ToxSci Advance Access originally published online on November 8, 2006
Toxicological Sciences 2007 95(2):321-330; doi:10.1093/toxsci/kfl160
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Mitogenic Signal Transduction Caused by Monomethylarsonous Acid in Human Bladder Cells: Role in Arsenic-Induced Carcinogenesis
,1
* Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona
Department of Carcinogenesis, University of Texas M.D. Anderson, Smithville, Texas
2To whom correspondence should be addressed at College of Pharmacy, 1703 E. Mabel, PO Box 210202, Tucson, Arizona 85721. Fax: (520) 626-2466. E-mail: eblin{at}pharmacy.arizona.edu.
Received August 21, 2006; accepted October 30, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have shown that human bladder cells (UROtsa), a target of arsenic-induced cancer, can biotransform arsenite to monomethylarsonous acid (MMA(III)), which is more cytotoxic and capable of transforming the UROtsa cells following long-term, low-level exposure. Cyclooxygenase-2 (COX-2) causes hyperplasia in bladder cells and is considered a key biomarker in bladder cancer. To investigate the role of mitogenic pathway stimulation in MMA(III)-induced transformation, UROtsa cells were treated with 50nM MMA(III) for 12, 24, or 52 weeks and analyzed by Western blot for COX-2 expression. Elevations in COX-2 expression were noted following chronic MMA(III) exposure, and this induction increased with duration of exposure, suggesting that COX-2 or the signal transduction pathways responsible for COX-2 protein expression may play a role in MMA(III)-induced transformation. Acute exposure studies found MMA(III) treatment (10, 50, and 100nM, 4 h) induced COX-2 in UROtsa cells with the lowest doses (10 and 50nM) causing the strongest induction. Using pharmacological inhibitors of various pathways, it was shown that epidermal growth factor receptor (EGFR), extracellular signal–regulated kinase (ERK-1/-2), phosphoinositide 3-kinase (PI3K), and src were important in the induction of COX-2 by MMA(III). ERK-2 phosphorylation was verified by Western blot analysis with a peak at 15 min, and c-jun was translocated to the nucleus following 50nM MMA(III) treatment. To determine MMA(III) targets, receptors of the erythroblastosis oncogene family (ErbB) family were further investigated. Chronic MMA(III) exposure led to upregulation of the EGFR or ErbB1. Short-term MMA(III) treatment caused the phosphorylation of ErbB2 in its autophosphorylation site. To verify the importance of these signaling pathways to the growth of the MMA(III)-transformed UROtsa cells in soft agar, various inhibitors were used to block pathways and monitor cells growth. Pathways of importance in anchorage-independent growth of UROtsa cells chronically exposed to MMA(III) are src, PI3K, and COX-1 and -2. As COX-2 is an important mediator that contributes to carcinogenesis via promotion of cell proliferation, inhibition of cell death, induction of angiogenesis, and facilitation of invasion, and it is highly upregulated both acutely and chronically in the MMA(III)-transformed cells, it is likely that activation of the mitogen-activated protein kinase pathway and increased COX-2 expression is a plausible mechanism for MMA(III) bladder carcinogenesis.
Key Words: monomethylarsonous acid; COX-2; UROtsa.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Epidemiological studies demonstrated that environmental arsenic exposure causes bladder cancer (Guo et al., 1997
; Hopenhayan-Rich et al., 1996; Smith et al., 1998). Since previous work demonstrated that the arsenic metabolite monomethylarsonous acid (MMA(III)) can induce malignant transformation in human bladder (UROtsa) cells, it is of interest to identify possible mechanism(s) that drive malignant transformation. URO-MSC52 cells, a UROtsa cell line transformed by chronic MMA(III) exposure, formed squamous cell carcinoma (SCC) tumors when heterotransplanted into severe combined immuno-deficiency (SCID) mice (Bredfeldt et al., 2006). This correlates with previous data in the literature as SCC characteristics were seen in nude mice after heterotransplantation of cells transformed by As(III) (Sens et al., 2004). SCC of the bladder is a relatively rare form of bladder cancer though, with the most common form of bladder cancer induced by arsenicals in humans and rodents being urothelial or transitional cell carcinoma (TCC). The development of SCC after transformed UROtsa cells heterotransplantation into mice could be an artifact of the model system, as both transformed cell lines were derived from the parental cell line, UROtsa. Most likely though, it has to do with the actual process of malignant transformation in these cells giving them properties that more closely resemble SCC than TCC (Sens et al., 2004). Regardless of specific pathology of tumors, in western countries, TCC is the fourth most common cancer in men and the fifth most common cancer in women, making TCC and even the total disease of bladder cancer important to study (Johansson and Cohen, 1997).
The focus of this study is early events in the promotion of arsenic-induced carcinogenesis in in vitro models. The most likely mechanism behind the promotion of arsenic-induced carcinogenesis in UROtsa cells is chronic hyperproliferation. Increased cell proliferation may result in higher rates of spontaneous mutation that contribute to carcinogenesis (Cohen and Ellwein, 1991). Hyperproliferation is caused by the stimulation of mitogenic signaling pathways. As(III) stimulates mitogenic signal transduction pathways and generates reactive oxygen species, which are responsible for its tumor promoter action and can also cause the induction of genes expression, such as cyclooxygenase-2 (COX-2) (Germolec et al., 1996; Hamadeh et al., 2002; Trouba and Germolec, 2004; Vane et al., 1998; Vega et al., 2001).
COX-2 is an inducible enzyme responsible for eicosanoid synthesis and is also a molecular marker of oxidative stress. COX-2 expression is induced by mitogens and proinflammatory cytokines (Vane et al., 1998). Elevated expression of COX-2 is frequently observed in human malignancies, including bladder tumors (Eltze et al., 2005; Fosslien, 2000; Wadhwa et al., 2005). Wadhwa et al. (2005) investigated the expression of COX-2 in human bladder tumor specimens and found that approximately 84% of tumors tested had elevated COX-2 expression. In addition, COX-2 expression correlated with advancing T stage and grade of tumor, highlighting the importance of COX-2 not only as a mediator of bladder carcinogenesis but also as a key biomarker for disease state. Klein et al. (2005) recently demonstrated that COX-2 overexpression is able to cause transitional cell hyperplasia and TCC in vivo. In this study, COX-2 overexpression was achieved via keratin 5 promoter in transgenic mice. The impact of COX-2 overexpression was apparent as only transgenic mice developed transitional cell hyperplasia and TCCs of the bladder. Since COX-2 has been implicated as being a key mediator of bladder carcinogenesis, it is a potentially interesting target for chemotherapy or prevention. Several groups recently demonstrated that COX-2 inhibitors inhibit cell growth and induce apoptosis in bladder cancer cell lines (Gee et al., 2006; Mohseni et al., 2004). Thus, not only does COX-2 expression appear to be causative of bladder malignancies but also necessary for sustained survival.
Since COX-2 leads to increased cellular proliferation and appears to be crucial for bladder carcinogenesis, a mechanism by which MMA(III) may induce transformation of bladder cells is through aberrant COX-2 induction. Both mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways have been implicated in bladder carcinogenesis. Activation of either PI3K or MAPK pathways can cause induction of COX-2 protein. Either PI3K or members of the MAPK family of signal transduction proteins, c-jun NH2–terminal kinases (JNKs), extracellular signal–regulated kinases (ERKs or p42/44 MAPK), and p38 MAPK/stress-activated protein kinases can phosphorylate transcription factors responsible for COX-2 expression (Lasa et al., 2000; Volanti et al., 2005). A number of studies demonstrated that As(III) can stimulate MAPK signal transduction pathways (Barchowsky et al., 1999; Drobna et al., 2003; Qu et al., 2002; Simeonova et al., 2002). In addition, As(III) activates PI3K in human prostate carcinoma (DU145) cells (Gao et al., 2004). Like As(III), MMA(III) activates MAPK pathway, specifically ERK-2 (Drobna et al., 2003). Simultaneous activation of either MAPK or PI3K followed by induction of COX-2 by MMA(III) has not been demonstrated in any human cell lines.
Chronic MMA(III) exposure causes UROtsa cells to form tumors in SCID mice (Bredfeldt et al., 2006). Therefore, it is likely that MMA(III) activates mitogenic signal transduction pathways that elevate COX-2 protein in UROtsa cells, which contributes to increased cellular hyperproliferation and malignant transformation. To address this hypothesis, COX-2 expression was investigated in URO-MSC52 cells and UROtsa cells acutely exposed to biologically relevant concentrations of MMA(III). Furthermore, pharmacological inhibitors were used to identify the signal transduction pathway responsible for COX-2 induction and to determine if inhibition of that pathway would attenuate the growth of a MMA(III)-transformed human bladder cell line, URO-MSC52.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Sodium arsenite, sodium vanadate (Na3VO4), Tris-HCl sodium chloride (NaCl), sodium fluoride (NaF), potassium chloride (KCl), sodium pyrophosphate (Na2H2P2O7), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), phenylmethylsulphonyl fluoride (PMSF), sodium deoxycholate (C24H39NaO4), protease inhibitor cocktail, epidermal growth factor (EGF), insulin, indomethacin, methanesulfonamide (NS-398), and dimethyl sulfoxide were purchased from Sigma Chemical Company (St Louis, MO). H89, BIM, OK, PP2, JNKi, SB205380, 4557W, and Wortmannin were purchased from Calbiochem (San Diego, CA). PD98059 and LY294002 were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Inhibitors were prepared according to manufacturer's protocol and frozen at – 20°C. Dulbecco's modified Eagle medium (DMEM), DMEM:F12, fetal calf serum (FBS), antibiotic-antimycotic, and 1X trypsin–ethylenediaminetetraacetic acid (EDTA) (0.25%) were acquired from Gibco Invitrogen Corporation (Carlsbad, CA). Noble agar was purchased from Amersham Biosciences (Piscataway, NJ). Diiodomethylarsine (MMA(III) iodide, CH3AsI2) was prepared by the Synthetic Chemistry Facility Core (Southwest Environmental Health Sciences Center, Tucson, AZ) using the method of Millar et al. (1960)
. Water used in studies was distilled and deionized.
Cell culture.
UROtsa cells were generously provided by Drs Mary Ann and Donald Sens (University of North Dakota). Stock cell cultures were grown on 75-mm2 plastic plates using DMEM enriched with 5% FBS and 1% antibiotic-antimycotic at 37°C in 5% CO2. For studies investigating signal transduction, cells were fed with a serum-free growth medium made up of 1:1 mixture of DMEM and Ham's F-12 supplemented with 1% antibiotic-antimycotic at least 24 h prior to dosing. Media was sterile filtered before use, and fresh growth media was given once every three days. At confluence, cells were removed from plastic using 0.25% trypsin:EDTA (1mM) and subcultured at a ratio of 1:4. Cells were allowed to become 70–85% confluent before experiments were conducted. MMA(III)-transformed UROtsa cells were obtained from the chronic exposure of UROtsa cells to 50nM MMA(III) for 52 weeks (Bredfeldt et al., 2006).
MMA(III) acid exposures.
Pure MMA(III) iodide was stored in ampules at 4°C. Fresh stock solutions of 25mM MMA(III) were made in distilled, deionized water. As previously reported by Gong et al. (2001), MMA(III) solutions in distilled, deionized water were stable for approximately 4 months at 4°C with no degradation observed when monitored using high-performance liquid chromatography–Inductively Coupled Plasma Mass Spectrometry. Stock solution was diluted to final concentrations of 1, 5, and 10µM for dosing. Cells were treated with 30 µl of dosing solution per 3 ml of media per well in six-well plates. Media containing MMA(III) was changed daily to ensure constant exposure to MMA(III).
Western blot analysis for COX-2 protein in cell lysates of UROtsa cells chronically exposed to MMA(III).
UROtsa cells were plated on 100-mm tissue culture plates during chronic exposure to 0.05µM MMA(III) (Bredfeldt et al., 2006). Cells were removed from media supplemented with MMA(III) for at minimum two passages before experimentation. After exposure of 12, 24, or 52 weeks, cells were rinsed with cold phosphate-buffered saline (PBS), removed from plates with trypsin:EDTA, and centrifuged. Cell pellet was snap-frozen in liquid nitrogen. Then, cell pellet was resuspended in radioimmunoprecipitation (RIPA) lysis buffer containing 50mM Tris-HCl (pH 8.6), 1% NP-40, 0.25% C24H39NaO4, 150mM NaCl, 1mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1mM NaF, 1mM Na3VO4, 1mM EDTA, and 10 µg/ml protease inhibitor cocktail. The lysates were sonicated and centrifuged at 14,000 rpm for 10 min at 4°C. Supernatant protein concentrations were determined by the Bicinchoninic Acid Kit for protein determination (BCA, Sigma-Aldrich, St Louis, MO). Thirty micrograms of each sample was loaded onto 8–12% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Samples were separated via SDS–polyacrylamide gel electrophoresis (PAGE) with Mini-Protean II (BioRad, Hercules, CA) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) and blocked overnight at 4°C with 5% nonfat dry milk in Tris-Buffered Saline Tween-20 (TBST). Blots were incubated for 3 h at room temperature or overnight at 4°C with primary antibodies for COX-2 (Cayman Chemical, Inc., Ann Arbor, MI) and glyceraldehyde phosphate dehydrogenase (GAPDH) (Calbiochem) at manufacturer's recommended dilution. The appropriate secondary antibody linked to horseradish peroxidase was used for detection of primary antibody. Chemiluminescent detection was performed with enhanced chemiluminescence (ECL) Western blotting substrate (Pierce Biotechnology, Inc., Rockford, IL or GE Healthcare, Piscataway, NJ). Images were scanned with a Scanjet 5370C (Hewlett Packard, Palo, Alto, CA) at maximum resolution and prepared in Adobe Photoshop 3.0 (San Jose, CA).
Western blot analysis of COX-2 protein in UROtsa cells acutely exposed to MMA(III) with or without pretreatment with kinase inhibitors.
UROtsa cells were plated 8 x 104 per well on six-well plates for Western blots. For studies using kinase inhibitors, cells were treated with pharmacological inhibitors 2 h prior to MMA(III) treatment. After MMA(III) (0–4 h) exposure, cells were rinsed with cold PBS and directly scraped into RIPA lysis buffer. The above protocol was used for Western blotting. Blots were incubated for 3 h at room temperature with primary antibodies for COX-2 (Cayman Chemical, Inc.) and GAPDH (Calbiochem) at manufacturer's recommended dilution. Proteins were detected via chemiluminescence, and images were scanned and prepared in Adobe Photoshop 3.0 as previously described in methods section. Recombinant COX-2 protein was purchased from Cayman Chemical, Inc. and resuspended in water following manufacturer's protocol.
Western blot analysis for Epidermal Growth Factor Receptor and ERK activation in UROtsa cells acutely treated with MMA(III).
For MAPK analysis, UROtsa cells were grown in medium without the presence of EGF or any stimulatory growth compound for 2 days prior to experimentation. UROtsa cells were plated in six-well plates (8 x 104) and treated with 0.05µM MMA(III) for 0–240 min. Cells were washed with ice cold PBS and scraped into RIPA buffer. Cell lysates were centrifuged at 14,000 rpm for 10 min at 4°C. Thirty micrograms of each sample were separated on 8–12% polyacrylamide gels and transferred to PVDF membranes. Following transfer, membranes were blocked with 5% nonfat milk and were incubated overnight at 4°C with phospho-p44/42 (phospho-ERK-1/-2), ERK-1/-2, or phospho-ErbB2 (Cell Signaling Technology, Inc.). The appropriate secondary antibody linked to horseradish peroxidase was used for detection of primary antibody. Chemiluminescent detection was performed as above described in methods section.
Nuclear fractionation for detection of nuclear translocation of c-jun.
UROtsa cells were seeded at a density of 8 x 104/dish in 36-mm plates and grown to 80–90% confluence. Cells were dosed with 0.05µM MMA(III) for 0–2 h. Nuclear fractionation protocol was adapted from Kosugi et al. (2001). Briefly, treated cells were removed from plates with trypsin and collected via centrifugation. Cell pellet was resuspended in hypotonic buffer containing 20mM HEPES, pH 7.4, 5mM KCl, 2mM MgCl2, 0.1% NP-40, 40mM Na2H2P2O7, 0.5% ß-mercaptoethanol, 1mM PMSF, 40mM ß-glycerophosphate, and 1mM Na3VO4 and incubated for 10 min on ice. Nuclei and other debris were pelleted via centrifugation through a 20% sucrose gradient at 10,000 rpm for 1 min. Pellet was resuspended in 800 µl of hypotonic solution and spun through sucrose gradient two more times. Final pellet was collected and resuspended in RIPA lysis buffer prior to Western blot analysis for nuclear translocation of c-jun. Membranes from Western blot analysis were incubated overnight with c-jun primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The appropriate horseradish peroxidase–conjugated secondary was used for chemiluminescent detection and data were collected and analyzed, as previously described in this section.
Western blot analysis for Epidermal Growth Factor Receptor protein in cell lysates of UROtsa cells chronically exposed to MMA(III).
UROtsa cells were collected during chronic exposure to 0.05µM MMA(III) (Bredfeldt et al., 2006). Following 12, 24, or 52 weeks of exposure, cells were rinsed with cold PBS, removed from plates with trypsin:EDTA, and centrifuged. Cell pellet was snap-frozen in liquid nitrogen. Then, cell pellet was resuspended in RIPA lysis buffer with protease inhibitor cocktail (Sigma), sonicated, and centrifuged at 14,000 rpm for 10 min at 4°C. Supernatant protein concentrations were identified via BCA assay. Thirty micrograms of each sample was loaded onto 8–12% SDS/polyacrylamide gels and separated via SDS-PAGE, transferred onto PVDF membranes, and blocked overnight at 4°C with 5% nonfat dry milk in TBST. Blots were incubated overnight at 4°C with primary epidermal growth factor receptor (EGFR) antibody at a 1:1000 dilution (Santa Cruz Biotechnologies, Inc.). As previously described, GAPDH was used as a loading control. For detection of primary EGFR antibody, blots were exposed to horseradish peroxidase–linked goat antimouse secondary antibody for 1 h at a 1:2000 dilution. Similarly, GAPDH primary antibody was detected with horseradish peroxidase linked with goat antimouse secondary antibody for 1 h at a 1:5000 dilution. Chemiluminescent detection was performed as mentioned using ECL Western blotting substrate. Images were scanned with a Scanjet 5370C (Hewlett Packard) at maximum resolution and prepared in Adobe Photoshop 3.0 (San Jose, CA).
Effect of kinase or COX-2 inhibitors on URO-MSC52 cell anchorage-independent growth.
Anchorage-independent growth was detected by colony formation in soft agar. For colony formation in soft agar, cell were removed from culture flask with trypsin and suspended in culture medium supplemented with 0.3% agar. The agar enriched with cells was overlaid onto 0.6% agar medium in a 24-well plate with a density of 1 x 104 cells per well. Cells were treated with inhibitors, PP2 (10µM), LY294002 (10µM), NS-398 (100µM), or indomethacin (100µM), every 2 days. After 14 days of incubation, colonies were manually counted with an Olympus CK2 microscope (Olympus America, Inc. Melville, NY). Data represent colonies formed in single plane of agar. Photographs were obtained with an Olympus IX70 microscope coupled to an Olympus camera (Olympus America, Inc). Analysis of photographs was conducted with Magnafire software (Optronics, Goleta, CA).
Effect of kinase or COX-2 inhibition on URO-MSC52 cell viability.
Alterations in mitochondrial activity were used as an indicator of cell viability. The methylthiazoletetrazolium (MTT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide) assay measures mitochondrial activity (Loveland et al., 1992). Cells were plated in six-well plates and treated with PP2 (0–50µM), LY294002 (0–50µM), indomethacin (0–100µM), or NS-398 (0–100M) for 48 h.
Statistical methods.
Data analysis was carried out using GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA). Graphs were generated in Microsoft Office Excel (Microsoft Corp., Redmond, WA). Data from the MTT assay and soft agar assay are expressed as the average of three experiments. These data are represented as the mean ± SEM. Specific statistical tests used to determine significance are described in figure legends.
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RESULTS |
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MMA(III) Concentration Selection
The concentration range for these studies was 0.01–0.1µM MMA(III), which was chosen for several reasons. Previous research demonstrated that 0.05µM MMA(III) was sufficient to induce malignant transformation in UROtsa cells (Bredfeldt et al., 2006
). Also, 0.01–0.1µM MMA(III) are concentrations found in human urine and make this study biologically relevant (Aposhian et al., 2000a; Mandal et al., 2001). These concentrations fall within the subcytotoxic range of MMA(III) to UROtsa cells.
COX-2 Is Induced in UROtsa Cells Chronically Treated with MMA(III)
Previous work has demonstrated that many bladder cancer tumor specimens have increased COX-2 expression (Eltze et al., 2005; Vollmer et al., 1998; Wadhwa et al., 2005). In addition to being an important biomarker in bladder cancer, inhibition of COX-2 inhibits bladder cancer cell growth in vitro and in vivo (Farivar-Mohseni et al., 2004; Gee et al., 2006; Mohammed et al., 2006). Western blot analysis of cell lysates harvested from UROtsa cells during malignant transformation via MMA(III) treatment, known as URO-MSC cells, illustrates that COX-2 is upregulated in a time-dependent fashion (Fig. 1). Normal UROtsa cells at high passage number similar to the URO-MSC cells were spot checked for increased COX-2 protein levels. These levels were consistent with low passage UROtsa cell levels of COX-2 protein (data not shown). This suggests that COX-2 induction is due to changes in the constitutive activity of signal transduction pathway responsible for induction.
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3) shows the time-dependent increase in COX-2 protein (72 kDa). Following COX-2 Western blot analysis, membranes were stripped and reprobed for GAPDH protein, which served as a loading control. Normal UROtsa cells at high passage number similar to the URO-MSC cells were spot checked for increased COX-2 protein levels. These levels were consistent with control cell levels (data not shown).Short-Term MMA(III) Treatment Induces COX-2 in UROtsa Cells
To demonstrate that COX-2 induction occurs due to MMA(III) treatment, naïve UROtsa cells were treated acutely with 0.01–0.1µM MMA(III) for 2 and 4 h. The strongest induction occurred following the 4-h treatment (Fig. 2). As(III) (1 and 10µM) was used as a positive control since previous studies have demonstrated that short-term treatment with As(III) causes COX-2 induction in normal human epidermal keratinocytes and vascular endothelial cells (Trouba and Germolec, 2004
; Tsai et al., 2002). Concentrations as low as 0.01 and 0.05µM MMA(III) induced COX-2. However, a reduction in COX-2 protein was observed at 0.1µM MMA(III), suggesting that COX-2 induction may be reduced by higher concentrations of MMA(III) or that the induction of COX-2 protein by higher concentrations of MMA(III) peaks at a time different from that of the lower concentrations.
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Signal Transduction Pathways Downstream of Epidermal Growth Factor Receptor Are Responsible for MMA(III)-Induced COX-2 Expression in UROtsa Cells
In order to determine which signal transduction pathway was responsible for MMA(III)-induced COX-2 expression, UROtsa cells were pretreated for 2 h with pharmacological inhibitors of various pathways, EGFR/ErbB2, src, PI3K, protein kinase A (PKA), protein kinase C (PKC), MAP/ERK kinase (MEK)-1 and -2, p38, JNK, and then exposed to 0.05µM MMA(III) for 4 h (Table 1). Following treatment, cell lysates were collected and analyzed via Western blot to determine if COX-2 induction could be blocked, thusly identifying the responsible signal transduction pathway. PP2, an inhibitor of src, PD98059, an inhibitor of MEK-1 and -2, 4557W and of both ErbB2 and EGFR, and LY294002, an inhibitor of PI3K, robustly blocked the induction of COX-2. Thus, MMA(III) appears to stimulate ligand-independent activation of EGFR, subsequent ERK-1 and -2 phosphorylation via MEK-1 and -2 as well activation of PI3K, which leads to elevations in COX-2 protein. Similar observations were previously made in UROtsa cells exposed to arsenic. Simeonova et al. (2002)
demonstrated that As(III) activates EGFR via src activation and subsequent ERK-1 and -2 phosphorylation in UROtsa cells. In addition similar observations were made in vivo where functional src is required for As(III)-induced activation of EGFR and ERK-1 and -2 in mouse urinary bladder following chronic arsenic exposure (Simeonova et al., 2002).
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MMA(III) Activates ERK Pathway in UROtsa Cells
As it appeared from the inhibitor pathway studies, low-level MMA(III) stimulates the EGFR ligand-independent pathways. To verify that ERK-1 and -2 phosphorylation did indeed lead to the COX-2 activation and increase, UROtsa cells were treated with MMA(III) (0.05µM, 0–120 min) and lysates were collected and analyzed via Western blot for ERK-1/-2 phosphorylation (Fig. 3A). Phosphorylation of ERK-1 and -2 in response to MMA(III) treatment (1µM) was observed by Drobna et al. (2003)
. In this study, ERK-2 was strongly activated, having a 30-fold increase in phosphorylation. ERK-1 was weakly activated with a fourfold increase in phosphorylation status. Although the antibody used was to both ERK-1 and -2, only ERK-2 activation was identified in the present study. At the concentration of 0.05µM MMA(III), ERK-2 is rapidly phosphorylated. Phosphorylation peaks following 15 min of treatment and then falls almost to baseline levels of activity. At 60 min, ERK-2 phosphorylation begins to increase again and stays consistently increased. This finding appears to reflect the effect of the low concentration of MMA(III) since the higher concentrations (1µM) cause phosphorylation status of ERK-2 to reach a maximum approximately 90 min after administration (Drobna et al., 2003).
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Further support for the activation of ligand-independent EGFR was collected via observation of increases in activator protein-1 (AP-1), a transcription factor downstream of ERK-1 and -2, nuclear translocation. UROtsa cells were treated with 0.05µM MMA(III) for 15–120 min. Following treatment, cell nuclei were separated from cytosol and analyzed via Western blot for c-jun, a component of AP-1 transcription factor. Figure 3B illustrates the observed increase in nuclear c-jun protein and suggests that AP-1 is activated following 0.05µM MMA(III) treatment.
EGFR Is Upregulated in URO-MSC52 Cells and Activated in UROtsa Cells Acutely Treated with MMA(III)
Following the MAPK pathway further upstream, it became necessary to take a closer look at the EGFR. To determine if changes in EGFR-associated signal transduction occurred with chronic MMA(III) exposure, URO-MSC52 cell lysates were isolated and Western blot analysis was performed to detect increases in EGFR protein. In URO-MSC52 cells, the relative amount of EGFR, also known as the oncogene ErbB1, was significantly elevated (Fig. 4A). This increase in EGFR/ErbB1 occurred in a time-dependent fashion, with a more apparent increase in protein at 52 weeks when compared with that at 24 weeks. This observation is consistent with gene array data collected from URO-MSC52 cells, which found ErbB to be elevated 10-fold compared with control UROtsa cells (data not shown). Numerous studies identified ErbB to be upregulated in bladder cancer tumor samples. In addition, bladder tumors with elevated ErbB or EGFR are more likely to be aggressive and invasive (Vollmer et al., 1998).
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To further demonstrate that MMA(III) causes the long-term induction seen in EGFR/ErbB1, acute studies were undertaken to determine alterations in signaling through the ErbB protein family phosphorylation status. Relative amounts of phospho-ErbB2 at site Y1221/1222 increased, suggesting activation of the receptor, which occurred via autophosphorylation (Fig. 4B). Phosphorylation of this site corresponds to activation of the MAPK/ERK cascade in a ligand-independent manner. The peak increase in phosphorylation was seen after exposure to MMA(III) for 1 and 4 hr, suggesting cyclical increases in protein levels.
Inhibitors of EGFR-Associated Signal Transduction Are Cytotoxic to URO-MSC52 Cells
To determine if signal transduction downstream of EGFR was necessary for URO-MSC52 cell viability and survival, cells were treated with inhibitors of src (PP2, 0–50µM), PI3K (LY294002, 0–50µM), COX-1 and -2 (indomethacin, 0–100µM), and COX-2 (NS-398, 0–100µM) for 72 h. Cytotoxicity of various inhibitors was evaluated via MTT assay (Fig. 5). Inhibition of PI3K and src significantly altered viability of URO-MSC52 cells. Further supporting the previous inhibitor data shown with the COX-2 induction, the PI3K inhibitor LY294002 was the most efficacious inhibitor with an IC50 of approximately 3µM. PP2, and inhibitor of src, was the second most effective inhibitor with an IC50 of roughly 10µM. Chiang et al. (2005) also found PP2 to be cytotoxic to a panel of 6 bladder cancer cell lines (J82, T24, UM-UC-3, EJ, KK47, and HU456). Inhibitors of COX-1 and/or -2, indomethacin, and NS-398 were not overtly cytotoxic, having IC50 values of > 100µM. Evaluation of treated URO-MSC52 cells via light microscopy revealed that COX inhibitors appear to either arrest cell division or induce apoptosis. However, PI3K and src appear to have a more dominant role in survival and growth.
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Inhibitors of EGFR-Associated Signal Transduction Block Tumorigenic Qualities of Chronically Treated Cells
Since PI3K, src, and COX-1 and -2 maintain functional growth and survival in URMSC52 cell, it is important to determine whether inhibitors of this EGFR-associated signal transduction pathway also alter the tumorigenic qualities of URO-MSC52 cells. Colony formation, or anchorage-independent growth, in soft agar is a quality frequently possessed by cancer cell lines. Previous work demonstrated that URO-MSC52 cells grow in soft agar (Bredfeldt et al., 2006
). URO-MSC52 and control cells were treated with PP2, LY294002, indomethacin, and NS-398 (Fig. 6). In all treatment groups, colonies were smaller and fewer in number. However, similar to cytotoxicity studies, LY294002 was again the most efficacious inhibitor of colony formation in soft agar, inhibiting colony formation approximately 90%. PP2 inhibited colony formation by 50% with all colonies being significantly smaller than untreated URO-MSC52 cells. Indomethacin also inhibited colony formation. In general, indomethacin-treated colonies were smaller and less dense in population. Similarly, NS-398 reduced colony formation in soft agar. The effects of indomethacin and NS-398 were somewhat different than PP2 and LY294002. While PP2 and LY294002 appeared to cause cytotoxicity to URO-MSC52 cells, indomethacin and NS-398 simply block anchorage-independent growth. Thus, cells treated with indomethacin or NS-398 were frequently alive, but not forming colonies. This reveals that src and PI3K play more important roles in the malignant transformation of UROtsa cells than do the COX-1/2 pathways. COX-1 and -2 are more important for sustaining the rapid proliferation and anchorage-independent growth important in the actual process of metastasis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Since chronic MMA(III) treatment causes UROtsa cells to hyperproliferate and form tumors in SCID mice (Bredfeldt et al., 2006
), it is of interest to investigate whether this arsenical promotes stimulation of mitogenic pathways in bladder cells in vitro. An important point of this study is that the levels of MMA(III) used closely resemble the levels seen in human urine of exposed populations throughout the world (Aposhian et al., 2000). Therefore, this study investigated the role of an environmentally relevant concentration of MMA(III) in the alteration of signal transduction. The role of the identified pathways in the maintenance of tumorigenic qualities of URO-MSC52 cells was also investigated. It is hypothesized if MMA(III) does promote hyperproliferation in UROtsa cells, MMA(III)-induced signaling alterations through the elevation of COX-2 protein levels may be a key mechanism of arsenic-induced bladder cancer.
Elevated expression of COX-2 is frequently observed in tumors of the urinary bladder (Eltze et al., 2005; Fosslien, 2000; Wadhwa et al., 2005). Recently, COX-2 overexpression was shown to cause transitional cell hyperplasia and TCC of the bladder in transgenic mice (Klein et al., 2005). In URO-MSC52 cells, COX-2 expression increases in a time-dependent fashion. The increased COX-2 expression correlates with phenotypic alterations, wherein UROtsa cells become malignant. For example, COX-2 expression is detected in URO-MSC cells following 12 weeks of exposure to 0.05µM MMA(III). The relative amount of COX-2 continued to increase following 24 and 52 weeks exposure to MMA(III). Since cell lysates were isolated from UROtsa cells following chronic treatment, it was unknown if COX-2 induction was exclusively due to MMA(III) or constitutive over activity in signal transduction. To address this question, UROtsa cells were treated with MMA(III) (0–0.1µM, 4 h) short term. Western blot analysis demonstrated that MMA(III) induced COX-2 protein at concentrations as low as 0.01µM. This is the first study to demonstrate that MMA(III) induces COX-2 expression in both short-term and chronic treatment models. However, previous work observed that As(III) treatment causes elevated COX-2 expression and prostaglandin E2 secretion (Trouba and Germolec, 2004; Tsai et al., 2002). Trouba and Germolec (2004) demonstrated that COX-2 was induced by ERK-1/-2 activation in Normal human epidermal keratinocytes (NHEK) cells. Although Drobna et al. (2003) observed that 1µM MMA(III) increased ERK-1/-2 levels, it was previously unknown if low nanomolar dose of MMA(III) could activate ERK.
Similar to work from Trouba and Germolec (2004), pharmacological inhibitors identified that MMA(III)-induced expression occurs via src activation, ERK-1/-2 phosphorylation, and PI3K. In this study, UROtsa cells were pretreated with kinase inhibitors 2 h prior to exposure with MMA(III). Western blot analysis of COX-2 protein revealed that inhibitors of EGFR/ErbB2, src, PI3K, and MEK-1 and -2 blocked COX-2 induction. Thus, MMA(III) activates src and ERK-1 and -2. Further evidence of this activation was shown by ERK-2 phosphorylation following 15 min of 0.05µM MMA(III) treatment. AP-1, a transcription factor downstream of ERK, was also activated by 0.05µM MMA(III) treatment, as demonstrated by increases in nuclear translocation of c-jun. Recently, Hour et al. (2006) detected increased c-fos, a protein that may be present in AP-1 dimers, in bladder tissues of humans drinking arsenic-contaminated water. Thus, not only is there in vitro evidence that this pathway is important in arsenic-exposed cells, there is also in vivo evidence that perturbations in this pathway occur in the human bladder following arsenic exposure.
The stimulation of ERK following arsenic treatment is believed to occur via EGFR/ErbB-mediated signaling (Simeonova et al., 2002; Wu et al., 1999). Acute MMA(III) exposure was found to stimulate phosphorylation of ErbB2 at an autophosphorylation site in UROtsa cells after 30 min of arsenical exposure. In contrast to previous studies in the literature, the time of ERK activation occurs before the ErbB2 activation as well as after. An increase in ERK phosphorylation occurred at both 15 and 60 min, suggesting that ERK is being activated in two different manners, possibly by MMA(III) directly and then again by the signaling cascade. The mechanism of MMA(III)- or As(III)-induced ERK activation is unknown and needs to be further investigated to determine the explicit role of ErbB2 and ERK-2 activation in MMA(III)-induced cellular transformation.
EFGR, also know as ErbB1, is upregulated in bladder cancer, suggesting that short-term and long-term perturbation in the activity in this signal transduction pathway are important in arsenic-induced carcinogenesis (Eltze et al., 2005; Lonn et al., 1993; Simeonova et al., 2002; Vollmer et al., 1998). Western blot analysis was performed on URO-MSC cells during transformation with MMA(III) to determine if relative amounts of this protein were increased. The amount of EGFR protein was increased in a time-dependent fashion, suggesting that EGFR and downstream effectors are important to the growth and survival of URO-MSC cells. Western blot analysis was also performed on acute exposure of UROtsa cells to 0.05µM MMA(III), which showed phosphorylation of ErbB2 at a distinct autophosphorylation site. Another important factor is that inhibition of ErbB2/EGFR also results in the decrease of COX-2 protein levels, even after MMA(III) exposure, suggesting that it is important in the signaling cascade that occurs after MMA(III) exposure.
To determine if the hypothesis that EGFR-related signaling was indeed crucial to URO-MSC52 cell growth, EGFR and downstream signaling targets of EGFR (src, PI3K, and COX-1 and/or -2) were inhibited via pharmacological inhibitors. First, the toxicity of inhibitors of src, PI3K, and COX-1 and/or -2 were assessed via MTT assay to determine if any of these signaling proteins were necessary for URO-MSC52 cell growth and survival. Inhibitors of src (PP2) and PI3K (LY204002) were the most toxic to URO-MSC52 cells, having approximate IC50 values of 10 and 3µM. Only higher concentrations (> 100µM) of COX-1/-2 (indomethacin) and COX-2 (NS-398) were significantly toxic to URO-MSC52 cells.
The inhibitors of src, PI3K, and COX-1 and/or -2 were further investigated to determine which would prevent anchorage-independent growth of the URO-MSC52 cells. The ability to form colonies in soft agar was used as a measure of anchorage-independent growth. All four inhibitors reduced the ability of URO-MSC52 cells to form colonies in soft agar, which was observed as smaller and fewer colonies. A number of studies have investigated the role of inhibitors of src and COX-1 and/or -2 in bladder cancer cells. Previous studies by Mohseni et al. (2004) found similar results, wherein COX-1 and/or -2 inhibitors reduced the proliferation of bladder cancer cells via apoptosis. Similarly, Gee et al. (2006) found that COX-2 inhibitor NS-398 significantly inhibited growth of UM-UC-1 and UM-UC-6 transitional carcinoma cells. Chiang et al. (2005) demonstrated that inhibition of src reduced the invasive capacity of a number of bladder cancer cell lines, including J82, T24, UM-UC-3, EJ, KK47, and HU56. In that study, invasion was measured with the matrigel invasion assay wherein cancer cells migrate through a membrane coated with matrigel. Like src, COX-2 is also important for anchorage-independent growth. Choi et al. (2005) found that COX-2 expression is necessary for the prevention of anoikis, or detachment-induced apoptosis, in the human bladder cancer cell line EJ. The authors hypothesized that COX-2 specifically activates PI3K kinase/Akt pathway to promote cell survival and invasiveness. The study presented herein does not completely agree with that hypothesis because it appears that PI3K pathway has developed autonomy that is independent of COX-2 expression in URO-MSC52 cells. This observation may be due to the overexpression of EGFR, which would independently activate PI3K pathway and provide a mechanism that bypasses the dependency of this pathway on COX-2 expression. This hypothesis is further supported by the observation that src and PI3K, proteins directly downstream of EGFR, appear to be more important for URO-MSC52 cell survival and anchorage-independent growth. From these data, it is apparent that PI3K, src, and COX-1 and -2 are necessary for the survival and growth of URO-MSC52 cells.
In summary, this study identifies a relationship between MMA(III)-induced perturbation in EGFR-related signal transduction wherein COX-2 protein is expressed following EGFR activation and ERK phosphorylation (Fig. 7). COX-2 is an important inflammatory mediator that contributes to carcinogenesis in various organs via promotion of cell proliferation, inhibition of cell death, induction of angiogenesis, and facilitation of invasion. In this study, the overexpression of COX-2 appears to be tied to mitogenic signaling pathways, thereby stimulating hyperproliferation. Since MMA(III) causes UROtsa cells to constitutively express COX-2 at early stages during MMA(III)-induced malignant transformation of UROtsa cells, it is a possible biomarker and target for chemoprevention of arsenic carcinogenesis (Castano et al., 1997; Marks and Furstenberger, 2000; Trouba and Germolec, 2004).
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NOTES |
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1 These are the first authors.
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ACKNOWLEDGMENTS |
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The authors would like to thank Drs Donald and Maryann Sens for their generous donation of UROtsa cells. The authors would also like to thank Dr Eugene Mash and Dr B. Jagadish for the synthesis of MMA(III) through the Southwest Environmental Health Sciences Core. The research herein was made possible by the National Institute of Environmental Health Sciences–supported Superfund Basic Research Program Grant (NIH ES04940) and the SWEHSC (ES06694). T.B. and K.E. are funded by an NIEHS training grant (ES07091).
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