Naunyn-Schmiedebergs Arch Pharmacol (2004) 369 : 312321 DOI 10.

1007/s00210-004-0864-2

312

O R I G I N A L A RT I C L E

Se Young Park Hyun Hee Ko Jin Ho Song Eun Sook Han Chung Soo Lee

Differential effect of nitrogen species on changes in mitochondrial membrane permeability due to mitomycin c in lung epithelial cells
Received: 14 June 2003 / Accepted: 19 December 2003 / Published online: 7 February 2004 Springer-Verlag 2004

Abstract The effect of reactive nitrogen species (RNS) against the cytotoxicity of mitomycin c (MMC) in lung epithelial cells was assessed by measuring the effect on mitochondrial membrane permeability. RNS had a differential effect against cytotoxicity of MMC depending on concentration. Viability loss in cells exposed to MMC was decreased by inhibitors of caspase-3, -8 and -9 and attenuated by antioxidants (N-acetylcysteine, dithiothreitol, ascorbate and rutin). Addition of 3-morpholinosydnonimine (SIN-1) differentially affected the MMC-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 M. Ascorbate, superoxide dismutase and haemoglobin prevented the inhibitory effect of 150 M SIN-1 on 10 g/ml MMC-induced cell death. SIN-1 inhibited the MMC-induced nuclear damage, loss in mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, caspase-3 activation, increase in reactive oxygen species (ROS) formation and depletion of GSH. SIN-1 also attenuated cell death due to H2O2. The cytotoxicity of MMC in the presence of oxidants or RNS producers was much less than the sum of the each effect of MMC and producer. SIN-1 may inhibit the MMC-induced viability loss in lung epithelial cells by suppressing the mitochondrial membrane permeability change and by interaction of its products with MMC. Keywords Mitomycin c Reactive nitrogen species Lung epithelial cells Mitochondrial membrane permeability Cell injury Differential inhibition

Introduction
Membrane permeability transition of mitochondria has been shown to be involved in a variety of toxic and oxidative

S. Y. Park H. H. Ko J. H. Song E. S. Han C. S. Lee () Department of Pharmacology, College of Medicine, Chung-Ang University, 156-756 Seoul, South Korea Tel.: +82-2-8205659, Fax: +82-2-8153856, e-mail: leecs@cau.ac.kr

forms of cell injury as well as apoptosis. Opening of the mitochondrial permeability transition pore causes depolarization of the transmembrane potential, release of Ca2+ and cytochrome c, osmotic swelling and loss of oxidative phosphorylation, which results in the loss of cell viability (Bernardi 1996; Mignotte and Vayssire 1998). Mitomycin c (MMC) is used commonly in combination with other drugs for the treatment of breast, lung and prostate cancers. Enzymatic bioactivation produces reactive oxygen species (ROS) such as superoxide radicals and hydrogen peroxide (Nakano et al. 1984; Pritsos and Sartorelli 1986). In EMT6 mouse mammary carcinoma cells MMC damages the DNA and mitochondrial membrane integrity (Pritsos et al. 1997). On the other hand, in human lung adenocarcinoma A549 cells morphological changes of mitochondria due to MMC have not been demonstrated (Simamura et al. 2001). It is also uncertain whether MMC-induced cell death is mediated by caspase-3 activation (Kobayashi et al. 2000; Pirnia et al. 2002). It is thus necessary to clarify whether opening of the mitochondrial membrane permeability pore induces activation of the caspases. Nitric oxide (NO) can exert protective or destructive effects depending on the available concentration, target cell type and interactions with ROS, metal ions and proteins (Hofseth et al. 2003). Inhibition of NO production can either suppress or exacerbate tumour formation. NO and peroxynitrite (ONOO) cause cell death through damage to chromatin by cross-linking of DNA (Nguyen et al. 1992) and induction of the mitochondrial permeability transition (Ghafourifar et al. 1999). In contrast to these findings, there is evidence that NO may also act as an antioxidant to oxidative stress. NO protects Chinese hamster V79 lung fibroblasts from the cytotoxicity of t-butyl hydroperoxide and cumene hydroperoxide (Wink et al. 1993) and in rat hepatocytes it reduces apoptosis by suppressing the loss of mitochondrial membrane potential and activation of caspase-3 due to tumour necrosis factor- plus actinomycin D (Li et al. 1999). Most anticancer drugs, including MMC, exhibit toxic effects that often prohibit continuation of therapy. MMC

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causes pneumonitis, which can progress to interstitial lung fibrosis (Beinert et al. 1999). In addition to DNA, mitochondria are postulated to be a cellular target for MMC. Nevertheless, the effect of MMC on the mitochondrial membrane permeability and apoptotic processes has not been clarified. As previously mentioned, NO may have either antioxidant or pro-oxidant effects. The purpose of the present study was to investigate the effect of reactive nitrogen species (RNS) on the cytotoxicity of MMC in relation to mitochondrial membrane permeability. We examined the effect of RNS against the toxicity of MMC in human lung epithelial cells by measuring the effect on the mitochondrial transmembrane potential, cytochrome c release, ROS formation, GSH content and caspase-3 activity.

MAX 340, Molecular Devices, Sunnyvale, Calif., USA). Cell viability was expressed as a percentage of the value in control cultures. Measurement of apoptosis. Apoptosis was assessed by measuring the DNA fragmentation, which occurs following the activation of endonucleases. Cells (1105 cells/ml) were treated with MMC in the presence of SIN-1 for 24 h at 37 C, washed with PBS and fixed with formaldehyde solution. Nucleotide (dNTP) was incorporated at the 3-ends of DNA fragments using terminal deoxynucleotidyl transferase (TdT) and the nucleotide detected using horseradish peroxidase and TACS-Sapphire according to the TiterTACS protocol. Data are expressed as absorbance at 450 nm. Morphological observation of nuclear change. Cells (1106 cells/ ml) were treated with MMC in the presence of SIN-1 for 24 h at 37 C and morphological changes in the nucleus assessed using the Hoechst dye 33258 (Oberhammer et al. 1992). The cells were incubated with 1 g/ml Hoechst 33258 for 3 min at room temperature and nuclei visualized using an Olympus Microscope with a WU excitation filter (Tokyo, Japan). Flow cytometric measurement of mitochondrial transmembrane potential. Changes in the mitochondrial transmembrane potential during the MMC-induced apoptosis in lung epithelial cells were quantified by flow cytometry with the cationic lipophilic dye DiOC6(3) (Lizard et al. 2000). Cells (1106/ml) were treated with MMC in the presence of SIN-1 for 4 h at 37 C, DiOC6(3) (40 nM) added to the medium and the cells incubated for 15 min at 37 C. After centrifugation at 1,500 g for 5 min, the supernatants were removed and the pellets resuspended in phosphate-buffered saline (PBS) containing 0.5 mM EDTA. For analysis, a FACScan cytofluorometer (Becton-Dickinson, San Jose, Calif., USA) with argon laser excitation at 501 nm was used to assess 10,000 cells from each sample. Measurement of cytochrome c release. The release of cytochrome c from mitochondria into the cytosol was assessed by using a solid-phase, enzyme-linked immunosorbent assay (ELISA) kit for the detection of human cytochrome c. Cells (5105/ml) were harvested by centrifugation at 800 g for 10 min, washed twice with PBS, resuspended in buffer (in mM): 250 sucrose, 20 HEPESKOH (pH 7.5), 10 KCl, 1.5 MgCl2, 1 EDTA, 1 EGTA, 0.5 dithiothreitol and 0.1 PMSF and homogenized further by successive passages through a 26-G hypodermic needle. The homogenates were centrifuged at 100,000 g for 30 min and the supernatant was used for analysis of cytochrome c. The supernatants were added to the 96-well microplates coated with monoclonal antibody specific for human cytochrome c that contains cytochrome c conjugate. The procedure was performed according to the manufacturers instructions. Absorbance of samples was measured at 450 nm in a microplate reader. A standard curve was constructed by adding diluted solutions of cytochrome c standard, handled like samples, to the microplates coated with monoclonal antibody. The amount was expressed as nanograms/millilitre by reference to the standard curve. Measurement of caspase-3 activity. Apoptosis in lung epithelial cells was assessed by measuring the activity of caspase-3, an apoptotic factor (Mignotte and Vayssire 1998). Cells (2106 cells/ml) were treated with MMC in the presence of SIN-1 for 24 h at 37 C. The effect of SIN-1 on apoptosis in the MMC-treated cells was determined according to the users manual for the ApoAlert CPP32/ caspase-3 assay kit. The supernatant obtained by centrifugation of lysed cells was added to the reaction mixture containing dithiothreitol and caspase-3 substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide) and incubated for 1 h at 37 C. Absorbance of the chromophore p-nitroanilide produced was measured at 405 nm. The standard curves were obtained from the absorbance of p-nitroanilide standard reagent diluted with cell lysis buffer (up to 20 nM). One unit of the enzyme was defined as the activity producing 1 nM chromophore.

Materials and methods

Materials. MMC, 3-morpholinosydnonimine (SIN-1), 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA NONOate), N-acetylcysteine, dithiothreitol, haemoglobin, rutin trihydrate, glutathione (oxidized form GSSG, reduced form GSH), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), z-Leu-Glu-(O-Me)-His-Asp(O-Me) fluoromethylketone (z-LEHD.fmk), Hoechst 33258, 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)], 2,7-dichlorofluorescin diacetate (DCFH2-DA), phenylmethylsulphonylfluoride (PMSF), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), glutathione reductase (from spinach), 1,3(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dulbeccos modified Eagles medium (DMEM) were from Sigma-Aldrich (St. Louis, Mo., USA). z-Asp-(O-Me)-Gln-MetAsp(O-Me) fluoromethyl ketone (z-DQMD.fmk) and z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (z-IETD.fmk) were from Calbiochem-Novabiochem (La Jolla, Calif., USA), the colorimetric apoptosis detection kit (TiterTACS) was from Trevigen (Gaithersburg, Md., USA), the human cytochrome c assay kit (Quantikine M) was from R&D systems (Minneapolis, Minn., USA), the CPP32/caspase-3 assay kit (ApoAlert) was from Clontech (Palo Alto, Calif., USA), and fetal bovine serum (FBS) from Life Technologies (Gibco-BRL, Grand Island, N.Y., USA). The polystyrene 24 and 96-well plates were from Corning (Corning, N.Y., USA), and the 15-ml polypropylene tubes (Falcon) used in the isolation of cells were from Becton-Dickinson (Franklin Lakes, N.J., USA). Culture of lung epithelial cells. Normal human embryo lung epithelial cells (WI-26 VA4) were obtained from the Korean cell line bank (Seoul, South Korea) and maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 g/ml streptomycin in a 5% CO2 atmosphere at 37 C. The culture medium was changed every 3 days and the cells were subcultured about once a week. Cells (1107) were plated on polystyrene 6015-mm cell culture dishes (Corning) 4872 h before experiments. Cells were washed with DMEM containing 1% FBS and replated onto 96-well plates at a density of 4104 cells/well in a volume of 200 l (or varying numbers of cells/ml in 24-well plates). Cells were treated with MMC in DMEM containing 1% FBS for 24 h at 37 C. Cell viability assay. Cell viability was measured by using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases (Mosmann 1983). Cells (4104) were treated with MMC in the presence of SIN-1 for 24 h at 37 C. The medium (200 l) was incubated with 10 l of 10 mg/ml MTT solution for 2 h at 37 C. The culture medium was removed and 100 l DMSO added to each well to dissolve the formazan. Absorbance was measured at 570 nm using a microplate reader (Spectra

314 Measurement of intracellular ROS formation. The dye DCFH2-DA, which is oxidized to fluorescent DCF by hydroperoxides, was used to measure relative levels of cellular peroxides (Fu et al. 1998). Cells (4104) were treated with MMC for 24 h at 37 C, washed, suspended in FBS-free DMEM, incubated with 50 M dye for 30 min at 37 C and washed with DMEM. The cell suspensions were centrifuged at 412 g for 10 min, the medium removed, the cells lysed with 1% Triton X-100 and fluorescence measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (Spectrafluor, Tecan, Salzburg, Austria). Measurement of total glutathione. The total glutathione (GSH plus GSSG) was determined using glutathione reductase (van Klaveren et al. 1997). Cells (4104) were treated with MMC for 24 h at 37 C, centrifuged at 412 g for 10 min in a microplate centrifuge, the medium removed, the cells lysed with 2% 5-sulphosalicylic acid (100 l) and then incubated in 100 l of reaction mixture containing 22 mM sodium EDTA, 600 M NADPH, 12 mM DTNB and 105 mM NaH2PO4, pH 7.5 at 37 C. Glutathione reductase (20 l, 100 U/ml) was added and the mixture incubated for a further 10 min. Absorbance was measured at 412 nm using a microplate reader. The standard curve was obtained from absorbance of the diluted commercial GSH incubated in the mixture as in samples. Preparation of peroxynitrite. ONOO was made according to (Beckman et al. 1994). Aliquots (5 ml) of ice-cold 50 mM NaNO2 and 50 mM H2O2 were mixed rapidly in a 25 ml-flask. A 2.5-ml aliquot of 1 M HCl was added to the nitrite/peroxide solution and 2.5 ml 1.5 M NaOH was added to the mixture about 1 s later. Residual hydrogen peroxide was eliminated by passage of the solution through a MnO2 column. The concentration of ONOO formed was determined using the extinction coefficient of 1670 M1 cm1 at 302 nm. Statistical analysis. Data are expressed as meansSEM. Data were analysed using one-way ANOVA. When significance was detected, post-hoc comparisons between the different groups were made using Duncans test for multiple comparisons. P<0.05 was considered significant. Depiction and expression of the data were based on previous reports.

Results
Effect of SIN-1 on cell death and nuclear damage due to MMC The depressant effect of RNS on the MMC-induced loss of cell viability was investigated in human lung epithelial cells. MMC caused cell death in these cells in a dose-dependent manner. The incidence of cell death after exposure to 10 g/ml MMC for 24 h was about 62% (Fig. 1A). MMCinduced cell death was inhibited by z-DQMD.fmk (a cellpermeable inhibitor of caspase-3), z-IETD.fmk (a cell permeable inhibitor of caspase-8) or z-LEHD.fmk (a cell permeable inhibitor of caspase-9) (each 40 M). z-DQMD.fmk was itself cytotoxic. SIN-1, which liberates NO and superoxide radicals, exhibited a differential effect on the MMC-induced cell death (Fig. 1B). The maximum inhibition of cell death due to MMC was achieved with 150 M SIN-1; beyond this concentration the inhibitory effect declined and 500 M SIN-1 had no inhibitory effect at all. To assess the cytotoxic effect of SIN-1 itself, lung epithelial cells were treated with SIN-1 in the absence of MMC. Treatment with SIN-1 (300 or 500 M) for 24 h caused 20 and 27% cell viability loss, respectively.
Fig. 1A, B Effect of 3-morpholinosydnonimine (SIN-1, SIN) on mitomycin c (MMC)-induced loss of cell viability in lung epithelial cells as assessed by the 1,3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A Cells (4104) were exposed to the caspase inhibitors z-DQMD.fmk (DQMD), z-IETD.fmk (IETD) or z-LEHD.fmk (LEHD) (all 40 M) for 24 h at 37 C in the absence (light columns) or presence (dark columns) of 10 g/ml MMC. B Cells (4104) were treated with various concentrations of SIN-1 (M, open columns), with various concentrations of MMC (g/ml, light columns), or with 10 g/ml MMC in the presence of SIN-1 (dark columns) for 24 h at 37 C. MeansSEM of six replicate values in two separate experiments. +P<0.05 vs. control (percentage of control); *P<0.05 vs. MMC alone

We also determined whether the cytotoxic effect of MMC was mediated by oxidative stress. Cells were incubated with 10 g/ml MMC in the presence of various antioxidants for 24 h. The addition of 1 mM sulphhydryl compounds (N-acetylcysteine or dithiothreitol), 1 mM ascorbate or 50 M rutin (a scavenger of NO and inhibitor of lipid peroxidation) depressed cell death due to MMC, while 10 g/ml SOD (a scavenger of superoxide radicals)

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To assess the inhibitory effect of SIN-1 against the cytotoxicity of MMC further, we investigated its effect on the nuclear morphological changes observed in the MMCtreated cells. Nuclear staining with Hoechst 33258 demonstrated that control lung epithelial cells had regular and round-shaped nuclei. In contrast, condensation and fragmentation of nuclei, characteristic of apoptotic cells, were evident in cells treated with 10 g/ml MMC for 24 h at 37 C (Fig. 3A). SIN-1 (150 M) decreased the MMC-induced nuclear damage, while the nuclear morphology in cells exposed to 150 M SIN-1 alone was similar to that in the control cells. During the process of apoptosis, DNA fragmentation is caused by activation of endonucleases. Fragmented DNA was assessed by measuring the binding of dNTP to the 3-ends of DNA fragments and detection by a quantitative colorimetric assay. Cells (1105 cells/ml) were treated with 10 g/ml MMC in the presence of SIN-1 for 24 h at 37 C. Control cells showed an absorbance of 0.2390.019 (n=6), whilst exposure to 10 g/ml MMC for 24 h increased the absorbance about twofold (Fig. 3B). SIN-1 (150 M) depressed the MMC-induced increase in absorbance, while absorbance in cells treated with 150 M SIN-1 alone was not significantly different from that in control cells. Effect of SIN-1 on MMC-induced mitochondrial membrane potential change, cytochrome c release and caspase-3 activation By investigating effect on the mitochondrial membrane permeability we assessed the cytotoxic effect of MMC. Changes in the mitochondrial transmembrane potential in lung epithelial cells treated with MMC were quantified by flow cytometry using the cationic lipophilic dye DiOC6(3). Exposure of lung epithelial cells to 10 g/ml MMC for 4 h at 37 C increased the percentage of cells with depolarized mitochondria (characterized by low values of the mitochondrial transmembrane potential). The MMC-induced loss of the mitochondrial transmembrane potential was inhibited by 1 mM N-acetylcysteine or 1 mM ascorbate. SIN-1 (150 M) inhibited the MMC-induced increase in cells with depolarized mitochondria, while the mitochondrial transmembrane potential in cells treated with 150 M SIN-1 alone was not significantly different from that in control (Fig. 4). Loss of the mitochondrial membrane potential causes the release of cytochrome c from mitochondria into the cytosol, where it then activates the proapoptotic caspases (Mignotte and Vayssire 1998). MMC-induced cell death was assessed by measuring the release of cytochrome c into the cytosol. Treatment of lung epithelial cells with 10 g/ml MMC for 4 h increased in the level of cytochrome c in the cytosol significantly (Fig. 5A). SIN-1 (150500 M), 1 mM N-acetylcysteine or 1 mM ascorbate reduced the MMCinduced release of cytochrome c. SIN-1 (300 and 500 M) alone also caused significant cytochrome c release. Apoptosis in lung epithelial cells was also assessed by measuring the activity of caspase-3. Control cells had a

Fig. 2A, B Effect of antioxidants on loss of cell viability in lung epithelial cells due to MMC. A Cells (4104) were treated with 10 g/ml MMC in the presence of antioxidants N-acetylcysteine (NAC), dithiothreitol (DTT), ascorbate (each 1 mM), superoxide dismutase (SOD 10 g/ml), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO, 50 M) or rutin (50 M) for 24 h at 37 C. B Cells (4104) were treated with 10 g/ml MMC and 150 M SIN-1 in the presence of antioxidants, including 100 g/ml haemoglobin, for 24 h at 37 C. The values are expressed as a percentage of control. MeansSEM of six replicate values in two separate experiments. +P<0.05 vs. control; *P<0.05 vs. MMC alone (A) or MMC plus SIN-1 (B)

or 50 M carboxy-PTIO (a scavenger of NO) had no inhibitory effect (Fig. 2A). We also assessed the inhibitory effect of superoxide radicals and NO against the cytotoxicity of MMC. Treatment with 1 mM ascorbate, 10 g/ml SOD or 100 g/ml haemoglobin (a scavenger of NO), but not 50 M carboxy-PTIO, depressed the inhibitory effect of SIN-1 on the cytotoxicity of MMC. N-acetylcysteine (1 mM), 1 mM dithiothreitol and 50 M rutin did not attenuate the inhibitory effect of SIN-1 and still exhibited a protective effect (Fig. 2B).

316 Fig. 3A, B Effect of SIN-1 on nuclear damage induced by MMC in lung epithelial cells. A Cells (1106) exposed to 10 g/ml MMC and 150 M SIN-1 for 24 h at 37 C were observed by fluorescence microscopy after nuclei staining with Hoechst 33258. a Control cells, b cells treated with MMC alone, c cells treated with MMC and SIN-1, d cells treated with SIN-1 alone. ad are representative of four different experiments. B Cells (1105) were treated with 10 g/ml MMC and 150 M SIN-1 for 24 h at 37 C. The 3-ends of DNA fragments were detected as described in Materials and methods. Data are expressed as absorbance and represent meansSEM of six replicate values in two separate experiments. +P<0.05 vs. control; *P<0.05 vs. MMC alone Fig. 4 Effect of SIN-1 on MMC-induced loss of the mitochondrial transmembrane potential in lung epithelial cells as shown by 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)] fluorescence. Cells (1106) were treated with 10 g/ml MMC, 150 M SIN-1 (SIN) and antioxidants N-acetylcysteine or ascorbate (both 1 mM) for 4 h at 37 C and then exposed to 40 nM DiOC6(3). MeansSEM of the percentage of cells with depolarized mitochondria in three independent experiments are also given; +P<0.05 vs. control; *P<0.05 vs. MMC alone

caspase-3 activity of 1.69 U per 2106 cells, whilst treatment with 10 g/ml MMC for 24 h increased caspase-3 activity to 7.56 U. SIN-1 (150 M), 1 mM N-acetylcysteine or 1 mM ascorbate attenuated the increase in caspase-3 activation due to MMC. SIN-1 (300 and 500 M) also increased caspase-3 activity and did not attenuate the MMCinduced activation of caspase-3 significantly (Fig. 5B). Effect of SIN-1 on ROS production and GSH depletion due to MMC To assess the involvement of ROS in the MMC-induced cell death, we investigated the production of ROS within cells by monitoring conversion of DCFH2-DA to DCF.

Exposure of lung epithelial cells to 10 g/ml MMC for 24 h increased in DCF fluorescence significant, a response that was inhibited by 1 mM N-acetylcysteine or 1 mM ascorbate (Fig. 6A). SIN-1 (150 M) depressed the MMCinduced increase in DCF fluorescence, while 500 M SIN-1 did not reduce the MMC-induced increase in DCF fluorescence but rather increased it. SIN-1 (500 M) alone also slightly increased the DCF fluorescence, but the increase was not significantly differently from the control and not affected by 1 mM N-acetylcysteine or 1 mM ascorbate. The depletion or oxidation of cellular GSH by toxic substances correlates with loss of cell viability (Tan et al. 1998; Rahman et al. 1999). The cytotoxic effect of MMC was assessed by its effect on GSH content. The thiol content in

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Fig. 5A, B Effect of SIN-1 on the release of cytochrome c and activation of caspase-3 due to MMC in lung epithelial cells. Cells were treated with SIN-1 (light columns) or with 10 g/ml MMC in the presence of the antioxidants N-acetylcysteine or ascorbate (each 1 mM) or SIN-1 (dark columns). Cells (5105) were treated with MMC for 4 h at 37 C for the assay of cytochrome c (A); 2106 cells were treated with MMC for 24 h for the assay of caspase-3 activity (B). MeansSEM of between four and six replicate values in one or two experiments. +P<0.05 vs. control; *P<0.05 vs. MMC alone

lung epithelial cells was 16.560.49 nmol/4104 cells. Treatment with 10 g/ml MMC for 24 h depleted GSH in cells by 10.82 nmol. The MMC-induced depletion of GSH was inhibited by 40 M z-DQMD.fmk, z-IETD.fmk or z-LEHD.fmk (Fig. 6B). z-DQMD.fmk (40 M) alone also decreased the GSH content. SIN-1 had its maximum inhibitory effect on GSH depletion at 150 M, beyond this concentration the inhibitory effect declined (Fig. 6C). SIN-1 alone (300 and 500 M) also reduced the GSH content significantly.

Fig. 6AC Effect of SIN-1 on the formation of reactive oxygen species (ROS) and glutathione (GSH) depletion due to MMC in lung epithelial cells. Cells (4104) were treated with SIN-1 (75500 M), caspase inhibitors z-DQMD.fmk, z-IETD.fmk or z-LEHD.fmk (each 40 M) or antioxidants N-acetylcysteine or ascorbate (each 1 mM) alone (light columns) or together with 10 g/ml MMC (dark columns), for 24 h at 37 C. Values are expressed as arbitrary units (a.u.) of fluorescence for ROS formation (A) or nmol for GSH contents (B, C). MeansSEM of six replicate values in two separate experiments. +P<0.05 vs. control; *P<0.05 vs. MMC alone

318 Fig. 7AD Effect of various oxidants on cell death due to MMC in lung epithelial cells. Cells (4104) were treated with various oxidants either alone (light columns) or together with 10 g/ml MMC (dark columns) for 24 h at 37 C. Oxidants used were: A hypoxanthine (X, 100 M) or xanthine oxidase (XO, 13.4 mU); B, t-butyl hydroperoxide (HP, 10100 M); C 1-propanamine-3-(2-hydroxy2-nitroso-1-propylhydrazine (PAPA NONOate, NO, 75 300 M); D peroxynitrite (PN, 18150 M). MeansSEM of six replicate values in two separate experiments. +P<0.05 vs. control; *P<0.05 vs. MMC alone

Role of ROS and RNS in SIN-1-induced inhibition of MMC toxicity The foregoing results suggested that the inhibition of MMC toxicity by SIN-1, at a concentration that showed a small cytotoxic effect of its own, appeared to be attributable to interaction of ROS or RNS with MMC. To examine the modulation of MMC toxicity due to the interaction, we examined the effect of various oxidants on MMC-induced cell death. The effect of ROS on the cytotoxicity of MMC was assessed in a reaction mixture containing hypoxanthine and xanthine oxidase, which effectively produces ROS, including superoxide radicals (Halliwell and Gutteridge 1999). Cells treated with 100 M hypoxanthine plus 13.4 mU xanthine oxidase for 24 h showed a significant incidence of cell death. The toxic effect of 10 g/ml MMC was not significantly different from the effect of MMC in the presence either of hypoxanthine plus xanthine oxidase or of xanthine oxidase alone (Fig. 7A). Xanthine oxidase alone did not cause cell death. We also examined the effect of t-butyl hydroperoxide on the cytotoxicity of MMC. t-Butyl hydroperoxide markedly induced cell death in lung epithelial cells in a dose-dependent manner (Fig. 7B). The toxic effect of 10 g/ml MMC was not significantly different from the effect of MMC in

the presence of 10 M t-butyl hydroperoxide. Cell death due to MMC plus t-butyl hydroperoxide (50 or 100 M) was less than the sum of the each effect of MMC and t-butyl hydroperoxide. PAPA NONOate (a NO producer) also caused significant cell death in a dose-dependent manner (Fig. 7C). However, the toxic effect of 10 g/ml MMC alone was not significantly different from the effect of MMC in the presence of PAPA NONOate (75300 M). A strong oxidant ONOO (18150 M) caused cell death in lung epithelial cells. The cytotoxic effect of 10 g/ml MMC plus ONOO (1875 M) was similar to the effect of MMC alone (Fig. 7D). Cell death due to MMC plus 150 M ONOO was similar to the effect of the same concentration of ONOO. The inhibitory effect of SIN-1 against the cytotoxicity of MMC was elucidated by measuring the scavenging action on ROS. The incidence of cell death after exposure to 200 M H2O2 for 3 h at 37 C was 34%, a response that was inhibited completely by 2.5 g/ml catalase. Despite the cytotoxic effect, SIN-1 attenuated cell death due to H2O2 in a concentration-dependent manner (Fig. 8A). We next examined the effect of ONOO on the cytotoxicity of H2O2. Cells treated with ONOO (25250 M) for 3 h at 37 C showed significant incidence of cell death. Under

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Fig. 8A, B Effect of SIN-1 or peroxynitrite on hydrogen peroxideinduced cell death in lung epithelial cells. A Cells (4104) were treated with 200 M H2O2, various concentrations of SIN-1 or 2.5 g/ml catalase for 3 h at 37 C and then sequentially with 10 g/ml catalase and MTT. B Cells (4104) were treated with peroxynitrite (25250 M) alone (light columns) or together with 200 M H2O2 (dark columns) for 3 h at 37 C. MeansSEM of between six and eight replicate values in two separate experiments. +P<0.05 vs. control; *P<0.05 vs. H O alone 2 2

these experimental conditions, the cytotoxic effect of 200 M H2O2 plus ONOO (25 or 50 M) was similar to the effect of H2O2 alone (Fig. 8B). Cell death due to H2O2 plus 250 M ONOO was not significantly different from the cytotoxic effect of 250 M ONOO.

Discussion
The MMC-induced DNA damage and subsequent cell death are mediated by ROS produced during the reduction of the drug (Pritsos and Sartorelli 1986; Millard and Hopkins 1993). Evidence suggests that mitochondrial dys-

function and increased formation of ROS are involved in cell death in various cell types (Fleury et al. 2002). Apoptotic cell death is thought to be mediated by interaction of ligand with the cell surface CD95 receptor followed by caspase-8 activation or mitochondrial dysfunction, resulting in the release of cytochrome c and subsequent activation of caspase-9 and -3 (Chandra et al. 2000). MMC causes disruption of the mitochondrial transmembrane potential in normal human AHH-1 lymphocytes, leading to activation of caspase-3 (Guillouf et al. 1999). In contrast to the present study, the cytotoxic effect of MMC on MCF-7 human breast cancer cells line is not mediated by the activation of caspase-3 (Pirnia et al. 2002) and MMC can induce caspase-8 activation and apoptosis in the absence of CD95 receptor interaction (Wesselborg et al. 1999). The mechanism by which MMC causes cell death is thus uncertain. In the present study, the inhibitory effect of the specific caspase inhibitors z-DQMD.fmk, z-IETD.fmk and z-LEHD.fmk suggests that MMC induces cell viability loss in lung epithelial cells by both caspase-8 activation and mitochondrial dysfunction, leading to activation of caspases-9 and -3. Previous reports have suggested that MMC cytotoxicity may be mediated differently by apoptotic processes depending on the cell type. In the present study we have demonstrated a significant cytotoxic effect of MMC on cell viability in lung epithelial cells using the MTT assay and by observing nuclear morphological changes and increase in DNA fragments. Loss of the mitochondrial membrane potential causes the release of cytochrome c from mitochondria to the cytosol, followed by the activation of caspase-3, which is involved in apoptotic cell death (Chandra et al. 2000). The condensation and fragmentation of nuclei (Fig. 3A and B) and a significant increase in caspase-3 activity (Fig. 5) were evidence for apoptotic death following exposure to MMC. Anticancer drugs may also cause cell injury by altering the mitochondrial membrane permeability (Amarante-Mendes et al. 1998; Guillouf et al. 1999). However, because some anticancer drugs cause apoptosis without the cytosolic accumulation of cytochrome c, the role of cytochrome c in cell death due to anticancer drugs has not been elucidated clearly (Tang et al. 1998). One of the aims of this study was thus to clarify whether the cytotoxic effect of MMC is mediated by the mitochondrial membrane permeability change and subsequent release of cytochrome c. The present results suggest that the loss of the mitochondrial membrane potential due to MMC causes the release of cytochrome c into the cytosol, leading to activation of caspase-9 and -3. This process may elicit apoptotic cell death in lung epithelial cells. Inhibition of mitochondrial respiration diminishes ATP production and increases ROS formation, resulting in apoptotic or necrotic cell death (Fleury et al. 2002). To explore the role of ROS in the cytotoxic effect of MMC, we investigated the formation of intracellular ROS by monitoring the increase in DCF fluorescence. The inhibitory effect of antioxidants such as N-acetylcysteine, ascorbate and rutin on cell viability and ROS formation in lung epithelial cells suggests that the cytotoxicity of MMC is mediated by oxidative

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stress. On the other hand, the lack of effect of SOD or carboxy-PTIO implies that MMC itself does not liberate free radicals, and low concentrations of NO are not involved in the toxicity of MMC. GSH effectively defends cells against ROS and regulates the redox state of many cellular substances (Rahman et al. 1999). The oxidation of both GSH and NADPH in mitochondria due to oxidative stress appears to induce the mitochondrial membrane permeability transition (Constantini et al. 1996). A decrease in GSH and a concomitant increase in ROS are found during the apoptotic process (Tan et al. 1998). Depletion of GSH increases the lethality of the DNA topoisomerase I inhibitor CPT-11 in V79 hamster lung fibroblasts (Sawyer and Bonner 1996). The present study showed that the MMC-induced depletion of GSH content, probably mediated by activation of caspases, led to a viability loss in lung epithelial cells. Because GSH is the main antioxidant system in cells, the depletion and oxidation of GSH due to MMC may enhance ROS accumulation and induce the mitochondrial membrane permeability change in these cells. NO can act as an antioxidant or pro-oxidant (Chiueh 1999; Joshi et al. 1999). Peroxynitrite, a strong oxidant, induces the mitochondrial membrane permeability transition followed by release of cytochrome c (Ghafourifar et al. 1999). In contrast, NO reduces the mitochondrial swelling and loss of plasma membrane integrity due to t-butyl hydroperoxide in astrocytes (Robb and Conner 2002). The present results showed that SIN-1 had a differential effect on the cytotoxic effect of MMC depending on concentration. SIN-1 at 150 M most effectively reduced the cytotoxicity of MMC, whilst other concentrations showed a lesser or no effect. A significant cytotoxic effect of SIN-1 itself at 300500 M may contribute to the lesser or lack of the inhibitory effect of SIN-1 on cytotoxicity of MMC. Ascorbate, SOD and haemoglobin nullified the inhibitory effect of SIN-1 on MMC cytotoxicity. This finding implies that the depressant effect of SIN-1 is mediated by the inhibitory effect of superoxide radicals and nitrogen species on the cytotoxicity of MMC. Although toxicity of MMC was apparently mediated in part by caspase-8 activation, the purpose of the present study was to assess the effect of SIN-1 on the cytotoxicity of MMC in relation to the mitochondrial membrane permeability change. The present results suggest that SIN-1 depressed MMC-induced cell death by attenuating the loss of mitochondrial transmembrane potential, cytochrome c release and activation of casapse-3, which may be induced by intracellular ROS formation and depletion of GSH. Unlike its inhibitory effect on cytochrome c release, 300 or 500 M SIN-1 did not inhibit MMC-induced caspase-3 activation significantly. The suppression of caspase-3 activity by 150 M SIN-1 in the MMC-treated cells thus appears not to be mediated chiefly by the inhibition of cytochrome c release. The decrease in caspase-3 activity may be mediated partly by inhibition of caspase-8 activity. The depressant effect of ascorbate, SOD and rutin suggests that the inhibitory effect of SIN-1 on MMC toxicity can probably be ascribed to interaction of ROS or RNS

with MMC. Despite the significant toxicity of the oxidant itself, the cytotoxicity of MMC in the presence of various oxidants or NO producer was not significantly different from that that of MMC alone. This implies that the toxicity of MMC appears to be attenuated by its interaction with ROS or RNS. The inhibitory effect of SIN-1 on the cytotoxicity of MMC was assessed by observing the scavenging action on hydrogen peroxide and was compared with the effect of ONOO. Unlike the effect of ONOO, SIN-1 at the stated concentrations continually reduced cell death due to hydrogen peroxide in lung epithelial cells. This finding suggests that the inhibition of MMCinduced cell death by SIN-1 may not be due to interaction of SIN-1 with MMC, nor by scavenging of hydrogen peroxide. High concentrations of NO induce apoptosis in susceptible cells, but low concentrations can be anti-apoptotic (Hofseth et al. 2003). NO is thought to inhibit apoptosis due to tumour necrosis factor- plus actinomycin D in cultured hepatocytes by suppressing mitochondrial dysfunction and caspase activation (Li et al. 1999). SIN-1 thus appears to attenuate the cytotoxicity of MMC by suppressing mitochondrial damage that otherwise would lead to activation of caspases and by inducing the interaction of MMC with ROS or RNS. In conclusion, our results show that RNS had a differential effect on the cytotoxicity of MMC depending on concentration. SIN-1 may interfere with the MMC-induced viability loss in lung epithelial cells by suppressing the mitochondrial membrane permeability change and by interaction of its products with MMC. Modulation of ROS and RNS production at the target tissue seems to affect the toxicity of MMC on normal or tumour cells.
Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3-20802-0013) to C.S.Lee.

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