Suppression of heat- and polyglutamine-induced cytotoxicityby nonsteroidal anti-inflammatory drugs Keiichi Ishihara, Nobuyuki Yamagishi and Takumi Hatayama Department of Biochemistry, Ky
Trang 1Suppression 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 drugs (NSAIDs), we examined the effects of
a cyclooxygenase inhibitor, indomethacin, and a
lipoxyg-enase inhibitor, nordihydroguaiaretic acid Both inhibitors
up-regulated the hsp promoter at 37C through the
acti-vation of heat shock factors, and increased cellular levels
of hsps in mammalian cells, although the degree of the
expression of hsps and thermotolerance of cells differed
depending on the drugs Furthermore, NSAIDs such as sodium salicylate and indomethacin suppressed the protein aggregation and apoptosis caused by an expanded poly-glutamine tract in a cellular model of polypoly-glutamine dis-ease These findings suggest that NSAIDs generally induce the expression of hsps in mammalian cells and may be used for the protection of cells against deleterious stressors and neurodegenerative diseases
Keywords: heat shock proteins; indomethacin; nonsteroidal anti-inflammatory drugs; nordihydroguaiaretic acid; poly-glutamine disease
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 biosynthesis 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 long-term use of NSAIDs was shown to
prevent the occurrence of Alzheimer’s disease without COX
inhibition [1] In addition to having these anti-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 levels 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, especially Hsp70, play important roles in its
development [4] Furthermore, in several polyglutamine
(polyQ) diseases such as Huntington’s disease and spino-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 Hsp105a reduces the aggregation of proteins and cellular toxicity 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 SBMA phenotypes in mice [12] Addition-ally, geldanamycin (a benzoquinone ansamycin that induces 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 the treatment of 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 do 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 gene [2] and African green monkey kidney COS-7 cells (Riken cell bank) were cultured
in Dulbecco’s modified Eagle’s medium (Nissui Pharma-ceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (Equitech-Bio, Kerriville, TX, USA) in a humidified atmosphere of 5% (v/v) CO2in air at 37C IND and NDGA (Nacalai Tesque, Kyoto, Japan) were dissolved in dimethylsulfoxide at 250 m , and 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, revised 27 September 2004,
accepted 6 October 2004)
Trang 2Tesque) was dissolved in phosphate-buffered saline
(NaCl/Pi) at 1M just before use Cells were incubated in
the medium with or without drugs at 37C for 1 h, washed
with medium three times and further incubated at 37C
without the drug
Measurement of hsp promoter activity
To measure hsp promoter activity in mammalian pGL105/
C3H cells (2· 105cells per 35 mm dish), they were treated
with a drug at 37C for 1 h and further incubated for 6 h
without the drug Then, the cells were washed three times
with NaCl/Pi, and lysed in 50 lL of Cell Lysis Reagent
(Promega, Madison, WI, USA) The cell lysates were
centrifuged at 20 000 g for 10 min, and the supernatants
recovered as cell extracts Aliquots (5 lL) of the extracts
were added to 50 lL of luciferase assay reagent (Promega),
and luciferase (luc) activity was measured using a TD-20/20
luminometer (Turner Designs, Sunnyvale, CA, USA)
Gel mobility shift assay
C3H10T1/2 cells (5· 105cells per 60 mm dish), treated
with or without a drug or heat-shocked, were washed with
NaCl/Pi, and quickly frozen at)80 C Frozen cells were
suspended in 100 lL of extraction buffer [20 mM Hepes/
KOH, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM
phenylmethanesulfonyl fluolide, 0.5 mM dithiothreitol,
0.42M NaCl and 25% (v/v) glycerol], incubated at 4C
for 15 min, and vortexed for 15 min at 4C After
centrifugation at 50 000 g for 5 min, aliquots of the
supernatant (15 lg protein) were incubated in 25 lL of
buffer containing 10 mMTris/HCl, pH 7.8, 1 mMEDTA,
50 mM NaCl, 0.5 mM dithiothreitol, 5% (v/v) glycerol,
0.2 mgÆmL)1bovine serum albumin, 40 lgÆmL)1
poly(dI-dC)] and 0.4 ngÆmL)1 32P-labeled heat shock element (HSE)
corresponding to nucleotides)115 to )81 of the human
hsp70gene [14] at 25C for 20 min The mixtures were then
electrophoresed on a native 4% polyacrylamide gel, and the
gel was dried and subjected to autoradiography To define
the specific HSF–HSE complex, unlabeled HSE was added
to the reaction mixture in a 100-fold molar excess of the
labeled HSE Under the experimental conditions, the
activated mouse HSF binds to the HSE of the human
hsp70gene, as does the activated human HSF [15]
Western blot analysis
C3H10T1/2 or COS-7 cells (5· 105 and 6· 105cells per
60 mm dish, respectively) were lysed in 200 lL of 0.1%
(w/v) SDS Cellular proteins (15 lg) were separated by
SDS/PAGE, and blotted onto a nitrocellulose membrane
The membrane was washed with Tris-buffered saline [0.1M
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) antibody at room temperature for 1 h
After washes with TTBS, the membrane was further
incubated with horseradish peroxidase-conjugated
anti-(rabbit or anti-(mouse IgG) 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 intoADOBE PHOTOSHOP5, and the intensity of the bands was quantified on a Macintosh computer using the public domain NIHIMAGE program (developed at US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ nih-image/)
Thermotolerance of cells C3H10T1/2 cells (7· 104cells per well) grown in 24-well plates containing collagen-coated coverslips were incubated with or without drugs at 37C for 1 h These cells were heat-shocked at 45C for 45 min after incubation at 37 C for 6 h in the absence of drugs, and further incubated at
37C At appropriate times, cells were then washed three times with NaCl/Pi, fixed with 4% (v/v) paraformaldehyde
at room temperature for 20 min, and observed under a phase-contrast microscope (Nikon, Tokyo, Japan) The viability of cells was also assessed based on the ability
of living cells to incorporate neutral red into lysozomes For the neutral red uptake assay, C3H10T1/2 cells (7· 104cells per well) in 24-well plates were treated with or without
a drug or heat-shocked, then incubated at 37C for 3 h with 50 lgÆmL)1of neutral red, and fixed with 1% (v/v) formaldehyde containing 1% (w/v) CaCl2 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 coverslips to 70–80% confluence were treated with or without drugs at 37C for 1 h and incubated at 37C for 3 h without the drug The cells were then transfected with the expression plasmid of a truncated androgen receptor containing 97 glutamine repeats fused
to green fluorescence protein (GFP) (tAR97) [9] with DMRIE-C reagent (Invitrogen, Carlsbad, CA, USA) After incubation at 37C for 72 h, the cells were washed with NaCl/Pi, fixed with 4% (v/v) paraformaldehyde for 30 min
at room temperature, and stained with 10 lM Hoechst
33342 for 15 min at room temperature The cells were observed using a confocal laser scanning microscope (Zeiss, 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 Hoechst
33342 and the TdT-mediated dUTP-biotin end labeling (TUNEL) method [11] The TUNEL method was per-formed using a DeadEndTMapoptosis 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
FEBS 2004 Suppression of cytotoxicity by NSAIDs (Eur J Biochem 271) 4553
Trang 3treated with various concentrations of IND at 37C for 1 h
and further incubated for 6 h without the drug, luc activity
increased depending on the concentration, and an
approxi-mately 25-fold increase was observed in cells pretreated with
0.75 mMIND compared to control cells (Fig 1A) Under
these conditions, cell viability was not reduced by IND at
concentrations up to 1 mM(Fig 1B) On the other hand,
luc activity was increased approximately fivefold in cells
pretreated with 0.2 mM NDGA compared to the control,
whereas cell death was observed at concentrations of
NDGA of more than 0.2 mM
We next examined whether these drugs enhance 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 37C for 1 h,
HSF was activated in cells treated with 0.5 and 0.75 mM
IND or 0.2 m NDGA, as in the heat-shocked cells,
although the activation of HSF was at higher levels in cells treated with IND than with NDGA Furthermore, when C3H10T1/2 cells were treated with 0.75 mMIND at 37C for 1 h and further incubated for 6 h, the cellular levels of Hsp105a and Hsc70 (a constitutive isoform of Hsp70) increased, and the expression of Hsp70 (an inducible isoform of Hsp70) was markedly induced (Fig 2B) On the other hand, the increase in the levels of Hsp105a and Hsc70 and the induction of Hsp70 expression were observed
at low levels in the cells treated with 0.2 mMNDGA Thus, 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 of IND or NDGA on hsp promoter in pGL105/C3H
cells (A) pGL105/C3H cells were incubated with or without IND and
NDGA at 37 C for 1 h, and further incubated at 37 C for 6 h
without the drug Then luc activity was assayed, with relative activity
shown as a ratio to that of untreated control cells 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 cells (B) Viability of cells was assessed by the neutral
red uptake assay Values represent the mean ± SE of three
inde-pendent experiments.
Fig 2 Effect of IND or NDGA on activation of HSF and accumulation
of hsps in mammalian cells (A) C3H10T1/2 cells were incubated with
or without IND or NDGA at 37 C for 1 h, or heat-shocked at 42 C for 1 h (HS) as a positive control Cell extracts from these cells were subjected to a gel mobility shift assay using 32 P-labeled HSE Specific HSF–HSE complexes were determined by adding a 100-fold excess of unlabeled HSE Arrows indicate specific HSF–HSE complexes (B) C3H10T1/2 cells were incubated with or without IND or NDGA at
37 C for 1 h, and then further incubated at 37 C for 6 h without the drug As a positive control, cells were heat-shocked at 41 C for 6 h (HS) Cellular proteins (15 lg) were separated by 10% SDS/PAGE, blotted onto nitrocellulose membranes, and immunostained using anti-Hsp105 or anti-Hsp70.
Trang 4induced resistance against subsequent heat shock (Fig 3A).
The treatment of cells with 0.5 and 0.75 mMIND or 0.1 and
0.2 mM NDGA at 37C for 1 h did not cause marked
changes of cell morphology, and immediately after exposure
to a lethal heat shock, the number of cells attached to culture
dishes was not significantly altered However, when these
cells were further incubated at 37C for 48 h, the number of
cells attached to the dishes was markedly decreased in
untreated controls However, many cells remained attached
to dishes when pretreated with 0.75 mMIND but not 0.1
and 0.2 mMNDGA Furthermore, the viability of cells was
assessed with the neutral red uptake assay (Fig 3B) The
uptake of dye 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 45C for
45 min, while pretreatment of cells with 0.75 mMIND but
not 0.2 m NDGA significantly suppressed the inhibition
Thus, cellular resistance to a subsequent heat stress seemed
to be enhanced by the drug that markedly induced the expression and accumulation of 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 caused by 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 tAR97, approximately 50% of cells expressing GFP fluorescence of tAR97 were found to contain protein aggregates and also condensed
Fig 3 Effect of IND or NDGA on thermotolerance of cells C3H10T1/2 cells were incubated with or without IND or NDGA at 37 C for 1 h (a), and incubated at 37 C for 6 h without the drug Then, these cells were heat-shocked at 45 C for 45 min (b), and further incubated at 37 C for
48 h (c) or 72 h (d) (A) Cells in (a), (b) and (c) were fixed with 4% (v/v) paraformaldhyde and observed using a phase contrast microscope (B) Viability of cells at (b) and (d) was assessed by neutral red uptake assay Values represent the mean ± SE of three independent experiments Statistical significance was determined with Student’s t-test; *P < 0.001 vs respective control cells.
FEBS 2004 Suppression of cytotoxicity by NSAIDs (Eur J Biochem 271) 4555
Trang 5chromatin, 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 mM and above
significantly suppressed the protein aggregation and
apop-tosis in a dose-dependent manner (Fig 4B) Under these
conditions, Hsp105a, Hsc70 and Hsp70 were markedly
accumulated in cells treated with 0.75 and 1 mM IND (Fig 4C) Furthermore, SA (a potent hsp inducer [2]) at concentrations of 40 and 80 mMsignificantly suppressed the aggregation of tAR97 and apoptosis, with a concomitant marked induction of Hsp105a and Hsp70 (Fig 5) How-ever, NDGA did not suppress the aggregation and apop-tosis caused by an expanded polyQ tract (data not shown)
Fig 4 Effects of IND on protein aggregation and cytotoxicity caused by an expanded polyQ tract (A) COS-7 cells were transfected with tAR97 expression plasmid and incubated further at 37 C for 72 h Cells were then fixed, stained with Hoechst 33342, and observed using a confocal laser scanning microscope Typical images of cells expressing tAR97 (GFP) diffusely (upper panels) or as aggregates (lower panels) are shown Apoptotic cells were identified based on the nuclear morphology (Hoechst) and by the TUNEL method (B) COS-7 cells were treated with 0, 0.5, 0.75 and 1.0 m M IND for 1 h, incubated at 37 C for 3 h without the drug, and then transfected with tAR97 expression plasmid After 72 h, cells were fixed, stained with Hoechst 33342 and observed using a confocal laser scanning microscope The arrowheads represent typical cells containing aggregated tAR97 or condensed chromatin Proportions of cells containing tAR97 aggregates or condensed chromatin in GFP-positive cells represent the mean ± SE of four independent experiments Statistical significance was determined with Student’s t-test; *P < 0.01 vs respective controls (C) Western blots of Hsp105a and Hsp70/Hsc70 in cells treated with or without IND.
Trang 6SA activates the hsp promoter and induces the expression of
hsps with a concomitant increase in the thermotolerance of
cells [2] Here, we further examined whether other NSAIDs
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 cells [17] A
pharmacological dose of IND 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 localization of Hsc70 in mammalian
cells [19] In this study, we revealed that IND and NDGA
activate HSF at 37C 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 and LOX These drugs may activate HSF and induce hsp expression independently of the inhibition of these enzymes However, arachidonate, an essential fatty acid, is released from phospholipids of the cell membrane by phospholipase A2
in response to extracellular signals Arachidonate 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 enzymes, by which the cellular concentration of arachidonate increases IND induced thermotolerance of cells, whereas NDGA did not significantly induce tolerance Because hsps play an important role in the acquisition of cellular resistance to various forms of stress [4,22,23], the difference between IND and NDGA seems to be due to their ability to induce 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 cytotoxicity caused by an
Fig 5 Effects of SA on protein aggregation and cytotoxicity caused by an expanded polyQ tract (A) COS-7 cells were treated with 0, 20, 40 and
80 m M SA at 37 C for 1 h, incubated at 37 C for 3 h without the drug, and then transfected with tAR97 expression plasmid After 72 h, cells were fixed, stained with Hoechst 33342 and observed using a confocal laser scanning microscope The arrowheads represent the cells containing aggregated tAR97 or condensed chromatin Proportions of cells containing tAR97 aggregates or condensed chromatin in GFP-positive cells represent the mean ± SE of four independent experiments Statistical significance was determined with Student’s t-test; *P < 0.01 vs respective controls (B) Western blots of Hsp105a and Hsp70/Hsc70 in cells treated with or without SA.
FEBS 2004 Suppression of cytotoxicity by NSAIDs (Eur J Biochem 271) 4557
Trang 7expansion of the polyQ tract As the cellular toxicity caused
by an expanded polyQ tract is suppressed by overexpression
of Hsp105a or Hsp70 in the cellular model [9,11], the
suppression by NSAIDs seemed to be due to the
accumu-lation of hsps by these drugs These findings strongly
support an additional role for NSAIDs as a cytoprotective
agent through the induction of hsps, and suggest that
NSAIDs may be useful for the treatment of stress and
neurodegenerative diseases
Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific
Research (T H.) from the Ministry of Education, Science, Culture and
Sports of Japan.
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