15-Deoxy-{Delta}-12,14-prostaglandin J2 induces programmed cell death of breast cancer cells by a pleiotropic mechanism

Miguel Pignatelli1, Jinny Sánchez-Rodríguez1,2, Angel Santos3,4 and Ana Perez-Castillo1,4

1 Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, 4, 28029, Madrid, Spain, 2 Sección de Investigaciones Metabólicas y Nutricionales, Instituto de Medicina Experimental, Universidad Central de Venezuela, Venezuela and 3 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

4 To whom correspondence should be addressed Email: aperez{at}iib.uam.es or piedras3{at}med.ucm.es


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activation of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) has been found to induce cell death in a variety of cells. In this regard, we reported recently that 15-deoxy-{Delta}-12,14-prostaglandin J2 (15dPG-J2), a specific ligand of the nuclear receptor PPAR{gamma}, inhibits proliferation and induces cellular differentiation and apoptosis in the breast cancer cell line MCF-7. In addition to PPAR{gamma} activation other proteins, such as NF-{kappa}B and AP1, have been shown to be targets of 15dPG-J2. However, the mechanism by which 15dPG-J2 triggers cell death is still elusive. Our results demonstrate that 15dPG-J2 initiates breast cancer cell death via a very rapid and severe impairment of mitochondrial function, as revealed by a drop in mitochondrial membrane potential ({Delta}{Psi}m), generation of reactive oxygen species (ROS) and a decrease in oxygen consumption. In addition, 15dPG-J2 can also activate an intrinsic apoptotic pathway involving phosphatidyl serine externalization, caspase activation and cytochrome c release. Bcl-2 over-expression and zVADfmk, albeit preventing caspase activation, have no effect on 15dPG-J2-mediated mytochondrial dysfunction and loss of cell viability. In contrast, the addition of radical scavengers or rotenone, which prevent 15dPG-J2-induced ROS production, block the loss of cell viability induced by this prostaglandin. Finally, 15dPG-J2-induced cell death appears to involve disruption of the microtubule cytoskeletal network. Together, these results suggest that PG-J2-induced mitochondrial dysfunction and ROS production inevitably leads to death, with or without caspases.

Abbreviations: 15dPG-J2, 15-deoxy-{Delta}-12,14-prostaglandin J2; FCCP, p-(trifluoromethoxy)phenylhydrazone; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; TMRE, tetramethylrhodamine methyl ester perclorate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
15-Deoxy-{Delta}-12,14-prostaglandin J2 (15dPG-J2) is a naturally occurring cyclopentenone formed via dehydration of the 11-hydroxy group from the cyclopentane ring of prostaglandin D2 (1). This compound is involved in regulating many physiological processes, including cell division, immune responses, ovulation, adipogenesis and bone development. 15dPG-J2 also plays a role in the pathophysiology of carcinogenesis as well as in the inflammatory process (2,3). The molecular mechanisms underlying the effects of 15dPG-J2 are still poorly defined. The discovery that 15dPG-J2 is an activating ligand of the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) {gamma} allowed a better understanding of the function of this compound (4). PPARs are a family of at least three nuclear receptors ({alpha}, {delta} and {gamma}) that regulate transcription of distinct genes through heterodimerization with the retinoid X receptors (5). PPAR{gamma} is of great current interest because it participates in biological pathways of intense basic and clinical interest, such as differentiation, insulin sensitivity, type 2 diabetes, atherosclerosis and cancer (reviewed in refs 68).

Recently, PPAR{gamma} ligands were shown to inhibit the growth of a variety of transformed cells. Activation of PPAR{gamma} induces terminal differentiation in human liposarcoma cells (9) and colon carcinoma cells (10). Also, we and others have obtained evidence that treatment of breast cancer cells with 15dPG-J2 results in a more differentiated, less malignant state, a reduction in growth rate and an enhancement of apoptosis (1113). Specifically, we have shown that 15dPG-J2 almost completely blocks phosphorylation of some receptor protein tyrosine kinase and, therefore, play a suppressive regulatory role in the tumor growth of human breast carcinoma cells that express these receptors. Activation of PPAR{gamma} has also been reported to be involved in apoptosis regulation of other cell types such as synoviocytes (14), endothelial cells (15), macrophages (16) and malignant lymphocytes (17,18).

In addition to 15dPG-J2, PPAR{gamma} can be activated by other compounds, including the anti-diabetic thiazolidinediones (PPAR{gamma}-selective) ligands, polyunsaturated fatty acids and certain affinity tyrosine derivatives (7). Current knowledge indicates that 15dPG-J2 may also exert some of its effects via mechanisms unrelated to activation of PPAR{gamma} (1921). In this context, it was demonstrated that this compound could directly interfere with NF-{kappa}B signaling at different levels. Many studies have suggested that 15dPG-J2 represses NF-{kappa}B activation by inhibiting the I{kappa}B-kinase complex activity, thereby preventing I{kappa}B{alpha} degradation (22,23), and also by reducing NF-{kappa}B binding through alkylation of p50/p65 dimers (24). 15dPG-J2 is also a direct inhibitor of the ubiquitin isopeptidase in the proteasome pathway, which was shown to result in the initiation of cell death (25). Finally, cyclopentenone prostaglandins can induce apoptosis of macrophages, human hepatic myofibroblasts and neuroblastoma cells by a mechanism unrelated to PPAR{gamma} and involving oxidative stress (2628). However, the precise pathway of apoptosis induction of breast cancer cells by these metabolites is still unclear.

In the present study, we have investigated the mechanisms underlying 15dPG-J2-mediated cell death in MCF-7 human breast cancer cells. We show that 15dPG-J2 induces breast cancer cell death via a severe impairment of mitochondrial function and an increase in ROS production. Although 15dPG-J2 also activates an apoptotic intrinsic pathway involving cytochrome c release and caspase activation, our results suggest that this pathway is not required for the effects of PG-J2 on MCF-7 cell death. Furthermore, we demonstrate that 15dPG-J2 has a profound effect on microtubule organization, which could represent another pathway leading to MCF-7 cell death.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture and transfection
MCF-7 cells were propagated and maintained in RPMI medium (Life Technologies) containing 10% fetal bovine serum (FBS) at 5% CO2 and 37°C in saturated humidity. SKBR3 cells were cultured in DMEM medium containing 10% FBS. Experimental cultures were usually grown in serum-starved medium and, after 12 h of growth in these conditions, cells were stimulated with the appropriate ligand, as indicated.

For transient transfection experiments [3xKBtk-luc (29)], semi-confluent MCF-7 cells were transfected using Transfast (Innogenetics, Madison, WI), according to the manufacturer's recommendations. Twenty-four hours post-transfection, cells were harvested for determination of luciferase and ß-galactosidase (to determine transfection efficiency) activities by using a reporter assay system (Innogenetics). Each transient transfection experiment was repeated at least three times in triplicate.

For stable transfection (p65), MCF-7 cells were transfected with Transfast as indicated above. After 12 h of transfection, the medium was replaced and the cells were incubated for 24 h, after which they were subcultured at 1:15 dilution with the addition of geneticin (1 mg/ml). The growth medium was renewed every 3 days, and fresh geneticin was added. Cultures were expanded and then screened for p65 expression.

The MCF-7 cells over-expressing bcl-2 (MCF-7bcl2) were provided by Dr Lopez-Rivas (30).

Antibodies
Mouse monoclonal antibodies to caspase 7 and bcl-2 were from Stressgen (San Diego, CA). Mouse anti-PARP monoclonal antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Monoclonal anti-{alpha}-tubulin, anti-cytochrome c and anti-caspase 3 were from Sigma (St Louis, MO), Pharmingen Bioscience and Cell Signaling (Hitchin, Hertfordshire), respectively.

Western blot analysis
Equal amounts of total cellular protein were separated in 10% SDS–PAGE. After electrophoresis, proteins were transferred to BioTrace PVDF membranes (GelmanSciences, Ann Arbor, MI). Blots were blocked with 5% dry milk in PBS containing 0.5% Tween 20 for 60 min and probed with appropriate antibodies. After washing, membranes were incubated with peroxidase-conjugated secondary antibodies and specific proteins were detected with the enhanced chemiluminescence system (Amersham Pharmacia Biotech). In some cases, blots were reprobed with different antibodies after stripping for 30 min in a buffer of 62.5 mM Tris–HCl (pH 6.7), 100 mM ß-mercaptoethanol and 2% SDS.

Confocal microscopy
Confocal microscopy was used to measure depolarization of the mitochondrial membrane potential ({Delta}{Psi}m), cytochrome c release and cytoskeleton organization. MCF-7 cells were plated on glass coverslips in 24-well cell culture plates and grown in regular medium for 24 h before switching to serum-starved medium for 12 h. The cells were then treated or not with 15dPG-J2 (10 µM), loaded with 20 nM Mitotracker Red CMXRos (Molecular Probes, Leiden, The Netherlands) for 45 min, fixed for 10 min with methanol at –20°C, and washed with PBS. After a 1-h incubation with the appropriate primary antibody, cells were washed and incubated with an Alexa 488-labeled (Molecular Probes) secondary antibody for 45 min at 37°C. Subcellular localization was determined using a TCS SP2 laser scanning spectral confocal microscope (Leica Mycrosystems). The images were obtained using a series of 0.5 µm (depth) spaced cell fluorescent slices (Z axis).

Cell viability
Impaired cell viability was measured using the MTT assay (Roche Diagnostic, GmbH), based on the ability of viable cells to reduce yellow MTT to blue formazan. Briefly, cells were cultured in 96-well microtitre plates and exposed to 15dPG-J2 for various periods of time, then cells were incubated with MTT (0.5 mg/ml, 4 h) and subsequently solubilized in 5% DMSO/5 mM HCl for at least 2 h in the dark. The extent of reduction of MTT was quantified by absorbance measurement at 550 nm according to the manufacturer's protocol. Cell viability was also assessed by propidium iodide staining and subsequent FACS analysis.

Determination of apoptotic cells
To calculate the extent of cell death, MCF-7 cells were treated or not with 15dPG-J2 and phosphatidylserine exposure on the surface of apoptotic cells was detected by flow cytometry after staining with Annexin V-FITC (Bender MedSystems, Vienna, Austria). Flow cytometry was performed on a FACScan cytometer using the Cell Quest software (Becton Dickinson).

Analysis of mitochondrial transmembrane potential ({Delta}{Psi}m)
Variations of the mitochondrial transmembrane potential {Delta}{Psi}m in vivo during apoptosis of MCF-7 cells were studied using tetramethylrhodamine methyl ester perclorate (TMRE, Molecular Probes). TMRE is a cationic, membrane-permeant dye that accumulates in the negatively charged mitochondrial matrix in response to {Delta}{Psi}m. MCF-7 cells were treated for the indicated periods of time with 15dPG-J2 and incubated for 30–45 min at 37°C in the presence of 50 or 40 nM TMRE, as indicated, followed by analysis in a FACScan flow cytometer and confocal microscopy.

Measurement of cytochrome c release
For analysis of cytochrome c release, MCF-7 cells were trypsinized, washed with TD buffer and resuspended in 30 µl of PBS containing 80 mM KCl, 250 mM sucrose, 1 mM DTT, protease inhibitors and 500 µg/ml digitonin. Cells were incubated in the lysis buffer for 8 min at 4°C and collected at 10 000 g for 5 min. The obtained pellet represented the mitochondria-containing nuclear-heavy membrane fraction. The supernatant was respun for a further 5 min at 10 000 g. The second supernatant, representing the cytosol including the light-membrane fraction, was loaded on a 10% polyacrylamide gel, and cytochrome c release was analyzed by immunoblotting. In order to visualize cytochrome c in cells that had been labeled with 20 nM Mitotracker Red CMXRos (Molecular Probes), MCF-7 cells were grown in glass cover slips and immunofluorescence was performed as described above.

Measurement of ROS formation
Generation of ROS was assessed by using the oxidation-sensitive fluorescent probe 2',7-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes) at 10 µM, which is rapidly oxidized by ROS into its fluorescent derivative. Following a 60-min incubation with DCFH-DA, MCF-7 or MCF-7bcl2 cells were washed with PBS and incubated with 10 µM 15dPG-J2 or 30 µM rosiglitazone for 30 or 60 min, as indicated. In some cases cells were pre-treated with 5 µM rotenone for 30 min before the addition of 15dPG-J2. Cells were then harvested and analyzed on a FACScan flow cytometer.

Mitochondrial oxygen consumption
Mitochondrial respiration of whole cells was recorded at 30°C with a Clark-type oxygen electrode. Cells were incubated with or without 15dPG-J2 for the indicated times and resuspended in a buffer containing 1 mM sucrose, 50 mM KCl, 5 mM KH2PO4, 0.5 mM EDTA, 5 mM MgCl2 and 30 mM Tris–HCl at pH 7.4, before determination of oxygen consumption. In some experiments 2 µg/ml oligomycin (Sigma) or 5 µM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma) were added, as indicated.

Statistical analysis
The data shown are the means ± SD of at least three independent experiments. Statistical comparisons for significance between cells with different treatments were performed using the Student's test, with P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of 15dPG-J2 on MCF-7 cell viability and apoptosis
Treatment of MCF-7 cells with 15dPG-J2 or rosiglitazone, a member of the thiazolidinedione family of drugs, induced a significant loss of cell viability, measured by MTT, propidium iodide staining and subsequent flow cytometry analysis (Figure 1A and B). Analysis of an early marker of apoptosis, Annexin V staining, indicated that 25% (15dPG-J2) and 17% (rosiglitazone) of the MCF-7 cell population was induced to undergo apoptosis within 16 h after treatment with these compounds (Figure 1C). Similar results were obtained with the SKBR3 breast cancer cell line (data not shown).



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Fig. 1. Effect of 15dPG-J2 on MCF-7 cell viability and apoptosis. MCF-7 cells were incubated in the presence or absence of 10 µM 15dPG-J2 or 30 µM rosiglitazone for 24 and 48 h. Cell viability was determined by MTT assay (A) or propidium iodide staining (B), as indicated in the Materials and methods. (C) MCF-7 cells were treated with 10 µM 15dPG-J2 or 30 µM rosiglitazone for 16 h, afterwards the number of apoptotic cells were determined by Annexin V-FITC staining and FACS analysis.

 
Effects of 15dPG-J2 on mitochondrial transmembrane potential ({Delta}{Psi}m)
There is increasing evidence that a decrease in the mitochondrial transmembrane potential is associated with mitochondrial dysfunction, and an altered mitochondrial function is linked to apoptosis and loss of cell viability. Thus, we next examined the effect of 15dPG-J2 treatment on the mitochondrial transmembrane potential. We measured {Delta}{Psi}m using the fluorescent probe TMRE and monitored using flow cytometry and confocal microscopy. As shown in Figure 2A, upon addition of 15dPG-J2 the mitochondrial fluorescence starts to decay and is very low at 6 h of treatment. This 15dPG-J2-induced depolarization is relatively transient and between 12 and 24 h there is a partial recovery in fluorescence. Changes in in situ mitochondrial membrane potential can be also monitored by confocal microscopy using TMRE loaded at low concentration. TMRE was loaded at 40 nM into MCF-7 cells for 45 min, afterwards cells were exposed to 15dPG-J2 and images were taken every 10 min. As can be seen in Figure 2B, a general decrease in {Delta}{Psi}m was initially observed, however after 16 h of incubation with 15dPG-J2 some cells clearly recover the mitochondrial potential, indicating a heterogeneous response of MCF-7 cells to treatment with 15dPG-J2.



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Fig. 2. Loss of {Delta}{Psi}m in MCF-7 cells. (A) MCF-7 cells were cultured in the presence or absence of 10 µM 15dPG-J2 for the indicated periods of time. Thirty minutes before harvesting, cells were treated with 50 nM TMRE and analyzed by flow cytometry. (B) MCF-7 cells were loaded with 40 nM TMRE for 45 min and then treated with 15dPG-J2. Confocal images were captured at 10-min intervals. Bar scale, 10 µm. Representative results of three independent experiments are shown.

 
15dPG-J2 leads to subcellular translocation of cytochrome c
Mitochondrial factors, including cytochrome c, are released following the induction of apoptosis. Immunoblot analysis of cytosolic extracts prepared from control and 15dPG-J2-treated cells demonstrated an increase in the presence of cytosolic cytochrome c in cells incubated in the presence of 15dPG-J2, compared with controls (Figure 3A). To further confirm these results we performed confocal microscopy studies using specific anti-cytochrome c antibodies. As can be seen in Figure 3B, the anti-cytochrome c antibody revealed a diffuse extra mitochondrial localization of cytochrome c in the majority of the cells after treatment with 15dPG-J2, whereas in all untreated cells, cytochrome c had a punctuate distribution and co-localized with the mitochondrial marker Mitotracker Red CMXRos.



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Fig. 3. Subcellular localization of cytochrome c. (A) Representative western blot analysis of cytochrome c carried out in basal conditions or after 4 or 12 h incubation with 10 µM 15dPG-J2. Cytoplasmic extracts were obtained as described in the Materials and methods. (B) Confocal images of MCF-7 cells stained with anti-cytochrome c antibody. MCF-7 cells were grown on glass coverslips, treated or not for 12 h with 15dPG-J2, and incubated with 20 nM MitoTracker red CMXRos for 45 min. After fixing, immunofluorescence analyses were performed and fluorescence was visualized by confocal microscopy. Bar scale, 10 µm. Representative results of three independent experiments are shown.

 
15dPG-J2 causes mitochondrial redistribution and microtubule disruption
In the experiments described above, we also observed a profound change in mitochondrial morphology. The mitochondria of non-treated control MCF-7 cells displayed a characteristic elongated cigar-like phenotype. However, mitochondria from 15dPG-J2-treated cells had lost their usual cigar-like morphology, assumed a more rounded morphology and a perinuclear distribution pattern. In this regard, it is known that mitochondrial distribution and shape in many cell types appears to depend on the docking of mitochondria to microtubules. Moreover, the spatial distribution of mitochondria is important for their biological function, and cytoskeletal changes that disrupt their distribution can cause cell death. Analysis of the cellular tubulin network, identified by labeling with anti-{alpha}-tubulin antibody and subsequent immunofluorescence microscopy, showed that, in control MCF-7 cells, microtubule fibers appeared as an ordered network throughout the cytosol and mitochondria were radially arranged and stretched out along microtubules, whereas in cells treated with 15dPG-J2 microtubules were significantly fragmented (Figure 4). Treatment of cells with the destabilizing drug nocodazole produced a pattern of mitochondria's morphology and distribution similar to that of 15dPG-J2-treated cells. Accordingly, mitochondria in nocodazole-exposed cells presented the same rounded morphology and dissociation from microtubule organization as cells treated with 15dPG-J2. These results suggest that intact cytoskeletal organization is required for the distribution and shape of the mitochondria observed in control cells and that the cytoskeletal organization is a target of 15dPG-J2.



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Fig. 4. Effect of 15dPG-J2 and nocodazole on the subcellular distribution of mitochondria. MCF-7 cells were grown on glass coverslips, treated or not for 12 h with 10 µM 15dPG-J2 or 1 µM nocodazole and incubated with 20 nM MitoTracker red CMXRos for 45 min. After fixing, cells were stained using an anti-{alpha}-tubulin antibody and fluorescence was visualized by confocal microscopy. Bar scale, 5 µm. Representative results of three independent experiments are shown.

 
15dPG-J2-induced cleavage of PARP and stimulation of caspase 7
We next measured caspase activation in response to 15dPG-J2 treatment. Since MCF-7 cells are deficient in caspase 3, we measured caspase 7 processing to determine if it could function similarly to caspase 3 as an effector caspase that can cleave PARP (Figure 5). Western blot analysis of protein extracts derived from 15dPG-J2-treated MCF-7 cells revealed an increase in cleaved caspase 7. The activation of effector caspase 7 was substantiated by the demonstration of PARP cleavage following 15dPG-J2 exposure. Treatment of MCF-7 cells with 15dPG-J2 for 24 h caused a proteolytic cleavage of PARP, with accumulation of the 85-kDa fragment and a concomitant disappearance of the full-length 116-kDa protein. In order to know if 15dPG-J2 was also able to activate caspase 3, we next treated the SKBR3 breast cancer cell line, which possesses a functional caspase 3, with 15dPG-J2 for 24 and 48 h. As can be seen in Figure 5, 15dPG-J2 was also able to produce activation of caspase 3 in these cells. Addition of the caspase inhibitor N-benzoylcarbanyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk) inhibited caspase activation and PARP cleavage (Figure 6A) but was not able to block cell death measured by the MTT assay and Annexin V-FITC staining (Figure 6B and C), suggesting that caspase activation is not essential to induce MCF-7 cell death by 15dPG-J2.



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Fig. 5. Time course of caspase activation and PARP cleavage. MCF-7 and SKBR3 cells were treated or not with 10 µM 15dPG-J2 for the indicated times. Protein lysates were used for western blot analysis using antibodies against PARP, caspase 7 and caspase 3, as indicated in the Materials and methods. Representative results from three independent experiments are shown.

 


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Fig. 6. Effect of caspase inhibition on 15dPG-J2-induced cell death. (A) MCF-7 cells were treated or not with 10 µM 15dPG-J2 and zVADfmk for the indicated times and PARP cleavage and caspase 7 activation was measured as indicated in the legend of Figure 5. Representative results from three independent experiments are shown. (B) MCF-7 cells were treated or not with 10 µM 15dPG-J2 and zVADfmk for 48 h and cell viability was assessed by the MTT assay, as indicated in the Materials and methods. (C) MCF-7 cells were treated or not with 10 µM 15dPG-J2 and zVADfmk for 16 h, afterwards the number of apoptotic cells were determined by Annexin V-FITC staining and FACS analysis.

 
Influence of anti-apoptotic bcl-2 protein on 15dPG-J2-mediated apoptosis
Bcl-2 and Bcl-XL have been reported to block the caspase pathway. To examine the role of bcl-2 in 15dPG-J2-induced cell death, we used a subclone of MCF-7 cells transfected with human bcl-2 (30), which expressed high hbcl-2 protein levels, as evidenced by western blot analysis (Figure 7A). This clone was resistant to apoptosis induced by 15dPG-J2 or rosiglitazone treatment (Figure 7B), even over the extended incubation period of 48 h. Prevention of apoptosis correlated with inhibition of caspase 7 activity and PARP cleavage (Figure 7C). However, although over-expression of bcl-2 blocked the effect of rosiglitazone on MCF-7 cell viability we did not observe any effect on loss of MCF-7 cell viability (Figure 7D) and only a partial reversion of the lowering in {Delta}{Psi}m (Figure 7E) triggered by 15dPG-J2. Together, the data suggest that this prostaglandin induces a caspase-dependent apoptosis of MCF-7 cells as well as cell death triggered by a mechanism independent of caspases but possibly dependent on mitochondrial dysfunction.



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Fig. 7. Effect of bcl-2 over-expression on 15dPG-J2-induced cell death. (A) Representative western blot showing expression of bcl-2 protein on a human bcl-2-transfected clone of MCF-7 cells (MCF-7bcl2). (B) MCF-7bcl2 cells were incubated or not with 10 µM 15dPG-J2 or 30 µM rosiglitazone for 16 h and the number of apoptotic cells were determined by Annexin V-FITC staining and FACS analysis. (C) MCF-7bcl2 cells were incubated or not with 10 µM 15dPG-J2 for the indicated periods of time and PARP cleavage and caspase 7 activation were measured as indicated in the legend of Figure 5. Representative results from three independent experiments are shown. (D) MCF-7bcl2 cells were incubated or not with 10 µM 15dPG-J2 or 30 µM rosiglitazone for 48 h and cell viability was determined by the MTT assay. (E) Control MCF-7 and MCF-7bcl2 cells were treated or not with 10 µM 15dPG-J2 and 30 minutes before harvesting, cells were treated with 50 nM TMRE and analyzed by flow cytometry to determine {Delta}{Psi}m.

 
Effect of NF-{kappa}B on 15dPG-J2-mediated apoptosis
Previous studies suggested that 15dPG-J2 can down-regulate NF-{kappa}B via activation of PPAR{gamma} or independently of its binding to this receptor (22,24). Then, we next tested whether 15dPG-J2 would affect NF-{kappa}B activity in MCF-7 cells and if over-expression of NF-{kappa}B could alter the effect of 15dPG-J2 on cell viability. To this end we performed transient transfections of parental MCF-7 cells with a reporter construct that contains three copies of the consensus NF-{kappa}B response element (3xKBtk-luc). As shown in Figure 8A, the expression of the reporter construct was significantly inhibited by 15dPG-J2. A similar reduction was observed in MCF-7 cells stably over-expressing the p65 protein (Figure 8B). These results show that 15dPG-J2 inhibits NF-{kappa}B activity in MCF-7 cells, as it has been demonstrated previously in other cell types. Finally, we investigated whether a reduction in NF-{kappa}B activity could be involved in the MCF-7 cell death induced by 15dPG-J2. As can be seen in Figure 8C, the reduction in cell viability triggered by 15dPG-J2, determined with the MTT assay, was unaffected in MCF-7p65 cells, suggesting that NF-{kappa}B, as shown above for bcl-2, has no effect on the loss of MCF-7 cell viability induced by this prostaglandin.



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Fig. 8. Effect of NF-{kappa}B on 15dPG-J2-induced cell death. SKBR3 and parental (A) or p65 over-expressing MCF-7 cells (MCF-7p65) (B) were transiently transfected with a NF-{kappa}B luciferase reporter construct, treated or not for 24 h with 15dPG-J2, and luciferase activity was measured. Data represent the mean of luciferase activity determined in triplicate in at least three independent experiments and expressed relative to the value obtained with non-treated cells. (C) MCF-7p65 cells were treated or not with 10 µM 15dPG-J2 and cell viability was measured by the MTT assay.

 
ROS generation
We further examined other possible mechanisms involved in the MCF-7 cell death induced by 15dPG-J2. To this end, we next determined the reactive oxygen species (ROS) generation by flow cytometry, looking for the conversion of the non-fluorescent DCFH-DA into a fluorescent molecule through oxidation. Treatment of MCF-7 cells with 15dPG-J2 for 30 or 60 min evoked a very rapid ROS formation (Figure 9A). We delineated the source of ROS production by applying the NAD(P)H oxidase inhibitor rotenone at a concentration of 5 µM. ROS production elicited by 15dPG-J2 was highly attenuated in the presence of rotenone, suggesting that the main source of oxygen species was the mitochondria through the activity of respiratory complex I. Rosiglitazone, although to a much lesser extent than 15dPG-J2, was also able to induce a moderate ROS generation. Since it has been described that the bcl-2 protein can inhibit the production of oxygen species during apoptosis induced by a variety of stimuli, we next measured ROS formation in MCF-7blc2 cells after treatment with 15dPG-J2. As depicted in Figure 9A, over-expression of the bcl-2 protein did not interfere with ROS production. Also, addition of the pan-caspase inhibitor zVADfmk did not have any effect on PG-J2-induced ROS production (data not shown). Importantly, rotenone, at a dose, which does not compromise the viability of MCF-7 cells (data not shown), was able to prevent the decrease in MCF-7 cell viability induced by 15dPG-J2 (Figure 10A). Moreover, the presence of the radical scavengers ebselen or ascorbate completely blocked the decrease in cell viability elicited by 15dPG-J2 (Figure 10B). Altogether, the data suggest that the generation of oxygen radicals, as a consequence of an alteration in the respiratory chain activity, plays a critical role in the initiation of the 15dPG-J2-induced cell death independent of caspase activation.



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Fig. 9. Effect of 15dPG-J2 on ROS generation. MCF-7 or MCF-7bcl2 cells were exposed to the oxidative-sensitive dye DCFH-DA probe for 1 h. After washing with PBS, cells were treated with 10 µM 15dPG-J2 or 30 µM rosiglitazone. Rotenone (5 µM) was added to some cultures 30 min before the addition of 15dPG-J2. At the indicated times cells were harvested and analyzed by flow cytometry to determine the percentage of cells displaying an increase in ROS production (reflected by a rightward shift of the histogram). Control cells were analyzed in parallel. A representative histogram of three independent experiments is shown.

 


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Fig. 10. Effect of rotenone and free radicals scavengers on MCF-7 cell viability after 15dPG-J2 treatment. (A) MCF-7 cells were incubated for 24 h with 15dPG-J2 in the presence or absence of 5 µM rotenone and cell viability was assayed by the MTT assay. (B) MCF-7 cells were incubated for 24 h with 15dPG-J2 in the presence or absence of 100 µM ascorbate or 40 µM ebselen and cell viability was assayed by the MTT assay.

 
Effect of 15dPG-J2 on oxygen consumption
To further investigate the mean bioenergetic state of the mitochondrial population in 15dPG-J2-exposed MCF-7 cells, oxygen consumption of intact cells was determined (Table I). We first measured the rate of oxygen consumption in whole cells (basal respiration). Addition of the complex V (ATP synthase) inhibitor oligomycin, allowed us to estimate oxygen consumption in the absence of oxidative phosphorylation, and the use of an uncoupler (FCCP) indicated the maximal respiratory rate. The ratio of these two values is defined as the respiratory control (31). In the absence of 15dPG-J2, the rates of basal oxygen consumption in control MCF-7 cells were 77.5 nmol O2/min/mg protein and declined ~20% 2 h after the addition of the prostaglandin. In the presence of oligomycin, basal oxygen consumption decreased 50% and no significant differences were observed between control and 15dPG-J2-treated cells. On the contrary, the rate of respiration uncoupled by the addition of FCCP decreased with 15dPG-J2 exposure in a very rapid manner (40% of the control rate within the first hour). As a result, the respiratory control value was significantly lower in MCF-7 cells as early as 60 min after treatment with 15dPG-J2. These results show that 15dPG-J2 is a potent inhibitor of the mitochondrial respiratory chain and are also in agreement with unpublished results from our laboratory showing an inhibition of respiratory complex I activity by 15dPG-J2 in isolated mitochondria.


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Table I. Effects of 15dPG-J2 on oxygen consumption

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The data presented in this paper support the conclusion that 15dPG-J2-induced MCF-7 cell death is due to an early and profound mitochondrial dysfunction including production of ROS, a significant reduction on O2 consumption, a lowering in the {Delta}{Psi}m, and a striking alteration in the mitochondrial morphology. Although 15dPG-J2 also induces caspase activation, this pathway does not seem to be required for the effect of 15dPG-J2 on MCF-7 cell death.

Previous studies showed that 15dPG-J2 induces apoptosis of various human malignant cells, including leukemic cells, breast carcinoma, prostatic carcinoma, malignant B-lineage cells and colorectal carcinoma (10,11,15,18,32). These effects are, in part, mediated by an activation of PPAR{gamma} by 15dPG-J2, although the precise mechanisms are not well understood. It has been shown that activation of PPAR{gamma} can down-regulate PI3-kinase signaling in part by inducing the expression of the PTEN tumor suppressor gene and, as a result, induces pancreatic cancer cell apoptosis (33). Inhibition of E2F/DP DNA binding (34), induction of p21 (35), down-regulation of cyclin D1 expression (36) or inhibition of phosphorylation/activation of the erbB's receptor family (12) have all been suggested as potential molecular mechanisms implicated in the control of cell cycle progression and activation of apoptotic pathways by 15dPG-J2. However, there is still a significant lack of knowledge on the precise mechanisms how 15dPG-J2 induces cell death.

The data presented here demonstrate that 15dPG-J2 induced several cellular events, including caspase 7 and caspase 3 activation, release of cytochrome c and breakdown of mitochondrial membrane potential, which are all indicative for the involvement of paradigmatic mitochondrial apoptotic pathway. The interpretation that 15dPG-J2-induced apoptosis is mediated via cytochrome c release and subsequent caspase 7 activation was further substantiated by the finding that over-expression of bcl-2 was able to block 15dPG-J2-induced apoptosis, as measured by Annexin V staining. It is very likely, that the induction of this mitochondrial apoptotic pathway by 15dPG-J2 can be mediated by PPAR{gamma} activation since rosiglitazone, a specific ligand of this receptor, also induces apoptosis in MCF-7 cells. However, activation of this pathway is not sufficient to explain the cytotoxic effects of this prostaglandin as bcl2 over-expression, albeit blocking the cytotoxic action of rosiglitazone, does not inhibit 15dPG-J2-induced cell death. In addition, our results suggest an implication of the transcription factor NF-{kappa}B in the apoptosis induced by 15dPG-J2 in MCF-7 cells. These results are in line with other reports indicating 15dPG-J2 inhibition of NF-{kappa}B activity in other systems (2224).

Our results also show that 15dPG-J2-mediated cell death, as measured by the MTT assay, was not inhibited by bcl-2 and p65 over-expression or treatment with the pan-caspase inhibitor zVADfmk, suggesting an alternative mechanism of MCF-7 cell death. In this regard, 15dPG-J2 and other cyclopentenone prostaglandins have been shown to induce ROS in diverse cell types, which could mediate their effects on cell death (28,3739). Therefore, in this study we have performed a detailed analysis of the effects of 15dPG-J2 on mitochondrial functionality and its implication in cell death. Our results showing a very early increase in ROS production and a concomitant inhibition of oxygen consumption after 15dPG-J2 treatment of MCF-7 cells, suggest that a profound mitochondrial dysfunction could be the earliest event in 15dPG-J2-induced cell death in these cells. It is interesting to point out that rotenone, at a dose which does not interfere with cell viability, markedly decreases ROS production induced by 15dPG-J2 and restores cell viability to basal values. In addition, the presence of the free radical scavengers, ebselen and ascorbate completely blocked the decrease in cell viability elicited by 15dPG-J2, suggesting that its effect in MCF-7 cell death is mainly mediated by ROS production. These results are in agreement with those performed in human neuroblastoma SH-SY5Y cells and human hepatic fibroblasts showing that the increase in ROS production and cell death induced by 15dPG-J2 can be prevented by treatment with the antioxidant N-acetylcysteine (27,28). The induction of hepatic fibroblasts cell death was prevented by the caspase inhibitor zVADfmk, suggesting an involvement of the caspase pathway in the action of 15dPG-J2 (28). In contrast, our results show that 15dPG-J2-induced MCF-7 cell death is independent of caspase activation. In agreement with the data shown here, it has been shown that when mitochondrial function is impaired, severe energy deficits occur and large amounts of oxygen free radicals are generated, which can induce the arrest of cell proliferation and the apoptotic process independent of caspase activation (40,41). It has also been suggested that a caspase-independent mechanism exits for commitment to cell death, which is likely to involve mitochondria (42). The mechanism by which 15dPG-J2 decreases oxygen consumption and induces ROS production in MCF-7 cells is very likely the consequence of a direct inhibition of the respiratory chain activity. In fact, recent results from our laboratory demonstrate a direct inhibition of respiratory complex I activity followed by an increase in ROS production after 15dPG-J2 treatment of isolated mitochondria (manuscript in preparation). Another link between cell death and mitochondrial physiology is suggested by the observation that respiratory chain deficiency predisposes cells to apoptosis (43). Wang et al. have demonstrated that mouse embryos with homozygous disruption of the mitochondrial transcription factor A gene (Tfam) present a massive cell death at embryonic day 9.5 and an increased apoptosis in the heart of tissue-specific Tfam knockouts, suggesting that respiratory chain deficiency is associated with increased in vivo apoptosis.

Our results also show a very striking change in mitochondrial morphology in MCF-7 breast cancer cells after exposure to 15dPG-J2. These changes are associated with a depolymerization of the microtubules similar to the one observed in cells treated with nocodazole. Alterations in mitochondrial distribution and morphology have been associated with various diseases, including cancer (44), Alzheimer's disease (45) and muscular dystrophy (46). Microtubules are dynamic components of the cytoskeleton and are critical for a wide variety of functions in eukaryotic cells. Microtubules are involved in the maintenance of cell shape, cell signaling, mitosis, mRNA localization and vesicle and organelle trafficking mechanisms (reviewed in ref. 47). Microtubules are the principal target in cells for a family of anticancer drugs, the so-called anti-tubulin agents. These factors strongly suppress microtubule dynamics that are essential for progression throughout mitosis (48) leading to sustained mitotic arrest and apoptosis (49). In this regard, unpublished data from our laboratory show that 15dPG-J2 induces a potent arrest of the cell cycle on G2/M phase in MCF-7 cells. In addition, we have demonstrated that 15dPG-J2 enhances the breast and ovarian cancer susceptibility gene BRCA1 gene expression in these cells (50), a protein necessary to induce G2/M arrest of the cell cycle and apoptosis induced by taxol or vincristine (51). The results presented here are in agreement with the data and suggest that 15dPG-J2 is a potent cytoskeleton-disrupting agent, which can be one of the causes leading to apoptosis of breast cancer cells.

In conclusion, our findings indicate that 15dPG-J2 triggers MCF-7 breast cancer cell death mainly by inducing early mitochondrial alterations, such as production of oxygen free radicals, a significant reduction on O2 consumption, and a lowering in the {Delta}{Psi}m by mechanisms probably independent of PPAR{gamma} activation. Moreover, our data suggest that a disruption of the cytoskeleton could also be involved in the effects of 15dPG-J2 on MCF-7 cell death.


    Acknowledgments
 
This work has been supported by Grants from the Dirección General de Enseñanza Superior e Investigación Científica, grants BMC2001-2342 (APC) and PM99-0057 (AS) and by FIS, grant 03C03/10 (APC). M.P. is a fellow of the Fondo de Investigaciones Sanitarias de la Seguridad Social. J.S.-R. is supported by the Agencia Española de Cooperación Internacional and by the research project 03C03/10. We thank B.Martinez for her help with the O2 consumption measurements. We also thank Manolo Espinosa (Centro de Biología Molecular, Madrid) for providing the 3x{kappa}Bluc reporter plasmid and Dr Abelardo Lopez-Rivas (Instituto de Parasitología y Bioquímica, CSIC, Granada) for the kind gift of MCF-7bcl2 cells.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 19, 2004; revised September 9, 2004; accepted October 1, 2004.