DBPIA-NURIMEDIA - PDF 무료 다운로드

J. ENVIRON. TOXICOL. Vol. 21, No. 3, 245~253 (26) Protein kinase A 억제제인 KT572 이글루카곤매개성항산화효소의발현감소에미치는영향 오수진 1,2, 조재훈 1, 박창식 2, 김상겸 1,2, 김봉희 1, * 1 충남대학교약학대학, 2 충남대학교형질전환복제돼지센터 Effects of the Protein Kinase A Inhibitor KT572 on -Mediated Decrease in Expression of Antioxidant Enzymes Soo Jin Oh 1,2, Jaehoon Jo 1, Chang Sik Park 2, Sang Kyum Kim 1,2 and Bong-Hee Kim 1, * 1 College of Pharmacy, 2 Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 35-763, Korea ABSTRACT We reported previously that glucagon decreased alpha- and pi-class glutathione S-transferases (GSTs) and microsomal epoxide hydrolase (meh) protein levels in primary cultured rat hepatocytes. The present study examines the effects of protein kinase A (PKA) inhibitor, KT572, on the glucagon-mediated decrease in expression of GSTs and meh. To assess cell viability, lactate dehydrogenase release and MTT activity were examined in hepatocytes treated KT572. Cell viability was significantly decreased in a concentrationdependent manner after incubation with KT572 at the concentrations of 1 µm or above for 24 h, which was inhibited by the cytochrome P45 inhibitor SKF-525A. In contrast, another PKA inhibitor H89 (up to 25 µm) was not toxic to hepatocytes. The glucagon-mediated decrease in expression of alpha- and pi-class GSTs and meh was completely inhibited by 25 µm H89 and attenuated by.1 µm KT572. This study demonstrates that KT572 may cause cytotoxicity in rat hepatocytes through cytochrome P45-dependent bioactivation. The present study implicates PKA in mediating the inhibitory effect of glucagon on expression of alpha- and piclass GSTs and meh. Key words : KT572, glucagon, protein kinase A, glutathione S-transferase, microsomal epoxide hydrolase 서 론 대표적인대사질환인당뇨병 (diabetes mellitus) 에서심혈관계질환, 신장질환, 신경계질환등다양한 To whom correspondence should be addressed. Tel: +82-42-821-5935, E-mail: bhkimnh@cnu.ac.kr 조직손상이합병증으로나타난다. 당뇨상태에서관찰되는산화적스트레스 (oxidative stress) 는만성적인당뇨합병증의주요한원인으로보고되고있다 (Baynes and Thorpe, 1999; West, 2). 또한당뇨와인슐린저항성 (insulin resistance) 은비알코올성지방간염 (non-alcoholic steatohepatitis) 환자에게서빈번히관찰되어이들질환의관련성이주목을 245

246 J. ENVIRON. TOXICOL. Vol. 21, No. 3 받고있다 (Neuschwander-Tetri and Caldwell, 23). 산화적스트레스는산화성물질 (oxidant and prooxidant) 과항산화물질 (antioxidant) 의평형이교란되어산화적반응이우세하고결과적으로조직손상을유발할수있는상태로정의된다 (Sies, 1985). 생체의항산화체계는저분자의항산화물질과항산화효소로구성되며각각의항산화물질과효소는협동적으로산화적스트레스에대항한다 (Yu, 1994). 특히, glutathione (GSH) 와 GSH 를기질로사용하는 GSH S-transferase (GST) 및 GSH peroxidase/reductase 등은활성산소종 (reactive oxygen species) 과친전자성물질 (electrophile) 을무독화시킬뿐만아니라산화된단백질, vitamin C 와 E 등을환원시킴으로써체내항산화활성의중심축을구성한다 (Meister, 1994). Microsomal epoxide hydrolase (meh) 역시 epoxide 등반응성이강한물질에물분자를 trans-addition 시킴으로써항산화작용을매개한다. 유전적다형성 (genetic polymorphism) 연구결과는 GST 와 meh 의활성변화가다양한암및간장질환의발생율과관련이있는것으로보고하였다 (To-Figueras et al., 21; Sonzogni et al., 22). 당뇨병은탄수화물을대표로하는영양소의대사뿐만아니라외인성물질의대사역시교란시킨다. Cytochrome P45 (CYP) 2B1, 2E1, 3A 그리고 4A 의활성및단백질함량이당뇨에서증가하며반면 CYP2C 와 meh 는감소한다 (Kim et al., 24a). 당뇨상태에서 phase II 반응을매개하는효소중 UDP-glucuronosyltransferase 1A1 은증가하며 sulfotransferase 2A1 은감소한다 (Kim et al., 24b). 당뇨병은인슐린 (insulin) 의분비또는반응성의감소와함께글루카곤 (glucagon) 등다양한호르몬의변화가동반된다. 일차배양랫트간세포실험계에서인슐린의처리는 alpha-class GST (Kim et al., 23a, 26a), meh (Kim et al., 23b) 및 GSH 합성의속도결정단계를촉매하는 gamma-glutamylcysteine ligase (Kim et al., 24b) 의발현을증가시키며글루카곤은상반된조절기전을가지는것으로보고되었다. 이상의실험결과는당뇨병에서관찰되는산화적스트레스가산화성물질의증가뿐만아니라항산화효소의감소와관련이있음을시사한다. 글루카곤은 adenylate cyclase 를활성화시켜세포 에서 cyclic AMP 의생성을증가시키며증가된 cyclic AMP 는인슐린신호전달체계를억제하고 protein kinase A (PKA) 를활성화시킨다 (Kimball et al., 24). 따라서대부분의글루카곤효과는 cyclic AMP 를경유한 PKA 의활성화에기인한다. H89 와 KT572 은 PKA 의억제를위해가장광범위하게사용되고있다. 두억제제는모두세포투과성을가지며효소억제실험에서 5~6 nm 의 inhibition constant 값을가지는것으로보고되었다 (Chijiwa et al., 199; Gadbois et al., 1992). 세포배양실험에서 PKA 를완전히억제하기위해 H89 는주로 25 µm KT572 은 5 µm 이주로사용된다. 본연구에서는일차배양랫트간세포를이용하여두 PKA 억제제의세포독성과글루카곤에의한항산화효소의발현감소에미치는영향을실험하였다. 1. 시약 재료및방법 Modified Chee s medium과 L-glutamine은 Invitrogen (Carlsbad, CA) 에서인슐린은 Insulin (Novolin R) 은 Novo Nordisk Pharmaceuticals Inc. (Princeton, NJ) 에서구입하였다. Collagenase (type I) 은 Worthington Biochemicals (Freehold, NJ) 에서 vitrogen (95~98% type I collagen, 2~5% type III collagen) 은 Cohesion Technologies Inc. (Palo Alto, CA) 에서구입하였다. Class-specific GST 항체와 meh 항체는선행연구에서이미보고한것을사용하였다 (Kim et al., 23a, b). H89와 KT572은 Calbiochem (La Jolla, CA) 에서 과기타시약은 Sigma-Aldrich (St. Louis, MO) 에서구입하였다. 2. 랫트간세포의분리및배양 간세포 (hepatocytes) 는웅성 Sprague-Dawley 랫트 (2~3 g) 에서 collagenase 를관류하여분리하였다 (Kim et al., 25). 간세포를 6 mm 배양접시에 3 1 6 cells 로가하고 1 µm 의인슐린이첨가된배지에서 4 시간동안배양하여간세포를배양접시에부착시켰다. 인슐린이없는배지로 2 회세척하

September 26 Oh et al. : Effects of KT572 on Cell Viability and Antioxidant Enzyme Expression 247 여미부착된세포와인슐린을제거하고 2 시간동안안정시킨후억제제나글루카곤을처리하였다. 이후세포는인슐린이없는배지에서배양하였으며매 24 시간마다배양액을교체하였다. 은멸균증류수에녹여냉장실에보관하였으며억제제는 DMSO 에녹여 -2 C 에서보관하였다. 예비실험결과최대글루카곤효과가관찰된 1 nm 을선택하였으며 PKA 억제제는글루카곤을처리하기 1.5 시간전에, SKF-525A 는 KT572 을처리하기 3 분전에배지에첨가하였다. 세포의생존을확인하기위해 MTT assay 와 LDH (lactate dehydrogenase) assay 를수행하였다. 실험결과는정상대조군의값을 1 으로환산하여 % 로표시하였다. 3. Immunoblot analysis 선행연구에서보고된방법에의거하여실험을수행하였다 (Kim et al., 25). 세포를 3mL 의 4 C phosphate-buffered saline (ph 7.4) 으로세척한후 5 mm HEPES (ph 7.2), 15 mm NaCl, 1.5 mm MgCl 2, 1 mm EGTA, 1% glycerol, 1% Triton X-1, 1 mm sodium pyrophosphate, 1 mm MnCl 2, 1 mm sodium orthovanadate, leupeptin (1 µg/ml), aprotonin (1 µg/ml) 과 2 mm phenylmethylsulfonyl fluoride 로구성된 lysis buffer 를첨가하였다. 세포를긁어 1.5 ml 의 Eppendorf tubes 에모은후 25- gauge needle 을끼운주사기로 5 회통과시켰다. 시료를 3 분간얼음에서방치하고 16, g 에서 2 분간원심분리하여상등액을얻었다. 상등액에서단백질함량을 bicinchoninic acid protein assay (Sigma- Aldrich) 로측정하고실험에사용하였다. Immunoblot analysis 를위해 5 (alpha-class GST), 15 (meh) 또는 3 (pi-class GST) µg 의단백질을 loading buffer 에희석시킨후 sodium dodecyl sulfate-polyacrylamide gel 에서전기연동하고 nitrocellulose membrane (Bio-Rad, Inc., Hercules, CA) 로이동시켰다. 단백질이결합된 nitrocellulose membrane 을 2 시간동안 5% milk powder 를함유하는 phosphate-buffered saline (ph 7.4) 에서반응시키고각각의일차항체를가한후상온에서 16 시간동안반응시켰다. 일차항체가결합된 nitrocellulose membrane 을 3 시간동안 horseradish peroxidase 포합되 어있는이차항체와반응시키고 enhanced chemiluminescence 시약과 Kodak X-OMAT film (Sigma- Aldrich) 을이용하여단백질을검출하였다. 검출된단백질을정량하기 Molecular Dynamics scanning laser densitometer 와 ImageQuant analysis program (Amersham Biosciences, Inc.) 을사용하였다. 4. GSH 정량 세포의 GSH 함량은전에보고된방법을사용하였다 (Kim et al., 26b). 세포를 6% perchloric acid 에서긁어 1.5 ml tube 에모으고원심분리하여상등액을시료로사용하였다. 상등액에 phosphate 완충액 (phosphate.125 M, EDTA 6.3 mm, ph 7.5) 을사용하여 GSH 농도가표준검량선농도범위에들도록희석하였다. Eppendorf tubes 에.3 mm NADPH 용액.7 ml, 6 mm DTNB (5, 5-dithio-bis- (2-nitrobenzoic acid)) 용액.1 ml, 검체또는 GSH 표준액.2 ml 을가하여잘섞은후상온에서 4 분간방치하였다. 반응액에 2 units/ml 농도의 GSH reductase 를가하고잘섞은후 412 nm 에서약 2 분간흡광도의변화를측정하여 linear 한 1 분간의기울기변화를구하고검량선으로부터 GSH 의농도를계산하였다. 실험에사용한 H89 와 KT572 은 in vitro 조건에서 1 mm 농도까지 GSH 의측정에유의적인변화를미치지못하였다. 5. 통계분석 실험군사이의통계적차이는 Student s t-test 또는 analysis of variance 후 Newman-Keuls multiple comparison test (p.5) 로검사하였다. 실험결과의재현성은 2~4 회의간세포분리를통하여확인하였다. 모든실험결과는평균 ± 표준편차로표시하였다. 결 과 1. H89 와 KT572 이세포생존에미치는영향 일차배양랫트간세포실험계에서 H89 와 KT 572 이세포독성에미치는영향을 MTT assay 와 LDH assay 를이용하여실험하였다 (Fig. 1). H89 는

248 J. ENVIRON. TOXICOL. Vol. 21, No. 3 Cell viability (% of control) Cell viability (% of control) 2 A 16 8 4 2 16 8 4 B 1 day 1 day Control KT572 (.1 µm) KT572 (.1 µm) KT572 (1 µm) KT572 (5 µm) H89 (25 µm) 2 day 3 day 25 µm 농도에서글루카곤의유무와무관하게 MTT 의활성 (Fig. 1) 과 LDH 의유리 (data not shown) 에변화를유발하지못하였다. 이결과는 H89 가간세포에서독성이낮은물질임을시사한다. KT572 은.1 µm 까지 MTT 활성및배지의 LDH 활성에변화를유발하지못하였으나 1 µm 농도에서일일이내에약 3% 의 MTT 활성감소를유발하였으며배지에서 LDH 의활성을정상대조군에비하여약 5% 증가시켰다 (Fig. 1A). KT572 는 5 µm 에서약 Control KT572 (.1 µm) KT572 (.1 µm) KT572 (1 µm) KT572 (5 µm) H89 (25 µm) 2 day 3 day Fig. 1. Effect of KT572 on viability of primary cultured rat hepatocyte cultured in the absence of glucagon (A) or in the presence of glucagon (B). After the 4-h plating period in the presence of 1 µm insulin, medium was replaced with insulin-free medium. Hepatocytes were then treated with KT572 or H89 for 1.5 h before addition of 1 nm glucagon, and these treatments were repeated every 24 h for 3 days. Values are shown as a percentage of the level monitored in control hepatocytes. Data are means± S.D. with three preparations of cell lysates. *,Significantly different than levels monitored in control hepatocytes. Cell viability (% of control) 15 9 6 3 a a a a a Control cells SKF-525A pretreated cells.1.1 1 5 1 KT572 (µm) 4% 의 MTT 활성감소와 7% 의배지 LDH 활성증가를초래하여농도의존적인독성을유발하였다 (Fig. 1A). KT572 의독성은시간경과에따라증가하여 3 일째에는 1 µm 과 5 µm 에서 MTT 활성이각각정상대조군의 26% 와 16% 로감소하였다 (Fig. 1A). 세포의기능을평가하기위해 24 시간동안 KT572 이처리된세포에서 GSH 의농도를측정하였다. 대조군에서 GSH 의농도는 33.2±2.1 nmol/mg 이었으며 KT572 의농도가.1,.1, 1 과 5 µm 로증가함에따라 GSH 함량은 34.2±1.9, 33.2±1.7, 3.2±.8 과 27.2±2.3 nmol/mg 으로감소하였다. 따라서 KT572 이농도의존적으로세포의 GSH 농도를감소시킴을확인하였다. 글루카곤과함께 KT572 을배양하고세포독성을실험하였다 (Fig. 1B). 배지에 1 nm 의글루카곤이첨가되었을때 24 시간동안 5 µm 로처리된 KT572 은일차배양랫트간세포에서독성을유발하지못하였다. 그러나 KT572 의배양시간이증가함에따라 MTT 활성의감소가관찰되었다. 또한배지의 LDH 활성역시 KT572 을처리하고 2 일부터유의적으 a Fig. 2. Effect of the cytochrome P45 inhibitor SKF-525A on the KT572-induced cytotoxicity in primary cultured rat hepatocytes. After the 4-h plating period in the presence of 1 µm insulin, medium was replaced with insulin-free medium. Hepatocytes were then treated with SKF-525A (1 µm) for.5 h before addition of KT572 for 24 h. Values are shown as a percentage of the level monitored in control hepatocytes. Data are means±s.d. with three preparations of cell lysates. Values with different letters are significantly different from each other, P.5. c a d a,b e b,c

September 26 Oh et al. : Effects of KT572 on Cell Viability and Antioxidant Enzyme Expression 249 UT KT572 1 1 (1 nm) KT572 (nm) A (1 nm) GSTA3/5 GSTA1/2 15 B 9 #, GSTA1/2 (%) 6 3 GSTA3/5 (%) 9 6 3 # * UT KT572 1 1 (1 nm) KT572 (nm) UT KT572 1 1 (1 nm) KT572 (nm) (1 nm) (1 nm) UT KT572 1 1 (1 nm) KT572 (nm) (1 nm) C 9 GSTP1 UT KT572 1 1 (1 nm) KT572 (nm) (1 nm) 15 D meh GSTP1 (%) 6 3, * meh (%) 9 6 3, * UT KT572 1 1 (1 nm) KT572 (nm) (1 nm) UT KT572 1 1 (1 nm) KT572 (nm) (1 nm) Fig. 3. Immunoblot analysis of the effect of the protein kinase A inhibitor KT572 on the glucagon-mediated decreases in GSTA1/2 (A), GSTA3/5 (B), GSTP1 (C) and meh (D) protein levels in primary cultured rat hepatocytes. After the 4- h plating period in the presence of 1 µm insulin, medium was replaced with insulin-free medium. Hepatocytes were then treated with KT572 for 1.5 h before addition of 1 nm glucagon, and these treatments were repeated every 24 h for 3 days. Values are shown as a percentage of the level monitored in untreated hepatocytes (UT). Data are means ±S.D. of immunoblot band densities of four preparations of cell lysates. #, Significantly different than levels monitored in UT, P.5 or P.1, respectively. *,Significantly different than levels monitored in hepatocytes treated with glucagon only, P.5 or P.1, respectively. 로증가하였다 (data not shown). 이결과는배지에글루카곤이첨가되었을때부분적으로 KT572 의독성이억제됨을시사한다. 간세포는다른세포에비하여외인성물질에대한대사활성이높은특징을가지고있다. 따라서 KT572 의독성에대사활성화가관여하는가를확인하기위해대표적인 CYP 억제제인 SKF-525A 를전처리하고 KT572 의독성을관찰하였다 (Fig. 2). SKF-525A 가전처리된세포에서 1 µm KT572 은약 28% 의 MTT 활성감소와 35% 의배지 LDH 증

25 J. ENVIRON. TOXICOL. Vol. 21, No. 3 GSTA3/5 GSTA1/2 UT H89+ A 15 B 9 GSTA1/2 (%) 6 3 GSTA3/5 (%) 9 6 3 UT H89+ UT H89+ GSTP1 meh UT H89+ C 15 UT H89+ D 9, GSTP1 (%) 6 meh (%) 9 6 3 3 UT H89+ UT H89+ Fig. 4. Immunoblot analysis of the effect of the protein kinase A inhibitor H89 on the glucagon-mediated decreases in GSTA1/2 (A), GSTA3/5 (B), GSTP1 (C) and meh (D) protein levels in primary cultured rat hepatocytes. After the 4- h plating period in the presence of 1 µm insulin, medium was replaced with insulin-free medium. Hepatocytes were then treated with 25 µm H89 for 1.5 h before addition of 1 nm glucagon, and these treatments were repeated every 24 h for 3 days. Values are shown as a percentage of the level monitored in untreated hepatocytes (UT). Data are means±s.d. of immunoblot band densities of four preparations of cell lysates. #, Significantly different than levels monitored in UT, P.5 or P.1, respectively. Significantly different than levels monitored in hepatocytes treated with glucagon only, P.1. 가를유발하였다 (Fig. 2). 반면 5 µm 의 KT572 에의해유발된세포독성은 SKF-525A 에의해완벽하게차단되었다 (Fig. 2). 이실험결과는 KT572 에의해매개되는세포독성이대사활성화와관련이있을가능성을시사한다. 따라서본결과는간세포와같이약물대사활성이높은세포에있어서 KT572 의사용은주의가필요함을제안한다. 2. KT572와 H89가글루카곤에의한항산화효소의발현감소에미치는영향일차배양랫트간세포실험계에서글루카곤의처리는 alpha-class GST, pi-class GST 및 meh의단백질발현을억제하는것으로보고되었다 (Kim et al., 23a, b). 본연구에서 PKA의억제제인 KT572과 H89가글루카곤의존성항산화효소의

September 26 Oh et al. : Effects of KT572 on Cell Viability and Antioxidant Enzyme Expression 251 발현억제에미치는영향을실험하였다 (Figs. 3 and 4). 1 nm 로처리된글루카곤은 alpha-class GST, pi-class GST 및 meh 의단백질발현을억제하여이전연구결과를확인하였다 (Fig. 3). 세포생존에영향이없는농도인 1 과 1 nm 로 KT572 를전처리한후글루카곤에의한변화에미치는영향을관찰하였다. 1 nm 로처리된 KT572 은글루카곤에의한 GSTA1/2 의감소에유의적인변화를유발하지못하였으나 1 nm 에서부분적인글루카곤억제효과가관찰되었다 (Fig. 3A). 또한글루카곤에의한 GSTA3/5, GSTP1 및 meh 의감소는 1 nm 의 KT572 의전처리에의해약화되었다 (Fig. 3B, C and D). 이상의연구결과는글루카곤에의한일부항산화효소의발현감소가 PKA 를경유하여발생할가능성을시사한다. 그러나사용한 KT572 의농도가 PKA 의활성을완전히억제하지못하여글루카곤의영향을약화시켰을가능성이있다. PKA 가글루카곤에의한 alpha-class GST, pi-class GST 및 meh 의감소를매개하였는가를확인하기위해 H89 를전처리하였다 (Fig. 4). 글루카곤에의한 alpha-class GST (Fig. 4A, B) 와 meh (Fig. 4D) 의감소는 25 µm H89 에의해완전히억제되었다. 또한 H89 는글루카곤에의한 pi-class GST 의감소를현격하게약화시켰다. 이상의결과는글루카곤에의한 alpha-class GST, pi-class GST 및 meh 의발현감소가 PKA 에의해매개되었음을시사한다. 고 찰 본연구결과는일차배양랫트간세포실험계에서글루카곤의처리가 alpha-class GST, pi-class GST 및 meh 의발현을감소시키고 PKA 의억제제인 H89 와 KT572 이글루카곤의영향을억제또는약화시킴을보인다. 또한 KT572 은약물대사활성이높은간세포에서 1 µm 부터독성을유발하며 CYP 억제제인 SKF-525A 의처리는 KT572 에의한독성으로부터간세포를보호하였다. 본연구논문은간세포에서 KT572 의세포독성을관찰한첫보고이다. 산화적스트레스는당뇨환자에게서증가하며당뇨합병증의중요한원인으로보고되고있다. 당뇨에서산화적스트레스발생기전을규명하기위 한연구는대부분산화성물질의생성증가에초점이맞추어져있다. 실제로본연구자는당뇨상태에서증가하는 ketone body 중 acetoacetae 가간세포에서농도의존적으로 GSH 의감소및활성산소종의증가를유발함을보고하였다 (Abdelmegeed et al., 24). 그러나산화적스트레스는산화성물질의증가뿐만아니라항산화활성의감소에의해서도발생한다 (Sies, 1985). 인슐린의처리는글루카곤과반대로 alpha-class GST 와 meh 의발현감소및 GSH 의합성감소를유발하였다 (Kim et al., 23a, b and 24b). 이상의실험결과는당뇨환자에게서관찰되는산화적스트레스의증가에항산화효소의발현감소가관여할가능성을시사한다. 첨가적으로당뇨의합병증을예방하기위해항산화효소의발현을정상화시킬수있는약물요법의적용가능성에대한추가적인연구가필요함을제안한다. H89 와 KT572 은모두 PKA 의억제를위해가장빈번히사용되는화학적억제제로세포투과성을가지며 5~6 nm 의 inhibition constant 값을가진다 (Chijiwa et al., 199; Gadbois et al., 1992). 비선택적인 protein kinase 억제제인 staurosporine 의유도체인 KT572 은 PKA 에높은선택성은가지며세포배양실험에서 1~5 µm 농도로사용된다 (Kase et al., 1987; Wang et al., 26). 논문검색결과 KT572 의세포독성과관련된논문은확인할수없었으며 PKA 의다른억제제인 H89 가 25 µm 까지독성을유발하지않는것을고려할때 KT57 의독성은 PKA 의억제효과와는무관한것으로판단된다. 이상의연구결과는 KT572 의세포독성의기전을규명하기위한추가적인연구결과가필요함을시사한다. 첨가적으로 staurosporine 은신경교세포 (neuronal glial cell) 에서 caspase 의활성화를통한 apoptosis 를유발하는것으로보고되었다 (Shih et al., 23; MacCormac et al., 24). 결론적으로본연구는당뇨병에서관찰되는산화적스트레스에 PKA 를경유하는글루카곤에의한항산화효소의활성감소가관여할가능성을시사한다. 또한 PKA 억제제인 KT572 은약물대사활성이높은간세포에서독성을유발하며따라서이억제제의사용에있어서주의가필요함을시사한다.

252 J. ENVIRON. TOXICOL. Vol. 21, No. 3 감사의글 이논문은 24 년도한국학술진흥재단 (KRF- 24-41-E393) 및한국과학재단형질전환복제돼지 ERC 프로그램 (grant R11-22-1--) 의지원을받았음. 참고문헌 Abdelmegeed MA, Kim SK, Woodcroft KJ and Novak RF. Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes: role of oxidative stress. J Pharmacol Exp Ther. 24; 31(2): 728-736. Baynes JW and Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999; 48: 1-9. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T and Hidaka H. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H- 89), of PC12D pheochromocytoma cells. J Biol Chem. 199; 265(9): 5267-5272. Gadbois DM, Crissman HA, Tobey RA and Bradbury EM. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proc Natl Acad Sci USA. 1992; 89(18): 8626-863. Kase H, Iwahashi K, Nakanishi S, Matsuda Y, Yamada K, Takahashi M, Murakata C, Sato A and Kaneko M. K- 252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases.biochem Biophys Res Commun. 1987; 142(2): 436-44. Kim SK, Abdelmegeed MA and Novak RF. Identification of the insulin signaling cascade in the regulation of alphaclass glutathione S-transferase expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther. 26a; 316(3): 1255-1261. Kim SK, Abdelmegeed MA and Novak RF. The mitogenactivated protein kinase kinase (mek) inhibitor PD9859 elevates primary cultured rat hepatocyte glutathione levels independent of inhibiting mek. Drug Metab Dispos. 26b; 34(4): 683-689. Kim SK, Woodcroft KJ and Novak RF. Insulin and glucagon regulation of glutathione S-transferase expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther. 23a; 35(1): 353-361. Kim SK, Woodcroft KJ and Novak RF. Insulin and growth factor signaling: Effects on drug-metabolizing enzymes, in Drug metabolism and transport (Lash LH ed), Humana Press, Totowa (24a), pp. 45-83. Kim SK, Woodcroft KJ, Khodadadeh SS and Novak RF. Insulin signaling regulates gamma-glutamylcysteine ligase catalytic subunit expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther. 24b; 311(1): 99-18. Kim SK, Woodcroft KJ, Kim SG and Novak RF. Insulin and glucagon signaling in regulation of microsomal epoxide hydrolase expression in primary cultured rat hepatocytes. Drug Metab Dispos. 23b; 31(1): 126-1268. Kim SK, Woodcroft KJ, Oh SJ, Abdelmegeed MA and Novak RF. Role of mechanical and redox stress in activation of mitogen-activated protein kinases in primary cultured rat hepatocytes. Biochem Pharmacol. 25; 7(12) : 1785-1795. Kimball SR, Siegfried BA and Jefferson LS (24) represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase. J Biol Chem 24; 279: 5413-5419. MacCormac LP, Muqit MM, Faulkes DJ, Wood NW and Latchman DS. Reduction in endogenous parkin levels renders glial cells sensitive to both caspase-dependent and caspase-independent cell death. Eur J Neurosci. 24; 2(8): 238-248. Meister A. Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem. 1994; 269(13): 9397-94. Neuschwander-Tetri BA and Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 23; 37: 2-1219. Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA and Murphy TH. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 23; 23(8): 3394-346. Sies H. Oxidative stress: Introductory remarks. In: H. Sies, Editor, Oxidative Stress, Academic Press, San Diego, New York, London (1985), pp. 1-8. Sonzogni L, Silvestri L, De Silvestri A, Gritti C, Foti L, Zavaglia C, Bottelli R, Mondelli MU, Civardi E and Silini EM. Polymorphisms of microsomal epoxide hydrolase gene and severity of HCV-related liver disease. Hepatology 22; 36: 195-21.

September 26 Oh et al. : Effects of KT572 on Cell Viability and Antioxidant Enzyme Expression 253 To-Figueras J, Gene M, Gomez-Catalan J, Pique E, Borrego N and Corbella J. Lung cancer susceptibility in relation to combined polymorphisms of microsomal epoxide hydrolase and glutathione S-transferase P1. Cancer Lett 21; 173: 155-162. Wang Y, Kim PK, Peng X, Loughran P, Vodovotz Y, Zhang B and Billiar TR. Cyclic AMP and Cyclic GMP suppress TNFalpha-induced hepatocyte apoptosis by inhibiting FADD up-regulation via a protein kinase A- dependent pathway. Apoptosis. 26 Mar; 11(3): 441-51. West IC. Radicals and oxidative stress in diabetes. Diabet Med 2; 17: 171-18. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev. 1994; 74(1): 139-162.