Báo cáo khoa học: Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells pptx

Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells Keiichi Ishihara, Kenji Horiguchi, Nobuyuki Yamagi

Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells Keiichi Ishihara, Kenji Horiguchi, Nobuyuki Yamagishi and Takumi Hatayama

Department of Biochemistry, Kyoto Pharmaceutical University, Japan

As heat shock proteins (Hsps) are involved in protecting cells

and also in the pathophysiology of diseases such as

inflam-mation, cancer and neurodegenerative disorders,

modula-tors of Hsp expression in mammalian cells would seem to be

useful for the treatment of various diseases In this study, we

isolated mammalian cell lines for screening of Hsp

modu-lators; mouse C3H10T1/2 cells stably transfected with a

plasmid containing the mouse Hsp105 or human Hsp70B

promoter upstream of a luciferase or b-galactosidase

reporter gene, respectively Using these cells, we examined

the effect of sodium salicylate (SA), which may induce the

transcription of hsp genes, on stress response in mammalian

cells When these cells were treated with SA for 1 h at 37
C,

both promoter activities were up-regulated by SA at con-centrations of more than 45 mM The activation of heat shock factor and the subsequent accumulation of Hsp105a and Hsp70 were detected in cells treated with SA at con-centrations of more than 20 and 45 mM, respectively Fur-thermore, SA induced resistance against a subsequent lethal stress These findings suggested that SA is a potent hsp inducer, and may be used to protect cells against deleterious stressors

Keywords: cytoprotection; heat shock factor; heat shock promoter; heat shock proteins; sodium salicylate

Heat shock proteins (Hsp) are a set of highly conserved

proteins that are produced in response to physiological and

environmental stress [1] Hsps are also expressed under

physiological conditions and play important roles in normal

cellular events such as the synthesis, translocation and

degradation of proteins [2] Hsps protect cells from the

cytotoxic effects of aggregated proteins produced by various

types of stress, and play a vital role in cell survival under

both physiological and stressed conditions Because cellular

resistance against stress appears to be regulated by the

expression levels of Hsps, selective modulators of Hsp

expression could have medicinal applications For instance,

the induction of Hsps seems to improve the prognosis of

patients after a massive operation Geranylgeranylacetone,

a nontoxic Hsp70 inducer, is suggested to prevent acute liver

failure after massive hepatectomy, at least in part, by

enhancing cellular levels of Hsp70 [3] Moreover, as Hsp70

and Hsp40 protect against the aggregation of mutated

proteins and cell death in neurodegenerative disorders such

as Parkinson’s and Huntington’s disease [4,5], Hsp inducers are expected to be useful for the treatment of these diseases

On the other hand, a major problem with hyperthermia, which is one of the therapies applied for advanced cancers,

is the development of a transient thermoresistance in cancer cells with recurrent heat treatments [6,7] The acquisition of thermotolerance is expected to be suppressed by reducing the expression levels of Hsps in the cells

In mammalian cells, the transcription of hsp genes is mediated by the conversion of a pre-existing heat shock factor (HSF) from an inactive to an active form [8] HSF presents as an inactive monomeric form in the cytoplasm under physiological conditions, and is converted to an active trimeric form that has sequence-specific DNA-binding activity under stressed conditions Activated HSF relocalizes to the nucleus and binds to heat shock element (HSE) in the 5¢-flanking region of hsp genes, resulting in the trans-activation of hsp genes [8] In the present study, we isolated mouse C3H10T1/2 cells stably transfected with a plasmid containing the mouse Hsp105 or human Hsp70B promoter upstream of a luciferase or b-galactosidase repor-ter gene, respectively, as a simple system for screening Hsp modulators

Furthermore, although sodium salicylate (SA) is widely used as a nonsteroidal anti-inflammatory drug, the mech-anism of action of SA is still a subject of debate Several suggestions such as inhibition of cyclooxygenase, which is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins [9] and inhibition of the activation of transcription factor nuclear factor-kappa B [10], have been made to describe how SA exerts its anti-inflammatory effects and also its side effects In addition, SA has been found to activate HSF in mammalian cells, although the induction of transcription of hsp genes may not be induced by SA [11]

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: SA, sodium salicylate; hsp(s), heat shock protein(s);

Hsp70, 70 kDa heat shock protein; Hsp40, 40 kDa heat shock

pro-tein; Hsp105, 105 kDa heat shock propro-tein; Hsp105a, a isoform

of Hsp105; HSF, heat shock factor; HSE, heat shock element;

Luc, luciferase; b-gal, b-galactosidase; DMEM, Dulbecco’s

modified Eagle’s medium.

(Received 14 April 2003, revised 30 June 2003,

accepted 3 July 2003)

Here we examined the effects of SA on stress response

in mammalian cells using a simple screening system, and

revealed that SA is a potent Hsp inducer in mammalian

cells, thereby protecting cells against deleterious stress

Experimental procedures

Cell culture

Mouse fibroblast C3H10T1/2 and mouse embryonic F9 cell

lines were cultured in Dulbecco’s modified Eagle’s medium

(DMEM) (Nissui Pharmaceutical, Tokyo, Japan)

supple-mented with 10% foetal bovine serum in a humidified

atmosphere of 5% (v/v) CO2in air at 37
C Human HeLa

cells were grown in Eagle’s Minimum Essential medium

(Nissui Pharmaceutical) containing 10% bovine serum in a

CO2incubator at 37
C

Screening for Hsp modulators

A reporter plasmid containing the Hsp105 promoter

upstream of a luciferase (luc) reporter gene was constructed

by subcloning a 1.2-kb fragment of the 5¢-flanking region of

the hsp105 gene [12] to pGL2-basic vector (Promega) The

p173OR plasmid, which contains the hsp70B promoter

upstream of the b-galactosidase (b-gal) reporter gene, was

obtained from StressGen Biotechnologies (San Diego, CA,

USA) pGL105 or p173OR plasmid (7 lg each) and pBK/

neo plasmid (Stratagene) containing a geneticin resistant

gene (3 lg) were cotransfected into C3H10T1/2 cells

(1· 107cells per 100 mm dish) with 30 lL lipofectAMINE

reagent (Invitrogen) according to the manufacturer’s

instructions, and incubated for 48 h Cells were then

maintained in DMEM containing 0.4 mgÆmL)1 G418

antibiotic reagent (Wako Pure Chemical, Osaka, Japan)

for 3 weeks, and C3H10T1/2 cell lines stably transfected

with pGL105 or p173OR plasmid, designated as pGL105/

C3H and p173OR/C3H, respectively, were obtained and

maintained in DMEM containing 0.2 mgÆmL)1G418

Measurement of Luc activity

pGL105C3H cells (2· 105cells per 35 mm dish) were

washed with NaCl/Pithree times, lysed in 50 lL Cell Lysis

Regent (Promega), and centrifuged at 20 000 g for 10 min

Aliquots (5 lL) of cell extracts were added to 50 lL

Luciferase Assay Reagent (Promega), and the Luc activity

was measured using a Turner Designs model TD-20/20

Luminometer

Measurement of b-gal activity

p173OR/C3H cells (2· 105cells per 35 mm dish) washed

with NaCl/Pi were suspended in 50 lL 0.25M Tris/HCl

pH 8.0, and lysed by freeze-thawing (frozen at)80
C for

30 min and thawed at 37
C for 3 min, five times) After

centrifugation at 20 000 g for 10 min, aliquots of cell

extracts (5 lg protein) were added to a final volume of

125 lL Z buffer (0.2Msodium phosphate buffer pH 7.5,

10 mM KCl, 1 mMmagnesium sulfate, 0.05 mM

2-merca-ptoethanol) Then, 25 lL 15 mM

chlorophenolred-b galactosidase were added, and the mixture was incubated

at 37
C for 30 min The reaction was stopped by adding

60 lL 1M Na2CO3, and absorbance at 574 nm was measured

Gel mobility shift assay C3H10T1/2 cells (5· 105cells per 60 mm dish) were washed with NaCl/Pi, and quickly frozen at)80
C Frozen cells were suspended in 100 lL extraction buffer (20 mM

Hepes/KOH pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mMphenylmethanesulfonyl fluoride, 0.5 mM dithiothre-itol, 0.42MNaCl and 25% glycerol, v/v), kept at 4
C for

15 min, and vortexed for 15 min at 4
C After centrifuga-tion at 50 000 g for 5 min, aliquots of the supernatant (15 lg protein) were incubated in 25 lL buffer containing

10 mM Tris/HCl pH 7.8, 1 mM EDTA, 50 mM NaCl, 0.5 mM dithiothreitol, 5% (v/v) glycerol, 0.2 mgÆmL)1 BSA, 40 lgÆmL)1poly[dI-dC] and 0.4 ngÆmL)1 32P-labelled HSE corresponding to nucleotides )115 to )81 of the human hsp70 gene [13] 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

To define the specific HSF–HSE complex, unlabelled HSE was added to the reaction mixture in a 100-fold molar excess

of the labelled HSE

Western blot analysis C3H10T1/2, F9 or HeLa cells were lysed in 100 lL 0.1% SDS Cellular proteins (15 lg) were separated by SDS/ PAGE, and blotted onto nitrocellulose membrane The membrane was washed with Tris-buffered saline (0.1M

Tris/HCl pH 7.5, 0.9% NaCl) containing 0.1% Tween 20 (TTBS), and reacted with rabbit anti-Hsp105 [14,15] or mouse anti-Hsp70 Ig (Sigma) at room temperature for 1 h After a wash with TTBS, the membrane was further incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) at room temperature for 1 h Hsp105a and Hsp70 were detected using enhanced chemiluminescence reagent (Santa Cruz Biotechnology) For quantification, films were digit-ized by scanning into AdobePHOTOSHOP5 (Adobe Systems), and the intensities of the bands (Hsp105 and Hsp70) were quantified using the software programNIH IMAGE(http:// rsb.info.nih.gov/nih-image/)

Examination of cell morphology C3H10T1/2 cells (7· 104 cells per well) grown in 24-well plates containing collagen-coated coverslips were washed with NaCl/Pithree times, fixed with 4% paraformaldehyde

at room temperature for 20 min, and then observed using

a phase-contrast microscope

Neutral red uptake assay C3H10T1/2 cells (7· 104cells/well) in 24-well plates were incubated for 3 h in the presence of 50 lgÆmL)1neutral red, and fixed with 1% formaldehyde containing 1% CaCl2for

1 min The dye incorporated into viable cells was extracted with 50% ethanol containing 1% acetic acid, and absorb-ance at 540 nm was measured

Isolation of mammalian cell lines for screening of Hsp

modulators

To facilitate the measurement of heat shock promoter

activity, we isolated mouse C3H10T1/2 cells that were

stably transfected with pGL105 reporter plasmid containing

the luc gene linked to a 1.2 kb fragment of the 5¢-flanking

region of the hsp105 gene [12] or p173OR reporter plasmid

containing the b-gal gene linked to the Hsp70B promoter,

and designated them pGL105/C3H and p173OR/C3H cells,

respectively (Fig 1A,C)

Under nonstressed conditions, the Luc activity in

pGL105/C3H cells was detected at low levels (Fig 1B)

When pGL105/C3H cells were incubated at 37
C for 6 h

after heat shock at 39, 41 or 43
C for 1 h, Luc activity was

enhanced approximately 4 and 10 times in cells

heat-shocked at 41 and 43
C, respectively, compared to control

levels During continuous heat shock at 39, 41 or 43
C for

6 h, Luc activity was only enhanced in cells treated at 41
C Because firefly luciferase is thermosensitive and may be rapidly inactivated at high temperature, we analyzed the amount of Luc protein in the soluble and insoluble fractions

of these cells treated at 39, 41 and 43
C for 6 h (Fig 1B, part b, upper panel) Luc protein was detected in the soluble fractions but not in the insoluble fractions under these conditions, and the amounts of the protein were directly proportional to the Luc activity in cells, suggesting that levels of Luc activity at high temperatures also reflect the levels of transcription and translation of Hsp105

Furthermore, when these cells were treated with chem-ical stressors such as sodium arsenite, cupric chloride and zinc chloride, Luc activity was also enhanced in a dose-dependent manner As Hsp105a, a major product of hsp105gene, is constitutively expressed and also induced by various forms of stress in mammalian cells, the expression

of Luc activity in pGL105/C3H cells seemed to reflect the

Fig 1 Stress-inducible Hsp105 and Hsp70B promoters in pGL105/C3H and p173OR/C3H cells (A) The structure of pGL105 plasmid containing the Hsp105 promoter upstream of the luciferase reporter gene is shown schematically (B) pGL105/C3H cells were incubated at 37
C for 6 h after heat shock at various temperatures for 1 h (a), incubated at various temperatures for 6 h (b), or treated with sodium arsenite (c), cupric chloride (d)

or zinc chloride (e) at 37
C for 6 h Then, Luc activity was assayed, and relative activities are shown as ratios to that of untreated control cells For detection of Luc protein, cells incubated at 37–43
C for 6 h were lysed and the lysates were centrifuged at 20 000 g for 15 min, Luc protein in the supernatant (s) and pellet (p) fractions were detected by Western blotting using anti-Luc Ig [upper panels in (b)] (C) The structure of p173OR plasmid containing the Hsp70B promoter up-stream of the b-galactosidase reporter gene is shown schematically (D) p173OR/C3H cells were incubated at 37
C for 6 h after heat shock at various temperatures for 1 h (a), incubated at various temperatures for 6 h (b), or treated with sodium arsenite (c), cupric chloride (d) or zinc chloride (e) at 37
C for 6 h Then, b-gal activity was assayed, and relative activities are shown (arbitrary unit, AU).

expression of endogenous Hsp105a in mammalian cells

[15,16]

In p173OR/C3H cells, b-gal activity was not detected

under nonstressed conditions (Fig 1D) The b-gal activity

in the cells was induced by heat shock but not by chemical

stressors The induction of the enzyme activity was

consis-tent with that of the hsp70B gene in mammalian cells [17]

Thus, the promoter activities of Hsp105 and Hsp70B can be

measured easily using the pGL105/C3H and p173OR/C3H

cells, and pGL105/C3H cells seemed to be more useful for

screening modulators of stress response in mammalian cells

Induction of stress response by SA

Enhancement of heat shock promoter activity by SA SA

induces the activation of HSF but does not enhance the

transcription of hsp genes in human HeLa cells and

Drosophila [11,18], whereas the drug is also shown to

induce Hsp70 synthesis in mouse L929 cells [19] Because

transcription of hsp genes may be induced by SA in

mammalian cells, we first examined the effect of SA on the

heat shock promoter using pGL105/C3H or p173OR/C3H

cells (Fig 2) When pGL105/C3H cells were treated with

15–60 mM SA at 37
C for 1 h and incubated further at

37
C for 6 h without SA, the Luc activity increased

approximately 10- and 30-fold in cells treated with 45 and

60 mM SA, respectively, compared with that of untreated

cells (Fig 2A) When the amounts of Luc protein in cells

treated with SA at 37
C were examined by Western

blotting, the amounts of the protein were directly

propor-tional to the Luc activity in cells, suggesting that the

increase of Luc activity by SA reflect the levels of

transcription and translation of Hsp105, not due to an

indirect effect of SA on the basal activity of Luc

Enhance-ment of Luc activity was also detected at 45 and 60 mMSA,

when pGL105/C3H cells were incubated at 39
C for 6 h

after the SA treatment However, the enzyme activity was

not enhanced in cells incubated at 41
C for 6 h after

treatment with 60 mM SA, due to the markedly reduced

viability of the cells

Furthermore, when p173OR/C3H cells were treated with

15–60 mM SA at 37
C for 1 h and incubated further at

37
C for 6 h without SA, the activity significantly increased

in cells treated with 45 and 60 mMSA (Fig 2B)

Enhance-ment of b-gal activity was also observed in cells incubated at

39 or 41
C for 6 h after SA treatment similarly to the Luc

activity in pGL105/C3H cells The enzyme activity was also

not enhanced in cells incubated at 41
C for 6 h after

treatment with 60 mm SA, due to the markedly reduced

viability of the cells These results suggested that Hsp105

and Hsp70B promoters were activated even at 37
C in cells

treated with 45 and 60 mMSA, followed by transcription

and translation of the gene products

When the effects of other known Hsp-inducing

com-pounds such as geldanamycin, curcumin and

geranylgera-nylacetone on the heat shock promoter were examined

using pGL105/C3H cells [20–22], the Luc activity increased

approximately two- to fivefold in cells treated with these

compounds at 37
C for 6 h compared with that of

untreated cells SA seemed to activate the heat shock

promoter markedly than these Hsp inducers in mammalian

cells (Table 1)

Fig 2 Effect of SA on Hsp promoters in pGL105/C3H and p173OR cells pGL105/C3H (A) or p173OR (B) cells were incubated with or without 15, 30, 45 and 60 m M SA at 37
C for 1 h, and further incu-bated at 37, 39 or 41
C for 6 h without SA Then, luciferase or b-galactosidase activity was assayed Each value represents the mean ± SD of three independent experiments Statistical significance was determined with Student’s t-test; *, P < 0.01 vs control cells incubated at 37, 39 or 41
C for 6 h Upper panel in (A) is Western blot

of Luc protein in the supernatant of cells treated with or without 30,

45 and 60 m M SA at 37
C.

Table 1 Effect of geldanamycin, curcumin, or geranylgeranylacetone on Hsp105 promoter in pGL105/C3H cells Cells were treated with com-pounds at 37
C for 1 h, and further incubated at 37
C for 6 h Each value is average of results from two independent experiments.

Relative luciferase activity

Activation of HSF by SA and accumulation of Hsp105a

and Hsp70

To examine whether SA enhances heat shock promoter

activity by activating HSF, a gel mobility shift assay using

32P-labelled HSE was performed When C3H10T1/2 cells

were treated with 10–60 mMSA at 37
C for 1 h, HSF was

activated in cells treated with SA at concentrations of more

than 20 mM(Fig 3A) The kinetics of activation of HSF by

SA revealed that the HSF–HSE complex was detected

immediately after 30 mM SA treatment, and quickly

diminished in 1–2 h at 37
C (Fig 3B) Furthermore, SA

affected the activation of HSF by heat shock (Fig 3C)

When activated by heat shock at 41
C for 1 h, the HSF

decreased to the basal level within 2 h However, when cells

treated with 30 mM SA at 37
C were continuously

heat-shocked at 41
C, the SA-activated HSF remained at a high

level for 2 h, then diminished to the basal level within 3 h of

heat shock Thus, SA seems not only to activate HSF at

37
C, but also to enhance the activation of HSF by heat

shock

Next, we examined whether Hsp105a and Hsp70 proteins accumulated in cells treated with SA (Fig 4) Mouse C3H10T1/2, mouse F9 and human HeLa cells were treated with 15–60 mMSA at 37
C for 1 h and further incubated for 6 h at 37
C The levels of Hsp105a and Hsp70 significantly increased in cells treated with 45 and 60 mM

SA However, the levels remained unchanged or decreased, when these cells were incubated at 41
C for 6 h after the SA treatment These results suggested that SA treatment at

37
C induced the expression of endogenous heat shock proteins such as Hsp105a and Hsp70 in various mammalian cells

Enhancement of thermoresistance of cells by SA Upon exposure to a sublethal heat treatment, mammalian cells acquire transient resistance to a subsequent heat shock that would normally be lethal [6,7] The phenomenon is known as thermotolerance, and much evidence supports the idea that Hsps, especially Hsp70, play important roles in its development Since SA seemed to induce the expression of Hsp105a and Hsp70 in mammalian cells, we examined whether SA induced resistance against subsequent lethal heat shock As shown in Fig 5A, the treatment of cells with

30 or 45 mMSA for 1 h did not induce marked changes of cell morphology and numbers of cells attached to culture dishes, although the attached cells were slightly reduced in number by 60 mM SA treatment When these cells were incubated at 37
C for 6 h and exposed to a lethal heat shock at 45
C for 45 min, cells attached to dishes markedly decreased in number regardless of SA treatment However, when these cells were further incubated at 37
C for 24 h, numbers attached to culture dishes were increased in cells treated with 45 or 60 mMSA, but not 30 mMSA Furthermore, the viability of cells was assessed based on the ability of living cells to incorporate neutral red into lysozomes (Fig 5B) The uptake of neutral red into cells decreased gradually after heat shock at 45
C for 45 min, although uptake of the dye was only slightly affected by 30–60 mM SA at 37
C However, the uptake again increased in cells pretreated with 45 and 60 mMSA for 24 and 120 h, respectively, after the heat shock Thus, the resistance of cells against a subsequent heat shock seemed to

be enhanced by 45 or 60 mMSA treatment

Discussion

As Hsps play important roles in the folding, regulation and degradation of cellular proteins and also cellular resistance against stress as molecular chaperones, drugs that can regulate the expression levels of Hsps in cells seem to have various medicinal applications In the present study, we isolated cell lines for screening of stress response modula-tors: mouse pGL105/C3H and p173OR/C3H cells pGL105/C3H cells have a plasmid containing the Hsp105 promoter upstream of a luc reporter gene, while p173OR/ C3H cells have a plasmid containing the Hsp70B promoter upstream of a b-gal reporter gene The Luc or b-gal activity

in these cells was expressed and induced similarly to endogenous Hsp105a or Hsp70B, respectively, in mamma-lian cells Using these cells, the activities of Hsp105 and Hsp70B promoters could be easily measured

Fig 3 Effect of SA on activation of HSF in C3H10T1/2 cells (A)

C3H10T1/2 cells were treated with or without 10, 20, 30 and 60 m M

SA at 37
C for 1 h, or heat-shocked at 42
C for 3 h as a positive

control (B and C) C3H10T1/2 cells were incubated with or without SA

at 37
C for 1 h, then further incubated at 37
C (B) or at 41
C (C)

without SA for up to 6 h Cell extracts from these cells were subjected

to gel mobility shift assay using a radioactive HSE probe HSF–HSE

complexes were determined by adding a 100-fold excess of unlabelled

HSE Arrows indicate specific HSF–HSE complexes.

Using these cells, we examined the effects of SA, a

nonsteroidal anti-inflammatory drug, on the stress response

of mammalian cells Jurivich et al have shown that 20 mM

SA induces activation of HSF but not transcription of hsp

genes in human cells [11] In contrast, Liu et al have shown

that 60 mMSA induces not only activation of HSF but also

accumulation of Hsp70 in mouse cells [19] Here we showed

that relatively high doses of SA (45–60 mM) induced the

activation of promoter activities of Hsp105 and Hsp70 as

well as the accumulation of Hsp105a and Hsp70 in mouse

and human cells, although relatively low doses of SA (20–

30 mM) induced the activation of HSF but not transcription

of hsp genes in cells Thus, relatively high doses of SA

seemed to be required for the induction of the transcription

of hsp genes and the accumulation of Hsp in mammalian

cells

HSF is a transcription factor which is converted to a

trimeric and hyper-phosphorylated active form from

inac-tive monomers in response to various forms of stress and

induces the transcription of hsp genes [8] SA is shown to

trigger HSF differently than heat shock [20] The

SA-induced form of HSF is not hyperphosphorylated like the

heat-induced form, and SA-induced threonine

phosphory-lation of HSF, whereas heat shock led to a predominance

of HSF serine phosphorylation [23] However, the HSF

activated by relatively high doses of SA did induce

transcription of hsp genes in cells The HSF activated by

SA may be somehow different at levels of modification such

as phosphorylation depending on the doses of SA, although

further study is needed to elucidate the different effects of

the SA-activated HSF on the induction of transcription of hspgenes

Hsps are suggested to play important roles in the acquisition of resistance of cells against various forms of stress [1] Here we showed that SA induced the activation

of HSF, the transcription of hsp genes and the accumu-lation of Hsps in various mammalian cells and a concomi-tant increase of thermoresistance of cells Thus, SA may

be used for the protection of cells against deleterious stressors Furthermore, several neurodegenerative disorders including Alzheimer’s, polyglutamine and Parkinson’s disease are though to be caused by an accumulation of protein aggregates in the brain [24], and Hsps such as Hsp70 and Hsp40 are shown to suppress the toxicity of these diseases [25,26] Recently, long-term use of non-steroidal anti-inflammatory drugs was shown to prevent the occurrence of Alzheimer’s disease [27,28] Our finding that SA induces the expression of Hsps in mammalian cells may explain the protective effect of SA on Alzheimer’s disease

SA activated heat shock promoter and induced the expression of Hsps in mammalian cells at concentrations higher than those used for its anti-inflammatory effects However, long-term use of therapeutic doses of the drug may induce the expression of Hsps in mammalian cells SA has a potent anti-inflammatory effect mediated by suppres-sion of the production of inflammatory mediators by inhibition of cyclooxygenase and nuclear factor-kappa B activation [9,10] In addition to the anti-inflammatory effects, it is noteworthy that SA which can activate HSF

Fig 4 Effect of SA on accumulation of Hsp105 and Hsp70 in mammalian cells C3H10T1/2 (A), F9 (B), and HeLa cells (C) were treated with or without SA at 37
C for

1 h, then further incubated at 37 or 41
C for

6 h without SA Cellular proteins (15 lg) were separated by SDS/PAGE (10% polyacryl-amide), blotted onto nitrocellulose membranes, and immunostained using anti-Hsp105 or anti-Hsp70 Ig (upper panels) Bands were quantified by densitometry, and relative levels of Hsp105a or Hsp70 are shown

as ratios to that of untreated cells at 37
C or cells heat-shocked at 41
C for 6 h, respect-ively (lower graphs) Each value in (A) and (B) represents the mean ± SD of three independent experiments Statistical significance was determined with Student’s t-test; *, P < 0.01 vs control cells incubated

at 37 or 41
C for 6 h.

Fig 5 Induction of thermoresistance of cells by SA (A) C3H10T1/2 cells were treated with or without 30, 45 and 60 m M SA at 37
C for 1 h (a) These cells were further incubated without SA at 37
C for 24 h (b) or heat shocked at 45
C for 45 min after incubation at 37
C for 6 h (c) The heat-shocked cells were further incubated at 37
C for 24 h (d) Then, these cells were fixed with 4% paraformaldhyde and observed using a phase contrast microscope (· 100) (B) C3H10T1/2 cells treated with or without 30, 45 and 60 m M SA at 37
C for 1 h were incubated at 37
C for 6 h without SA Then, these cells were heat-shocked at 45
C for 45 min, and further incubated at 37
C for the indicated periods Viability of cells was assessed by neutral red uptake assay.

and induce the transcription of hsp genes may be used as

an Hsp inducer for treatment of diseases

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