_138926 SON DNA binding protein 003.10 BM802700 nudix (nucleoside diphosphate linked moiety X)-type motif 2 002.86 BX647177 Bcl-2 inhibitor of transcription 002.68 NM_002759 eukaryotic translation initiation factor 2-alpha kinase 2 002.52 D79987 extra spindle poles like 1 (S. cerevisiae) 002.29 AB011178 PH domain and leucine rich repeat protein phosphatase 000.48 NM_057178 ring finger and FYVE-like domain containing 1 000.35 AB209394 tumor necrosis factor receptor superfamily, member 21 000.34 NM_014739 BCL2-associated transcription factor 1 000.24 NM_000041 apolipoprotein E 000.10 BC070091 caspase recruitment domain family, member 9 000.09 AK122828 cell death-inducing DFFA-like effector b 000.02 NM_000077 cyclin-dependent kinase inhibitor 2A (melanoma, p16) 046.29 CR749329 pleiomorphic adenoma gene-like 1 034.08 NM_002576 p21/cdc42/rac1-activated kinase 1 (STE20 homolog, yeast) 020.53 NM_181847 adhesion molecule with Ig-like domain 2 013.00 AB096256 unc-5 homolog B (C. elegans) 011.88 AK223503 caspase 1, apoptosis-related cysteine peptidase 010.26 NM_002982 chemokine (C-C motif) ligand 2 009.76 NM_013232 programmed cell death 6 009.67 NM_000345 synuclein, alpha (non A4 component of amyloid precursor) 008.12 NM_000686 angiotensin II receptor, type 2 007.85 AF480462 sterile alpha motif and leucine zipper containing kinase AZK 006.62 NM_003877 suppressor of cytokine signaling 2 005.36 AB209147 caspase 9, apoptosis-related cysteine peptidase 005.05 AB005666 signal-induced proliferation-associated gene 1 004.96 NM_198287 inhibitor of growth family, member 4 004.03 NM_001015049 BCL2-associated athanogene 5 003.34 AK126377 protein phosphatase 1F (PP2C domain containing) 003.23 NM_014288 integrin beta 3 binding protein (beta3-endonexin) 003.00 NM_014288 integrin beta 3 binding protein (beta3-endonexin) 002.94 BX648811 inhibin, beta A (activin A, activin AB alpha polypeptide) 002.63 AK001497 death effector domain containing 002.60 AF480462 sterile alpha motif and leucine zipper containing kinase AZK 002.43 NM_007051 Fas (TNFRSF6) associated factor 1 002.39 NM_058183 SON DNA binding protein 002.12 NM_016222 DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 002.05 AY125488 death effector domain containing 2 002.04 AK127469 excision repair cross-complementing rodent repair deficiency 002.02 Biologic process Anti-apoptosis (up-regulated) Anti-apoptosis (down-regulated) Apoptosis (up-regulated)
328 / 대한응급의학회지 : 제 19 권제 3 호 2008 Table 1. Up-and Down- regulated gene expressions related with apoptotic function (continue) GenBank Accession No. Updated gene title Signal intensity Biologic process NM_001018159 amyloid beta precursor protein binding protein 1 000.50 NM_032515 BCL2-related ovarian killer 000.49 NM_016835 microtubule-associated protein tau 000.49 NM_014739 BCL2-associated transcription factor 1 000.48 NM_006785 mucosa associated lymphoid tissue lymphoma translocation gene1 000.47 NM_006849 protein disulfide isomerase family A, member 2 000.47 NM_006282 serine/threonine kinase 4 000.46 BC042844 caspase 10, apoptosis-related cysteine peptidase 000.46 BC063043 perforin 1 (pore forming protein) 000.46 NM_207111 TRIAD3 protein 000.45 NM_001316 CSE1 chromosome segregation 1-like (yeast) 000.45 DQ186648 tumor protein p53 (Li-Fraumeni syndrome) 000.45 AK131280 endoplasmic reticulum to nucleus signalling 2 000.44 NM_194271 ring finger protein 34 000.44 AK125880 tumor protein p53 inducible nuclear protein 1 000.43 AF173003 bifunctional apoptosis regulator 000.42 NM_007295 breast cancer 1, early onset 000.41 NM_033081 death inducer-obliterator 1 000.41 BX537544 inositol hexaphosphate kinase 2 000.41 NM_005406 Rho-associated, coiled-coil containing protein kinase 1 000.40 NM_138724 BCL2-like 14 (apoptosis facilitator) 000.39 NM_004346 caspase 3, apoptosis-related cysteine peptidase 000.39 Apoptosis NM_004938 death-associated protein kinase 1 000.38 (down-regulated) NM_004052 BCL2/adenovirus E1B 19kDa interacting protein 3 000.38 NM_171982 tripartite motif-containing 35 000.38 NM_004040 ras homolog gene family, member B 000.37 NM_002745 mitogen-activated protein kinase 1 000.37 BC032619 sema domain, transmembrane domain (TM), and 000.35 cytoplasmic domain, (semaphorin) 6A NM_001197 BCL2-interacting killer (apoptosis-inducing) 000.35 NM_000551 von Hippel-Lindau tumor suppressor 000.35 BC038505 BCL2-associated athanogene 4 000.34 NM_000551 von Hippel-Lindau tumor suppressor 000.32 NM_207111 TRIAD3 protein 000.32 NM_004783 TAO kinase 2 000.32 NM_020529 nuclear factor of kappa light polypeptide gene enhancer 000.32 in B-cells inhibitor, alpha NM_138762 BCL2-associated X protein 000.32 AB209211 TIA1 cytotoxic granule-associated RNA binding protein 000.30 NM_020529 nuclear factor of kappa light polypeptide gene enhancer 000.30 in B-cells inhibitor, alpha NM_198544 apoptosis-inducing, TAF9-like domain 1 000.30 AK127033 dynamin 2 000.29 NM_002111 huntingtin (Huntington disease) 000.28 NM_014977 apoptotic chromatin condensation inducer 1 000.28 BC002726 death-associated protein 000.27 AK001361 protein phosphatase 1, regulatory (inhibitor) subunit 15A 000.26 NM_002744 protein kinase C, zeta 000.26
김 @ 훈외 : Paraquat 로저항성을유발시킨인간간암세포주에서전사반응에대한 DNA microarray 분석 / 329 Table 1. Up-and Down- regulated gene expressions related with apoptotic function (continue) GenBank Accession No. Updated gene title Signal intensity NM_033307 caspase 4, apoptosis-related cysteine peptidase 000.25 NM_032797 apoptosis-inducing factor (AIF)-like mitochondrion-associated 000.22 inducer of death NM_033027 AXIN1 up-regulated 1 000.21 NM_005067 seven in absentia homolog 2 (Drosophila) 000.21 NM_020418 poly (rc) binding protein 4 000.20 AK126917 Apoptosis-inducing factor like 000.19 BX647064 seven in absentia homolog 1 (Drosophila) 000.18 NM_032797 apoptosis-inducing factor (AIF)-like mitochondrion-associated 000.18 inducer of death NM_014225 protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), 000.17 alpha isoform AY247738 tribbles homolog 3 (Drosophila) 000.16 AF061943 TAO kinase 2 000.15 NM_144781 programmed cell death 2 000.15 NM_021960 myeloid cell leukemia sequence 1 (BCL2-related) 000.12 CR936637 BCL2-like 1 000.10 NM_000312 protein C (inactivator of coagulation factors Va and VIIIa) 000.06 NM_001621 aryl hydrocarbon receptor 000.06 NM_000061 Bruton agammaglobulinemia tyrosine kinase 000.05 CR936637 BCL2-like 1 000.03 NM_033081 death inducer-obliterator 1 000.19 Biologic process Apoptosis (down-regulated) 아직까지임상적으로뚜렷한효과를보이는치료약물은거의없는상태이다. 또한 paraquat에유도된다양한유전자의발현, 특히 paraquat의저항성과관련된특정유전자의발현등과관련된연구는거의이루어지지않고있다. Cho 등 6) 의연구에서는 paraquat를처리한흙에서저항성을보이는미생물균 Streptomyces coelicolor를추출하여저항성을보이는원인에대해분석하여 paraquat 저항성유전자중 2개의유전자인 pqra (TetR-like DNAbinding motif 의전사단위 ) 와 pqrb (putative efflux pump of the major facilitator superfamily) 의변이와관련이있다고보고하고있다. 하지만이는미생물에서의내재적저항성연구이다. 본연구에서는약물대사의중요한기능을하는인간간조직기원의암세포주에치명적인농도의 paraquat를장기간처리후저항성암세포주를유도한후발현된유전자를분석하고이를통해내재적저항성과관련된유전자와단백질을찾고자하였다. 본실험에서간암세포주를활용한이유는실험동물의정상간조직기원세포주는장기간의계대배양이어렵고, 조작이쉽지않아, 정상간세포의중요작용인해독 ( 약물의분해 ) 과관련된유전적기능이있을것으로판단되는인간간암세포주로대체하여아직까지명확히밝혀지지않은 paraquat 대사과정연구와저항성유도관련기전, 이를통한다른조직기원세포로의적용을위해서이다. 전연구 3) 에서인간자궁경부암세포를활용한 in vitro 실 험에서 100 um 농도에서는 24시간내 95% 이상의세포손상이관찰되었고, 1 um 농도에서는약물처리후 48시간부터 50% 정도의세포독성이발생하기시작하였으며 10 um 농도에서는 48시간에서 95% 이상의세포손상이확인된바있다. 본실험에서는약물대사물질과관련된간세포주기원의인간간암세포를활용하였다. 100 um 농도의 paraquat에생존을유지하면서저항성을나타내는세포군을유도하기위해비치명적농도인 1 nm부터세포생존을유지하는범위에서점차 paraquat의농도를증가시키면서계대배양을실시하였다. 이후대략 100 um 농도에서생존을할때세포를모아 paraquat 미처리정상군과세포독성검사를시행해본결과저항성세포군에서 paraquat에매우높은저항성을보였다. 동시에세포형태관찰을해본결과세포는군집상태에서각각분리하여성장하였고모양또한길어지는양상을보였다. 이는 paraquat가세포에서세포로의이동을막기위한방어기전과함께세포의골격과관련된 actin, tubulin 단백질발현의변화, 세포간부착단백질관련유전자발현의변화에의한것으로사료된다. 이후이러한저항성과관련된유전자변이를관찰하기위해 DNA microarry 기법을활용하였다. DNA microarry는 Southern blotting이나 Northern blotting과같은 array based hybridization 원리를사용한것으로서수백개에서수천개의 DNA를 nitrocellulose membrane이아닌 slide glass와같은기판위에고
330 / 대한응급의학회지 : 제 19 권제 3 호 2008 밀도로정렬고정화한것을말한다 7,8). DNA microarray의장점은한번의실험으로동시에많은수의유전자들에대한발현변이양상을탐색할수있으며또한유전자의진단, 돌연변이, 의약품의효과확인및질병진단용등에널리응용될수있는새로운차원의 high-throughput screening 시스템이라고할수있다 9,10). 최근에는독성학분야에서도약물독성의기전, 적응, 효능등의분석에 DNA microarray 활용이시작되고있다 11). 본 paraquat 연구에 DNA microarray를활용된경우는 Satomi 등 12,13) 이쥐에 paraquat를처리후폐섬유화과정에서발현되는유전자를분석하여폐섬유화과정에 paraquat가미치는영향을조사한연구뿐이다. 향후 paraquat 독성연구시기존의항산화제효과의기전연구, paraquat의다양한세포군에서다른독성작용연구, 장기 paraquat 노출시에뇌세포를포함한특정장기의독성작용연구뿐만아니라, 새로운치료제개발에도 DNA microarray 기법은중요한 수단으로활용될수있을것이다. 본실험에서사용된 Operon Human Whole 35 K Oligo chip의각 spot은 control 유전자를포함하여총 36,912 개의 spot이포함되어있으며 Exon sequence oligo(31,387개 ), transcript sequence oligo(3,396개 ) 와 chip의 quality 확인과 normalization을위하여 control sample(1,877개 ) 로구성되어있고이들은 27개의 column과 29개의 row로 48개의 block에 769개씩분산되어포함되어있다. 이 DNA Microarray chip에정상세포와유도된저항성세포의 cdna를처리하여반응을시킨결과정상세포에서발현된유전자에비해유도된저항성세포에서의유전자변화중 2배이상차이를보이는수가 6,717개로서 Satomi 등 12,13) 등이 paraquat를처리한경우보다더많은유전자변화를보였다. 이는장기간 paraquat를 in vitro 상에서계속에서처리하였기때문으로사료된다. 본실험에서는 DNA Microarray chip의형 Table 2. Up-and Down-regulated gene expressions related with cytochrome P450 genes GenBank Accession No. Updated gene title Signal intensity NM_000104 cytochrome P450, family 1, subfamily B, polypeptide 1 332.38 NM_019885 cytochrome P450, family 26, subfamily B, polypeptide 1 018.01 AK056794 cytochrome P450, family 11, subfamily A, polypeptide 1 012.32 NM_030622 cytochrome P450, family 2, subfamily S, polypeptide 1 007.96 CD299556 Cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) 006.33 BC004391 cytochrome b, ascorbate dependent 3 003.80 NM_030622 cytochrome P450, family 2, subfamily S, polypeptide 1 003.72 BQ214619 Cytochrome c oxidase subunit VIIa polypeptide 2 (liver) 003.34 CR599916 Cytochrome c oxidase subunit VIIc 003.21 BQ214619 Cytochrome c oxidase subunit VIIa polypeptide 2 (liver) 003.09 NM_005333 holocytochrome c synthase (cytochrome c heme-lyase) 002.84 NM_178033 cytochrome P450, family 4, subfamily X, polypeptide 1 002.75 BG179412 Cytochrome c oxidase subunit VIIb 002.72 AK092584 cytochrome P450, family 2, subfamily R, polypeptide 1 002.67 CD517959 Cytochrome c oxidase subunit Vib polypeptide 1 (ubiquitous) 002.54 AW886288 Transcribed locus, strongly similar to XP_519866.1 002.37 BX641103 cytochrome b-561 domain containing 2 002.36 NM_006294 ubiquinol-cytochrome c reductase binding protein 002.33 BX537676 cytochrome P450, family 3, subfamily A, polypeptide 5 002.33 BF569085 Cytochrome c-1 002.14 BX537499 Similar to Ubiquinol-cytochrome C reductase complex 11 kda protein 002.09 AB209000 cytochrome b5 reductase 2 000.48 CD014011 P450 (cytochrome) oxidoreductase 000.41 X13897 cytochrome P450, family 2, subfamily A, polypeptide 6 000.39 BX647922 outer mitochondrial membrane cytochrome b5 000.39 CR600874 COX11 homolog, cytochrome c oxidase assembly protein (yeast) 000.38 NM_007326 cytochrome b5 reductase 3 000.37 AK094006 ubiquinol-cytochrome c reductase core protein II 000.32 AL832998 cytochrome P450, family 20, subfamily A, polypeptide 1 000.27 BX648730 cytochrome P450, family 4, subfamily V, polypeptide 2 000.26 NM_023944 cytochrome P450, family 4, subfamily F, polypeptide 12 000.23
김 @ 훈외 : Paraquat 로저항성을유발시킨인간간암세포주에서전사반응에대한 DNA microarray 분석 / 331 광스캐너분석에서정상세포군에녹색형광이, 저항성세포군에적색형광을부착하였으므로녹색형광이강할수록저항성세포군에서의유전자발현이정상세포군과비교하여상대적으로억제된것을의미하며, 적색형광이강할수록반대로유전자발현이상대적으로증가된것이다. 이러한형광발현의차이는두군간의유전자발현차이를의미하여이는전사후에두군간의단백질의변화, 즉세포의기능과형태의변화의차이와높은상관관계를보인다. 본실험에서 100배이상의변화가관찰된경우가 58개유전자가되었다. 이러한유전자의발현은 paraquat 노출에서세포가생존하기위한전략의일환으로판단된다. 이러한세포생존전략의하나로서기능성유전자발현에대한분석결과세포자멸유도와관련된유전자는 27개가발현이증가되고세포자멸억제유전자 7개가발현이증가된반면세포자멸억제유전자 13개가발현이증가되었고, 세포자멸유도유전자는 37개가발현이감소되어전체적으로세포자멸억제와관련된유전자의발현이더높았던사실은세포자멸유전자조절이 paraquat 독성시생존을위한방어기전의하나라고판단되며이들유전자의조절을통한새로운치료방법의고안이가능할것으로사료된다. Paraquat의독성유발과관련이있는 cytochrome P450 효소는현재 paraquat에전자전달하는기능을하여독성에중요한물질인 paraquat 이온의생성에중요한것으로알려져있다 2). 또한 cytochrome P450 효소는간세포등에서약물의대사를통한비독성화과정에서중요한단백질로서도알려져있다. 본연구에서는 cytochrome P450 관련효소중에 36개의유전자가의미있는발현의증가또는감소가있었다. 특히 cytochrome P450, family 1, subfamily B, polypeptide 1( 이하 CYB1B1) 은 332 배이상의유전자발현증가소견을보여 paraquat의내재적분해와관련된중요한단백질가능성이있을것으로판단된다. 이는 cytochrome P450 효소가현재알려진것처럼 paraquat의독성기전에핵심적인역할만하는것이아니라, subtype에따라 paraquat의대사와같은해독기전에도관여함을시사하는소견이다. 이는향후 CYB1B1 같은특정 subtype의 cytochrome P450 효소의발현을유도하여내재적 paraquat 독성극복방법으로응용될수있을것으로판단된다. 특히향후기존에CYB1B1의강력한유도물질로알려진 estrogen 등으로 paraquat의독성극복과관련된연구가병행되면본연구는새로운치료물질의개발에도도움을줄것으로사료된다. 또한 paraquat에의해유도된 glutathione S-transferase kappa 1, Glutathione S-transferase theta 2, microsomal glutathione S-transferase 3 등의 glutathione 관련유전자의발현도 3~10배증가된소견을보여 paraquat의저항성기전중에또다른역할로작용한 것으로사료된다. 특이한점은급성 paraquat 중독시발생되는활성산소종 (reactive oxygen species) 의제거에 SOD 효소가중요한역할을하는것으로알려져있다 14). 이효소는 superoxide을덜해로운 hydrogen peroxide 형태로전환하여산화적스트레스를줄이는데중요하다. 본연구에서는예상과달리 SOD가 2.3배의유전자가감소한소견이관찰되었다. 급성 paraquat에대한급성반응과달리만성적약물처리로인한방어기전은 paraquat의대사와관련된단백질의유전자가주로발현이증가또는감소하였기때문으로판단된다. 본연구의제한점으로는간암세포주만을대상으로하였다는점에서향후 paraquat의주독성장기인폐세포를대상으로도연구가이루어질필요가있으며더나아가 in vivo에서도연구가필요하다. 또한간암세포주를사용함으로서정상적인간세포주와 paraquat에대한유전적반응성의차이 ( 예, 자멸기전과관련된유전자발현등 ) 가있을수있다는점이다 15-17). 결론적으로유도된 paraquat 저항성은복합적인유전자발현의증가와감소에의해발생된것으로추정되며특히세포자멸관련유전자발현, cytochrome P450 효소계의유전자발현, glutathione 관련효소계유전자발현의변화가저항성의유도에중요한역할을한것으로사료된다. 향후이들유전자와관련하여특정유전자의발현증가또는감소를통한단백질의변화를유도함으로서 paraquat 치료에있어서내재적저항성을유발하는치료방법이새로운치료방법으로서연구될수있을것으로판단한다. 이를통해임상적으로는소량의 paraquat 음독시에만성적으로진행되는독성작용을극복하기위해기존에알려진약물을활용하여상기유전자발현을인위적으로유도함으로서치료적효과를볼수있는새로운접근이가능할것으로사료된다. 결론이번연구는 paraquat의장기간 preconditioning을통해유도된인간간암세포주의저항성과관련된 DNA microarray 분석을통해반세포자멸관련유전자의발현증대, 세포자멸관련유전자발현감소, xenobiotics 대사와관련된 cytochrome P450 효소계, glutathione 유전자증대등이저항성과관련이있을것으로판단된다. 특히 cytochrome P450은현재유해한자유산소기생성을위한단백질의역할을하는것으로알려졌으나 CYB1B1등의특정 subtype은반대로 paraquat의제거와관련된역할을할수있음을확인할수있었고, 향후이들 subtype cytochrome P450 계열효소의발현유도 ( 예, estrogen
332 / 대한응급의학회지 : 제 19 권제 3 호 2008 등의약물 ) 를통한 paraquat 치료, 특히만성또는소량의음독환자의치료에대한새로운접근방법이될수있을것으로사료된다. 참고문헌 01. Lee EY, Kim YT, Yang JO, Hong SY. Long-term prognosis of paraquat-induced lung injury. Korean J Med 2003; 65:308-14. 02. Suntres ZE. Role of antioxidants in paraquat toxicity. Toxicology 2002;180:65-77. 03. Kim H, Lee SW. The effects of quercetin on paraquatinduced cell damage. J Korean Soc Emerg Med 2007;18:41-8. 04. Eun SW, Han CH, Yoon YK, Yang DH, Cho SR, Kim WJ, et al. Effect of vitamin C on plasma total antioxidant status (TAS) in patients with paraquat intoxication. Korean J Med 2000;58:666-74. 05. Bonneh-Barkay D, Reaney SH, Langston WJ, Di Monte DA. Redox cycling of the herbicide paraquat in microglial cultures. Brain Res Mol Brain Res 2005;134:52-6. 06. Cho YH, Kim EJ, Chung HJ, Choi JH, Chater KF, Ahn BE, et al. The pqrab operon is responsible for paraquat resistance in Streptomyces coelicolor. J Bacteriol 2003;185:6756-63. 07. Wiltgen M, Tilz GP. DNA microarray analysis: principles and clinical impact. Hematology 2007;12:271-87. 08. Ehrenreich A. DNA microarray technology for the microbiologist: an overview. Appl Microbiol Biotechnol 2006; 73:255-73. 09. Jares P. DNA microarray applications in functional genomics. Ultrastruct Pathol 2006;30:209-19. 10. Anisimov SV. Application of DNA microarray technology to gerontological studies. Methods Mol Biol 2007;371: 249-65. 11. Lettieri T. Recent applications of DNA microarray technology to toxicology and ecotoxicology. Environ Health Perspect 2006;114:4-9. 12. Satomi Y, Tsuchiya W, Miura D, Kasahara Y, Akahori F. DNA microarray analysis of pulmonary fibrosis three months after exposure to paraquat in rats. J Toxicol Sci 2006;31:345-55. 13. Satomi Y, Tsuchiya W, Mihara K, Ota M, Kasahara Y, Akahori F. Gene expression analysis of the lung following paraquat administration in rats using DNA microarray. J Toxicol Sci 2004;29:91-100. 14. Li JR, Yu P. Expression of Cu, Zn-superoxide dismutase gene from Saccharomyces cerevisiae in Pichia pastoris and its resistance to oxidative stress. Appl Biochem Biotechnol 2007;136:127-39. 15. Diodovich C, Urani C, Maurici D, Malerba I, Melchioretto P, Orlandi M, et al. Modulation of different stress pathways after styrene and styrene-7,8-oxide exposure in HepG2 cell line and normal human hepatocytes. J Appl Toxicol 2006;26:317-25. 16. Reynaert H, Rombouts K, Vandermonde A, Urbain D, Kumar U, Bioulac-Sage P, et al. Expression of somatostatin receptors in normal and cirrhotic human liver and in hepatocellular carcinoma. Gut 2004;53:1180-9. 17. Farah IO, Begum RA, Ishaque AB. Differential protection and transactivation of P53, P21, Bcl2, PCNA, cyclin G, and MDM2 genes in rat liver and the HepG2 cell line upon exposure to pifithrin. Biomed Sci Instrum 2007;43: 116-21.