Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received January 7, 2004; accepted March 15, 2004
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ABSTRACT |
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Key Words: cyclooxygenase; arsenic; PD98059; SB202190; MAP kinase.
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INTRODUCTION |
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Arsenic exposure through drinking water is normally in the form of either arsenite (trivalent) or arsenate (pentavalent), and the increased cancer risk, particularly for skin cancer, is attributed primarily to the presence of inorganic trivalent arsenic (Rossman, 2003). This is supported by evidence that sulfhydryl groups, which have a high affinity for trivalent arsenic, are abundant in keratin (Leonard and Lauwerys, 1980
), and the production of organic arsenic metabolites that have demonstrated carcinogenecity (Hughes, 2002
) does not occur in human keratinocytes in vitro (Styblo et al., 1999
). Inorganic trivalent arsenic also modulates epidermal gene and protein expression (Germolec et al., 1996
; Hamadeh et al., 2002
) and behaves similar to tumor-promoting agents via its ability to modulate proliferation and mitogenic signaling (Simeonova and Luster, 2000
; Trouba et al., 2000
). However, unlike most tumor promoters, inorganic trivalent arsenic is a known carcinogen in humans. Although the mechanisms contributing to arsenic carcinogenesis are proposed to include genotoxicity, DNA methylation perturbations, oxidative stress, abnormal cell proliferation, cocarcinogenesis, and tumor promotion (Hughes, 2002
), proinflammatory mediators associated with arsenic skin carcinogenesis are hypothesized to contribute significantly to the tumor promoterlike activity of this metalloid (Germolec et al., 1996
; Vega et al., 2001
).
Expression of cyclooxygenase-2 (COX-2), an inducible proinflammatory enzyme that regulates eicosanoid synthesis, often is elevated during cancer and is induced by tumor promoters, inflammatory cytokines, and growth factors (Fosslien, 2000). COX-2 is procarcinogenic as demonstrated by experiments where tumorigenesis was inhibited in COX-2 knockout mice and cancer chemoprevention studies that employed nonsteroidal anti-inflammatory drugs (NSAIDs; Vainio, 2001
). Elevated eicosanoid (e.g., prostaglandin E2 [PGE2]) levels also occur in both basal and squamous cell carcinomas of the skin (Vanderveen et al., 1986
) and are associated with increased metastatic and invasive tumor behavior (Cuendet and Pezzuto, 2000
). Because COX-2 is proinflammatory and its overexpression sensitizes mouse skin for carcinogenesis (Muller-Decker et al., 2002
), the mechanism(s) by which inorganic trivalent arsenic modulates inflammatory and proliferative events in the skin may involve aberrant COX-2 expression and activity.
Mitogen-activated protein kinase (MAPK) pathways regulate cell growth, transformation, apoptosis, and inflammation and modulate COX-2 expression and activity. The MAPK family includes c-Jun NH2-terminal kinases (JNKs), extracellular signalregulated kinases (ERKs or p42/44 MAPK), and p38 MAPK/stress-activated protein kinases (SAPKs). MAPKs phosphorylate transcription factors (e.g., Elk-1) regulating cox-2 expression (Lasa et al., 2000) and accessory proteins (e.g., cPLA2) that play an active role in eicosanoid synthesis (Schmidlin et al., 2000
). Arsenic stimulates MAPK signaling events (Barchowsky et al., 1999
; Drobna et al., 2003
; Qu et al., 2002
; Simeonova et al., 2002
), possibly contributing to alterations in COX-2 expression/activity (Tsai et al., 2002
). However, simultaneous COX-2 modulation and MAPK activation by inorganic trivalent arsenic has not been demonstrated in epithelial cells, particularly in human keratinocytes, one of the primary targets for the induction of cancer by arsenic.
Because elevated COX-2 expression occurs in skin inflammation and cancer and keratinocytes are the primary site of COX-2 synthesis during these processes, we examined if sodium arsenite modulates COX-2 expression and PGE2 secretion in NHEK and the possible signaling mechanism(s) by which arsenite regulates COX-2 in keratinocytes.
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MATERIALS AND METHODS |
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Tissue culture.
Normal human mammary epidermal keratinocytes (NHEK), obtained from BioWhittaker/Clonetics, were cultured in monolayer at passage 6 in low calcium concentration (0.15 mM) growth medium (KBM-2) supplemented with EGF (5 ng/ml), bovine pituitary extract (BPE, 50 mg/ml), 0.5 mg/ml hydrocortisone, 50 µg gentamicin/ml, transferrin, and epinephrine. Cells were incubated at 37°C in a CO2-enriched atmosphere (5%). For all experiments, cells were cultured to 6080% confluence (2 to 3 days postplating). Subsequent treatment of cells with noncytotoxic concentrations of sodium arsenite (Hamadeh et al., 2002
) was performed in supplemented KBM-2 minus BPE and hydrocortisone (KBM-2
) to eliminate the mitogenic effects of BPE and anti-inflammatory effects of hydrocortisone. EGF present in KBM-2
did not interfere with MAPK activation by arsenite (unpublished data). PD98059, SB202190, or vehicle (DMSO) treatment was performed at the time of medium replacement (KBM-2
), 1 to 2 h prior to the addition of arsenite. In experiments employing PD98059, SB202190, aspirin, piroxicam, and NS-398, vehicle concentrations were maintained below 0.5%.
DNA synthesis.
NHEK were seeded into 24-well tissue culture plates (12,000 cells/well) in KBM-2 and grown to 6080% confluence. Medium was replaced with fresh KBM-2 containing 0, 1, 2.5, or 5 µM arsenite. Cells were labeled for 2 h with 1 µCi/ml [3H]-thymidine (Amersham Pharmacia, Piscataway, NJ) at 46 h of arsenite exposure and then washed several times with ice-cold Hank's balanced salt solution (HBSS). Following several washes with 10% trichloroacetic acid (Mallinckrodt, Phillipsburg, NJ), radioactivity was eluted using 0.3 N NaOH and [3H]-thymidine incorporation into DNA was quantified (counts per minute, CPM, are expressed as percentages of control) by scintillation counting. In experiments employing nonsteroidal anti-inflammatory drugs (NSAIDs), medium was replaced with fresh KBM-2
containing 2.5 µM arsenite in combination with 1000 µM aspirin, 10 µM NS-398, and 10 µM piroxicam. NSAID concentrations were based on therapeutic doses as previously reported (Dromgoole et al., 1983
; Futaki et al., 1993
; Hobbs, 1983
) and on cytotoxicity data for NHEK. NHEK cytotoxicity was quantified using the neutral red cytotoxicity assay and was not increased following coexposure to 2.5 µM arsenite plus aspirin, NS-398, or piroxicam at 1000 µM, 10 µM, and 10 µM, respectively (unpublished data).
Northern blotting.
NHEK were seeded into 150-mm tissue culture dishes (300,000 cells/dish) in KBM-2 and grown to 6080% confluence, at which time the medium was replaced with KBM-2 . Following a 1 to 2 h acclimation at 37°C, arsenite was added to a final concentration of 0, 1, 2.5, or 5 µM (in duplicate). After arsenite treatment, NHEK were washed with ice-cold HBSS, cell pellets were obtained from duplicate plates, and RNA was isolated using the RNeasy mini-prep system (Qiagen, Valencia, CA). Next, 20 µg aliquots of total RNA were fractionated by formaldehyde-agarose gel electrophoresis. Following transfer to nylon membranes and UV cross-linking, membrane-bound RNA was hybridized using QuikHyb solution (Stratagene, La Jolla, CA) to a 32P-labeled human probe for human cox-2 prepared using random primers methodology (Invitrogen). Membranes were exposed to phosphoimager screens and gene expression was visualized using a Molecular Dynamics phosphoimager and Image Quant software (Amersham Pharmacia).
Western blotting.
NHEK were seeded into 100-mm tissue culture dishes (100,000 cells/dish) in KBM-2 and grown to 6080% confluence, at which time the medium was replaced with KBM-2 . After a 1 to 2 h acclimation at 37°C, arsenite was added to the culture medium. Following arsenite treatment, cells were disrupted in ice-cold lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA [pH 8.0], 1% Triton X-100, 10% glycerol, 1 mM NaVO4, 1 mM NaF, 5 mM DTT, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) prior to protein quantification. Lysates were combined with NuPage LDS buffer (Invitrogen) under reducing conditions, and SDS-PAGE (15 µg cellular protein) was performed on 412% bis/tris gels (Invitrogen). Fractionated proteins were transferred onto Hybond (Amersham Pharmacia) nitrocellulose membranes and incubated with a goat polyclonal COX-2 antibody (Caymen Chemicals, Ann Arbor, MI). For MAPK analysis, membranes were incubated with polyclonal antibodies for either anti-phospho/active p42/44, p38, MEK-1, -2, nonphosphorylated p38 (Cell Signaling/New England Biolabs, Inc., Beverly, MA), or nonphosphorylated p42/44 MAPK (Upstate Biotechnologies, Waltham, MA). A rabbit secondary antibody to IgG conjugated to horseradish peroxidase was used for chemiluminescence detection and proteins were visualized using the ECL detection system (Amersham Pharmacia), according to the manufacturer's instructions. Protein expression was quantified using a Molecular Dynamics laser densitometer and Image Quant software (Amersham Pharmacia).
PGE2 measurements.
NHEK were seeded into 12-well tissue culture dishes (15,000 cells/dish) in KBM-2 and grown to 6080% confluence, at which time the medium was replaced with KBM-2 containing 0, 1, 2.5, or 5 µM arsenite. Following treatment, culture medium was collected, centrifuged at 12,000 x g for 5 min to remove cell debris, and frozen at 80°C prior to analysis. PGE2 in culture medium was measured by enzyme immunoassay (Oxford Biomedical Research Inc., Oxford, MI), according to the manufacturer's instructions. Quantification was performed using a Molecular Devices (Sunnyvale, CA) kinetic microplate reader and SoftMax Pro software.
Statistical analysis.
When appropriate, analysis of variance (ANOVA) was performed. Following ANOVA, significant differences were determined by application of the Tukey-Kramer Multiple Comparisons Test or the Students t-test (p 0.05). All experiments were performed a minimum of three times unless otherwise indicated.
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RESULTS |
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DISCUSSION |
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In NHEK, sodium arsenite stimulated time- and dose-dependent changes in COX-2 expression and PGE2 secretion. Our data are consistent with a recent report by Tsai and coworkers showing the induction of COX-2 protein, mRNA, and PGE2 secretion in human umbilical vascular endothelial cells (HUVECs) by arsenite (Tsai et al., 2002). These authors demonstrated that modulation of COX-2 by arsenite was mediated through NF-
B. In their study and another (Bunderson et al., 2002
), where arsenite-induced COX-2 expression was monitored in bovine aortic endothelial cells (BAECs), induction of COX-2 occurred at arsenite concentrations five to ten times higher than those used in our study. Our data indicate that arsenite modulates COX-2 expression in NHEK at concentrations (e.g., submicromolar to low micromolar) within the range of those found in contaminated drinking water and in the urine of humans consuming contaminated drinking water (Gebel, 2001
).
Similar to previous studies, our western analyses consistently showed the presence of a protein doublet that migrated at the appropriate COX-2 molecular weight (70 kDa; Habib et al., 1993). Both bands in this doublet are thought to be COX-2; however, the higher molecular weight band is believed to represent the active form of this enzyme since COX-2 contains several tyrosine groups that are substrates for phosphorylation and are important in its enzymatic activity (Shimokawa et al., 1990
). Chemical modification of specific tyrosine residues by nitration also abolishes COX-2 enzyme catalytic activity (Deeb et al., 2002
). In our experiments, the intensity of the lower molecular weight band diminished as the concentration of arsenite increased (8 and 12 h, Fig. 2). Also, the MAPK inhibition experiments revealed that the lower molecular weight band was present to a greater extent following arsenite and inhibitor treatment (2.5 and 5 µM, Fig. 6). These data are consistent with arsenite stimulating an increase in active/phosphorylated COX-2. Because human epidermis is a source of prostaglandins and PGE2 regulates epidermal cell proliferation and cytokine secretion, it is noteworthy that changes in COX-2 expression and/or prostaglandin level may play a significant role in arsenite's tumor promoterlike activities.
The potential to modulate COX-2 in keratinocytes is strengthened by the fact that numerous MAPKs are activated by arsenic. In NHEK, arsenite stimulated p42/44 MAPK phosphorylation at concentrations similar to those reported previously (Barchowsky et al., 1999; Drobna et al., 2003
) and at concentrations near those found in contaminated drinking water (Gebel, 2001
). LaRochelle and coworkers (1999)
have reported p42/44 MAPK phosphorylation kinetics in response to keratinocyte growth factor (KGF/FGF-7) stimulation similar to those of this study; yet, the mechanism(s) and biological significance of biphasic (transient and delayed) kinase phosphorylation are not well understood. Interestingly, the kinetics of p42/44 MAPK phosphorylation stimulated by arsenite are different than those for EGF and suggest that low concentrations of arsenite initially target EGF-independent signaling in NHEK. EGF receptormediated signaling, nonetheless, is proposed to be a target of arsenic (Simeonova et al., 2002
; Wu et al., 1999
). Many studies examining the effects on MAPK signaling have employed significantly higher concentrations of arsenic (
100 µM; Liu et al., 1996
; Ludwig et al., 1998
). Although novel data have been derived from these studies, high-dose arsenic exposure is likely to modulate stress-, apoptotic-, or death-related eventsevents that may be more related to acute toxicity. This is in contrast to repeated, long-term, low-dose arsenic exposure, which would be predicted to produce chronic toxicity or carcinogenesis.
Our data suggest that both p42/44 and p38 MAPK signaling contribute to the regulation of COX-2 by arsenite, as treatment with PD98059 or SB202190 prevented maximal COX-2 induction. Inhibition of MEK-1, -2, or p38 did not completely abrogate the increases in COX-2, indicating that additional events contribute to COX-2 regulation by arsenite. Chen and coworkers reported that p42/44 MAPKs do not play a role in the stimulation of cox-2 expression by UVB radiation in immortalized human keratinocytes, whereas activation of p38 is required for cox-2 induction (Chen et al., 2001). These data, along with ours, indicate that the mechanisms involved in skin carcinogenesis are unique to the environmental insult and/or cell type. The finding that p38 phosphorylation is not stimulated transiently by arsenite yet treatment with SB202190 leads to a reduction in arsenite-induced COX-2 expression is difficult to resolve. One possibility is that arsenite stimulates p38 phosphorylation at a later time point(s) than those examined or, alternatively, that it induces/represses the action of a secondary mediator(s) that modulates p38 activation in a delayed manner.
COX-2 induction and MAPK activation occurred at concentrations of arsenite that also stimulated DNA synthesis. NSAIDs that inhibit COX enzymes are promising chemopreventative agents for human cancers including skin cancer (Marks and Furstenberger, 2000), and it has been suggested that the inhibitory effect of some NSAIDs on carcinogenesis occurs during the tumor promotion step (i.e., during proliferation; Castano et al., 1997
). Our findings indicate that aspirin, piroxicam, or NS-398 partially suppressed the stimulatory effect of arsenite on DNA synthesis and implicated COX enzymes in the modulation of keratinocyte proliferation by arsenite. Specific NSAIDs not only inhibit cyclooxygenases but also modulate mitogenic events including MAPK (e.g., ERK and JNK) and transcription factor (e.g., AP-1) activation (Huang et al., 1997
; Liu et al., 2003
). In some cases, MAPK inhibition takes place when NSAID treatment precedes mitogenic stimulation (e.g., following UV irradiation) but not when treatment occurs concomitant with or following stimulation (Huang et al., 1997
). In our experiments, arsenite was added concurrently with aspirin, piroxicam, or NS-398, and the stimulation of p42/44 phosphorylation by arsenite was not inhibited by pretreatment with either 10 or 30 µM NS-398 (unpublished data). The latter suggests that inhibition of early MAPK/transcription factor activation is not critical in arsenite-induced DNA synthesis attenuation following NSAID treatment.
In summary, the relationship between COX-2 induction and MAPK activation presents a possible mechanism(s) involved in arsenic-induced dermatotoxicity, preneoplastic events, and skin cancer, and one that may be of particular toxicological relevance. Altered COX-2 expression, mediated partially by MAPKs, would be predicted to contribute to skin carcinogenesis by influencing inflammation, apoptosis, proliferation, differentiation, metabolism, and immunity.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Environmental Immunology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 541-0870. E-mail: germolec{at}niehs.nih.gov
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