Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells

Carl E. Clay1, Andrew M. Namen1, Gen-ichi Atsumi1, Mark C. Willingham2, Kevin P. High1, Timothy E. Kute2, Anthony J. Trimboli1, Alfred N. Fonteh1, Paul A. Dawson1 and Floyd H. Chilton1,3,4

1 Department of Internal Medicine,
2 Department of Pathology and
3 Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston Salem,NC 27157-1054, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPAR{gamma} investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPAR{gamma} is expressed in several transformed cells. However, the function of PPAR{gamma} activation in neoplastic cells is unclear. Activation of PPAR{gamma} with the known prostanoid agonist 15-deoxy-{Delta}12,14-prostaglandin J2 (15dPGJ2) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ2 was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ2 induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ2 initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPAR{gamma} activation in some breast cancer cells and further suggests a potential role for PPAR{gamma} ligands for the treatment of breast cancer.

Abbreviations: ATRA, all-trans-retinoic acid; DAPI, 4',6-diamidine-2'-phenylindole diHCl; {Delta}12-PGJ2, {Delta}12-prostaglandin J2; 15dPGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; PBS, phosphate-buffered saline; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PG, prostaglandin; RAR, retinoic acid receptor; RXR, retinoid X receptor; TGZ, troglitazone; TZD, thiazolidinedione.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A common feature of tumor cells is their failure to terminally differentiate in response to appropriate stimuli. The degree of tumor cell differentiation can dictate the rate of proliferation, malignant potential and sensitivity to therapeutic interventions. Therefore, there have been major efforts to uncover novel agents and mechanisms that control tumor cell differentiation. There is extensive literature on the use of retinoic acids and their derivatives, acting through their receptors [retinoic acid receptor (RAR) and retinoid X receptor (RXR)], to arrest or reverse cancer in both animals and humans (110). Another member of the nuclear hormone receptor family, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), is preferentially expressed in adipose tissue and has an important role in fat metabolism and adipocyte differentiation (1116). Ligands, including thiazolidinediones (TZDs) (17), certain fatty acids (18,19) and metabolites of arachidonic acid (20,21), induce activation of PPAR{gamma} and association with RXR to form a functional heterodimer (22).

Recent data reveal that PPAR{gamma} is expressed in colonic tumors and metastatic breast adenocarcinomas (2325). The discovery of PPAR{gamma} in neoplastic cells raises the critical question of its functional significance in human cancers. Ligand activation of PPAR{gamma} inhibits proliferation and causes lipid accumulation in cultured breast cancer cells and some breast cancer cell lines (24,26). A recent study suggested that addition of the RAR agonist all-trans-retinoic acid (ATRA) plus a PPAR{gamma} agonist induced apoptosis of human breast cancer cells in vitro and attenuated tumor growth in mice (27).

In the current study we show that addition of a known prostanoid PPAR{gamma} ligand, 15-deoxy-{Delta}12,14-prostaglandin J2 (15dPGJ2) (23,24), inhibits the proliferation of breast cancer cells by blocking cell cycle progression (G1 arrest) and inducing early and late events associated with apoptosis. Furthermore, pretreatment of MDA-MB-231 cells with 15dPGJ2 attenuates the capacity of these cells to induce tumors in nude mice. Together, these data show that prostanoids such as 15dPGJ2 may represent an important group of molecules that have the capacity to induce apoptosis of certain neoplastic cells and by this action inhibit tumorigenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells and reagents
MDA-MB-231 (28) and MCF-7 breast cancer epithelial cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal calf serum (Life Technologies, Grand Island, NY), 1% penicillin and 1% streptomycin (BioWhitaker, Walkersville, MD). All prostaglandins (PGs) were purchased from Cayman Chemical (Ann Arbor, MI). Troglitazone (TGZ) was a generous gift from Parke Davis/Warner Lambert (Plainsboro, NJ).

RT–PCR analysis of PPAR{gamma} mRNA
PPAR{gamma} primers (5'-primer CCTGGCTCAGATGGCTCGG and 3'-primer CCTACGTTTAGAAGGCC) were used with total RNA isolated from MDA-MB-231 cells using the Wizard Total RNA isolation kit (Promega, Madison, WI). RT–PCR was performed using the GeneAmp RNA PCR kit and AmpliTaq Gold DNA polymerase (Perkin Elmer, Foster City, CA). Total RNA (200 ng) was converted to single-stranded cDNA and then AmpliTaq Gold (5 U) was added. Following PCR (35 cycles of 94°C for 1 min, 52°C for 30 s and 72°C for 30 s), the products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Control reactions were performed in parallel in which RNA was omitted from the cDNA synthesis reaction. The cDNA band was excised from the gel and the DNA isolated using the Wizard DNA isolation kit (Promega). The DNA was sequenced and analyzed in the Core DNA Facility (Wake Forest University School of Medicine, Winston Salem, NC).

Luciferase reporter assay
MDA-MB-231 cells were seeded and allowed to grow to near confluence in 100 mm tissue culture dishes. Cells were then transiently transfected with the luciferase reporter plasmid (a generous gift from Dr Bruce Spiegleman's laboratory) and appropriate controls using Superfect (Qiagen, Valencia, CA) according to the manufacturer's protocol. Cells were then washed using phosphate-buffered saline (PBS), provided with various concentrations of 15dPGJ2 and incubated for 48 h at 37°C and 5% CO2. After the incubation period, cells were transfered into 15 ml conical tubes using a rubber policeman, centrifuged to pellet the cells and washed twice in PBS. The medium was removed and cells were assayed for luciferase activity using the Luciferase Activity kit (Promega) and a Turner TD-20E luminometer (Sunnyvale, CA).

Analysis of cell cycle
Cells were incubated in the presence of the indicated concentrations of PGs, including PGF2{alpha}, PGJ2, {Delta}12-PGJ2 and 15dPGJ2. After an 18 h incubation, cells were pelleted and resuspended in 70% ice-cold ethanol, pelleted again and stained with propridium iodide for cell cycle analysis. Ploidy status was determined using a Coulter Epics XL-MCL flow cytometer (Hileah, FL) and percentage of cells in S phase of the cell cycle was determined using ModFit (Verity Software House Inc., Topsham, ME). In separate experiments, incubations were allowed to proceed for 48 h, cells were pelleted, resuspended and counted on a hemocytometer.

Video time-lapse microscopy
Apoptosis in neoplastic cells is characterized by specific morphological events that can be observed by video time-lapse microscopy. Previous studies have documented the specific morphological events associated with apoptosis in neoplastic cells (2931). These specific apoptotic events begin with adherent cells becoming round (cellular rounding), followed by membrane blebbing. A proportion of cells send out echinoid-type protrusions, or spikes, followed by the cessation of movement. Finally, cells blister and expel their cytoplasmic contents into the medium. In these experiments, MDA-MB-231 cells were treated with 15dPGJ2 (10 µM) or TGZ (100 µM) and placed on a Zeiss Axiovert 135 phase contrast microscope equipped with a warm stage heater/recirculator device that maintained the cell culture at 37°C temperature and 5% CO2/95% air atmosphere. Cells were illuminated with red light and images were obtained using a Dage-MTI 100 CCD camera and recorded using a Panasonic AG-6740 video time-lapse recorder at a fixed rate of 1 frame/10 s (600:1 final time-lapse). The numbers of cells showing apoptotic morphology, including cellular rounding, blebbing, production of echinoid spikes, cessation of movement, blistering and cellular lysis, were assessed and the events of apoptosis were characterized and recorded for each cell in the field of view (2931). The percentage of cells in the field of view that undergo each specific event of apoptosis was graphed as a function of time for each agonist.

Histochemical methods
Nuclear condensation and segmentation are specific events observed during apoptosis (3234). As an additional marker, nuclear morphology was examined using histochemical staining with 4',6-diamidine-2'-phenylindole diHCl (DAPI) (Sigma, St Louis, MO), a fluorescent dye that selectively labels DNA. Briefly, 1.0x106 MDA-MB-231 cells were incubated with 15dPGJ2 (10 µM) or TGZ (100 µM) in 35 mm culture dishes. After a 36 h incubation, suspended cells contained in the medium were centrifuged onto a glass slide using a Cytospin 3. Both adherent cells in culture dishes and suspended cells centrifuged onto slides were then fixed in 10% v/v formalin in PBS (15 min, 23°C). This was followed by incubation with 0.1 µg/ml DAPI in methanol (15 min, 23°C). After washing, cells were mounted under a coverslip in glycerol and viewed using a Zeiss Axioplan-2 epifluorescence microscope equipped with UV excitation filters. Digital images of suspended and adherent cells were recorded using a Spot camera.

Tumor growth in nude mice
Three-week-old female BALB/C nude mice were purchased from Charles River (Raleigh, NC) and housed in the Animal Care Facility (Wake Forest University School of Medicine, Winston Salem, NC). The nude mouse model for the study of invasion and metastasis of tumors in vivo (28,35) was used to determine the progression of MDA-MB-231 cells after treatment with PPAR{gamma} agonists. Briefly, MDA-MB-231 cells were incubated with 15dPGJ2 (10 µM) for 18 h and viability was determined by trypan blue exclusion. Viable cells (7.5x105) in 100 µl culture medium were transplanted by s.c. injection into the right breast pad and viable vehicle-treated cells (7.5x105) in 100 µl culture medium were transplanted by s.c. injection into the left breast pad of mice. Tumor area was measured every third day beginning 7 days post-injection.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of PPAR{gamma} and cellular proliferation
Initial studies investigated the expression of PPAR{gamma} mRNA in the estrogen receptor-negative breast cancer cell line MDA-MB-231. RT–PCR analysis readily detected the PPAR{gamma} mRNA in MDA-MB-231 breast cancer cells (data not shown). DNA sequencing confirmed that the expected sized PCR product (1002 bp) was identical to the human PPAR{gamma} cDNA sequence.

To determine the effect of PGs on cell proliferation, MDA-MB-231 cells were incubated with 10 µM PGF2{alpha}, PGJ2, {Delta}12-prostaglandin J2 ({Delta}12-PGJ2) or 15dPGJ2 and total cell number as well as the capacity of cells to move into S phase of the cell cycle was determined as a measure of cell proliferation. The number of cells traversing the S phase of the cell cycle (Figure 1AGo) was markedly reduced after 18 h of exposure to a known PPAR{gamma} agonist, 15dPGJ2 (20,21). In contrast, a PG that does not bind PPAR{gamma}, PGF2{alpha} (1821), did not influence either marker of cellular proliferation. Total cell number was also determined after incubation with the test compounds for 48 h (Figure 1BGo). Cell number was significantly decreased by the PGJ2 metabolites {Delta}12-PGJ2 and 15dPGJ2. Together, these data reveal that MDA-MB-231 breast cancer cells express mRNA encoding PPAR{gamma} and that PGs of the J series reduce the proliferation of these cells.



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Fig. 1. Influence of PGs on breast cancer cell growth. Total cell number and the percentage of cells traversing the S-phase of the cell cycle was observed as markers of cell cycle progression in MDA-MB-231 breast cancer cells after the addition of the exogenous prostaglandins PGJ2, its metabolites {Delta}12-PGJ2 and 15dPGJ2 and PGF2{alpha}. MDA-MB-231 cells (1.0x106) were incubated with 10 µM of the various PGs for 18 (A) or 48 h (B) for analysis of cellular proliferation. Percentage of cells in S phase was determined by flow cytometry and total cell number was assessed using a Coulter cell counter. Data are representative of means ± SEM of four separate experiments.

 
The next experiments investigated the dose–response relationship of two known PPAR{gamma} agonists on cellular proliferation (Figure 2Go). Estrogen receptor-negative MDA-MB-231 and estrogen receptor-positive MCF-7 cells were incubated with 15dPGJ2 (0.1–20 µM) or TGZ (0.1–100 µM) for 18 h and the percentage of cells in the S phase of the cell cycle was determined by flow cytometry. Exposure of both cell lines to high concentrations of either PPAR{gamma} agonist blocked cell cycle progression. However, the prostanoid agonist 15dPGJ2 inhibited cell cycle at concentrations 10-fold lower than TGZ in the MDA-MB-231 cell line. 15dPGJ2 was a potent inhibitor of the cell cycle in MCF-7 cells and TGZ was a more potent inhibitor of the cell cycle in MCF-7 than in MDA-MB-231 cells.



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Fig. 2. Dose-dependent response of PPAR{gamma} agonists on cell cycle progression in MDA-MB-231 and MCF-7 cells. MCF-7 (A) and MDA-MB-231 (B) cells (1.0x106) were incubated for 18 h with 15dPGJ2 at concentrations ranging from 0.1 to 20 µM or with TGZ at concentrations ranging from 0.1 to 100 µM for 18 h. The percentage of cells in S phase was determined by flow cytometry. Data are representative of means ± SEM of three separate experiments.

 
In order to test the transcriptional activation of PPAR{gamma} by 15dPGJ2 we performed luciferase reporter assays by transient transfection of MDA-MB-231 cells with a plasmid containing the PPAR{gamma} response element upstream of the luciferase reporter construct. Increasing concentrations of 15dPGJ2 resulted in a dose-dependent increase in luciferase activity (Table IGo). These results suggest that 15dPGJ2 induces cell cycle inhibition by direct interaction with PPAR{gamma}.


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Table I. Luciferase reporter activity of MDA-MB-231 cells exposed to various concentrations of 15dPGJ2
 
Influence of 15-deoxy-{Delta}12,14-PGJ2 on differentiation and apoptosis
Several investigators have demonstrated that PPAR{gamma} agonists can induce differentiation in neoplastic cells, including breast adenocarcinoma and liposarcoma cells (2326). A hallmark of cellular differentiation is the accumulation of intracellular neutral lipid which can be stained with the fluorescent lipophilic stains Nile Red or Oil Red O and easily visualized by fluorescent microscopy (3638). Thus, we used these stains in the next experiments and examined whether addition of 15dPGJ2 to MDA-MB-231 cells also induces differentiation. Unlike the reported action of TZDs, which increase lipid accumulation in breast cancer cells (24,26), treatment of MDA-MB-231 cells with 15dPGJ2 (10 µM) for 24, 48 or 96 h had no significant effect on accumulation of cellular lipid (data not shown). However, treatment of MDA-MB-231 cells with 15dPGJ2 (10 µM) was associated with a consistent decrease in cell viability and proliferation. This finding raised the question of whether 15dPGJ2 may induce signaling events that lead to the induction of apoptosis.

Chromatin condensation is an early event of apoptosis (2934). MDA-MB-231 cells treated with 15dPGJ2 (10 µM) or TGZ (100 µM) for 36 h show a marked increase in nuclear condensation, as indicated by the dense staining pattern of DAPI. 15dPGJ2- and TGZ-treated cells that remained attached to the cell culture dish show the characteristic staining morphology of apoptotic cells (Figure 3Go). In addition, cells that were suspended in medium also underwent apoptosis (Figure 4Go). The changes in cell surface morphology of 15dPGJ2-treated cells was viewed by phase contrast (Figures 3C and 4CGoGo) and nuclear condensation of the same cell population was viewed by UV excitation (Figures 3C' and 4C'GoGo). TGZ-treated cells also showed nuclear changes associated with apoptosis as seen by phase contrast microscopy (Figures 3B and 4BGoGo) and by UV excitation (Figures 3B' and 4B'GoGo). Arrows indicate cell nuclei that are in the process of apoptosis. In contrast, control cells did not undergo apoptosis (Figures 3A and A' and 4A and A'GoGo). Rather, two cells can be seen in the early stages of mitosis and anaphase of mitosis as indicated by arrowheads (Figure 4A'Go). These data reveal that agonists of PPAR{gamma} can induce apoptosis in MDA-MB-231 cells.



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Fig. 3. Nuclear changes associated with exposure of MDA-MB-231 cells to 15dPGJ2 or TGZ. MDA-MB-231 cells (1.0x106) were treated with vehicle alone (A), 100 µM TGZ (B) or 10 µM 15dPGJ2 (C) for 36 h. Medium was collected and cells which remained attached to the cell culture dish were fixed with 10% v/v formamide and stained with DAPI (0.1 µg/ml). Nuclear morphology was examined as described in Materials and Methods. Arrows indicate apoptotic cells. These data are representative of three separate experiments.

 


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Fig. 4. Nuclear changes associated with exposure of MDA-MB-231 cells to 15dPGJ2 or TGZ. MDA-MB-231 cells (1.0x106) were treated with vehicle alone (A), 100 µM TGZ (B) or 10 µM 15dPGJ2 (C) for 36 h. Medium was collected and cells, which were suspended in culture medium, were centrifuged onto a glass slide. Cells were then fixed with 10% v/v formamide and stained with DAPI (0.1 µg/ml). Nuclear morphology was examined as described in Materials and methods. Arrows indicate apoptotic cells and arrowheads indicate cells in mitosis. These data are representative of three separate experiments.

 
In addition to DNA condensation and segmentation during apoptosis, specific changes in cell surface morphology can be recorded by video time-lapse microscopy. The morphological changes associated with the progression of apoptosis include initial cell rounding, surface membrane blebbing, cessation of movement and extension of echinoid spikes followed by membrane blistering and lysis (2931). Video time-lapse microscopy was used to record these events in MDA-MB-231 cells exposed 15dPGJ2 or TGZ (Figure 5Go). Treatment of MDA-MB-231 cells with 15dPGJ2 (10 µM) induced all of the characteristic morphological changes associated with apoptosis (Figure 5AGo). Cellular rounding and membrane blebbing, the earliest features of apoptosis, were observed from 8 to 30 h after treatment, but cells remained attached to the culture dish and were still viable by trypan blue exclusion. Cells ceased movement and produced echinoid spikes beginning at 20 h post-treatment. By 48 h >60% of cells had undergone blistering and lysis, the last events of apoptosis. Treatment of MDA-MB-231 cells with TGZ (100 µM) induced a slower and more modest response (Figure 5BGo). Only 30% of cells had completed apoptosis by 50 h. In contrast, treatment of MDA-MB-231 cells with vehicle alone did not induce any of the events of apoptosis. Rather, all of the cells in this group underwent at least one mitosis and the cell population doubled after 24 h.



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Fig. 5. Video time-lapse microscopy of MDA-MB-231 cells treated with 15dPGJ2 or TGZ. MDA-MB-231 cells (1.0x106) were treated with 10 µM 15dPGJ2 (A) or 100 µM TGZ (B) and images were recorded using a video time-lapse recorder at the rate of 1 frame/10 s (final time lapse equals 600:1). Each cell in the field of view was followed from the time of seeding to the indicated stop time. The events associated with apoptosis or mitosis for each cell were recorded from the time of seeding. These data are representative of three separate experiments.

 
In vivo inhibition of tumorigensis
The next experiments were designed to determine whether a brief in vitro exposure of breast cancer cells to 15dPGJ2 could influence the capacity of these cells to generate tumors in nude mice. MDA-MB-231 cells were treated for 18 h with 15dPGJ2 (10 µM) and 7.5x105 viable cells were transplanted by s.c. injection into the right breast pad and 7.5x105 control cells were transplanted by s.c. injection into the left breast pad of 3-week-old female nude mice (Figure 6Go). Untreated control cells rapidly developed visible tumors and dramatic growth was observed throughout the time course. In contrast, a one time treatment of cells with 15dPGJ2 markedly attenuated their capacity to develop visible tumors. Moreover, these tumors grew to a significantly smaller size compared with untreated control tumors.



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Fig. 6. Tumor progression in nude mice. MDA-MB-231 cells were treated with 10 µM 15dPGJ2 for 18 h. Control treatment consisted of vehicle without drug. Viable control-treated cells (7.5x105) were injected s.c. into the right breast pad and viable 15dPGJ2-treated cells (7.5x105) were injected s.c. into the left breast pad of female nude mice. Tumor area (mm2) was determined every 3 days after the onset of visible tumor growth. Open circles represent vehicle-treated cells and closed circles represent 15dPGJ2-treated cells. These data represent the mean areas ± SEM of three separate experiments (n = 7 mice/experiment).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we have demonstrated that MDA-MB-231 cells express PPAR{gamma} mRNA and that treatment of MDA-MB-231 and MCF-7 cells with the PPAR{gamma} agonists 15dPGJ2 and TGZ reduces their proliferative capacity. Furthermore, 15dPGJ2 and TGZ induce morphological changes in MDA-MB-231 cells that are associated with the onset and progression of apoptosis. 15dPGJ2 is associated with strong transciptional activation of a PPAR{gamma} response element in a reporter gene assay system. Finally, brief exposure of MDA-MB-231 cells to 15dPGJ2 markedly attenuated tumor formation in a nude mouse model. Together, these results suggest that apoptosis, not differentiation, is the primary biological response for PPAR{gamma} activation by 15dPGJ2 in some breast cancer cell lines.

Many developing anticancer therapies focus on two cellular pathways, differentiation and apoptosis. Differentiation agents, such as retinoids, have been used in many types of cancer and have proven clinical utility in leukemia and Kaposi's sarcoma (110). Like retinoids, agonists of PPAR{gamma} induce formation of a functional heterodimer with RXR (22). Moreover, activation of PPAR{gamma} in liposarcoma cells has been shown to induce differentiation (1116), indicating that PPAR{gamma} activation may be a promising therapeutic approach for cancers of mesenchymal cell origin. It is not clear, however, how PPAR{gamma} activation affects cell growth in cancers of endodermal origin. Treatment of several breast cancer cell lines (21MT, MCF-7, T47D, MDA-MB-231 and BT474), infiltrating primary intraductal carcinoma and normal murine breast epithelial cells with the PPAR{gamma} agonist TZD drugs has been shown to inhibit cell growth in some cases and induce events associated with terminal differentiation (15,23,24,26). Our data reveal that treatment of MDA-MB-231 cells with the PPAR{gamma} ligand 15dPGJ2 inhibits cell proliferation by blocking entry into the S phase of the cell cycle. Furthermore, this block is not associated with differentiation, but apoptosis. Based on video time-lapse microscopy, this process requires 36–48 h to complete. Recently, Elstner et al. (27) demonstrated apoptosis of MCF-7 cells treated with PPAR{gamma} agonists, but only after the addition of both ATRA and 15dPGJ2 or TGZ for 96 h.

By incorporating the technology of video time-lapse microscopy, we have been able to follow the events of apoptosis from onset to completion. These events include cellular rounding, blebbing, the production of echinoid protrusions and blistering, followed by cell lysis, but did not include the formation of DNA ladders, as is traditionally seen by gel electrophoresis. Collins et al. (30) have shown that DNA ladder formation is a very late event in some cells that follows the events we have documented by video time-lapse microscopy. This technology allows for a more precise examination of events that occur after exposure of breast cancer cells to PPAR{gamma} agonists. Early nuclear condensation followed by surface morphological changes in response to 15dPGJ2 support the contention that MDA-MB-231 cells undergo many of the classical events associated with the onset and progression of apoptosis. Furthermore, similar to findings that have been reported for 21PT breast cancer cells (24), once these cells had been exposed to a PPAR{gamma} agonist, removal of the agonist did not result in a renewed capacity for growth and tumorigenesis in vivo. This suggests that brief activation of PPAR{gamma} by 15dPGJ2 may be sufficient to stimulate an irreversible apoptotic pathway.

It is interesting to note that TGZ was a less potent inducer of apoptosis in our studies than 15dPGJ2. The reasons for these differences have yet to be uncovered. One explanation may be that a novel receptor for endogenous J series PGs may be involved in the signal transduction events that lead to apoptosis in these cells, similar to the adenylyl cyclase-coupled PGE2 and PGF1 receptors which have been described in liver cells (45). However, PPAR{gamma} reporter assays and published data suggest a strong correlation between 15dPGJ2 and PPAR{gamma} activation (1821,24,27). Secondly, the fact that 15dPGJ2 has been used sparingly in many of the previous studies of neoplastic cells may explain why a strong correlation between PPAR{gamma} activation and apoptosis has not yet been made. Thirdly, because 15dPGJ2 is a potent PPAR{gamma} agonist, the high degree of PPAR{gamma} activation observed in this study may be higher than that which has been reported using TZDs in other studies. To our knowledge this is the first neoplastic cellular system where activation of PPAR{gamma} alone is associated with apoptosis.

It is becoming clear that activation of the same receptor with different ligands may result in different responses. Differentiation is the dominant biological response to activation of PPAR{gamma} with TZDs in liposarcoma cells, whereas in our studies with breast cancer cell lines, 15dPGJ2 induces apoptosis. In colonic tumors, PPAR{gamma} activation with synthetic ligands has shown both an increase in colon polyp formation and a reversal of malignant changes (3941) under different experimental conditions. In activated monocytes and macrophages, PPAR{gamma} activation with natural agonists inhibits the production of inflammatory cytokines and the expression of inducible nitric oxide synthase (42,43) that may be key events in the induction of apoptosis in human macrophages (44). Clearly, the molecular mechanisms that lead to these differing results must be elucidated to facilitate more rational approaches for the treatment of different cancers that express PPAR{gamma}. The current study reveals the potential to utilize prostaglandin agonists of PPAR{gamma} for the treatment of breast cancers that express PPAR{gamma} and the exciting possibility that these compounds may induce apoptotic cell death.


    Acknowledgments
 
The authors gratefully acknowledge Dr Pasha Sarraf and Dr Bruce Spiegleman's laboratory for the luciferase reporter plasmid. The authors would also like to thank Park Davis/Warner Laboratories for the gracious gift of troglitazone as well as Dr Michael Seeds for his technical assistance and helpful discussions and Nora Zbieranski for her technical assistance.


    Notes
 
4 To whom correspondence should be addressed Email: fchilton{at}wfubmc.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 17, 1999; revised May 28, 1999; accepted June 17, 1999.