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Carcinogenesis Advance Access originally published online on August 19, 2005
Carcinogenesis 2006 27(2):328-336; doi:10.1093/carcin/bgi213
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Carcinogenesis vol.27 no.2 © Oxford University Press 2005; all rights reserved.

Potentiation of tumor formation by topical administration of 15-deoxy-{Delta}12,14-prostaglandin J2 in a model of skin carcinogenesis

Olga Millán 1, 2, {dagger}, Daniel Rico 1, 2, {dagger}, Héctor Peinado 3, Natasha Zarich 4, Konstantinos Stamatakis 5, Dolores Pérez-Sala 5, José M. Rojas 4, Amparo Cano 3 and Lisardo Boscá 1, 2, *

1 Instituto de Bioquímica, CSIC-UCM, 28040 Madrid, Spain, 2 Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain, 3 Instituto de Investigaciones Biomédicas, CSIC-UAM, Departamento de Biqouímica, 28029 Madrid, Spain, 4 Unidad de Biología Celular, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain and 5 Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain

* To whom correspondence should be addressed. Tel: +914531200; Fax: +914531245; Email: lbosca{at}cnic.es


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of prostaglandins on the development of papillomas has been investigated in mice receiving prostaglandins E2 (PGE2) or the cyclopentenone 15-deoxy-{Delta}12,14-PGJ2 (15dPGJ2) topically, using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced tetradecanoylphorbol acetate (TPA)-promoted model of skin carcinogenesis. The presence of 15dPGJ2 during DMBA and TPA treatment inhibited apoptosis and increased the rate, number, size and vascularization of the papillomas, some of them progressing into carcinomas. Moreover, skin sections from mice treated for one week with DMBA and 15dPGJ2 showed a much reduced rate of apoptotic cells, and an enhanced expression of vascular epithelial growth factor when compared with animals receiving DMBA, with or without PGE2. The analysis of molecular events in the MCA3D keratinocyte cell line showed that 15dPGJ2 activated Ras and improved cell viability by inhibiting DMBA-dependent apoptosis. In addition to this, cell adhesion was impaired in MCA3D keratinocytes co-treated with 15dPGJ2 and DMBA, at the same time when the expression of cyclooxygenase-2 (COX-2) was observed under these conditions. These effects mediated by 15dPGJ2 might contribute to understand the role of COX-2 metabolites in carcinogenesis, leading to an increase of cell viability after mutagenic injury and therefore in the progression of tumors.

Abbreviations: COX-2, cyclooxygenase-2; CyPG, cyclopentenone PG; 15dPGJ2, 15-deoxy-{Delta}12,14-PGJ2; DMBA, 7,12-dimethylbenz[a]anthracene; MMP9, matrix metalloproteinase-9; PG, prostaglandins; PGE2/PGJ2, prostaglandin E2/J2; TPA, tetradecanoylphorbol acetate; VEGF, vascular epithelial growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Mouse skin is one of the best characterized epithelial tissues and possesses an organized cell population mainly derived from epidermal stem cells (1–3). The study of carcinogenesis in this model has identified a series of cellular changes, with transitions that define stages in the tumorigenic process—hypoplasia, dysplasia, benign papillomas and finally the appearance of carcinomas—with each step characterized by specific genetic alterations (3). The conclusion from these studies is that various cell types from the epidermal stem cell compartment can originate tumors of differing malignancy. A protocol for chemical carcinogenesis is the well-defined two-stage induction in which a low dose of mutagen, usually 7,12-dimethylbenz[a]anthracene (DMBA), acts as the initiator, and repetitive exposure to the tumor promoter tetradecanoylphorbol acetate (TPA) generates the appearance of papillomas (4,5). Most of these (>90%) exhibit mutations in H-ras (6–8), although only a few progress to transformation into carcinomas (9,10).

In the course of papilloma formation several changes have been identified that may contribute to their development. One such change is the expression of cyclooxygenase-2 (COX-2), which appears to be overexpressed in papillomas. Biochemical, pharmacological and genetic approaches indicate that overexpression of COX-2 and local delivery of certain prostaglandins (PG) promote the development of papillomas, not only in response to chemical carcinogens, but also after the exposure to other skin insults, such as UV radiation (11–18). In this regard, inhibition of COX-2, or of certain specific PG receptors such as EP2 (19), protects the skin against the development of papillomas (8,12,13,17,20,21). In addition, H-Ras is activated in the early stages of skin carcinogenesis and may also contribute to the development of papillomas. We have shown previously that the cyclopentenone PG (CyPG)—15-deoxy-{Delta}12,14-PGJ2 (15dPGJ2)—modifies (by alkylation) and specifically activates H-Ras through the formation of a Michael's adduct on cysteine 184 (22). In the present work we have analyzed the effects of the topical application of this CyPG on the initiation and promotion phases of skin carcinogenesis in terms of papilloma formation. Our data show that the presence of 15dPGJ2 during DMBA treatment significantly enhances the rate of formation, size and vascularization of the papillomas. Moreover, some carcinomas were evident at the end of the study in mice treated with 15dPGJ2 after DMBA initiation. In addition to this, we have used a keratinocyte cell line, MCA3D, which exhibits normal H-Ras, as a cellular model to analyze the biochemical changes that occur during the early stages of DMBA and PG treatment. These studies suggest that 15dPGJ2 induces H-Ras activation and cell protection of the apoptosis originated by DMBA, at the same time when the cell adhesion is impaired. These effects mediated by 15dPGJ2 might contribute to the selection of additional transformed cells by DMBA and TPA treatment.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
Swiss albino mice (n = 50) were housed individually under controlled environmental conditions in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Tumor promotion experiments
Six-week-old female Swiss mice were randomly divided into five groups of 10 mice. The dorsal skin in the inter-scapular area was shaved 2 days before tumor initiation, and animals showing no hair re-growth were used in the experiment. DMBA (32 µg dissolved in 200 µl of acetone per mouse) was used as a tumor initiator and was applied to the skin of all mouse groups. The DMBA was combined with PGE2 (60 µM for group 3) and 15dPGJ2 (60 µM for groups 5 and 6) (Table I). Commencing one week after the tumor initiation, mice were treated twice a week for 20 weeks with TPA (12.5 µg TPA dissolved in 200 µl of acetone) combined with PGE2 (60 µM, groups 2 and 3) or 15dPGJ2 (60 µM, groups 4 and 5). Mice from groups 1 and 6 were treated with TPA plus the vehicle (DMSO). Tumor promoting activity was evaluated by both the proportion of tumor-bearing mice and the number of tumors (>2 mm in diameter) per mouse (5,23). Animals were killed after the termination of the treatments by inhalation of an excess of carbon dioxide. Skin flaps from the inter-scapular area of the back of mice (1.5 x 2.0 cm) were excised, and papilloma samples (>5 mm) were taken for histopathology and biochemical analyses. Skin for biochemical studies was immediately frozen at –80°C, until used for the experiments.


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  		Table I. Experimental protocol of skin carcinogenesis

 
Histopathology examination and vascular epithelial growth factor measurement
Excised tumors were fixed in 10% neutral buffered formalin and then embedded in paraffin. Skin samples were excised and stained with hematoxylin and eosin. Alternatively, tumors were excised and immediately frozen in liquid-nitrogen-cooled isopentane and embedded in OCT for cryostat sectioning and immunofluorescence. When the levels of vascular epithelial growth factor (VEGF) were determined, skin samples were rapidly homogenized in lysis buffer (see below) and assayed using an ELISA kit (R&D Systems, MN).

Immunofluorescence analysis of tumors
Sections of 5–10 µm of the OCT-embedded samples from tumors were fixed in methanol and acetone (–20°C) and then incubated with primary antibodies. The primary antibodies used were rat monoclonal anti-mouse-CD31 (1:100) (BD-Pharmingen, Palo Alto, CA) and rabbit anti-mouse-MMP9 (Chemicon International, Temecula, CA). Secondary antibodies were goat anti-rat and goat anti-rabbit IgG coupled to Alexa 594 and Alexa 488, respectively (1:1000) (Molecular Probes, Eugene, OR). Apoptosis in skin sections was evaluated using TUNEL, following the instructions from the commercial supplier (Roche, Barcelona, Spain).

Keratinocyte cell line
Murine keratinocytic MCA3D cells were maintained at subconfluence in Ham's F-12 medium (Gibco, Madrid, Spain), supplemented with 10% fetal bovine serum (FBS), antibiotics (10 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml gentamicin) and 2 mM glutamine (24–26). The medium was replaced with the fresh medium containing 0.5% FBS 1 h before stimulation. When 15dPGJ2 was used, it was administered 15 min prior to DMBA addition.

Measurement of cell proliferation
Cells were seeded on flat bottomed 96-well plates. After 24 h of treatment, the incorporation of bromodeoxyuridine (BrdU) into the DNA of proliferating cells was measured by using the Cell Proliferation Elisa Kit No. 1 647 229 (Roche, Barcelona, Spain). The time allowed for BrdU incorporation was 6 h.

Measurement of cell migration
MCA3D transmigration assays were performed in 8-µm pore Transwell chambers (Costar, Bethesda, MD). Cells were resuspended in a medium containing 0.5% bovine fetal serum and seeded at 5 x 104 cells/well on collagen-coated filters, in the presence of the different treatments in the lower chamber. Cells were stained with propidium iodide (PI) and MCFDA (Molecular Probes, Eugene, OR) after 48 h of migration. Four fields of each transwell were analyzed by using confocal microscopy and the cells were counted in different planes. The transmigration distance was calculated by taking the upper surface of the porous membrane as a reference.

Caspase activation
In vitro caspase activity was measured in the soluble fraction after lysis of the cells in 200 µl of lysis buffer (20 mM EDTA, 0.5% Triton X-100, 5 mM Tris–HCl, pH 8.0) and spinning the lysate at 13 000 g for 5 min at 4°C (27,28). The following fluorescent probes were used according to the manufacturer's instructions (BD-Pharmingen, Palo Alto, CA): N-acetyl-DEVD-AMC for caspase 3, Ac-IETD-AFC for caspase 8 and N-acetyl-LEHD-AFC for caspase 9. To assess the specificity of the reaction the activity was measured in the presence of the corresponding peptide aldehyde inhibitor or z-VAD.fmk. Linearity of the caspase activity in the reaction was assessed over a 30 min period. In vivo caspase activity was measured by using the confocal microscopy analysis on a Radiance 2100 microscope (Bio-Rad, San Diego, CA). Cells were maintained in phosphate buffered saline (PBS) and loaded with the following fluorescent caspase substrates: CaspGlow green, caspase-3; CaspGlow red, caspase-8; and CaspGlow red, caspase-9. In addition to this, the binding of annexin V (Bender, Barcelona, Spain), and fluorescence of Hoechst 33342 (Sigma, St Louis, MO) were also measured. A 488 nm argon laser line was used to excite CaspGlow green, a 543 nm He–Ne laser line for CaspGlow red, a 635 nm diode laser for Annexin-V-APC and a 405 nm diode laser for Hoescht 33342. Fluorescences were recovered with 515/30 BP, 560 LP, 640 LP and 440/40 BP, respectively, and electronically analyzed.

Preparation of cell extracts and analysis by immunoblot
Cells seeded on 100 mm plates were stimulated for the indicated times and lysed in 400 µl of lysis buffer. Protein content was assayed using the Bio-Rad protein reagent. The relative amounts of IAP-1 and IAP-2, Bax, Bcl-2, plakoglobin, ß-catenin, E-cadherin, p53, COX-2 and ß-actin were determined in extracts by size-fractionating equal amounts of the total protein on 10–15% SDS–PAGE and transferred to Hybond P membranes (Amersham, Bucks, UK) (27). After blocking with 4% non-fat dry milk, membranes were incubated with the corresponding antibodies (1:1000 dilutions, except 1:500 for E-cadherin and 1:5000 for ß-actin). The blots were developed after incubation with horseradish peroxidase conjugated IgG (1:2000 to 1:25 000) using the standard ECL protocol (Amersham, Bucks, UK). Antibodies against COX-2, IAPs, Bax, Bcl-2, p53 and ß-actin were obtained from Santa Cruz Biotechnology. Antibodies against plakoglobin, ß-catenin, integrin-ß1 and E-cadherin were from Transduction Laboratories. Rat monoclonal anti-E-cadherin ECCD-2 (1:100) was provided by Dr M. Takeichi, Kyoto University, Japan.

Ras-GTP loading
Ras-GTP loading was performed as described previously (22,29). Ras-GTP was affinity retained with GST-Raf RBD (amino acids 1–149). Immunoblot analyses were performed as described above, using the anti-pan Ras antibody (Transduction Laboratories, Carpinteria, CA). All Ras-GTP levels were related to total Ras protein levels as determined by anti-Ras immunoblotting of the corresponding total-cell lysates.

FACS staining and analysis
After incubation of the plates with the indicated stimuli, floating cells were collected, stained and assayed in a CyAn flow cytometer (DakoCytomation, Carpinteria, CA). In vivo caspase 3 staining was achieved using the CaspGlow green caspase-3 staining kit. PI staining was performed using 0.005% PI. Apoptosis was analyzed by plotting PI fluorescence against caspase 3 fluorescence. Upper-left and upper-right quadrants correspond to necrotic cells, the lower-left quadrant corresponds to live cells, and the lower-right quadrant to apoptotic cells, as assessed by cell sorting of these populations as described previously (30).

Statistical analysis
Data were analyzed using the SPSS software (Chicago). ANOVA was used to evaluate statistical significance. Results are expressed as the mean ± SD of the indicated number of experiments. Statistical significance was estimated with Student's t-test for unpaired observations. A P-value of <0.05 was considered significant. For the analysis of the western blots, we used linear correlations between increasing amounts of protein and signal intensity.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
15dPGJ2 increases tumor formation in a model of mouse skin carcinogenesis
15dPGJ2 has been suggested to contribute to cell proliferation by enhancing H-Ras activation via a chemical modification of Cys 184 (22). To explore whether 15dPGJ2 would act similarly during cell transformation we investigated its effect on tumorigenesis in the well-defined two-step model of mouse skin carcinogenesis (5,23). In this model, one of the earliest events triggered by DMBA treatment is the mutation of H-Ras alleles (8). We therefore examined the effect of 15dPGJ2 during this initiation process. For this, we used the traditional protocol, in which tumor formation is initiated using DMBA and TPA acts as a tumor promoter (Table I). Papilloma appearance scores are shown in Figure 1A. The number of tumors in mice treated with 15dPGJ2 during tumor initiation (Group 5) was markedly increased from the eighth week of treatment as compared with Groups 1–4. We also observed 2 carcinomas among 20 papillomas after 20 weeks, suggesting an acceleration of malignant tumor transformation. When 15dPGJ2 was omitted from the promotion step (Group 6) the score profile converged progressively than that of Group 4. The other mouse groups showed similar rates of tumor formation, including the mice treated with the non-CyPG prostaglandin PGE2. However, the number of tumors in animals from Groups 3, 4 and 6 were slightly higher than in Groups 1 and 2.


Figure 1
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  		Fig. 1. Time course of papilloma formation and sample analysis. Animals were treated as described in Table I. The tumors in mouse dorsal skin were assigned scores weekly and are presented as the number of tumors per mouse (A). Tumors derived from the different groups were analyzed by immunostaining for CD31. See the higher number of blood vessels in group 5 derived carcinoma and papillomas (panels a and b, respectively) versus the other groups (B). Papillomas (pp) and the surrounding skin (sk) were homogenized and the levels of Ras-GTP, phospho-Erk and total Erk, and p38 MAPK were determined by ‘pull-down’ and immunoblot analyses (C). Results show one representative experiment of the 12 papillomas analyzed, and the levels of Ras-GTP and P-Erk (P-Erk/Erk) with respect to lane 1 (mean ± SD; n = 12). *P< 0.05; ** P < 0.005 versus the PGE2-treated group; aP < 0.01 versus the control (group 1) condition.

 
During this tumorigenic experiment the mice in Group 5 started to die 17–20 weeks after the treatment. In addition, the tumors in this group showed an irrigated phenotype, with very frequent bleeding zones. Analysis of the distribution of the angiogenic marker CD31 revealed an increased angiogenic response in papillomas from Group 5 mice (Figure 1B, panel b). We also analyzed a carcinoma from this group, which showed strongly enhanced angiogenesis in all the tumor extensions (Figure 1B panel a, data not shown). The angiogenic responses in papillomas from all the other mouse groups were weaker, and these animals exhibited no evident differences in phenotype or vessel formation from Group 1 control mice (Figure 1B, panels c and d versus e). In addition to the increase in CD31 expression, a parallel accumulation of matrix metalloproteinase (MMP)-9 levels was also observed around the angiogenic vessels (data not shown).

The effects of 15dPGJ2 on tumor number and malignant behavior led us to measure its effect on molecular and cellular parameters associated with tumor formation. Activation of Ras and Erk and p38 MAPKs has been well characterized in the mouse skin-carcinogenesis model (8). Erk activation was slightly more intense in tumors from animals treated with 15dPGJ2 (Figure 1C), but the activation of p38 was not evident at the time of the assay under any of the conditions assayed. Analysis of the apoptotic response in skin sections showed that the number of apoptotic cells was markedly reduced by treatment with 15dPGJ2 for 1 week, compared with treatment with DMBA alone or DMBA plus PGE2 (Figure 2A). This was accompanied by an enhanced expression of the pro-angiogenic factor VEGF (Figure 2B).


Figure 2
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  		Fig. 2. Inhibition of apoptosis and enhancement of VEGF in skin treated with 15dPGJ2/DMBA. Animals were treated for one week with the indicated PGs and DMBA at the same concentrations as in Table I and the skin samples were collected to evaluate apoptosis using TUNEL (green) (A), and to determine the local levels of VEGF using ELISA (B). Results show a representative TUNEL and the mean ± SD of three sections from five animals of each condition (apoptotic cells/100 nuclei (Hoechst)), or the corresponding values of VEGF. *P < 0.05; **P < 0.005 versus the control; aP < 0.001 versus DMBA.

 
15dPGJ2 protects cells from DMBA-induced apoptosis
To gain insight into the mechanism of action of 15dPGJ2 on skin-cell tumorigenesis, we used the mouse keratinocyte cell line MCA3D (24–26). Ras was inactive in untreated MCA3D cells and in cells treated with DMBA alone (Figure 3A). In contrast, 15dPGJ2 increased the levels of Ras-GTP. A quantitative analysis of these data is shown in Figure 3B. In agreement with these results, the phosphorylation of Erk and Akt—potential downstream targets activated by Ras—was notably enhanced by 15dPGJ2. Analysis of MCA3D proliferation in response to DMBA revealed a significant decrease in the number of cells incorporating BrdU (70% reduction), at the same time when there was a 4-fold increase in apoptosis (Figure 3C and D). Incubation with 15dPGJ2 did not significantly affect proliferation or apoptosis. However, in cells co-treated with 15dPGJ2 and DMBA, proliferation was decreased by only 23%, and the cells were protected from DMBA-induced apoptosis (Figure 3C and D). Moreover, analysis of the activation of caspases in MCA3D cells showed that DMBA significantly activated caspases 8 and 3. In agreement with the protective effect against DMBA-induced apoptosis, this activation of caspases 8 and 3 was attenuated by 15dPGJ2 (Figure 3E). Interestingly, caspase 9 activity remained quite low in response to all the treatments. An analysis of apoptosis, measured after the binding of annexin V and in situ activation of caspase 3 is also shown and confirms the data observed in cell extracts (Figure 3F).


Figure 3
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  		Fig. 3. Activation of Ras by 15dPGJ2 in MCA3D cells and protection against DMBA-induced apoptosis. Cells were preincubated for 15 min with 3 µM 15dPGJ2, and then 200 nM DMBA was added. The levels of Ras-GTP, phospho-Erk and phospho-Akt were determined (A). The quantitative determination of the Ras-GTP exchange levels is shown in (B). Proliferation (C), apoptosis (D) and caspase activation (E and F) were determined after 18 h. The activities of caspases 8, 9 and 3 were determined in cell extracts (E). The percentage of caspase 8 and annexin V positive cells was determined by confocal microscopy with a specific permeant fluorescent substrate and FITC-labeled annexin V, respectively (F). Results show a representative experiment out of the three (A) or the mean ± SD (n = 4) (B–E). (F) A representative staining of the cells, and the fluorescence distribution was evaluated and quantified (mean ± SD) are shown. *P < 0.01 versus the control condition. aP < 0.01 versus the DMBA condition.

 
To evaluate the mechanisms governing DMBA-dependent apoptosis and the protection exerted by 15dPGJ2, the protein levels of the IAP and Bcl-2 families were determined (27). While cIAP-1, cIAP-2 and xIAP levels did not change significantly in these cells regardless of the treatment, Bcl-2 levels decreased in the presence of 15dPGJ2 plus DMBA, and Bcl-xL levels increased under these conditions. Interestingly, Bax levels decreased in cells treated with 15dPGJ2 alone. In contrast, the levels of p53 increased moderately after treatment with 15dPGJ2 or DMBA, and an additive effect was observed in cells treated with both the agents (Figure 4); this is consistent with reports that CyPGs induce accumulation of p53 protein in other cell types (31,32).


Figure 4
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  		Fig. 4. Protein levels of members of the IAP and Bcl-2 families in MCA3D cells treated with DMBA and 15dPGJ2. Total protein extracts were prepared from the cells treated for 24 h, as indicated in Figure 2. The cell extract was fractionated by SDS–PAGE and the levels of proteins were determined by western blot. Results show one representative experiment out of the four (blots) and the mean ± SD of the intensity of the corresponding bands with respect to that of ß-actin (graphs). *P < 0.01 versus the control condition.

 
15dPGJ2 promotes the expression of cyclooxygenase-2 in MCA3D cells
COX-2 expression and increased PG production constitute an important factor in skin carcinogenesis, and COX-2 metabolites have been reported to induce resistance to apoptosis in epithelial cancer cell lines and to sensitize these cells to the action of carcinogens (12,13,20,21). Consistent with this, we observed that 15dPGJ2 induced COX-2 expression in MCA3D cells, an effect not mimicked by PGE2 or DMBA. Cells treated with DMBA plus 15dPGJ2 exhibited enhanced COX-2 expression (Figure 5). This response might represent a positive feedback mechanism that would perpetuate COX-2 expression and Ras activation in keratinocytes, as has been proposed in keratinocytic cell lines (17). All these data are compatible with an increase in the survival of cells co-treated with PGs, in particular 15dPGJ2, and DMBA.


Figure 5
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  		Fig. 5. COX-2 expression in MCA3D cells. Cell extracts were prepared after treatment for the indicated time periods with 3 µM 15dPGJ2 (A) or for 24 h with the vehicle (none, control) 3 µM PGE2 or 200 nM DMBA, as indicated (B). Levels of COX-2 protein were estimated by image scanning and are expressed in arbitrary units. Values are means of three estimates ± SD, 20 µg of the total protein were loaded per lane. ECL exposures are representative of three experiments with similar results.

 
15dPGJ2 decreases cell adhesion of DMBA-treated cells
In the course of these experiments we observed that cells treated with DMBA, alone or in combination with 15dPGJ2, became detached and could be collected from the culture medium (Figure 6A). Analysis of these cells revealed that most of the DMBA-treated cells were positive for caspase 3 activity and TUNEL, whereas pre-incubation with 15dPGJ2 potently protected cells against DMBA-induced apoptosis. This was accompanied by an increase in the number of detached cells over a period of up to 72 h (Figure 6B). Moreover, an examination of the expression of molecules related to cell adhesion showed important differences at 24 h between cells treated with DMBA alone versus DMBA plus 15dPGJ2: ß-catenin was released into the cytosol and nucleus in cells treated with DMBA, a process that was attenuated in the presence of 15dPGJ2, and E-cadherin was de-organized in most of the cells after treatment with DMBA, whereas integrin ß1 was organized in cells treated with 15dPGJ2 plus DMBA (Figure 6C). Interestingly, the absolute levels of ß-catenin and plakoglobin decreased after the DMBA treatment. Total levels of these proteins were detected in the corresponding cell extracts (Figure 6D). Together, these results suggest that combined treatment with DMBA and 15dPGJ2 causes changes in cell adhesion properties. Furthermore, cell migration studies showed that cells co-treated with 15dPGJ2 and DMBA had a higher incidence of trans-well migration (Figure 6E).


Figure 6
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  		Fig. 6. Cells treated with 15dPGJ2 and DMBA exhibit reduced adhesion and enhanced cell migration. Cell culture medium was collected at the indicated times and the cell layers were washed with 1 ml of PBS. The density of detached cells was determined in a flow cytometer. The percentage of apoptotic cells was determined as described in the section Materials and methods (A and B). The distribution of ß-catenin, E-cadherin, plakoglobin and integrin ß1 was determined by confocal microscopy with specific antibodies (C). The total levels were determined by western blot using specific antibodies (D). Cell migration was determined and evaluated by microscopy (E). Results show a representative experiment out of the four or the mean ± SD (n = 4). *P < 0.01 versus the control condition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this work we have investigated the ability of PGs, in particular of the cyclopentenone 15dPGJ2, to promote papilloma and tumor formation by epithelial cells. We had previously described a specific activation of H-Ras by 15dPGJ2 in NIH 3T3 and Cos1 cells (22), and based on this evidence we investigated whether topical application of this PG might increase the appearance of papillomas and tumors in the well-defined model of DMBA-induced, TPA-promoted skin carcinogenesis. Our data show a significant antiapoptotic effect of 15dPGJ2 after administration of DMBA (first week), as well as an increase in the local levels of VEGF. In agreement with these data, the development of papillomas in animals that were treated with 15dPGJ2 before initiation and then with PG during promotion with TPA exhibited an earlier appearance and increased size and number. The continued presence of 15dPGJ2 enhanced the angiogenesis, as reflected by the frequent bleeding of the papillomas and by the increase in vascular markers. These observations are compatible with the occurrence of more viable lesions in the presence of the PG. However, when 15dPGJ2 was only present during the initiation step with DMBA, the number of papillomas shifted to the same as observed in animals treated with PGE2, probably due to the expression of COX-2 and the endogenous release of prostaglandins. Finally, the observation of the progression of some papillomas to carcinomas revealed a positive selection of malignant cells exerted by 15dPGJ2. Interestingly, all these effects were not observed with PGE2, a prostaglandin that is not a cyclopentenone and unable to bind H-Ras (22), suggesting a straightforward relation between H-Ras alkylation and tumor development.

To gain insight into the molecular mechanisms that might contribute to the development of the tumors in animals, we used a keratinocyte cell line with normal Ras activity, MCA3D (note that Ras was active in the papillomas analyzed at the end of the treatments in the in vivo model of carcinogenesis). We observed a marked resistance to apoptosis in cells co-treated with 15dPGJ2 and DMBA versus the corresponding cells treated only with DMBA. The mechanisms suggested to mediate this effect involve the impairment of pro-caspase processing. However, the levels of classic antiapoptotic genes (IAP and Bcl-2 family) were quite similar, although Bax levels remained lower versus the DMBA condition. In addition to this, the activation of H-Ras by 15dPGJ2 and the downstream antiapoptotic signaling via PI3kinase/Akt may contribute to inhibit apoptosis and to prevent anoikis, as it has been described by various groups (33–35). Alternatively, the redistribution of ß–catenin to the nucleus detected in MCA3D treated with DMBA, but not when 15dPGJ2 was included, might contribute to protection against apoptosis, as suggested previously (24). In addition, an increased migration capacity was noted when cells were co-treated with DMBA and 15dPGJ2, a condition that might favor dissemination of the cells.

Genetic and pharmacological evidence suggests that overexpression of COX-2 is critical for epithelial carcinogenesis, and it provides a major target for cancer chemoprevention by non-steroidal antiinflammatory drugs (NSAIDs) (11,15,20,21,36–40). COX-2 is constitutively upregulated in papillomas and carcinomas induced by either chemical initiation/promotion or UV-irradiation (13,36). Moreover, transgenic COX-2 overexpression sensitizes mouse skin for carcinogenesis and leads to high levels of epidermal PGE2, PGF2 and 15dPGJ2. This is insufficient for tumor induction but transforms epidermis into an ‘autopromoted’ state, i.e. dramatically sensitizes the tissue for genotoxic carcinogens (41). Supporting this, administration of COX-2 inhibitors has been shown to prevent skin carcinogenesis and to induce the regression of pre-existing tumors (13). COX-2 could contribute to carcinogenesis by promoting cell proliferation and angiogenesis, and by protecting cells from apoptosis. In this connection, COX-2 has been shown to inhibit apoptosis in epithelial cancer cell lines (42), and its overexpression efficiently counteracts the apoptosis induced by p53 or genotoxic stress (43). It is interesting to note that in some cell types CyPG have been reported to induce conformational alterations of p53 that lead to loss of transcriptional capacity and impairment of p53-dependent apoptosis (31). Contrasting with this situation, it has been found in neuroblastoma cells that 15dPGJ2 induces apoptosis and overexpression of functional p53 (32). Our data show that p53 protein levels increase in the presence of DMBA and 15dPGJ2, suggesting an interplay between p53 and COX-2 that might have a regulatory role on apoptosis in epithelial cells (43).

In order to evaluate the pathological significance of 15dPGJ2 further work is required to establish the conditions under which it is synthesized by COX-2 expressing cells. While some authors reported difficulties in the measurement of changes in 15dPGJ2 synthesis under inflammatory conditions (44), others reported an enhanced synthesis and accumulation during the resolution phase of the inflammatory response (45,46). We have not measured the serum or papilloma levels of PGs, but in macrophages expressing COX-2, and to a lesser extent in MCA3D (work in progress), a clear accumulation of 15dPGJ2 in the culture medium has been detected.

Taken together, the results reported in this study suggest that CyPGs enhance cell viability and migration under conditions of genotoxic stress (DMBA), favoring the development of papillomas, an angiogenic response, and progression to carcinomas. The activation of Ras by these CyPGs may play a role in this process, potentially linking Ras activation to an increase of cell protection of apoptosis upon mutagenic injury and therefore leading to the development of cellular transformation and tumor progression.


   Notes
 
{dagger} The authors contributed equally to this work Back


   Acknowledgments
 
The authors thank Dr S. Bartlett for the help in the preparation of the manuscript. O.M., D.R., H.P., K.S. and N.Z. were recipients of fellowships from CAM, MCYT, CSIC and ISCIII, respectively. This research was supported by the grants SAF2003-02604, 03713 and 02604, SAF2002-00783 and SAF2001-2819 from MCYT, and the grants 08.4/0025.1/2003 from Comunidad de Madrid, FIS03-C03/10 from FISS and ON03 180-02 and ON04/179 from the Fundació la Caixa.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 14, 2005; revised July 21, 2005; accepted August 12, 2005.


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