Role of heat shock proteins in oxygen radical–induced gastric apoptosis

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Role of heat shock proteins in oxygen radicaleinduced gastric apoptosis Anna M. Leung, MD,1 Maria J. Redlak, PhD, and Thomas A. Miller, MD* Department of Surgery, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

article info

abstract

Article history:

Background: The generation of reactive oxygen species (ROS) and their resultant oxidative

Received 15 November 2013

damage is a common pathway for gastric mucosal injury. Developing strategies to protect

Received in revised form

the gastric epithelium against oxygen free radical damage is of profound pathophysio-

27 June 2014

logical interest. We have previously shown caspase-mediated apoptosis as a major cause of

Accepted 8 July 2014

ROS-induced cell death in gastric mucosa. Because heat shock proteins (Hsps) confer

Available online 15 July 2014

protection against many cytotoxic agents, this study was undertaken to determine whether modulation of Hsps was protective against oxidative damage.

Keywords:

Materials and methods: AGS cells (human gastric mucosal cell line) received either no pre-

Heat shock proteins

treatment, heat shock pretreatment (1 h at 42
1 C), or pretreatment with an Hsp

Apoposis

modulating drug (geldanamycin or quercetin). Cells were then exposed to hydrogen

Gastric mucosal injury

peroxide (H2O2), a representative ROS (1 mM, a physiologically relevant concentration), for

Caspases

24 h. Caspase-3 activation and Poly ADP Ribose Polymerase (PARP) inactivation, as well as

Free oxygen radical

DNA-histone complex formation were used as measures of apoptosis. Inducible Hsps (Hsp70 and Hsp90) were detected using Western blot analysis. Results: Results showed heat shock pretreatment induced increased expression of Hsp70 without change in Hsp90. In response to H2O2 exposure alone, there was significant increase in DNA-histone complex formation as well as caspase-3 activation and PARP cleavage in gastric epithelium. Heat shock pretreatment resulted in statistically significant prevention in these measures of apoptosis. Geldanamycin increased Hsp70, but elicited cleavage of Hsp90 and subsequently resulted in an increase in H2O2-induced apoptosis. Quercetin decreased Hsp70 and resulted again in increased H2O2-induced apoptosis. Conclusions: These findings indicate that heat shock pretreatment protects gastric mucosal cells against H2O2-induced apoptosis and that Hsp70 and Hsp90 may play key roles in this process. These results further suggest that perturbations in Hsp metabolism may induce mucosal injury in response to oxygen free radicals. Published by Elsevier Inc.

1.

Introduction

Current knowledge implicates oxygen free radicals as important mediators of gastric mucosal injury in response to episodes of

ischemiaereperfusion, and following exposure to ethanol, bile acids, Helicobacter pylori, and nonsteroidal anti-inflammatory drugs [1e3]. The result of such injury involving the gastric epithelium can range from simple gastric irritation to frank

* Corresponding author. Department of Surgery, Medical College of Virginia Campus, Virginia Commonwealth University, Surgical Service (112), 1201 Broad Rock Blvd., Richmond, VA 23249. Tel.: þ1 804 675 5112; fax: þ1 804 675 5390. E-mail address: [email protected] (T.A. Miller). 1 Dr. Leung is currently associated with the Department of Surgery, Penn State Hershey Medical Center, Hershey, Pennsylvania 0022-4804/$ e see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jss.2014.07.013

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ulcer formation and the potential sequellae of hemorrhage and perforation. We have previously shown that the free radical hydrogen peroxide mediates its damaging effects on gastric mucosal cells through an apoptotic pathway [4]. Thus, targeting this method of death provides a potential strategy to protect the gastric mucosa against free radical-induced injury. One approach that has been used to protect gastric mucosa against oxygen radicaleinduced injury has focused on increasing intracellular antioxidants. In this regard, induction of heat shock proteins (Hsps) has been proposed as a novel means of preventing injury induced by free radicals [5]. Among the various Hsps, 70-kDA Hsp (Hsp70) appears to exhibit broad cytoprotective functions. Increased expression of Hsp70, for example, has been shown to protect against increased cellular damage induced by hyperthermia, endotoxin, ultraviolet radiation, nitric oxide, and ischemiaereperfusion [6e8]. The present study was designed to test the hypothesis that increased expression of Hsp70 protects cultured gastric cells against oxygen free radical damage. We used hydrogen peroxide (H2O2) as the reactive oxygen species (ROS) to induce injury in a concentration of 1 mM, which was shown previously to be an apoptosis-inducing concentration and having physiological relevance [4]. A human gastric surface cell line, known as AGS cells, was used to accomplish these goals. We have used this cell line for many years, as have many other investigators, to study various aspects of gastric epithelial biology [9e11].

2.

Materials and methods

2.1.

Gastric epithelial cell culture

A human gastric epithelial cell line (AGS), derived from gastric adenocarcinoma cells and purchased from American Type Culture Collection (Manassas, VA), was used in these experiments. The cells were seeded in 75-cm2 culture flasks (Corning, New York) and maintained in Ham’s F-12 (Cellgro) culture medium supplemented with 10% fetal bovine serum, 100-mg/ mL penicillin, 100-mg/mL streptomycin, and 0.25-mg/mL amphotericin B. The cells were grown at 37 C in a humidified atmosphere of 5% CO2. Experiments were performed in 3.5cm2 dishes (2.5 million cells per dish) or in six-well tissue culture plates, (300,000 cells per well; Costar; Sigma-Aldrich, St. Louis, MO). Before treatment, media were aspirated and cells were washed with phosphate-buffered saline.

2.2.

Chemicals and reagents

A stock solution of 9.8 M H2O2 (Fisher Scientific, Pittsburgh, PA) was maintained at 4 C and diluted freshly before each use. Geldanamycin (17-AAG), the Hsp90 inhibitor (Sigma, St. Louis, MO), and Quercetin (C15H10O7), the Hsp70 inhibitor (Sigma), were both dissolved in 100% dimethyl sulfoxide (DMSO) and maintained at 20 C. The stock solution of DMSO for these agents was 100 mM with the final concentration of DMSO in medium not exceeding 0.05% when used for experiments.

2.3.

Heat shock treatment

Cultured cells were counted and plated equally in six-well tissue culture plates overnight. They were then subjected to

hyperthermia of 42
1 C for 1 h with a water bath. As a control, cells were cultured under normal conditions without hyperthermia. Cells were then routinely allowed to recover for 6 h at 37 C in a humidified atmosphere containing 5% CO2. After recovery, cells were used for appropriate studies.

2.4.

Quercetin treatment

Cultured cells were counted and plated equally in six-well tissue culture plates overnight. They were then incubated with quercetin 100 mM for 4 h. As a control, cells were cultured under normal conditions without quercetin. Cells were then used for appropriate studies. The dose of quercetin used for these experiments is commonly used in cell culture studies [12].

2.5.

Geldanamycin treatment

Cultured cells were counted and plated equally in six-well tissue culture plates overnight. They were then incubated with geldanamycin 0.2e1 mM for 24 h. As a control, cells were cultured under normal conditions without geldanamycin. Cells were then used for appropriate studies. The dose range for geldanamycin used for these experiments is commonly used in cell culture studies [13].

2.6. Western blot analysis of caspase-3, Hsp70, Hsp90, b-actin, and Poly ADP Ribose Polymerase After H2O2 treatment, whole cell lysates were extracted with lysis buffer containing 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 20 mM Tris (pH 7.4), 1 mM EDTA, 1 mM ethylene glycol tetra-acetic acid, 1 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% NP-40. An equal amount of cell lysate (50 mg) was solubilized in sample buffer, boiled for 5 min, and electrophoresed on a 4%e20% Tris-glycine gel (Invitrogen; Fisher Scientific, Pittsburgh, PA). Proteins were then transferred to polyvinylidene difluoride membrane (BioRad Laboratories, Hercules, CA). Nonspecific binding was blocked with 20 mM Tris-HCl buffered saline (pH 7.6) plus 0.05% Tween-20 (TBS-T) containing 5% nonfat dry milk and 1% bovine serum albumin. After incubation with the appropriate primary antibody of Hsp70 (Cat no. 4876), Hsp90 (Cat no. 4877), b-actin (Cat no. 8457), caspase-3 (Cat no. 9665), or Poly ADP Ribose Polymerase (PARP) (Cat no. 9532) (Cell Signaling Technology, Danvers, MA), membranes were washed with TBS-T and then incubated with horseradish peroxidase anti-rabbit immunoglobulin G in the second reaction. Enhanced chemiluminescence reagent was used in accordance with the manufacturer’s recommendations, and the resulting membranes were exposed to Kodak AR film and developed using a Kodak (Eastman Kodak Company, Rochester, NY) X-OMAT processor. Caspases that are involved in the execution of apoptosis exist in living cells as inactive zymogens that become activated through cleavage of intracellular caspase cascades [14,15]. The effect of hyperthermia on H2O2-induced apoptosis was assessed by Western blot analysis on caspase-3 and PARP cleavage. This caspase was chosen for measurement because it is the most important member of the effector caspases responsible for orchestrating apoptosis [14,15].

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After immunoblotting, bands were quantified using MultiGauge (version 3.1), the Fuji camera accompanying software (Fujifilm, Fuji Medical Systems Stanford, CA). Bands were normalized with b-actin and compared with the corresponding control band.

2.7.

Protein concentration assay

Protein concentration was determined in all studies by the Bradford Method, which used the BioRad Protein Assay Reagent and bovine serum albumin as a standard. The Bradford assay is a colorimetric protein assay based on the absorbance shift of the dye Coomassie Brilliant Blue. Protein stabilizes the blue form of the dye, thus the amount of complex present in the solution is a measure for the protein concentration and can be estimated by the use of an absorbance reading.

2.8. Measurement of apoptosis (histone-associated DNA fragmentation) For detection of histone-associated DNA fragmentation, a cell death detection enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals, Indianapolis, IN) was used according to the manufacturer’s instructions. Histoneassociated DNA fragmentation directly correlates with an increase in apoptosis [16,17]. In the early stages of this type of cell death, the endogenous endonucleases cleave doublestranded DNA at the most accessible internucleosomal linker regions, generating mono-and oligo-nucleosomes. In contrast, the DNA of the nucleosomes is tightly complexed with the core histones H2A, H2B, H3, and H4, and therefore is protected from cleavage by endonucleases.

2.9.

Statistical analysis

Fig. 1 e Effect of heat shock on AGS cells and H2O2-induced apoptosis: Hsp expression, Caspase-3, and PARP cleavage. Heat shock pretreatment induces Hsp70 without increasing Hsp90. Caspase-3 and PARP are cleaved by H2O2, indicative of apoptosis. Heat shock pretreatment decreases H2O2-induced caspase-3 and PARP cleavage. b-Actin is used as a control for equal loading. Results are representative of three independent experiments. Numbers under each blot are densitometry results reported as percent change from control (C) after being normalized against b-actin.

All data are expressed as the mean
standard error. At least three independent experiments were performed for each component of our study. Statistical analysis was performed using the analysis of variance (SigmaStat 3.1 software; Systat Software, San Jose, CA) with significant differences determined at P < 0.05.

demonstrated that heat pretreatment decreased caspase-3 activation (i.e., cleavage) when compared with H2O2 exposure alone and maintained this decreased activation in the presence of H2O2 exposure. In addition, PARP, which is activated downstream from effector caspases, showed decreased inactivation (i.e., cleavage) into the 85-kDa cleaved protein with heat pretreatment, both alone and when challenged with H2O2.

3.

Results

3.3. Effect of hyperthermia on H2O2-induced apoptosis: histone-associated DNA fragmentation

3.1.

Effect of hyperthermia on AGS cells: Hsp expression

To determine whether hyperthermia induces Hsp expression in AGS cells, such cells received no pretreatment or were exposed to heat pretreatment. Western blot analysis of inducible Hsp70 and Hsp90 showed heat pretreatment markedly increased expression of Hsp70 without any change in Hsp90. Results are shown in Figure 1.

Enzyme-linked immunosorbent assay analysis showed a significant increase in histone-associated DNA fragmentation when compared with control when cells were treated with 1 mM H2O2. There was a statistically significant decrease in histone-associated DNA fragmentation when cells were first pretreated with heat and then treated with 1 mM H2O2 (18-fold increase versus fivefold increase). These results are shown in Figure 2.

3.2. Effect of hyperthermia on H2O2-induced apoptosis: caspase-3 and PARP cleavage

3.4. Effect of hyperthermia on H2O2-induced apoptosis: cell morphology

Cells either received no pretreatment or were exposed to heat pretreatment. Cells were then treated with 1 mM H2O2 for 24 h. As shown in Figure 1, Western blot analysis of caspase-3

To determine whether hyperthermia truly resulted in cell salvage, cell morphology was compared among cells treated with 1 mM H2O2 alone and those pretreated with heat shock

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3.5.

Quercetin studies

3.5.1. Effect of quercetin on H2O2-induced apoptosis: Hsp expression To determine the effect of quercetin on Hsp expression, cells received no pretreatment or were pretreated with quercetin (100 mM) for 4 h and then treated with 1 mM H2O2. Quercetin pretreatment caused a decrease in Hsp70 expression as shown in Figure 4.

3.5.2. Effect of quercetin on H2O2-induced apoptosis: caspase3 and PARP cleavage

Fig. 2 e Effect of heat shock on H2O2-induced apoptosis: histone-associated DNA fragmentation. Apoptosis measured as histone-associated DNA fragmentation was expressed as fold increase versus untreated cells. Results show control, treatment with 1 mM H2O2 at 24 h, heat shock, and heat shock pretreatment with subsequent treatment with 1 mM H2O2 at 24 h. Cells pretreated with heat shock showed statistically significant decrease in histoneassociated DNA fragmentation. Results are expressed as the mean ± standard error of three experiments. Statistical significance was determined using the analysis of variance test. *versus control; **versus 1 mM H2O2 24 h.

and then treated with 1 mM H2O2. Micrographs of AGS cells treated with 1 mM H2O2, with and without heat pretreatment, are shown in Figure 3. Cell damage was much more profound when cells were treated with H2O2 alone than when pretreated with heat and then treated with H2O2.

After ascertaining that quercetin resulted in a decrease in Hsp70 expression, we endeavored to determine whether this effect correlated with an increase in H2O2-induced apoptosis as measured by caspase-3 and PARP cleavage. We found that pretreatment with quercetin did in fact result in an increase in caspase-3 cleavage (activation) and PARP cleavage (inactivation) as shown in Figure 4. This pretreatment also enhanced caspase-3 cleavage and PARP cleavage when cells were challenged with H2O2.

3.5.3. Effect of quercetin on H2O2-induced apoptosis: histoneassociated DNA fragmentation Results obtained from Western blotting of caspase-3 and PARP after quercetin pretreatment correlated with histoneassociated DNA fragmentation data. H2O2 treatment resulted in a marked increase in histone-associated DNA fragmentation (40þ fold increase over control). Quercetin pretreatment by itself resulted in a small increase in histone-associated DNA fragmentation (approximately fivefold increase over control), and had no protective effect against H2O2 treatment. These results are depicted in Figure 5.

Fig. 3 e Effect of heat shock on H2O2-induced apoptosis: cell morphology. Micrographs of AGS cells treated with 1 mM H2O2 with and without heat pretreatment. Cell damage and injury were decreased by heat pretreatment.

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Fig. 4 e Effect of quercetin on AGS cells and H2O2-induced apoptosis: Hsp expression, caspase-3, and PARP cleavage. Quercetin causes a decrease in Hsp70 and Hsp90. Caspase3 and PARP are cleaved by H2O2, indicative of apoptosis. Quercetin pretreatment increases H2O2-induced caspase-3 and PARP cleavage. b-Actin is used as a control for equal loading. Results are representative of three independent experiments. Numbers under each blot are densitometry results reported as percent change from control (C) after being normalized against b-actin.

3.5.4. Effect of quercetin on H2O2-induced apoptosis: cell morphology To determine whether quercetin elicited any protective effects against H2O2 treatment when assessed morphologically, cell morphology was compared among control cells, cells treated with H2O2 alone, and cells pretreated with quercetin and then treated with H2O2. Characteristic micrographs are shown in Figure 6. Such results show that quercetin pretreatment actually increases the morphologic signs of cellular damage caused by H2O2.

3.6.

Geldanamycin studies

3.6.1. Effect of geldanamycin on H2O2-induced apoptosis: Hsp expression To determine the effect of geldanamycin on Hsp expression, cells either received no pretreatment or were pretreated with geldanamycin (0.2e1 mM) for 24 h before treatment with 1 mM H2O2. Geldanamycin pretreatment resulted in an increase in expression of Hsp70 and increased cleavage of Hsp90 as shown in Figure 7.

3.6.2. Effect of geldanamycin on H2O2-induced apoptosis: caspase-3 and PARP cleavage After determining that geldanamycin resulted in an increase in Hsp70 and an increase in cleavage of Hsp90, we determined the effect of geldanamycin pretreatment on apoptosis as measured by caspase-3 and PARP cleavage. Despite the increase in Hsp70, pretreatment with geldanamycin resulted in an increase in caspase-3 and PARP cleavage as shown in Figure 7.

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Fig. 5 e Effect of quercetin on H2O2-induced apoptosis: histone-associated DNA fragmentation. Apoptosis measured as histone-associated DNA fragmentation was expressed as fold increase versus untreated cells. Results show control, treatment with 1 mM H2O2 at 24 h, quercetin, and quercetin pretreatment with subsequent treatment with 1 mM H2O2 at 24 h. Cells pretreated with quercetin showed a trend toward increased histone-associated DNA fragmentation. Results are expressed as the mean ± standard error of three experiments. Statistical significance was determined using the analysis of variance test. *versus control; ** versus H2O2 and quercetin and H2O2. (Color version of the figure is available online.)

3.6.3. Effect of geldanamycin on H2O2-induced apoptosis: histone-associated DNA fragmentation Results obtained from Western blotting of caspase-3 and PARP correlated with histone-associated DNA fragmentation data. H2O2 treatment resulted in a marked increase in histoneassociated DNA fragmentation (19-fold increase over control). Geldanamycin pretreatment by itself resulted in a minimal increase in histone-associated DNA fragmentation (1.2-fold increase over control that did not achieve statistical significance). Although not statistically significant, geldanamycin pretreatment along with H2O2 treatment showed an increase in histoneassociated DNA fragmentation over H2O2 treatment alone (22fold increase over control). These results are depicted in Figure 8.

3.6.4. Effect of geldanamycin on H2O2-induced apoptosis: cell morphology To determine whether geldanamycin truly resulted in increased cellular damage caused by H2O2 treatment, cell morphology was compared among control cells, cells treated with H2O2, and cells pretreated with geldanamycin and then treated with H2O2. Representative micrographs are shown in Figure 9. These results show that geldanamycin pretreatment does not prevent apoptosis but actually increases the morphologic signs of cellular damage caused by H2O2.

4.

Discussion

The gastric epithelium is constantly exposed to a variety of noxious substances. Acute and chronic gastric mucosal injury

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Fig. 6 e Effect of quercetin on H2O2-induced apoptosis: cell morphology. Micrographs of AGS cells treated with 1 mM H2O2 with and without quercetin pretreatment. Cell damage and injury were increased by quercetin pretreatment.

secondary to such damaging agent exposure remains an important clinical problem. Oxidative damage from ROS has been implicated as the final common pathway of cellular injury and death in many pathologic conditions involving the gastrointestinal tract including ischemiaereperfusion injury, hemorrhagic shock, and H pylori exposure [18,19]. Consequently, there is a need to develop novel strategies to protect the gastric mucosa against such oxidative damage. One

Fig. 7 e Effect of geldanamycin on AGS cells and H2O2induced apoptosis: Hsp expression, caspase-3, and PARP cleavage. Geldanamycin induces Hsp70 while decreasing Hsp90 expression. Caspase-3 and PARP are cleaved by H2O2, indicative of apoptosis. Geldanamycin pretreatment increases H2O2-induced caspase-3 and PARP cleavage. bActin is used as a control for equal loading. Results are representative of three independent experiments. Numbers under each blot are densitometry results reported as percent change from control (C) after being normalized against b-actin.

strategy of cellular protection is called adaptive cytoprotection [20]. This process involves pretreatment of cells with a nonlethal dose of cellular stress resulting in a higher survival after subsequently applying a lethal dose of the same or other injury [21]. Heat shock pretreatment is one type of cytoprotection where heat treatment (i.e., hyperthermia) is used to induce a conserved heat shock response that protects

Fig. 8 e Effect of geldanamycin on H2O2-induced apoptosis: histone-associated DNA fragmentation. Apoptosis measured as histone-associated DNA fragmentation was expressed as fold increase versus untreated cells. Results show control, treatment with 1 mM H2O2 at 24 h, geldanamycin, and geldanamycin pretreatment with subsequent treatment with 1 mM H2O2 at 24 h. Cells pretreated with geldanamycin showed a trend toward increased histone-associated DNA fragmentation. Results are expressed as the mean ± standard error of three experiments. Statistical significance was determined using the analysis of variance test.

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Fig. 9 e Effect of geldanamycin on H2O2-induced apoptosis: cell morphology. Micrographs of AGS cells treated with 1 mM H2O2 with and without geldanamycin pretreatment. Cell damage and injury were increased by geldanamycin pretreatment.

living organisms against subsequent challenge from a lethal injury [22]. Currently, antiulcer drugs mediate their actions either through (a) the elimination of aggressive factors (mainly acid) as occurs with such agents as proton pump inhibitors [23] or histamine receptor blockers (H2 blockers) [24] or (b) the enhancement of defensive mechanisms (mainly mucus and bicarbonate secretion) as occurs with the agent sucralfate [25]. If another level of protection could be added through the induction of Hsps in the gastric mucosa, this would provide an additional means of defense against mucosal damage. The results of the present study support such a strategy. Heat shock pretreatment induces the synthesis of diverse Hsps with the capability of eliciting protective effects against a

Fig. 10 e Schematic of proposed pathway through which H2O2 triggers apoptosis and Hsp70 affects apoptosis. (Color version of the figure is available online.)

wide range of cellular stresses [26]. This group of substances comprises a protein family with a highly conserved structure. Hsps are present in both eukaryotic and prokaryotic cells and orchestrate their effects through fundamental cellular processes. Originally, they were discovered in cells after exposure to elevated temperatures, thus earning the nomenclature of Hsps [27]. However, many stress factors are able to induce these proteins including hypoxia, ultarviolet radiation, viral transformation, and cytotoxic agents [28,29]. Hsps are classified into different subfamilies based on their molecular weight and sequence homology [6]. Of particular, therapeutic interest is the Hsp70 family. This family is composed of molecular chaperones that have many roles in the cell including assistance in the folding and assembly of proteins, prevention of aggregation, participation in the immune response, protein degradation, and modulation of apoptosis [30e32]. Although the precise mechanisms of Hsp70 protection are unknown, there are several theories that have been proposed to account for its antiapoptotic actions. Hsp70 is thought to act at several levels including inhibition of the translocation of Bax, inhibition of mitochondrial release of cytochrome c, formation of apoptosome inhibitors of caspases, and modulation of c-Jun N-terminal kinases, nuclear factor kappa B, and Akt signaling pathways [33]. Our study demonstrated that heat shock pretreatment protects AGS cells against H2O2-induced apoptosis. This observation was supported by data from morphologic examination, caspase-3 and PARP cleavage, and histone-associated DNA fragmentation, all of which indicated protective effects against apoptosis. Although the specific mechanism is unclear, we propose that the cytoprotective effects of heat shock are secondary to increased Hsp70 expression. Such a contention is consistent with previous observations demonstrating that Hsp70 upregulation is protective against

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models of injury in cardiomyocytes [34,35], renal cells [36], pulmonary cells [37], and astrocytes [38,39]. Although heat shock itself is fairly nonspecific, our proposal of the protective nature of Hsp70 was further supported by our experiments where Hsp70 was downregulated with a corresponding obviation of its protective action from pretreatment with quercetin. Quercetin, a flavone group compound has been shown to inhibit Hsp70 expression by blocking the binding of heat shock factor 1 to the heat shock element [40]. Of further note, quercetin has been observed to sensitize pancreatic cancer cells [41], prostate cancer cells [42] and melanotic cells [43] to damage likely thorough its anti-Hsp70 activity. Our experiments are consistent with these findings by demonstrating that quercetin did in fact downregulate Hsp70 levels in AGS cells with a subsequent increase in apoptosis. Furthermore, the dose of quercetin (100 mM) used in our studies is similar to that used in these other reports. Finally, the observation that quercetin induced a modest degree of apoptosis under control conditions (see Fig. 5) raises the question of whether this occurred through targeting basal levels of Hsp70 or whether other apoptotic mechanisms were activated. The multitargeting effect of quercetin in activating apoptosis has been observed by other investigators [44e46]. Nonetheless, increased expression of Hsp70 did not completely protect cells against oxygen free radical damage in our model of injury. The Hsp mechanism is a complex process and it is difficult to manipulate one Hsp without affecting others. This is illustrated in our use of geldanamycin, a drug known to increase Hsp70, yet also decreasing Hsp90. Being one of the most abundant proteins in eukaryotic cells, upregulation of Hsp90 is known to increase cell survival through its effects on two different pathways. First, it interferes with apoptosis at the mitochondrial level, and second, it alters the function of survival proteins through various signal transduction pathways [47]. Interestingly, geldanamycin has been noted in several studies to elicit potent antitumor effects [48,49] secondary to its ability to directly bind to Hsp90 and inhibit the formation of Hsp90 multichaperone complexes [47]. Downregulation of Hsp90 therefore would lead to increased cellular damage with stress. In our model of cell death, we found no protection against cellular damage as evidenced by cell morphology, caspase-3 and PARP cleavage, and histone-associated DNA complex formation. Although geldanamycin increased Hsp70, it correspondingly decreased Hsp90, cleaving it into smaller fragments. We postulate that this is the reason that geldanamycin was unable to confer protection against free oxygen radical damage despite causing an increase in the expression of Hsp70. The findings demonstrated in our study clearly show that heat shock pretreatment protects gastric mucosal cells against H2O2-induced apoptosis; and that Hsp70 seemingly plays a key role in this process. Our results further suggest that inhibition of Hsp70 through quercetin treatment and decreases in Hsp90 through geldanamycin treatment may induce mucosal injury in response to oxygen free radicals. Such observations have potential clinical implications for at least two diseases. The first is the risk of gastroduodenal mucosal injury from substances and situations that mediate their effects through oxygen free radical damage. Thus, agents

that induce heat shock, by increasing Hsp70, may provide a novel approach to dealing with oxygen free radical-induced gastric mucosal damage. A second area of potential clinical relevance is in the prevention of gastric cancer. Germaine to this issue are studies that have attempted to use agents, which decrease Hsp70 and Hsp90 as treatment for cancers such as lymphoma and melanoma, as well as those arising from the pancreas, cervix, prostate, and breast [50]. Our findings suggest that agents that downregulate Hsp70 may be an adjunctive strategy in gastric cancer therapy. On the basis of the observations presented in this report, a schematic representation of the possible mechanism of heat shock protection against oxidative damage is shown in Figure 10. Oxidative damage, mediated by ROS, has been implicated as a major cause of cellular injury and death in various pulmonary disorders, cardiovascular disease, and neurodegenerative dysfunction, and in our laboratory gastroduodenal mucosal injury. Under physiological conditions, 95% of molecular oxygen undergoes controlled reduction in the mitochondrial cytochrome oxidase system to form water. The rest of molecular oxygen is univalently reduced to reactive oxygen intermediates, namely superoxide anion (O 2 ), hydrogen peroxide (H2O2), and the hydroxyl radical (OH). In situations where excessive amounts of ROS accumulate, cellular damage commonly occurs. Therapeutic targeting is hampered by an incomplete understanding of the etiologic factors underlying pathogenesis. One approach that has been used to protect gastric mucosa against oxygen radicaleinduced injury has focused on increasing intracellular antioxidants. In this regard, induction of Hsps has been proposed as a novel means of preventing injury induced by free radicals. The present study was undertaken to determine whether Hsps could be effective in mediating cellular death in gastric mucosal cells exposed to H2O2. H2O2 is generated from nearly all sources of oxidative stress and can diffuse freely in and out of cells and tissues. Our results demonstrated that Hsp70 appears to be protective with upregulation and detrimental to cell survival with downregulation. Translation from bench to bedside requires further study to investigate the mechanisms and potential therapeutic advantages of modulation of Hsp70 expression in gastric mucosa.

Acknowledgment These studies were supported in part by a grant from the AD Williams Foundation awarded to Dr T.A.M. The authors express their gratitude to Ms Dalila Marques and Dr William Pandak of the Gastroenterology Division for their help with the densitometry studies. This article is original research. Authors’ contributions: A.M.L., M.J.R., and T.A.M. contributed to study conception and design; A.M.L. and M.J.R. were responsible for acquisition of data; A.M.L., M.J.R., and T.A.M. analyzed and interpreted the data; A.M.L., M.J.R., and T.A.M. drafted the manuscript; A.M.L. and T.A.M. made the critical revision of the article.

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Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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