Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells

Life Sciences 80 (2007) 1759 – 1767 www.elsevier.com/locate/lifescie

Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells Chung Soo Lee ⁎, Yun Jeong Kim, Eun Sook Han Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156–756, South Korea Received 28 October 2006; accepted 5 February 2007

Abstract The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18β-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18β-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 μM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18β-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage. © 2007 Elsevier Inc. All rights reserved. Keywords: 3-morpholinosydnonime; Glycyrrhizin; 18β-Glycyrrhetinic acid; Mitochondrial permeability transition; Lung epithelial cells; Protection

Introduction Nitrogen species, including nitric oxide, play a critical role in physiological regulation of airway functions and are involved in airway disease (Ricciardolo et al., 2006). Inflammatory cells, alveolar epithelial cells and bronchial endothelial cells in response to external stimuli generate nitrogen species, which are involved in lung cell damage (Caramori and Papi, 2004). The cytotoxic properties of nitric oxide are ascribed to the formation of peroxynitrite, a strong oxidant produced by the rapid reaction of nitric oxide with superoxide (Muijsers et al., 1997). Nitrogen species provoke amplification of inflammatory processes in the airways and lung parenchyma causing protein dysfunction and cell damage (Ricciardolo et al., 2006). They are implicated in tissue injury and airway dysfunction in diseases such as asthma and chronic obstructive pulmonary disease (Redington, 2006; ⁎ Corresponding author. Tel.: +82 2 820 5659; fax: +82 2 815 3856. E-mail address: [email protected] (C.S. Lee). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.02.003

Ricciardolo et al., 2006), and in tissue damage due to carcinogens and drugs (Rahman et al., 2006). Membrane permeability transition of mitochondria has been shown to be involved in a variety of toxic and oxidative forms of cell injury as well as apoptosis. Opening of the mitochondrial permeability transition pore causes a depolarization of the transmembrane potential, releases of Ca2+ and cytochrome c, osmotic swelling and loss of oxidative phosphorylation, which results in loss of cell viability (Bernardi 1996; Mignotte and Vayssiere, 1998). Reactive nitrogen species inhibit the mitochondrial respiratory chain (Brown, 1999), increase the production of reactive oxygen species (Poderoso et al., 1996) and induce formation of the mitochondrial permeability transition (Ghafourifar et al., 1999), which leads to cell death. Licorice root is a traditional herbal remedy that has been used for the treatment of various pathologic conditions, including chronic hepatitis, and gastric ulcer (Shibata, 2000). Increasing evidences indicate that glycyrrhizin, a triterpenoid saponin found in Glycyrrhiza glabrata, and its hydrolyzed metabolite 18β-

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glycyrrhetinic acid reveal anti-inflammatory, anti-cancer, antihepatotoxic and anti-viral effects (Jeong et al., 2002; Matsui et al., 2004; Agarwal et al., 2005; Hoever et al., 2005). Glycyrrhizin and 18β-glycyrrhetinic acid have anti-oxidant ability and reduce tissue damage due to oxidative insults such as carbon tetrachloride and ischemia-reperfusion injury (Nagai et al., 1991; Jeong et al., 2002; Kinjo et al., 2003). However, treatment of glycyrrhizin and 18β-glycyrrhetinic acid alone shows a cytotoxic effect against cancer cells by inducing formation of reactive oxygen species (Hibasami et al., 2006; Makino et al., 2006). Furthermore, both compounds exhibit opposing effects against bile acid-induced cell death in rat hepatocytes (Gumpricht et al., 2005) and do not reduce the toxicity of cadmium against H4IIE rat-derived hepatocyte cell line (Kim et al., 2004). Despite the cytoprotective or anti-oxidant effect, 18βglycyrrhetinic acid and glycyrrhizin reveal an opposing or no effect depending on the kind of toxic insults. Furthermore, with respect to the mitochondrial permeability transition, the effect of licorice compounds against lung cell damage due to exposure of nitrogen species remains uncertain. The aim of the present study was therefore to assess the effect of glycyrrhizin and 18βglycyrrhetinic acid against the nitrogen species-induced lung cell damage in relation to the mitochondria-mediated cell death process and role of oxidative stress. Materials and methods Materials TiterTACS™ colorimetric apoptosis detection kit was purchased from Trevigen, Inc. (Gaithersburg, MD, USA), Quantikine® human cytochrome c immunoassay kit was from R&D systems (Minneapolis, MN, USA), anti-cytochrome c (A-8) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), horseradish peroxidase-conjugated anti-mouse IgG was from EMD-Calbiochem. Co. (La Jolla, CA, USA), SuperSignal® West Pico chemiluminescence substrate was from PIERCE Biotechnology Inc. (Rockford, IL, USA) and ApoAlert™ CPP32/ caspase-3 assay kit was from CLONTECH Laboratories Inc. (Palo Alto, CA, USA). 3-morpholinosydnonime HCl, glycyrrhizin, 18β-glycyrrhetinic acid, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)), dichlorofluorescin diacetate (DCFH2-DA), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), phenylmethylsulfonylfluoride (PMSF) and other chemicals were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).

were washed with DMEM medium containing 1% FBS 24 h before experiments and replated onto the 96- and 24-well plates. Numbers of cells used in these experiments were based on the journals and experimental protocols described in Materials and methods, and on journals related to the study subjects. 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). Lung epithelial cells (4 × 104 cells/200 μl) were treated with 750 μM 3morpholinosydnonime in combination with 0.5–50 μM licorice compounds for 24 h at 37 °C. The medium was incubated with 10 μl of 10 mg/ml MTT solution for 2 h. After centrifugation at 412 ×g for 10 min, the culture medium was removed and 100 μl dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 570 nm using a microplate reader (Spectra MAX 340, Molecular Devices Co., Sunnyvale, CA, USA). Cell viability was expressed as a percentage of the value in control cultures. Morphological observation of nuclear change Lung epithelial cells (1 × 106 cells/ml) were treated with 750 μM 3-morpholinosydnonime in combination with 10 μM glycyrrhizin or 1 μM 18β-glycyrrhetinic acid for 24 h at 37 °C and the nuclear morphological change was assessed using the Hoechst dye 33258 (Oberhammer et al., 1992). Cells were washed 1 ml phosphate-buffered saline (PBS), pH 7.4 and incubated with 1 μg/ml Hoechst 33258 for 3 min at room temperature. Nuclei were visualized using an Olympus Microscope with a WU excitation filter (Tokyo, Japan). Measurement of apoptosis in cells Apoptosis was assessed by measuring the DNA fragmentation, which occurs following the activation of endonucleases. Lung epithelial cells (4 × 104 cells/200 μl) were treated with 750 μM 3morpholinosydnonime in combination with 10 μM glycyrrhizin or 1 μM 18β-glycyrrhetinic acid for 24 h at 37 °C, washed with PBS and fixed with 3.7% buffered formaldehyde solution. Nucleotide (dNTP) was incorporated at the 3′-ends of DNA fragments using terminal deoxynucleotidyl transferase (TdT) and the nucleotide was detected using a streptavidine–horseradish peroxidase and TACS-Sapphire according to TiterTACS protocol. Data were expressed as absorbance at 450 nm.

Cell culture

Flow cytometric measurement of mitochondrial transmembrane potential

Normal human embryo lung epithelial cells (WI-26 VA4) were obtained from Korean cell line bank (Seoul, South Korea). Lung epithelial cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin in a 5% CO2 atmosphere at 37 °C as described in the manual of the cell line bank. Cells

Changes in the mitochondrial transmembrane potential during the 3-morpholinosydnonime-induced apoptosis in lung epithelial cells were quantified by flow cytometry with the cationic lipophilic dye DiOC6(3) (Berthier et al., 2004). Cells (1 × 106 cells/ml) were treated with 750 μM 3-morpholinosydnonime in combination with 1–10 μM licorice compounds for

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24 h at 37 °C, DiOC6(3) (40 nM) added to the medium and cells incubated for 15 min at 37 °C. After centrifugation at 412 ×g for 10 min, the supernatants were removed and the pellets suspended in 1 ml of PBS containing 0.5 mM EDTA. For analysis, a FACScan cytofluorometer (Becton Dickinson, San Jose, CA, 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 Western blot analysis and solidphase enzyme-linked immunosorbent assay kit. Lung epithelial cells (1 × 107 cells/ml for Western blotting and 5 × 105 cells/ml for ELISA) were harvested by centrifugation at 412 ×g for 10 min, washed twice with PBS, resuspended in buffer (in mM): sucrose 250, KCl 10, MgCl2 1.5, EDTA 1, EGTA 1, dithiothreitol 0.5, PMSF 0.1 and HEPES-KOH 20, pH 7.5 and homogenized further by successive passages through a 26-gauge hypodermic needle. The homogenates were centrifuged at 100,000 ×g for 30 min and the supernatant was used for analysis of cytochrome c. Protein concentration was determined by the method of Bradford according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA, USA). For Western blotting, supernatants were mixed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer and boiled for 5 min. Samples (30 μg protein/well) were loaded onto each lane of 15% SDSpolyacrylamide gel and transferred onto PVDF membranes (Amersham Biosciences Co., Piscataway, NJ, USA). Membranes were blocked for 2 h in TBS (50 mM Tris–HCl, pH 7.5 and 150 mM NaCl) containing 0.1% Tween 20 and 5% non-fat dried milk. The membranes were labeled with anti-cytochrome c (diluted 1:1000 in TBS containing 0.1% Tween 20) overnight at 4 °C with gentle agitation. After four washes in TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:2000) for 2 h at room temperature. After treatment with SuperSignal® West Pico chemiluminescence substrate, protein bands were identified by detecting the enhanced chemiluminescence in Luminescent image analyzer (Lite for Las-1000 plus version 1.1, Fuji Photo Film Co., Tokyo, Japan). For the ELISA-based quantitative analysis, 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 manufacturer's instructions. The 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 ng/ml by reference to the standard curve. Measurement of caspase-3 activity Lung epithelial cells (2 × 106 cells/ml) were treated with 750 μM 3-morpholinosydnonime in combination with 1–10 μM

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licorice compounds for 24 h at 37 °C and caspase-3 activity was determined according to the user's manual for the ApoAlert™ CPP32/caspase-3 assay kit. The supernatant obtained by a 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. The absorbance of the chromophore p-nitroanilide was measured at 405 nm. The standard curves were obtained from the absorbances 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 nmol of p-nitroanilide. Measurement of intracellular reactive oxygen species formation The dye DCFH2-DA, which is oxidized to fluorescent dichlorofluorescin (DCF) by hydroperoxides, was used to measure relative levels of cellular peroxides (Fu et al., 1998). Lung epithelial cells (4 × 104 cells/200 μl) were treated with 750 μM 3-morpholinosydnonime in combination with 0.5– 50 μM licorice compounds for 24 h at 37 °C, washed, suspended in FBS-free DMEM, incubated with 50 μM dye for 30 min at 37 °C and washed with PBS. The cell suspensions were centrifuged at 412 ×g for 10 min, and the medium was removed. Cells were dissolved with 1% Triton X-100 and fluorescence was 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 (reduced form GSH + oxidized form GSSG) was determined using glutathione reductase (van Klaveren et al., 1997). Lung epithelial cells (4 × 104 cells/ 200 μl) were treated with 750 μM 3-morpholinosydnonime in combination with 0.5–50 μM licorice compounds for 24 h at 37 °C, centrifuged at 412 ×g for 10 min in a microplate centrifuge and the medium was removed. The pellets were washed twice with PBS, dissolved with 2% 5-sulfosalicylic acid (100 μl) and incubated in 100 μl of the reaction mixture containing 22 mM sodium EDTA, 600 μM NADPH, 12 mM DTNB and 105 mM NaH2PO4, pH 7.5 at 37 °C. Glutathione reductase (10 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 that was incubated in the mixture as in samples. Measurement of nitrite/nitrate production Nitric oxide liberated from 3-morpholinosydnonime was measured by assaying nitric oxide metabolites, nitrite and nitrate (NOx). 3-morpholinosydnonime (750 μM) was added to a 200 μl of cell-free DMEM, incubated for 24 h at 37 °C and then the nitrate in the medium was reduced to nitrite by incubation with nitrate reductase (500 mU/ml), 160 μM NADPH and 4 μM flavin adenine dinucleotide at room temperature for 2 h. The medium

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was mixed with an equal amount of Griess reagent (from SigmaAldrich Inc.). Absorbance was measured at 540 nm, and the amount of nitrite was determined using sodium nitrite as the standard. The results were expressed as total nitrite equivalents (NOx). Statistical analysis Data are expressed as the mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance. When significance was detected, performing the Duncan's test for multiple comparisons the post hoc comparisons between the different groups were made. A probability less than 0.05 was considered to be statistically significant. Results Licorice compounds prevent 3-morpholinosydnonime-induced cell death and nuclear damage The preventive effect of licorice compounds glycyrrhizin and 18β-glycyrrhetinic acid on the cytotoxicity of 3morpholinosydnonime, a donor of nitric oxide and superoxide, was assessed in lung epithelial cells. 3-morpholinosydnonime liberates a strong oxidant peroxynitrite, which is formed by reaction of nitric oxide with superoxide (Muijsers et al., 1997). In a cell-free DMEM, 750 μM 3-morpholinosydnonime for a 24 h-incubation liberated 163.4 ± 1.0 μM NOx (nitric oxide metabolites nitrite and nitrate; mean ± S.E.M., n = 6). Lung epithelial cells exposed to 750 μM 3-morpholinosydnonime for 24 h exhibited about 48% of cell death. Treatment of 0.5–50 μM glycyrrhizin significantly reduced the 750 μM 3-morpholinosydnonime-induced cell death with a maximal inhibitory effect at 10 μM (57%); beyond this concentration the inhibitory effect declined (Fig. 1A). We also examined the effect of 18β-glycyrrhetinic acid, a metabolite of glycyrrhizin, against the 3-morpholinosydnonime-induced cell viability loss in lung epithelial cells, in which 1 μM 18β-glycyrrhetinic acid showed a maximal inhibitory effect (62%) (Fig. 1B). Although glycyrrhizin at 50 μM and 18 βglycyrrhetinic acid at 10 μM caused about 7 and 8% cell death, respectively, they attenuated the 3-morpholinosydnonimeinduced cell death. Meanwhile, 18β-glycyrrhetinic acid at 25 μM exhibited a significant cytotoxic effect (about 17% cell viability loss) and did not reveal the preventive effect (data not shown). We confirmed the involvement of nitrogen species in the toxic effect of 3-morpholinosydnonime against lung epithelial cells by using scavengers of reactive nitrogen species. Cells were treated with 750 μM 3-morpholinosydnonime in the presence of various scavengers for 24 h. The addition of 100 μM melatonin (a scavenger of reactive oxygen and nitrogen species), 25 μM carboxy-PTIO (a nitric oxide scavenger), 50 μM rutin (the scavenger of nitric oxide and inhibitor of lipid peroxidation) and 250 μM uric acid (a scavenger of peroxynitrite) reduced the 3-morpholinosydnonime-induced cell death (Fig. 2).

Fig. 1. Inhibition of 3-morpholinosydnonime-induced cell death by licorice compounds. Lung epithelial cells were pre-treated with 0.5–50 μM glycyrrhizin (A) or 0.5–10 μM 18β-glycyrrhetinic acid (B) for 15 min, exposed to 750 μM 3morpholinosydnonime in combination with licorice compounds for 24 h, and cell viability was determined. Data represent the mean ± S.E.M. (n = 6). +P b 0.05 compared to control (percentage of control) and ⁎P b 0.05 compared to SIN alone. 3-morpholinosydnonime was expressed as SIN, glycyrrhizin as GL, and 18β-glycyrrhetinic acid as GA.

To clarify the preventive effect of licorice compounds against the 3-morpholinosydnonime-induced apoptotic cell death, we investigated the effect of glycyrrhizin and 18βglycyrrhetinic acid on the nuclear morphological changes observed in the 3-morpholinosydnonime-treated cells. Nuclear staining with Hoechst 33258 demonstrated that the control lung epithelial cells had regular and round-shaped nuclei. In contrast, cells treated with 750 μM 3-morpholinosydnonime exhibited a condensation and fragmentation of nuclei, a characteristic of apoptotic cells, whose response was attenuated by the addition of 10 μM glycyrrhizin and 1 μM 18β-glycyrrhetinic acid (Fig. 3A). 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 detected by a quantitative colorimetric assay. Lung epithelial cells were treated with 750 μM 3morpholinosydnonime in the presence or absence of licorice compounds. Control cells showed absorbance of 0.213 ± 0.006

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glycyrrhetinic acid (Fig. 4B). We confirmed the preventive effect of licorice compounds on the 3-morpholinosydnonimeinduced mitochondrial damage by performing the ELISA-based quantitative analysis for the cytochrome c release and caspase3. Lung epithelial cells treated with 750 μM 3-morpholinosydnonime showed a significant increase in the cytochrome c release and the activation of caspase-3. The present data revealed that 10 μM glycyrrhizin and 1 μM 18β-glycyrrhetinic acid significantly attenuated the release of cytochrome c and increase in caspase-3 activity due to 3-morpholinosydnonime exposure (Fig. 4B and C).

Fig. 2. Effect of scavengers on 3-morpholinosydnonime-induced cell death. Lung epithelial cells were pre-treated with the scavengers [100 μM melatonin, 25 μM carboxy-PTIO [PTIO], 50 μM rutin and 250 μM uric acid] for 15 min, exposed to 750 μM 3-morpholinosydnonime in the presence of scavengers for 24 h, and cell viability was determined. Data represent the mean ± S.E.M. (n = 6). + P b 0.05 compared to control (percentage of control) and ⁎P b 0.05 compared to SIN. 3-morpholinosydnonime was expressed as SIN.

(mean ± S.E.M., n = 6), whilst exposure to 750 μM 3-morpholinosydnonime for 24 h increased the absorbance about 2.9-fold (Fig. 3B). Treatment of glycyrrhizin and 18β-glycyrrhetinic acid (10 and 1 μM each) significantly prevented the fragmentation of DNA induced by 3-morpholinosydnonime. Licorice compounds prevent 3-morpholinosydnonime-induced changes in mitochondrial membrane permeability Opening of the mitochondrial permeability transition pore causes the release of cytochrome c from mitochondria into the cytosol and subsequent activation of caspases as one of the mitochondria-mediated cell death signaling events (Kim et al., 2006). We assessed the preventive effect of licorice compounds against the nitrogen species-induced cell death by investigating the effect on changes in the mitochondrial membrane permeability. Changes in the mitochondrial transmembrane potential in lung epithelial cells exposed to 3-morpholinosydnonime were quantified by flow cytometry with the dye DiOC6 (3). When lung epithelial cells were treated with 750 μM 3morpholinosydnonime for 24 h, the percentage of cells with depolarized mitochondria (characterized by low values of the transmembrane potential) increased. The addition of glycyrrhizin and 18β-glycyrrhetinic acid (10 and 1 μM each) significantly prevented the 3-morpholinosydnonime-induced increase in cells with depolarized mitochondria (Fig. 4A). The 3-morpholinosydnonime-induced change in the mitochondrial membrane permeability was assessed by measuring the cytochrome c release and caspase-3 activation. In Western blot analysis, lung epithelial cells treated with 750 μM 3morpholinosydnonime for 24 h showed an increase in the cytosolic cytochrome c levels, whose response was attenuated by the addition of 10 μM glycyrrhizin and 1 μM 18β-

Fig. 3. Preventive effect of licorice compounds on 3-morpholinosydnonimeinduced nuclear damage. Lung epithelial cells were pre-treated with 10 μM glycyrrhizin or 1 μM 18β-glycyrrhetinic acid for 15 min, and exposed to 750 μM 3-morpholinosydnonime in combination with licorice compounds for 24 h. In experiment A, cells were observed by fluorescence microscopy after nuclei staining with Hoechst 33258. Figure represents microscopic morphology of the control cells (a), cells treated with 3-morpholinosydnonime (b), cells treated with 3-morpholinosydnonime + glycyrrhizin (c) and cells treated with 3-morpholinosydnonime + 18β-glycyrrhetinic acid (d). a–d are representative of four different experiments. In experiment B, the 3′-ends of DNA fragments were detected as described in Materials and methods. Data are expressed as absorbance and represent the mean ± S.E.M. (n = 6). +P b 0.05 compared to control and ⁎P b 0.05 compared to SIN alone. 3-morpholinosydnonime was expressed as SIN, glycyrrhizin as GL, and 18β-glycyrrhetinic acid as GA.

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Fig. 4. Preventive effect of licorice compounds on 3-morpholinosydnonime-induced loss of the mitochondrial transmembrane potential, release of cytochrome c and activation of caspase-3. Lung epithelial cells were treated with 750 μM 3-morpholinosydnonime in the presence of 10 μM glycyrrhizin, 1 μM 18β-glycyrrhetinic acid or 50 μM rutin for 24 h. Data are expressed as the percentage of cells with depolarized mitochondria for the mitochondrial membrane potential (A), ng/ml for cytochrome c release (B) and units for caspase-3 activity (C), and represent the mean ± S.E.M. (n = 3–6). +P b 0.05 compared to control and ⁎P b 0.05 compared to SIN alone. The levels of cytochrome c in the cytosolic fractions were also analyzed by Western blotting with anti-cytochrome c antibody (B). Data are representative of three different experiments. 3-morpholinosydnonime was expressed as SIN, glycyrrhizin as GL, and 18β-glycyrrhetinic acid as GA.

Licorice compounds prevent 3-morpholinosydnonime-induced formation of reactive oxygen species and depletion of GSH To determine whether the preventive effect of licorice compounds against the cytotoxicity of 3-morpholinosydnonime was ascribed to their effect on oxidative stress, we investigated the effect on the formation of reactive oxygen species and the depletion of GSH in lung epithelial cells. The formation of reactive oxygen species within cells was determined by monitoring a conversion of DCFH2-DA to DCF. Lung epithelial

cells treated with 750 μM 3-morpholinosydnonime showed a significant increase in DCF fluorescence. Glycyrrhizin at 10 μM and 18β-glycyrrhetinic acid at 1 μM exhibited 62– 63% of inhibitory effect against the 3-morpholinosydnonimeinduced increase in DCF fluorescence; beyond these concentrations the inhibitory effect declined (Fig. 5). Reduction of cellular GSH levels increases the sensitivity of neurons to the toxic insults and induces changes in mitochondrial function (Hall, 1999). The work conducted whether the preventive effect of licorice compounds on the cytotoxicity due

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species cause cell death by inducing suppression of the mitochondrial respiratory chain, which leads to increased formation of reactive oxygen species, and formation of the mitochondrial permeability transition (Brown, 1999; Ghafourifar et al., 1999). 3-morpholinosydnonime liberates nitric oxide and superoxide. The reaction between nitric oxide and superoxide is highly favorable and produces a strong oxidant peroxynitrite that is mainly involved in oxidative cell damage (Muijsers et al., 1997; Chandra et al., 2000). This finding was also observed in this study. Lung epithelial cells treated with 3morpholinosydnonime revealed the condensation and fragmentation of nuclei and significant increase in caspase-3 activity, which indicates apoptotic cell death. Opening of the mitochondrial permeability transition pore causes a release of cytochrome c from mitochondria into the cytosol, leading to the activation of caspase-3 that is involved in apoptotic cell death (Crompton, 1999; Kim et al., 2006). The present results

Fig. 5. Inhibition of 3-morpholinosydnonime-induced formation of reactive oxygen species by licorice compounds. Lung epithelial cells were pre-treated with 0.5–50 μM glycyrrhizin (A) or 0.5–10 μM 18β-glycyrrhetinic acid (B) for 15 min, and then exposed to 750 μM 3-morpholinosydnonime in combination with licorice compounds for 24 h. Data are expressed as arbitrary units of fluorescence (a.u.) and represent the mean ± S.E.M. (n = 6). +P b 0.05 compared to control and ⁎P b 0.05 compared to SIN alone. 3-morpholinosydnonime was expressed as SIN, glycyrrhizin as GL, and 18β-glycyrrhetinic acid as GA.

to nitrogen species was ascribed to the inhibitory effect on the depletion of GSH. The thiol content in the control lung epithelial cells was 4.09 ± 0.05 nmol/mg protein. Treatment of 750 μM 3-morpholinosydnonime for 24 h depleted GSH contents by 50%, whose response was prevented by the addition of glycyrrhizin and 18β-glycyrrhetinic acid (Fig. 6). Glycyrrhizin had a maximal inhibitory effect at 10 μM (60%) and 18βglycyrrhetinic acid at 1 μM (64%); beyond these concentrations their inhibitory effect declined. Although glycyrrhizin at 50 μM and 18β-glycyrrhetinic acid at 10 μM decreased GSH contents by 8–9%, they prevented the 3-morpholinosydnonime-induced depletion of GSH. Discussion Mitochondrial dysfunction and increased oxidative stress are involved in cell death process in various types of cells (Mignotte and Vayssiere, 1998; Fleury et al., 2002). Nitrogen

Fig. 6. Preventive effect of licorice compounds on 3-morpholinosydnonimeinduced decrease in the GSH contents. Lung epithelial cells were pre-treated with 0.5–50 μM glycyrrhizin (A) or 0.5–10 μM 18β-glycyrrhetinic acid (B) for 15 min, and then exposed to 750 μM 3-morpholinosydnonime in combination with licorice compounds for 24 h. Data are expressed as nmol of GSH/mg protein and represent the mean ± S.E.M. (n = 6). +P b 0.05 compared to control and ⁎P b 0.05 compared to SIN alone. 3-morpholinosydnonime was expressed as SIN, glycyrrhizin as GL, and 18β-glycyrrhetinic acid as GA.

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indicate that the 3-morpholinosydnonime-induced apoptotic cell death seems to be mediated by the loss of the mitochondrial membrane potential and cytochrome c release, resulting in activation of caspase-3. Licorice compounds glycyrrhizin and 18β-glycyrrhetinic acid have anti-oxidant ability and reduce oxidative damage due to carbon tetrachloride, tert-butyl hydroperoxide or ischemia– reperfusion injury (Nagai et al., 1991; Jeong et al., 2002; Kinjo et al., 2003). The treatment of 18β-glycyrrhetinic acid attenuates tumor necrosis factor-α, lipopolysaccharide-induced cell death in human hepatoblastoma cell line and cultured liver cells (Yoshikawa et al., 1999; Zheng and Lou, 2003). However, glycyrrhizin and 18β-glycyrrhetinic acid exhibit an opposing or no effect against the toxicity of bile acid and cadmium-induced cell death in hepatocytes (Kim et al., 2004; Gumpricht et al., 2005). Furthermore, glycyrrhizin and 18β-glycyrrhetinic acid can induce cell death in T cell lines or cancer cell lines without caspase activation (Ishiwata et al., 1999; Hibasami et al., 2006). Therefore, it is uncertain whether the preventive effect of licorice compounds comes from their inhibitory action on the mitochondrial permeability transition. The present study was conducted to assess the effect of glycyrrhizin and 18β-glycyrrhetinic acid against the nitrogen species-induced lung cell damage in relation to the mitochondria-mediated cell death process. As seen in the present data, glycyrrhizin less than 50 μM and its metabolite 18βglycyrrhetinic acid less than 10 μM significantly prevented the nitrogen species-induced cell viability loss in lung epithelial cells. Although there is a difference of inhibitory potency on the basis of concentration, a metabolite 18β-glycyrrhetinic acid as well as glycyrrhizin exhibits a cytoprotective effect. 18β-Glycyrrhetinic acid at low concentration seems to exert an effective protective effect against the nitrogen species-induced cell damage. The present data suggests that glycyrrhizin less than 50 μM and 18βglycyrrhetinic acid less than 10 μM may prevent the toxicity of nitrogen species against lung epithelial cells by suppressing disruption of the mitochondrial transmembrane potential that results in the release of mitochondrial cytochrome c and activation of caspase-3. Meanwhile, it has been demonstrated that glycyrrhizin and 18β-glycyrrhetinic acid alone cause cell death in various cancer lines (Hibasami et al., 2006; Makino et al., 2006). The toxicity at high concentration seems to nullify the protective of licorice compounds. The inhibition of mitochondrial respiratory chain due to the exposure of toxic substances causes the production of reactive oxygen species and nitrogen species (Brown, 1999; Chandra et al., 2000; Fleury et al., 2002). Reactive oxygen species act upon mitochondria, causing a disruption of mitochondrial membrane potential and the release of cytochrome c. The generation of reactive oxygen species and depletion of GSH may be involved in the formation of mitochondrial permeability transition in lung epithelial cells exposed to anti-cancer drugs (Hong et al., 2003; Park et al., 2004). Along with the previous reports, the inhibitory effect of scavengers, including melatonin and rutin, and the increased formation of reactive oxygen species strongly indicate that mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime are mediated by oxidative stress.

Treatment of glycyrrhizin and 18β-glycyrrhetinic acid attenuates either renal injury due to ischemia–reperfusion or FeCl2 plus ascorbate-induced lipid peroxidation in liver homogenates through scavenging action on free radicals (Yokozawa et al., 2000; Jeong et al., 2002). However, in human myeloid leukemia cell line HL60 treatment of 18βglycyrrhetinic acid is demonstrated to induce apoptosis by increasing the production of reactive oxygen species (Makino et al., 2006). Therefore, these results represent uncertainty whether licorice compounds have anti-oxidant ability and reveal a cytoprotective effect. We examined whether the preventive effect of licorice compounds against the mitochondrial damage was ascribed to their inhibitory effect on oxidative stress. Increase in reactive oxygen species and drops in GSH levels are involved in the apoptotic process (Mignotte and Vayssiere, 1998; Tan et al., 1998). The oxidation and depletion of GSH modulate opening of the mitochondrial permeability transition pore (Constantini et al., 1996; Hall, 1999). The mitochondrial GSH depletion is suggested to trigger the apoptotic pathway (Hall, 1999). As seen in the present study, glycyrrhizin and 18βglycyrrhetinic acid attenuated the formation of reactive oxygen species and depletion of GSH due to exposure of 3morpholinosydnonime. The inhibitory effect of licorice compounds on the 3-morpholinosydnonime-induced cell death approximately correlated with the effect on GSH depletion. Therefore, the treatment of glycyrrhizin and 18β-glycyrrhetinic acid seems to prevent the 3-morpholinosydnonime-induced changes in the mitochondrial membrane permeability by inhibiting the formation of reactive oxygen species and interfering with cellular GSH depletion. Overall, the results show that licorice compounds glycyrrhizin and 18β-glycyrrhetinic acid seem to prevent the 3morpholinosydnonime-induced viability loss in lung epithelial cells by suppressing the mitochondrial permeability transition, leading to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to exhibit a protective effect against lung cell injury, which is mediated by increased formation of nitrogen species. Acknowledgment This research was supported by the Chung-Ang University Research Grants in 2006. References Agarwal, M.K., Iqbal, M., Athar, M., 2005. Inhibitory effect of 18βglycyrrhetinic acid on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in mice. Redox Report 10, 151–157. Bernardi, P., 1996. The permeability transition pore. Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death. Biochimica et Biophysica Acta 1275, 5–9. Berthier, A., Lemaire-Ewing, S., Prunet, C., Monier, S., Athias, A., Bessède, G., Pais de Barros, J.P., Laubriet, A., Gambert, P., Nèel, D., 2004. Involvement

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