Attenuation of MG132-induced HeLa Cell Death by N-Acetyl Cysteine via Reducing Reactive Oxygen Species and Preventing Glutathione Depletion

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1 Attenuation of MG132-induced HeLa Cell Death by N-Acetyl Cysteine via Reducing Reactive Oxygen Species and Preventing Glutathione Depletion YONG HWAN HAN 1, YEON MI YANG 2, SUNG ZOO KIM 1 and WOO HYUN PARK 1 1 Department of Physiology, Medical School, Centers for Healthcare Technology Development, and 2 Institute for Medical Sciences and Pediatric Dentistry, School of Dentistry Chonbuk National University, JeonJu, , Republic of Korea Abstract. MG132 as a proteasome inhibitor can induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, the effects of N-acetyl cysteine (NAC; an antioxidant) on MG132-induced HeLa cell death in relation to ROS and glutathione (GSH) were investigated. MG132 induced cell growth inhibition and apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ m ), activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage. MG132 increased ROS levels, including O 2, and GSH depleted cell numbers of HeLa cells. NAC reduced the number of annexin V-positive cells and MMP (ΔΨ m ) loss by MG132. In addition, NAC significantly reduced the ROS level and prevented GSH depletion. In conclusion, NAC prevented MG132-induced HeLa cell death via decreasing ROS and preventing GSH depletion. Reactive oxygen species (ROS) include hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 ) and hydroxyl radical ( OH). ROS are formed as by-products of mitochondrial respiration or by oxidases such as nicotine adenine diphosphate (NADPH) oxidase and xanthine oxidase (XO) (1). A change in the redox state of a tissue implies a change in ROS generation or metabolism. Glutathione (GSH) is the main non-protein antioxidant in the cell and provides electrons for enzymes such as glutathione peroxidase, which reduce H 2 O 2 to H 2 O. GSH has been shown to be crucial for cell proliferation, cell cycle progression and apoptosis (2, 3) and is known to protect cells from toxic insult by detoxifying toxic metabolites of drugs and ROS (4). Although cells Correspondence to: Woo Hyun Park, Ph.D., Associate Professor, Department of Physiology, Medical School, Chonbuk National University, JeonJu, Republic of Korea. Tel: , Fax: , parkwh71@chonbuk.ac.kr Key Words: MG132, apoptosis, NAC, ROS, GSH. possess antioxidant systems to control their redox state, which is important for their survival, excessive production of ROS can be induced and gives rise to the activation of events that lead to death or survival in different cell types (5-7). The ubiquitin-proteasomal system represents the major nonlysosomal pathway through which intracellular proteins involved in cell cycling, proliferation, differentiation, and apoptosis are degraded in eukaryotic cells (8, 9). The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. The peptide aldehyde MG132 (carbobenzoxy-leu-leu-leucinal) effectively blocks the proteolytic activity of the proteasome complex (10). Proteasome inhibitors including MG132 have been shown to induce apoptotic cell death through the formation of ROS (11, 12). ROS formation and GSH depletion due to proteasome inhibitors may cause mitochondrial dysfunction and subsequent cytochrome c release, which leads to cell viability loss (13, 14). In the present study, it was demonstrated that MG132 inhibited the growth of HeLa cells and the effects of NAC on MG132-treated HeLa cells in relation to cell death, ROS and GSH were investigated. Materials and Methods Cell culture. Human cervix adenocarcinoma HeLa cells were obtained from the American Type Culture Collection (ATCC) and maintained in a humidified incubator containing 5% CO 2 at 37 C. HeLa cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (GIBCO BRL, Grand Island, NY, USA). Cells were routinely grown in 100- mm plastic tissue culture dishes (Nunc, Roskilde, Denmark). Reagents. MG132 purchased from Calbiochem (San Diego, CA, USA) was dissolved in DMSO at 10 mm as a stock solution. N-Acetyl-cysteine (NAC; Sigma, St. Louis, MO, USA) was dissolved in the buffer [20 mm HEPES (ph 7.0)] at 100 mm as a stock solution. Cells were co-incubated with MG132 with or without NAC, which was pretreated for 30 min prior to treatment with MG132. DMSO (0.2%) was used as a control vehicle. All stock solutions were wrapped in foil and stored at 20 C /2010 $

2 Cell growth assay. Cell numbers and cell growth inhibition was determined by trypan blue cell counting or by measuring 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye absorbance by living cells, as previously described (15). In brief, cells per well were seeded in 24-well plates (Nunc, Roskilde, Denmark) for cell counting, and cells per well were seeded in 96-well microtiter plates for MTT assays. After exposure to MG132 with or without NAC for 24 hours, cells in 24- or 96-well plates were collected with trypsin digestion for trypan blue exclusion cell counting or were used for an MTT assay. Twenty microliters of MTT (Sigma) solution (2 mg/ml in PBS) were added to each well of the 96-well plates. The plates were incubated for 4 additional hours at 37 C, after which the MTT solution in the medium was aspirated off and 200 μl of DMSO were added to each well to solubilize the formazan crystals formed in the viable cells. The optical density was measured at 570 nm using a microplate reader (Spectra MAX 340; Molecular Devices Co, Sunnyvale, CA, USA). Western blot analysis. The expression of proteins was evaluated using Western blot analysis as previously described (16). In brief, cells in 60 mm culture dish (Nunc, Roskilde, Denmark) were incubated with MG132 with or without NAC for 24 hours. The cells were then washed in PBS and suspended in 5 volumes of lysis buffer (20 mm HEPES. ph 7.9, 20% glycerol, 200 mm KCl, 0.5 mm EDTA, 0.5 % NP40, 0.5 mm DTT, 1% protease inhibitor cocktail). Lysates were then collected and stored at 20 C until further use. Supernatant protein concentration was determined by the Bradford method (17). Supernatant samples containing 40 μg total protein were resolved by 8 or 15% SDS-PAGE gel depending on the target protein sizes, transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting, and probed with anti-ubiquitin, anti-p27, anti-caspase-3, anti-parp and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with horseradish peroxidaseconjugated secondary antibodies. Blots were developed using an ECL kit (Amersham, Arlington Heights, IL, USA). Annexin V staining. Apoptosis was determined by staining cells with annexin V-fluorescein isothiocyanate (FITC; Ex/Em=488 nm/519 nm, PharMingen, San Diego, CA, USA) as previously described (18). In brief, cells in 60 mm culture dish (Nunc) were incubated with MG132 with or without NAC for 24 hours. Cells were washed twice with cold PBS and then resuspended in 500 μl of binding buffer (10 mm HEPES/NaOH ph 7.4, 140 mm NaCl, 2.5 mm CaCl 2 ) at a density of cells/ml. Five microliters of annexin V-FITC were then added to these cells, which were analyzed with a FACStar flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Measurement of MMP (ΔΨ m ). The MMP (ΔΨ m ) levels were measured using rhodamine 123 fluorescent dye (Ex/Em= 485 nm/535 nm; Sigma), as previously described (19). In brief, cells in 60 mm culture dish (Nunc) were incubated with MG132 with or without NAC for 24 hours. Cells were washed twice with PBS and incubated with rhodamine 123 (0.1 μg/ml) at 37 C for 30 min. Rhodamine 123 staining intensity was determined by flow cytometry. An absence of rhodamine 123 from cells indicated the loss of MMP (ΔΨ m ) in As4.1 cells. The MMP (ΔΨ m ) levels in the cells, excluding MMP (ΔΨ m ) loss cells, were expressed as the mean fluorescence intensity (MFI), which was calculated by CellQuest software version 2.0. (Becton Dickinson, San Jose, CA, USA). Figure 1. Effects of MG132 on the growth of HeLa cells. Exponentially growing cells were treated with the indicated doses of MG132 for 24 hours. A: Cell growth was assessed by an MTT assay. B: Samples (40 μg) of protein extracts were resolved by SDS-PAGE gel, transferred onto the PVDF membranes, and immunoblotted with the indicated antibodies against ubiquitin and β-actin. *p<0.05 Compared with MG132- untreated HeLa control group. Cell cycle analysis. Cell cycle distributions were determined by propidium iodide (PI; Ex/Em=488 nm/617 nm, Sigma-Aldrich, St. Louis, MO, USA) staining as previously described (20). In brief, cells in 60 mm culture dish (Nunc) were incubated with MG132 with or without NAC for 24 hours. Cells were then washed with PBS and fixed in 70% ethanol. Cells were washed again with PBS, then incubated with PI (10 μg/ml) with simultaneous RNase treatment at 37 C for 30 min. Cell DNA contents were measured using a FACStar flow cytometer (Becton Dickinson, San Jose, CA, USA) and analyzed using lysis II and CellFIT software version 2.0 (Becton Dickinson, San Jose, CA, USA). Detection of intracellular ROS and O 2 levels. Intracellular ROS such as H 2 O 2, OH, and ONOO were detected by means of an oxidation-sensitive fluorescent probe dye, 2,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA; Invitrogen Molecular Probes, OR, USA; Ex/Em=495 nm/529 nm) (21). As H 2 DCFDA is poorly selective for O 2, dihydroethidium (DHE; Invitrogen Molecular Probes; Ex/Em=518 nm/605 nm), which is highly selective for O 2, was used for its detection. In brief, cells in 60 mm culture dish (Nunc) were incubated with MG132 with or without NAC for 24 hours. Cells were then washed in PBS and incubated with 20 μm H 2 DCFDA or DHE at 37 C for 30 min according to the instructions of the manufacturer. DCF and DHE fluorescence intensities were detected using a FACStar flow cytometer (Becton Dickinson). ROS and O 2 levels were expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software version 2.0. Detection of the intracellular glutathione (GSH). Cellular GSH levels were analyzed using 5-chloromethylfluorescein diacetate (CMFDA; Ex/Em=522 nm/595 nm; Molecular Probes, Invitrogen, Carlsbad, CA, USA) as previously described (21). In brief, cells in 60 mm culture dish (Nunc) were incubated with MG132 with or without NAC for 24 hours. Cells were then washed with PBS and incubated with 5 μm CMFDA at 37 C for 30 min according to the instructions of the manufacturer. CMF fluorescence intensity was determined using a FACStar flow cytometer (Becton 2108

3 Han et al: Effect of NAC on MG132-induced HeLa Cell Death Figure 2. Effects of NAC on growth, apoptosis and MMP (ΔΨ m ) in MG132-treated HeLa cells. Exponentially growing cells were treated with 10 μm MG132 for 24 hours following 30 min pre-incubation of 2 mm NAC. Cell growth (A) and cell number (B) were assessed by an MTT assay and trypan blue exclusion cell counting, respectively. C: Graph shows the percents of annexin V staining cells in HeLa cells. D and E: Graphs show the percentage of rhodamine 123-negative (MMP (ΔΨm) loss) cells in HeLa cells and MMP (ΔΨ m ) levels (%) compared with MG132-untreated control cells, respectively. F: Samples (40 μg) of protein extracts were resolved by SDS-PAGE gel, transferred onto the PVDF membranes, and immunoblotted with the indicated antibodies against caspase-3, PARP and β-actin. *p<0.05 Compared with MG132-untreated HeLa control group, #p<0.05 compared with cells treated with MG132 only. Dickinson). Negative CMF staining (GSH-depleted) cells were expressed as the percentage of ( ) CMF cells. CMF levels in cells except GSH-depleted cells were expressed as MFI, which was calculated by CellQuest software version 2.0. Statistical analysis. The results given in this study represent the mean of three independent experiments (mean±sd). The data was analyzed using Instat software (GraphPad Prism4, San Diego, CA, USA). One-way analysis of variance (ANOVA) with post hoc analysis using Tukey s multiple comparison test was used for parametric data. The statistical significance was defined as p<0.05. Results Effects of NAC on cell growth and apoptosis in MG132- treated HeLa cells. When the growth of HeLa cells after treatment with MG132 was assessed by an MTT assay, dosedependent reduction of cell growth was observed in HeLa cells at 24 hours (Figure 1A). Moreover, a relative increase in anonymous ubiquitinated proteins in MG132-treated HeLa cells was observed compared with control cells (Figure 1B). The effect of NAC on cell growth and apoptosis of MG132-treated HeLa cells was then examined. For these experiments, 10 μm MG132 and 2 mm NAC were chosen as suitable doses to differentiate the levels of cell growth inhibition or death. NAC did not significantly change the growth of MG132-treated HeLa cells using an MTT assay and trypan blue cell counting (Figures 2A and B). MG132 significantly increased annexin V staining cells to about 14% compared with MG132-untreatd HeLa control cells (Figure 2C). NAC significantly reduced the number of annexin V- FITC positive cells induced by MG132 (Figure 2C). Effects of NAC on MMP (ΔΨ m ) and apoptotic-related proteins in MG132-treated HeLa cells. Apoptosis is closely related to the collapse of MMP (ΔΨ m ) (22). Therefore, the loss of MMP (ΔΨ m ) in MG132-treated HeLa cells was determined using a rhodamine 123 dye. As expected, a loss of MMP (ΔΨ m ) was observed in MG132-treated cells at 24 hours (Figure 2D). NAC significantly prevented MMP (ΔΨ m ) loss in MG132- treated HeLa cells (Figure 2D). NAC also in part restored the MMP (ΔΨ m ) level in these cells, and NAC alone reduced MMP (ΔΨ m ) level in MG132-untreated HeLa control cells (Figure 2E). 2109

4 Figure 3. Effects of MG132 and/or NAC on the cell cycle distribution in HeLa cells. Exponentially growing cells were treated with 10 μm MG132 for 24 hours following 30 min pre-incubation of 2 mm NAC. The changes of cell cycle phase distribution were assessed by DNA flow cytometric analysis. A: Each figure shows histograms for cell cycle analysis in HeLa cells. B: Samples (40 μg) of protein extracts were resolved by SDS-PAGE gel, transferred onto the PVDF membranes, and immunoblotted with the indicated antibodies against p27 and β-actin. Figure 4. Effects of MG132 and/or NAC on ROS and GSH levels in HeLa cells. Exponentially growing cells were treated with 10 μm MG132 for 24 hours following 30 min pre-incubation of 2 mm NAC. ROS and GSH levels in HeLa cells were measured using a FACStar flow cytometer. A: Representative histograms for DCF intensity in HeLa cells. B and C: Graphs indicate DCF (ROS) levels (%) from A (B) and DHE (O 2 ) levels (%) compared with MG132-untreated HeLa control cells (C). D and E: The graph shows ( ) CMF (GSH-depleted) cells (%) (D) and mean CMF (GSH) levels (%) compared with MG132-untreated HeLa control cells (E). *p<0.05 Compared with MG132-untreated HeLa control group. #p<0.05 compared with cells treated with MG132 only. Caspase-3 plays an essential role as an executioner in apoptosis (23). Therefore, it was determined whether MG132 and/or NAC affect the activity of caspase-3 in HeLa cells. In MG132-treated HeLa cells, the level of procaspase- 3 (32 kda precursor) was reduced (Figure 2F), suggesting that the activation of caspase-3 occurred in MG132-treated cells. NAC did not alter the level of procaspase-3 (Figure 2F). In addition, cleavage of PARP (poly (ADP-ribose) polymerase) protein provides one of the most recognizable examples of apoptosis (24). As shown in Figure 2F, the intact 116 kda moiety of PARP was decreased in MG132- treated HeLa cells and the cleavage form of PARP was increased. NAC did not change alter the cleavage form of PARP (Figure 2F). Effects of NAC on the cell cycle distributions in MG132- treated HeLa cells. Next, it was determined whether the cell cycle distribution in HeLa cells was changed by MG132 and/or NAC for 24 hours. As shown in Figure 3A, DNA flow cytometric analysis indicated that MG132 did not strongly alter the cell cycle distributions in HeLa cells and NAC did not change the distributions in MG132-treated HeLa cells. However, when the expression of p27, a cyclin-dependent kinase inhibitor (CDKI) was assessed, p27 was strongly increased in MG132-treated HeLa cells (Figure 3B). NAC reduced the p27 protein level up to the basal state (Figure 3B). Effects of NAC on ROS and GSH levels in MG132-treated HeLa cells. When intracellular ROS levels were determined 2110

5 Han et al: Effect of NAC on MG132-induced HeLa Cell Death in HeLa cells treated with MG132 and/or NAC at 24 hours, intracellular ROS (DCF) level was increased in MG132- treated cells (Figures 4A and B). NAC significantly reduced ROS (DCF) level in MG132-treated HeLa cells (Figures 4A and B). Red fluorescence derived from DHE, reflecting the intracellular O 2 level, was increased in MG132-treated HeLa cells (Figures 4C). However, NAC did not change the O 2 level in these cells (Figure 4C). When the changes of GSH levels were analysed in HeLa cells at 24 hours, MG132 increased the GSH depleted cell number about 9% compared with MG132-untreated HeLa control cells (Figure 4D). NAC slightly prevented GSH depletion in MG132-treated HeLa cells (Figure 4D). When CMF (GSH) levels in HeLa cells, apart from GSH-depleted cells, were assessed, MG132 significantly increased GSH levels and NAC reduced the GSH level in these cells (Figure 4E). Discussion The present study focused on the evaluation of the effects of MG132 and/or NAC on HeLa cells in relation to cell death, ROS and GSH. MG132 inhibited the growth of HeLa cells and increased anonymous ubiquitinated proteins in HeLa cells. These data suggest that the inhibition of ubiquitin-proteasomal system by MG132 is involved in the growth inhibition of HeLa cells. MG132 also increased annexin V-FITC-positive cells and reduced the level of procaspase-3 and PARP in HeLa cells, implying that MG132-induced HeLa cell death occurred via apoptosis. NAC did not significantly change the growth of MG132- treated HeLa cells but reduced the apoptotic level in these cells. Therefore, NAC treatment seems to be more involved in an anti-apoptotic effect in MG132-treated cells. In relation to the cell cycle distribution in HeLa cells, MG132 presumably induced non-specific arrests at all the phases of the cell cycle, since MG132 inhibited HeLa cell growth by approximately 50% under MTT assay but induced apoptosis by at most 14%. In addition, p27, which typically causes cells to arrest in the cell cycle, was increased in MG132-treated HeLa cells. NAC did not change the cell cycle distributions in MG132-treated HeLa cells, but strongly decreased p27 protein level in these cells. These data imply that NAC is not involved in the cell cycle distributions of MG132-treated HeLa cells, and the distributions between cells treated with MG132 only and cells co-treated with MG132 and NAC are controlled differently by cell cycle-regulated proteins such as p27. Proteasome inhibitors including MG132 are known to induce apoptotic cell death through formation of ROS (12, 13, 25). It is reported that ROS formation due to proteasome inhibitors may cause mitochondrial dysfunction and subsequent cytochrome c release, which leads to loss of cell viability (13, 14). Correspondingly, MG132 increased ROS (DCF) level including O 2 levels in HeLa cells. MG132 induced the loss of MMP (ΔΨm) and reduced MMP (ΔΨ m ) levels in HeLa cells. NAC decreased the ROS (DCF) level in MG132-treated HeLa cells, which was accompanied by the prevention of MMP (ΔΨ m ) loss in these cells. However, NAC did not affect O 2 levels in MG132-treated HeLa cells. Although the mechanism underlying the ROS generation after MG132 treatment is not clearly explained, these data suggest that changes in ROS (DCF) level, rather than O 2 level, are related to apoptotic cell death of HeLa. In addition, MTT reduction is considered to be an indirect measurement of mitochondrial activity (26). Because NAC alone significantly reduced MMP (ΔΨ m ) level in MG132- untreated HeLa control cells without cell growth inhibition, the changes in MMP (ΔΨ m ) levels are not fully correlated with those of MTT reduction in HeLa cells. The redox state of cellular GSH is an important modulatory element in the protein ubiquitination pathways (27). It is reported that GSH depletion due to proteasome inhibitors may lead to cell death (13, 14). Correspondingly, MG132 increased GSH depletion in HeLa cells. NAC slightly prevented GSH depletion in MG132-treated HeLa cells, which is accompanied by the reduction of annexin V-positive cells and MMP (ΔΨ m ) loss in these cells. It is of note that the CMF (GSH) level in HeLa cells was increased. Probably, the increased GSH level occurred in response to the increasing ROS produced by MG132 treatment, and therefore some HeLa cells taken beyond their capacity to resist ROS insults died. In addition, the relatively reduced GSH level by NAC seemed to result from its use for scavenging ROS by MG132. In conclusion, the proteasome inhibition by MG132 in HeLa cells induced cell growth inhibition, which was accompanied by increasing ROS level as well as triggering GSH depletion. NAC prevented MG132-induced HeLa cell death via reducing ROS and preventing GSH depletion. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( ) and a grant of the Korean Ministry of Education, Science and Technology (The regional Core Research Program/Centers for Healthcare Technology Development). References 1 Zorov DB, Juhaszova M and Sollott SJ: Mitochondrial ROSinduced ROS release: An update and review. 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