Suppression of heat- and polyglutamine-induced cytotoxicity by nonsteroidal anti-inflammatory drugs Keiichi Ishihara, Nobuyuki Yamagishi and Takumi Hatayama Department of Biochemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto, Japan We have shown that sodium salicylate activates the heat shock promoter and induces the expression of heat shock proteins (hsps), with a concomitant increase in the thermotolerance of cells. To determine whether these effects are generally displayed by nonsteroidal anti- inflammatory d rugs (NSAIDs), w e examined the effects of a cyclooxygenase inhibitor, indomethacin, and a lipoxyg- enase inhibitor, nordihydroguaiaretic acid. Both inhibitors up-regulated the hsp promoter at 37 °C through t he acti- vation of heat shock factors, and i ncreased cellular levels of hsps in mammalian cells, although the degree of the expression of hsps and t hermotolerance of cells differed depending on the d rugs. Furthermore, NSAIDs s uch as sodium salicylate and indomethacin s uppressed the protein aggregation a nd apoptosis caused by an expanded poly- glutamine tract in a cellular model of polyglutamine dis- ease. These findings suggest that NSAIDs generally induce the e xpression of hsps in mammalian c ells and m ay be used for the protection of cells against deleterious st ressors and neurodegenerative diseases. Keywords: heat s hock proteins; indomethacin; nonsteroidal anti-inflammatory drugs; nordihydroguaiaretic acid; poly- glutamine d isease. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sodium salicylate (SA) and indomethacin (IND) are widely used as analgesic or antipyretic agents for the clinical treatment of inflammatory diseases. Most NSAIDs exhibit an inhibitory effect on cyclooxygenases (COXs), which catalyze the b iosynthesis of prostaglandins and thromb- oxanes from arachidonic acid. On the other hand, nor- dihydroguaiaretic acid (NDGA) inhibits lipoxygenases (LOXs), which produce leukotrienes from arachidonic acid. Recently, the l ong-term u se of NSAIDs was s hown to prevent the occurrence of Alzheimer’s disease without COX inhibition [1]. In addition to having these a nti-inflammatory effects, SA activates the heat shock promoter and induces the expression of heat shock proteins (hsps) with a concomitant increase in the thermotolerance of cells [2]. Cellular resistance against deleterious stress seems to be regulated by the expression leve ls of hsps in cells [3]. Upon exposure to a sublethal heat treatment, mammalian cells acquire transient resistance to a subsequent heat shock that would be normally lethal, and much evidence supports the idea that hsps, e specially H sp70, play important roles in its development [4]. Furthermore, in several polyglutamine (polyQ) diseases s uch as Huntington’s disease and s pino- cerebellar ataxia type 3/Machado–Joseph disease, the polyQ-induced cytotoxicity was suppressed by the over- expression of Hsp70, Hsp40 and Hsp27 [ 5–10]. Recently, we have shown that H sp105a reduces the aggregation o f proteins and cellular t oxicity caused by an expansion of the polyQ tract using a cellular model of spinal and bulbar muscular atrophy (SBMA) [11]. Overexpression of Hsp70 also ameliorates S BMA phenotypes in mice [12]. A ddition- ally, geldanamycin ( a b enzoquinone ansamycin t hat i nduces the stress response of cells) inhibits huntingtin aggregation in a cellular model of Huntington’s disease [13]. Thus, selective hsp inducers may be useful for t he treatment o f some diseases and for medicinal applications. In this study, we examined whether NSAIDs generally induce the expression of hsps and the resistance of cells using a COX inhibitor IND and a LOX inhibitor NDGA, and revealed that NSAIDs d o induce the expression of hsps in mammalian cells and may be used for the protection of cells against deleterious stressors and diseases. Materials and methods Cell culture and drug treatment Mouse C3H10T1/2 fibroblast cells (Riken cell bank, Tsukuba, Japan), pGL105/C3H cells stably transfected with a plasmid containing the mouse Hsp105 promoter upstream of a luciferase reporter g ene [2] and African green monkey kidney COS-7 cells (Riken cell b ank) were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharma- ceutical, T okyo, Japan) supplemented with 10% fetal bovine serum (Equitech-Bio, Kerriville, TX, USA) in a humidified atmosphere of 5% (v/v) CO 2 in air at 37 °C. IND a nd NDGA (Nacalai Tesque, Kyoto, J apan) w ere dissolved in dimethylsulfoxide at 250 m M , a nd SA (Nacalai Correspondence to T. Hatayama, Department of Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607–8414, Japan. Fax: +81 75 595 4758, Tel.: +81 75 595 4653, E-mail: hatayama@mb.kyoto-phu.ac.jp Abbreviations: AA, arachidonate; COX, cyclooxygenase; GFP, green fluorescence protein; hsp(s), heat shock protein(s); HSF, heat shock factor; HSE, heat shock element; luc, luciferase; NSAIDs, nonsteroi- dal anti-inflammatory drugs; IND, indomethacin; LOX, lipoxygenase; NDGA, nordihydroguaiaretic acid; polyQ, polyglutamine; SA, sodium salicylate; SBMA, spinal and bulbar muscular atrophy. (Received 5 August 2004, rev ised 27 S eptember 2004, accepted 6 October 2004) Eur. J. Biochem. 271, 4552–4558 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04419.x Tesque) was dissolved in phosphate-buffered saline (NaCl/P i )at1 M just before use. Cells were incubated in the medium with or w ithout drugs at 3 7 °C for 1 h, washed with medium three times and further incubated at 37 °C without the drug. Measurement of hsp promoter activity To measure h sp promoter act ivity in mammalian pGL105/ C3H cells (2 · 10 5 cells per 3 5 mm dish), they w ere t reated with a drug at 37 °C for 1 h and further incubated for 6 h without the drug. Then, the cells were washed three times with NaCl/P i ,andlysedin50lL of Cell Lysis Reagent (Promega,Madison,WI,USA).Thecelllysateswere centrifuged at 20 000 g for 1 0 min, and the supernatants recovered as cell e xtracts. Aliquots (5 lL) of the extracts were added to 5 0 lL o f l uciferase assay reagent (Promega), and luciferase ( luc) activity was measured using a T D-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). Gel mobility shift assay C3H10T1/2 cells (5 · 10 5 cells per 60 mm dish), treated with or without a drug or heat-shocked, were washed with NaCl/P i , and qu ickly frozen a t )80 °C. Frozen cells were suspended in 100 lL of extraction buffer [20 m M Hepes / KOH, pH 7.9, 1.5 m M MgCl 2 ,0.2m M EDTA, 0.5 m M phenylmethanesulfonyl fluolide, 0.5 m M dithiothreitol, 0.42 M NaCl and 25% (v/v) g lycerol], incubated at 4 °C for 15 min, and vortexed for 15 min at 4 °C. After centrifugation at 50 000 g for 5 min, aliquots of the supernatant (15 lg protein) were incubated in 25 lLof buffer containing 10 m M Tris/HCl, pH 7.8, 1 m M EDTA, 50 m M NaCl, 0.5 m M dithiothreitol, 5% (v/v) glycerol, 0.2 mgÆmL )1 bovine serum albumin, 40 lgÆmL )1 poly(dI- dC)] and 0 .4 ngÆmL )132 P-labeled heat s hock e lement (HSE) corresponding to nucleotides )115 to )81 of the human hsp70 gene [14] at 25 °C for 20 min. The mixtures were then electrophoresed on a native 4% polyacrylamide gel, and the gel was dried and subjected to autoradiography. T o define the sp ecific HSF–HSE complex, unlabeled HSE was added to the reaction mixture in a 100-fold molar excess o f the labeled HSE. U nder the exp erimental conditions, t he activated mouse HSF binds to the HSE of the human hsp70 gene, as does the activated human HSF [15]. Western blot analysis C3H10T1/2 or COS-7 cells (5 · 10 5 and 6 · 10 5 cells per 60 mm dish, respectively) were lysed in 200 lLof0.1% (w/v) SDS. Cellular proteins (15 lg) were separated by SDS/PAGE, and blotted onto a n itrocellulose membrane. The membrane w as washed with Tris -buffered saline [0.1 M Tris/HCl, pH 7 .5 and 0.9% (w/v) NaCl] containing 0.1% (v/v) Tween 20 (TTBS), and reacted with rabbit anti- Hsp105 [16] or mouse anti-Hsp70 (Sigma Chemical, St. Louis, MO, USA) a ntibody at room temperature for 1 h . After w ashes with TTBS, the membrane was further incubated with horseradish peroxidase-conjugated anti- (rabbit or anti-(mouse I gG) Igs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. Hsp105a and Hsp70/Hsc70 were detected using electro- chemiluminescence reagent (Santa Cruz Biotechnology). Films were digitized by scanning into ADOBE P HOTO SHOP 5, and the intensity of the bands was quantified on a Macintosh computer using the public domain NIH IMAGE program (developed a t US National Institutes of H ealth and available on the Internet at http://rsb.info.nih.gov/ nih-image/). Thermotolerance of cells C3H10T1/2 cells (7 · 10 4 cells per well) grown in 24-well plates containing collagen-coated coverslips were incubated with or without drugs at 37 °C for 1 h. These cells were heat-shocked a t 4 5 °C f or 45 min a fter incubation at 37 °C for 6 h i n the absence of drugs, and further incubated at 37 °C. At appropriate times, cells were then washed three times with NaCl/P i , fixed with 4% (v/v) paraformaldehyde at room temperature for 20 min, and observed under a phase-contrast microscope (Nikon, Tokyo, Japan). The viability o f c ells was also assessed b ased on the ability of living cells to incorporate neutral red into lysozomes. For the neutral red uptake assay, C3H10T1/2 c ells (7 · 10 4 cells per well) in 24-well plates were treated with or without a drug or heat-shocked, then incubated at 3 7 °Cfor3h with 50 lgÆmL )1 of neutral red, and fixed with 1% (v/v) formaldehyde co ntaining 1% (w/v) CaCl 2 for 1 min. The dye incorporated into cells was extracted with 50% (v/v) ethanol containing 1% (v/v) acetic acid, and absorbance at 540 nm was measured. Cellular model of polyQ disease COS-7 cells grown on c overslips t o 7 0–80% confluence were treated with or without dr ugs at 3 7 °Cfor1hand incubated a t 3 7 °C f or 3 h without the d rug. The cells were then transfected with the e xpression plasmid of a t runcated androgen receptor containing 97 glutamine repeats fused to green fluorescence protein (GFP) (tAR97) [9] with DMRIE-C reagent (Invitrogen, Carlsbad, CA, USA). A fter incubation at 37 °C f or 72 h, the cells were washed with NaCl/P i , fi xed w ith 4% (v/v) paraformaldehyde f or 3 0 min at room temperature, and stained with 10 l M Hoechst 33342 for 15 min at room temperature. The cells were observed using a confocal laser s canning microscope (Z eiss, Jena, Germany). The numbers of transfected cells with and without visible aggregates were counted independently in randomly chosen microscopic fields in different areas of a coverslip. Approximately 300–600 transfected cells were analyzed for data in each experiment. Apoptotic cells were identified by their nuclear morphology stained with H oechst 33342 and the TdT-mediated dUTP-biotin end labeling (TUNEL) method [11]. The TUNEL method was per- formed using a DeadEnd TM apoptosis detection kit (Pro- mega) according to the manufacturer’s instructions. Results Induction of hsp promoter activity, activation of HSF and accumulation of hsps by IND and NDGA We first examined the effect of IND and NDGA on the hsp105 promoter (Fig. 1). When pGL105/C3H cells were Ó FEBS 2004 Suppression of cytotoxicity by NSAIDs (Eur. J. Biochem. 271) 4553 treated with various concentrations of IND a t 37 °Cfor1h and further incubated for 6 h with out the drug, luc a ctivity increased depending on the concentration, and an approxi- mately 25-fold i ncrease w as observed i n cells pretreated with 0.75 m M IND compared to control cells (Fig. 1A ). Under these conditions, cell viability was not reduced by IND at concentrations up to 1 m M (Fig. 1B). On t he other hand, luc activity was increased approximately fivefold in cells pretreated with 0.2 m M NDGA compared to the control, whereas cell death was observed at concentrations of NDGA of more than 0.2 m M . We next examined whether these drugs e nhance hsp promoter activity through activation of HSF, by perform- ing a gel mobility shift assay ( Fig. 2A). When C3H10T1/2 cells were treated with IND or NDGA at 37 °Cfor1h, HSF was activated in cells treated with 0.5 and 0.75 m M IND or 0.2 m M NDGA, as in the heat-shocked cells, although the activation of HSF was at high er levels in cells treated with IND than with NDGA. Furthermore, w hen C3H10T1/2 cells were treated with 0.75 m M IND at 37 °C for 1 h and furt her incubated for 6 h , the cellular levels of Hsp105a and Hsc70 (a constitutive isoform of Hsp70) increased, and t he expression of Hsp70 (an inducible isoform of Hsp70) was markedly induced (Fig. 2B). On the other hand, the increase i n the levels of Hsp105a and Hsc70 a nd the induction of Hsp70 e xpression were observed at low levels in t he cells treated with 0.2 m M NDGA. T hus, although NSAIDs seemed to commonly induce hsps at physiological temperatures, the levels of the expression of hsps differed depending on the drug. Induction of thermotolerance of cells by IND As IND and NDGA caused hsps to accumulate in mammalian cells, we next examined whether these drugs Fig. 1. Effect o f IND or NDGA on hsp promoter in pGL105/C3H cells. (A) pGL105/C3H cells were incubated with or w ithout IND and NDGA a t 3 7 °C for 1 h, and further incubated at 37 °Cfor6h without the d rug. Then luc activi ty was assayed, with relative activity shown as a ratio to that of u ntreated control c ells. Each value repre- sents the mean ± SE of three independent experiments. Statistical significance was determined with Student’s t-test; *P <0.01 vs. respective control c ells. (B) Viability of cells was a ssessed by the neutral red upt ake assay. Values re present the mean ± SE of three inde- pendent experiments. Fig. 2. Effect of IND or NDGA on activation of HSF a nd accumulation of hsps in mammalian cells. (A) C 3H10T1/2 cells were incubated w ith or withou t I ND or NDGA a t 3 7 °C for 1 h, or heat-shock ed at 42 °C for 1 h (HS) a s a positive control. Cell extracts from these cells were subjected to a gel mobility shift assay using 32 P-labeled HSE. S pecific HSF–HSE comp lexes we re d etermine d by a dding a 1 0 0-fold ex cess of unlabeled HSE. A rrows indicate specific HSF–HSE complexes. (B) C3H10T1/2 cells were incubated with or without IND o r N DGA a t 37 °C for 1 h , a nd then fu rther incubated at 37 °C f or 6 h withou t t he drug. As a positive control, cells were heat-shocked at 41 °Cfor6h (HS). Cellular proteins (15 lg) we re sep arate d by 10% S DS/ PAG E, blotted onto nitrocellulose membranes, and immunostained using anti-Hsp105 or anti-Hsp70. 4554 K. Ishihara et al.(Eur. J. Biochem. 271) Ó FEBS 2004 induced resistance against s ubsequent heat shock ( Fig. 3A). The treatment of cells with 0.5 and 0.75 m M IND or 0.1 and 0.2 m M NDGA at 37 °C for 1 h did not cause marked changes of cell morphology, and i mmediately a fter exposure to a lethal h eat shock, the number of cells attached to culture dishes was not significantly altered. However, when these cells were further incubated a t 37 °C for 48 h, the number of cells attach ed to the dishes was markedly dec reased in untreated controls. H owever, many c ells remained attached to dishes when pretreated with 0.75 m M IND but not 0.1 and 0.2 m M NDGA. Furthermore, the viability of c ells was assessed w ith the neutral red uptake assay (Fig. 3B). The uptake o f d ye was not affected by IND or NDGA treatment alone. However, the uptake was markedly suppressed in untreated control cells 72 h after heat shock at 45 °Cfor 45 min, while pretreatment of cells with 0.75 m M IND but not 0.2 m M NDGA significantly suppressed the inhibition. Thus, cellular r esistance to a subsequent heat stress seemed to be enhanced by the drug that markedly induced the expression and accumulation o f hsps. Suppression of the aggregation of protein containing an expanded polyQ tract by NSAIDs Hsps such as Hsp105a, Hsp70 and Hsp40 have been identified as potent modulators of aggregation and/or cell death c aused b y the expression of proteins with an expanded polyQ tract in cellular models of neurodegenerative diseases [9,11]. We then examined whether NSAIDs suppress the protein aggregation and apoptosis in a cellular model of SBMA (Figs 4 and 5 ). When COS-7 cells were transfected with an expression plasmid of t AR97, approximately 50% of c ells expressing GFP fluorescence of tAR97 were found to contain protein aggregates and also c ondensed Fig. 3. Effect o f I ND or NDG A o n t hermotolerance of cells. C3H 10T1/2 cells we re i ncubated w ith or without IND or NDGA a t 3 7 °C for 1 h (a), and incubated at 37 °C for 6 h with out the d rug. Then , these ce lls were hea t-shock ed at 45 °C for 45 min (b), and further incubated at 37 °Cfor 48 h (c) or 72 h (d). ( A) Cells in (a), (b ) and (c) w ere fixed with 4% ( v/v) paraformaldhyde and o bserved using a phase contrast m icroscope. ( B) Viability of cells at (b) and (d) was assessed b y neutral red uptake assay. Values repre sent the m ean ± SE of three independ ent exp eriments. Statistical significance was determined with Student’s t-test; *P < 0.001 vs. respective control cells . Ó FEBS 2004 Suppression of cytotoxicity by NSAIDs (Eur. J. Biochem. 271) 4555 chromatin, a characteristic of apoptosis. The cells contain- ing condensed chromatin were TUNEL-positive, another characteristic of apoptosis (Fig. 4A). Pretreatment of cells with IND at concentrations of 0.75 m M and above significantly suppressed the protein a ggregation and apop- tosis in a dose-dependent manner. (Fig. 4B). Under these conditions, Hsp105a, Hsc70 and Hsp70 we re marke dly accumulated in cells treated with 0.75 and 1 m M IND. (Fig. 4 C). Furthermore, SA (a potent hsp inducer [2]) at concentrations of 40 and 80 m M significantly suppressed t he aggregation of tAR97 and apopto sis, with a concomitant marked induction of Hsp105a and Hsp70 (Fig. 5). How- ever, NDGA did not suppress the aggregation and apop- tosis caused b y a n e xpanded polyQ tract (data not sho wn). Fig. 4. Effects of IND on protein aggregation and cytotoxicity caused by an expanded poly Q tract. (A) COS-7 cells were transfected with tAR97 expression p lasmid and incubated further at 37 °C for 72 h. Cells were t hen fi xed, stained with H oechst 33 342, and o bserved u sing a confocal laser scanning m icroscope. T ypical images of cells expressing tAR97 (GFP) d iffusely (upper pa nels) or a s aggregates (lower p anels) are sh own. Apoptotic cells were identified based on t he nuclear morphology (Hoechst) and by t h e TUNEL method. (B) C OS-7 cells were treated w ith 0, 0.5, 0 .75 and 1.0 m M IND f or 1 h, i ncubated at 37 °C for 3 h without the drug, and t hen transfected with tAR97 expression plasmid. After 72 h , cells we re fixed, stained w ith Hoechst 33342 and obser ved using a confocal l aser scanning mic roscope. The a rrowheads represent typical cells c ontaining aggregated tAR97 or c onde nsed c hromatin . Prop ortions o f cells contain ing tAR97 aggregates or c on densed ch roma tin i n G FP-positiv e c ells rep resen t t he mean ± SE of four independent expe rimen ts. Statistical signifi cance was dete rmined with S tudent’s t-test; *P < 0.01 vs. respective controls. (C) Western blot s of Hsp105a and Hsp70/Hsc70 in cells treated with or w it hout IND. 4556 K. Ishihara et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Discussion SA activates the hsp promoter and induces the expression o f hsps with a concomitant i ncrease i n t he thermotoleran ce of cells [2]. Here, we further examined whether other NSAID s such as IND (a COX inhibitor) and NDGA (a LOX inhibitor) induced the expression of hsps in mammalian cells. IND has been shown to activate HSF, but not to induce a stress response in mammalian c ells [17]. A pharmacological dose of I ND lowers the temperature threshold of HSF activation, and a complete heat shock response can be attained at a temperature that is otherwise insufficient. In contrast, IND induces the expression of Hsp70 in human umbilical vein endothelial cells [18] or induces the nuclear localiz ation of Hsc70 in mammalian cells [19]. I n this study, we revealed that IND and NDGA activate HSF at 37 °C and induce hsp expression in mammalian cells, suggesting that NSAIDs generally induce the expression of hsps at physiological temperatures. As to the mechanism by which the stress response is induced by NSAIDs, these drugs activated HSF at a higher dose than those needed for the inhibition of COX a nd LOX. These drugs may activate HSF and induce hsp exp ression independently of the i nhibition of these e nzymes. H owever, arachidonate, an essential f atty acid, is released f rom phospholipids o f the cell membrane b y phospholipase A 2 in response t o e xtracellular s ignals. A rachidonate is oxidized by COX and LOX to produce various eicosanoids such as prostaglandins, thromboxanes and leukotrienes [20]. As arachidonate induces the activation and phosphorylation of HSF in mammalian cells and in synergy with an elevated temperature [21], the activation of HSF by NSAIDs may be in part caused by the inhibition of these e nzymes, b y w hich the cellular concentration of arachidonate increases. IND induced thermotolerance of cells, whereas NDGA did no t s ignificantly induce tolerance. Because hsps play an important role in the acquisition of cellular resistance to various forms o f stress [ 4,22,23], the d ifference b etween IN D and NDGA seems to be due to their ability t o in duce the expression of hsps. Furthermore, in a cellular model of SBMA, NSAIDs such as SA and IND suppressed forma- tion of the protein aggregates and c ytotoxicity caused by a n Fig. 5. Effects of SA on pro tein aggregation a nd cytotoxicity c aused by an expanded polyQ tract. (A) COS-7 cells were treated with 0, 20, 4 0 and 80 m M SA at 37 °C for 1 h, incubated at 37 °C for 3 h without the drug, and then transfe cted with tAR97 expression plasmid. After 7 2 h, cells were fixed, stained with Hoechst 33342 and observed using a confocal laser scann ing mic roscope. The arrowh eads re present the c ells containing aggregated tAR97 or c onde nsed chromatin. Propo rtions of cells containing tAR97 aggregates or condensed chromatin in GFP-positive ce lls represent the mean ± SE of four ind epende nt experiments. S tatistical sign ifican ce was determine d with Stu dent’s t-test; *P < 0.01 vs. r esp ecti ve controls. (B) Western blots of Hsp105a and Hsp70/Hsc70 in cells treated with or without SA. Ó FEBS 2004 Suppression of cytotoxicity by NSAIDs (Eur. J. Biochem. 271) 4557 expansion of t he polyQ tract. 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Suppression of heat- and polyglutamine-induced cytotoxicity by nonsteroidal anti-inflammatory drugs Keiichi Ishihara, Nobuyuki Yamagishi and Takumi Hatayama Department of Biochemistry,. °C for 1 h and furt her incubated for 6 h , the cellular levels of Hsp105a and Hsc70 (a constitutive isoform of Hsp70) increased, and t he expression of Hsp70 (an inducible isoform of Hsp70) was. manufacturer’s instructions. Results Induction of hsp promoter activity, activation of HSF and accumulation of hsps by IND and NDGA We first examined the effect of IND and NDGA on the hsp105 promoter (Fig.