Arylamine N-acetyltransferases: a new inhibitor of apoptosis in HepG2 cells

Arylamine N -acetyltransferases: a new inhibitor of apoptosis in HepG2 cells 1 2 first-author 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:sygao2002@163.com †E-mail:sygao2002@163.com sygao2002@163.com Postdoctoral Research Station of the Institute of Materia Medica, Center for Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin 150076, China Postdoctoral Research Station of the Institute of Materia Medica, Center for Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin 150076, China MOE Engineering Research Center of Natural Anticancer Drugs, Harbin University of Commerce, Harbin 150076, China MOE Engineering Research Center of Natural Anticancer Drugs, Harbin University of Commerce, Harbin 150076, China Objective: 2 To explore how arylamine N -acetyltransferases (NATs) is related to cell apoptosis. Methods: 2 NAT activity in apoptotic HepG2 cells was measured using high performance liquid chromatography (HPLC); the apoptosis rate of HepG2 cells acted upon by an NAT inhibitor was measured using flow cytometry. Results: 2 NAT activity was lowered in apoptotic HepG2 cells; apoptosis rate induced by camptothecin (CAM) increased after inhibition of NAT activity in HepG2 cells. Conclusion: 2 NAT can inhibit apoptosis in HepG2 cells. Arylamine N -acetyltransferases (NATs) Apoptosis HepG2 Camptothecin (CAM) INTRODUCTION 1 Arylamine N -acetyltransferases (NATs), phase II-drug metabolizing enzymes, are a group of enzymes that catalyze the addition of an acetyl group from Acetyl coenzyme A (AcCoA) to a terminal nitrogen on suitable substrates such as arylamines, hydrazines (Weber and Hein, 1985 1985 ), arylhydroxylamines (Hein et al., 1993 1993 ) or arylhydrazines (Fig. 1 1 ). They play an important role in the metabolism of drugs and toxins as well as in detoxification (Hein et al., 1992 1992 ; 1994 1994 ; King et al., 1997 1997 ). The active center of NATs is a Cys 69 -His 107 -Asp 122 triad. In other words, the triplet of cysteine residue (Cys 69 ), the histidine residue (His 107 ), and the aspartic acid residue (Asp 122 ) have the same active center as cysteine proteases (Turk et al., 1998 1998 ). This structural similarity between NATs and cysteine proteases suggests that they share a common evolutionary origin. Their similar crystal structures also suggest that NATs exert their effects through a mechanism strategy partially common to cysteine proteases (Rodrigues-Lima et al., 2001 2001 ). Since cysteine proteases are an important kind of enzymes associated with cell apoptosis (Gao et al., 2005 2005 ; Ho and Hawkins, 2005 2005 ), we speculated that NATs may also be associated with cell apoptosis, and then designed the following experiments to test this speculation. MATERIALS AND METHODS 1 Cell line 2 Human hepatocarcinoma cell line HepG2 was originally purchased from American Type Culture Collection (ATCC), USA and was maintained by the Institute of Cancer Studies of Heilongjiang Cancer Hospital, China. Drugs and reagents 2 Acridine orange (AO) and ethidium bromide (EB) were purchased from Amresco, USA; Triton X-100, RNase A, propidium iodide (PI), 2-AAF, Tris, leupeptin, AcCoA and bovine serum albumin (BSA) were purchased from Sigma, USA; phenylmethylsulfonyl fluoride (PMSF) was purchased from Amresco, USA; dithiothreitol (DTT) was purchased from Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China; acetylcarnitine, 2-AF and carnitine acetyltransferase were purchased from Fluka, USA; RPMI 1640 culture medium was purchased from Hyclone, USA; pancreatin was purchased from Gibco, USA; fetal bovine serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., China. Apparatuses 2 Waters HPLC (2695 pump, 2487 ultraviolet (UV) detector, automatic sample-injector, and Empower Workstation) and SymmetryShield C 18 inverted chromatographic column (4.6 mm×250 mm, 5 μm) were from Waters Co., Ltd., USA; super clean table SW-CJ-2F was from Suzhou Purification Equipment Co., Ltd., China; carbon dioxide incubator CO-150 was from New Brunswick Scientific Co., Inc., USA; ultrasonic extractor AS-2060B was from Tianjin Autoscience Instruments Co., Ltd., China; invert microscope CKX-41-32 Olympus was from Japan; flow cytometer EPICS XL-MCL was from Beckman-Coulter, USA. Preparation of the reagents 2 Cell lysate (20 ml): 20 μl of 1 mol/L DTT, 5.845 ml of 10 mg/ml ethylenediaminetetraacetic acid (EDTA), 10 μl of 100 mmol/L PMSF, 23.280 μl of 2 mg/ml leupeptin, 14.356 ml of 20 mmol/L Tris-HCl. AcCoA cycle mixture (3 ml): 6 μl of 1 mol/L DTT, 17.535 μl of 10 mg/ml EDTA, 10.787 mg of acetylcarnitine, 6 U of carnitine acetyltransferase, 2.9177 ml of 50 mmol/L Tris-HCl. PI staining solution (50 ml): 0.2916 g of NaCl, 2.5 mg of PI, 0.5 mg of RNase A, 1 ml of Triton X-100, 50 mg of sodium citrate. These were dissolved with double-distilled water in a 50 ml volumetric flask. With its volume brought to scale, the solution was kept away from light at 4 °C. Changes of NAT activity in apoptotic HepG2 cells 2 1. Cell incubation 3 HepG2 cells of a suitable concentration were inoculated in a culture flask containing RPMI 1640 solution with 10% (v/v) of fetal bovine serum. The culture was then incubated at 37 °C, with 5% (v/v) CO 2 and saturated humidity. The cells were made to adhere to the inner wall of the flask as they grew, and culture transfer was performed once every 2~3 d. 2. Observation of apoptosis in HepG2 cells induced by CAM 3 (1) Grouping and drug administration 4 HepG2 cells in the phase of logarithmic growth were taken and digested with pancreatin, and RPMI 1640 culture medium containing 10% (v/v) of fetal bovine serum was added to adjust the cells to a concentration of 5×10 5 cells/ml. The cell solution was then divided and put in two 100-ml culture flasks as the negative control and the CAM group, respectively. The culture flasks were set in a CO 2 incubator and incubated for 24 h at 37 °C and 5% (v/v) CO 2 , after which CAM was added to the CAM group to reach a final concentration of 0.4 μg/ml, while an equal volume of RPMI 1640 culture medium was added to the negative control. The samples were then incubated in a CO 2 incubator for 24 h at 37 °C and 5% (v/v) CO 2 . (2) Apoptosis observed using flow cytometry (Wang et al., 2007 2007 ) 4 After digested with pancreatin, HepG2 cells were rinsed 3 times with phosphate buffered solution (PBS). The supernatant was discarded, and the residue was blown in a small amount of PBS and mixed well, after which 2 ml of cold ethanol (70%, v/v) was added to fixate the cells, and the sample was left at 4 °C for the night (the fixation needs more than 12 h). The sample was centrifuged the next day, the supernatant was discarded, and the residue was rinsed 3 times with PBS. About 0.1 ml of liquid was retained, and 800 μl of PI staining solution was added to and thoroughly mixed with it. The sample was then left to be stained in dark for 30 min. After it was filtered through a nylon net, the sample was measured with a flow cytometer, with an activation wavelength of 488 nm (Zhang et al., 2004 2004 ). 3. Measurement of NAT activity 3 HepG2 cells in the phase of logarithmic growth were taken and digested with pancreatin, and RPMI 1640 culture medium containing 10% (v/v) of fetal bovine serum was added to adjust the cells to a suitable concentration. The cell solution was then divided into 7 equal portions, with each in a 100-ml culture flask. The concentration of the cell solution was 5×10 5 cells/ml. After the culture flasks were incubated in a CO 2 incubator for 24 h at 37 °C and 5% (v/v) CO 2 , 2-AF as substrates and CAM were added, so that the final concentrations were 45 μmol/L and 0.4 μg/ml, respectively, but no CAM was added to the negative control group. After the samples were incubated in a CO 2 incubator for 24 h at 37 °C and 5% (v/v) CO 2 , the culture medium was drawn and centrifuged for 10 min at 3500× g , after which the supernatants were immediately extracted with equal volumes of the mixture of ethyl acetate:methanol (95:5, v/v). The supernatants were drawn and evaporation was allowed to proceed until the samples were dry, after which methanol was added to dissolve the residue thoroughly and to bring volume of the solution up to 2 ml. The sample was then measured using Waters HPLC with an automatic sample injection of 20 μl. Conditions used for the HPLC measurement were: SymmetryShield C 18 inverted chromatographic column (4.6 mm×250 mm, 5 μm); mobile phase (20 mmol/L KH 2 PO 4 (pH 4.5):CH 3 CN=53:47, v/v); UV detector with a detecting wavelength of 288 nm and a column temperature of (25±5) °C. The retention time was about 13.5 min for 2-AAF, and about 17 min for AF. NAT activity was studied through HPLC analysis of the yield of 2-AAF and expressed in nmol acetylated substrate/10 6 cells. Interference with apoptosis through the inhibition of NAT activity 2 1. Cell incubation 3 The procedure followed is the same as abovementioned in “Changes of NAT activity in apoptotic HepG2 cells”. 2. Grouping and drug administration 3 The experiment was performed on 5 groups, namely, the negative control, the CAM group, the CAM+100 μmol/L iodoacetamide group, the CAM+500 μmol/L iodoacetamide group, and the iodoacetamide group. After the cells had adhered to the flask wall, the negative control was treated with an equal volume of RPMI 1640 culture medium; the CAM group was treated with CAM (final concentration 0.08 μg/ml); the CAM+100 μmol/L iodoacetamide group was treated with CAM (final concentration 0.08 μg/ml) and iodoacetamide (final concentration 100 μmol/L); the CAM+500 μmol/L iodoacetamide group was treated with CAM (final concentration 0.08 μg/ml) and iodoacetamide (final concentration 500 μmol/L); and the iodoacetamide group was treated with iodoacetamide (final concentration 500 μmol/L). The samples were incubated at 5% (v/v) CO 2 and 37 °C for 48 h. 3. Apoptosis observed using flow cytometry 3 The procedure followed is the same as abovementioned in “Changes of NAT activity in apoptotic HepG2 cells”. Data analysis 2 Data were presented as mean± SD . Statistical significance was evaluated with Student’s t -test. RESULTS 1 Observation of apoptosis in HepG2 cells induced by CAM 2 Fig. 2 2 shows that cells in negative control had a round shape and their DNA structures kept intact, but the morphology of cells in 0.4 μg/ml CAM group was significantly different. A typical sign for apoptosis appeared in the 0.4 μg/ml CAM group. Fig. 3 3 shows that in 0.4 μg/ml CAM group, the G 2 /M peak disappeared, and a sub-G 0 peak of apoptosis appeared prior to the G 0 /G 1 phase. All these results show that 0.4 μg/ml CAM could induce the apoptosis of HepG 2 cells. %G 1 : Percent of G 1 phase; %S: Percent of S phase; %G 2 : Percent of G 2 phase; %Tot: Apoptosis rate, i.e., percent of sub-G 0 phase; Cell No.: Cell numbers determined Changes of NAT activity in apoptotic HepG2 cells 2 The results are shown in Fig. 4 4 and Table 1 1 . After 0.08 μg/ml of CAM was applied to HepG2 cells for 48 h, the amount of 2-AAF produced was lowered, which means that the activity of NAT was decreased. P < 0.01 compared with control Group Concentration of drug (µg/ml) 2-AAF peak areas (×10 3 AU·s) Control 773±1.89 CAM 0.08 489±1.61 ** ** Interference with apoptosis through the inhibition of NAT activity 2 As shown in Fig. 5 5 , no apoptosis peak appeared for the negative control; CAM in the concentration of 0.08 μg/ml induced apoptosis in HepG2 cells, with an apoptosis rate of 10.4%; the apoptosis rate for the 0.08 μg/ml CAM+100 μmol/L iodoacetamide group was 17.4%; the apoptosis rate for the 0.08 μg/ml CAM+500 μmol/L iodoacetamide group was 30.6%; but iodoacetamide alone failed to induce apoptosis in HepG2 cells. So iodoacetamide is an inhibitor of NAT activity. Our results show that when NAT activity was inhibited, the apoptosis rate induced by CAM was increased, which implies that NAT can act to inhibit apoptosis of HepG2 cells, and that the lowering or loss of NAT activity can promote apoptosis in tumor cells. %G 1 : Percent of G 1 phase; %S: Percent of S phase; %G 2 : Percent of G 2 phase; %Tot: Apoptosis rate, i.e., percent of sub-G 0 phase; Cell No., Cell numbers determined DISCUSSION 1 The structural similarity between NATs and cysteine proteases suggests that they may be derived from the same source. Their similar crystal structures also reveal that NATs exert their effect through the same mechanism as cysteine proteases (Sinclairet al., 2000 2000 ). Since cysteine proteases are closely associated to apoptosis (Fan et al., 2005 2005 ; Harwood et al., 2005 2005 ; Bozhkov et al., 2005 2005 ), NATs may also be suggested to be associated with apoptosis. To test this speculation, the AcCoA-HPLC assay was conducted, with 2-AF as the substrate, to observe the effect of CAM on the amount of 2-AF metabolized into 2-AAF by NATs in the cytoplasm of HepG2 cells, and hence indirectly to observe the activity of NATs. We designed two experiments: (1) CAM was applied to HepG2 cells over a range of concentrations and incubation times, and the change in NAT activity was observed; (2) observation was made on the change in the apoptosis rate in HepG2 cells induced by CAM after NAT inhibitor was added. The results show that in Experiment (1) NAT activity was lowered in the CAM group relative to the negative control, i.e., NAT activity was lowered in apoptotic HepG2 cells, and that in Experiment (2) after NAT activity was inhibited by iodoacetamide, an NAT inhibitor, the apoptosis rate in HepG2 cells induced by CAM was increased. Thus we conclude that NATs are associated with apoptosis to some extent, and that lowering NAT activity can promote apoptosis in HepG2 cells. References PV Bozhkov , MF Suarez , LH Filonova , G Daniel , AAJr Zamyatnin , S Rodriguez-Nieto , B Zhivotovsky , A Smertenko . Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis . Proc Natl Acad Sci USA , 2005 . 102 ( 40 ): 14463 - 14468 . DOI: 10.1073/pnas.0506948102 http://doi.org/10.1073/pnas.0506948102 . Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis TJ Fan , LH Han , RS Cong , J Liang . Caspase family proteases and apoptosis . Acta Biochim Biophys Sin (Shanghai) , 2005 . 37 ( 11 ): 719 - 727 . DOI: 10.1111/j.1745-7270.2005.00108.x http://doi.org/10.1111/j.1745-7270.2005.00108.x . Caspase family proteases and apoptosis JX Gao , YQ Zhou , RH Zhang , XL Ma , KJ Liu . Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine . Sheng Li Xue Bao , 2005 . 57 ( 6 ): 755 - 760 . (in Chinese) . Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine SM Harwood , MM Yaqoob , DA Allen . Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis . Ann Clin Biochem , 2005 . 42 ( 6 ): 415 - 431 . DOI: 10.1258/000456305774538238 http://doi.org/10.1258/000456305774538238 . Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis DW Hein , TD Rustan , MA Doll , KD Bucher , RJ Ferguson , Y Feng , EJ Furman , K Gray . Acetyltransferases and susceptibility to chemicals . Toxicol Lett , 1992 . 64-65 123 - 130 . DOI: 10.1016/0378-4274(92)90181-I http://doi.org/10.1016/0378-4274(92)90181-I . Acetyltransferases and susceptibility to chemicals DW Hein , MA Doll , TD Rustan , K Gray , Y Feng , RJ Ferguson , DM Grant . Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases . Carcinogenesis , 1993 . 14 ( 8 ): 1633 - 1638 . DOI: 10.1093/carcin/14.8.1633 http://doi.org/10.1093/carcin/14.8.1633 . Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases DW Hein , TD Rustan , RJ Ferguson , MA Doll , K Gray . Metabolic activation of aromatic and heterocyclic N -hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases . Arch Toxicol , 1994 . 68 ( 2 ): 129 - 133 . DOI: 10.1007/s002040050045 http://doi.org/10.1007/s002040050045 . Metabolic activation of aromatic and heterocyclic N-hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases PK Ho , CJ Hawkins . Mammalian initiator apoptotic caspases . FEBS J , 2005 . 272 ( 21 ): 5436 - 5453 . DOI: 10.1111/j.1742-4658.2005.04966.x http://doi.org/10.1111/j.1742-4658.2005.04966.x . Mammalian initiator apoptotic caspases CM King , SJ Land , RF Jones , M Debiec-Rychter , MS Lee , CY Wang . Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines . Mutat Res , 1997 . 376 ( 1-2 ): 123 - 128 . DOI: 10.1016/S0027-5107(97)00034-1 http://doi.org/10.1016/S0027-5107(97)00034-1 . Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines F Rodrigues-Lima , C Delomenie , GH Goodfellow , DM Grant , JM Dupret . Homology modelling and structural analysis of human arylamine N -acetyltransf-erase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop . Biochem J , 2001 . 356 ( 2 ): 327 - 334 . DOI: 10.1042/0264-6021:3560327 http://doi.org/10.1042/0264-6021:3560327 . Homology modelling and structural analysis of human arylamine N-acetyltransf-erase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop JC Sinclair , J Sandy , R Delgoda , E Sim , ME Noble . Structure of arylamine N -acetyltransferase reveals a catalytic triad . Nat Struct Biol , 2000 . 7 ( 7 ): 560 - 564 . DOI: 10.1038/76783 http://doi.org/10.1038/76783 . Structure of arylamine N-acetyltransferase reveals a catalytic triad D Turk , G Guncar , M Podobnik , B Turk . Revised definition of substrate binding sites of papain-like cysteine proteases . Biol Chem , 1998 . 379 ( 2 ): 137 - 147 . . Revised definition of substrate binding sites of papain-like cysteine proteases GY Wang , JW Zhang , QH Lü , RZ Xu , QH Dong . Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation . Journal of Zhejiang University SCIENCE B , 2007 . 8 ( 4 ): 248 - 255 . DOI: 10.1631/jzus.2007.B0248 http://doi.org/10.1631/jzus.2007.B0248 . Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation WW Weber , DW Hein . N -acetylation pharmacogenetics . Pharmacol Rev , 1985 . 37 ( 1 ): 25 - 79 . . N-acetylation pharmacogenetics YH Zhang , HY Peng , GH Xia , MY Wang , Y Han . Anticancer effect of two diterpenoid compounds isolated from Annona glabra Linn . Acta Pharmacol Sin , 2004 . 25 ( 7 ): 937 - 942 . . Anticancer effect of two diterpenoid compounds isolated from Annona glabra Linn 10.1631/jzus.B0820090 Project supported by the National Natural Science Foundation of China (No. 30400591), the Science Foundation of Heilongjiang Province, China (Nos. D2004-13 and D200505), and the Young Scientist Fund of Harbin City, China (No. 2004AFQXJ035) 2008-03-20 2008-07-29 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 09