prostaglandin-d2 and Breast-Neoplasms

Though the past few years have witnessed exciting achievements in targeted and immunotherapeutic treatments of all breast cancer subtypes, yet the decline in breast cancer mortality has been slowed, urging the need for further expanding options of high-quality treatments. Prostaglandin D2 synthase (PTGDS)/prostaglandin D2 (PGD2) play important roles in a variety of cancer types and show tissue-specificity, however, there are limited relevant reports in breast cancer. Therefore, the aims of the present study were to investigate the effects of PTGDS/PGD2 in breast cancer by large-scale bioinformatic analysis and in vitro experiments conducted on human breast cancer cell lines. Results of our study indicated that patients with high levels of PTGDS expression showed a reduced potential of tumor proliferation. PGD2 treatment significantly inhibited the proliferation and migration of breast cancer cells, which was mediated by the reduced expression of TWIST2. Overexpression of TWIST2 reversed the inhibitory effects of PGD2 on breast cancer cell proliferation. These results provided the novel evidence that PTGDS may play a significant role in modulating breast cancer growth, with implications for its potential use in treating breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Intramolecular Oxidoreductases; Lipocalins; Prostaglandin D2; Signal Transduction; Twist-Related Protein 2

Activation of peroxisome proliferator-activated receptor γ (PPARγ) serves as a key factor in the proliferation and invasion of breast cancer cells and is a potential therapeutic target for breast cancer. However, the mechanisms underlying this effect remain largely unknown. Heme oxygenase-1 (HO-1) is induced and overexpressed in various cancers and is associated with features of tumor aggressiveness. Recent studies have shown that HO-1 is a major downstream target of PPARγ. In this study, we investigated the effects of induction of HO-1 by PPARγ on TPAinduced MMP-9 expression and cell invasion using MCF-7 breast cancer cells. TPA treatment increased NF-κB /AP-1 DNA binding as well as MMP-9 expression. These effects were significantly blocked by 15d-PGJ2, a natural PPARγ ligand. 15d-PGJ2 induced HO-1 expression in a dose-dependent manner. Interestingly, HO-1 siRNA significantly attenuated the inhibition of TPA-induced MMP-9 protein expression and cell invasion by 15d-PGJ2. These results suggest that 15d-PGJ2 inhibits TPA-induced MMP- 9 expression and invasion of MCF-7 cells by means of a heme oxygenase-1-dependent mechanism. Therefore, PPARγ/HO-1 signaling- pathway inhibition may be beneficial for prevention and treatment of breast cancer. [BMB Reports 2020; 53(4): 212-217].

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression; Heme Oxygenase-1; Humans; Matrix Metalloproteinase 9; MCF-7 Cells; Neoplasm Invasiveness; NF-kappa B; PPAR gamma; Prostaglandin D2; Signal Transduction; Transcription Factor AP-1

15-Deoxy-Δ

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cysteine; Female; Humans; MCF-7 Cells; Mice; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Phosphorylation; Prostaglandin D2; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Reactive Oxygen Species; Signal Transduction

15-Deoxy-∆12,14-prostaglandin J2 (15d‑PGJ2) is a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that displays anticancer activity. Various studies have indicated that the effects of 15d‑PGJ2 are due to both PPARγ-dependent and -independent mechanisms. In the present study, we examined the effects of a biotinylated form of 15d‑PGJ2 (b‑15d‑PGJ2) on hormone-dependent MCF‑7 and triple‑negative MDA‑MB‑231 breast cancer cell lines. b‑15d‑PGJ2 inhibited cell proliferation more efficiently than 15d‑PGJ2 or the synthetic PPARγ agonist, efatutazone. b‑15d‑PGJ2 was also more potent than its non-biotinylated counterpart in inducing apoptosis. We then analyzed the mechanisms underlying this improved efficiency. It was found not to be the result of biotin receptor-mediated increased incorporation, since free biotin in the culture medium did not decrease the anti-proliferative activity of b‑15d‑PGJ2 in competition assays. Of note, b‑15d‑PGJ2 displayed an improved PPARγ agonist activity, as measured by transactivation experiments. Molecular docking analyses revealed a similar insertion of b‑15d‑PGJ2 and 15d‑PGJ2 into the ligand binding domain of PPARγ via a covalent bond with Cys285. Finally, PPARγ silencing markedly decreased the cleavage of the apoptotic markers, poly(ADP-ribose) polymerase 1 (PARP‑1) and caspase‑7, that usually occurs following b‑15d‑PGJ2 treatment. Taken together, our data indicate that biotinylation enhances the anti-proliferative and pro-apoptotic activity of 15d‑PGJ2, and that this effect is partly mediated via a PPARγ-dependent pathway. These results may aid in the development of novel therapeutic strategies for breast cancer treatment.

Topics: Binding Sites; Biotinylation; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Models, Molecular; Molecular Docking Simulation; PPAR gamma; Prostaglandin D2; Thiazolidinediones

Reports indicate that 15deoxydelta12,14prostaglandinJ2 (15dPGJ2) has anticancer activities, but its mechanisms of action have yet to be fully elucidated. We therefore investigated the effects of 15dPGJ2 on the human breast cancer cell lines, MCF7 (estrogen receptor ERα+/ERβ+) and MDAMB231 (ERα/ERβ+). Cellular proliferation and cytotoxicity were determined using the 3(4,5dimethylthiazol2yl)2,5diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays while apoptosis was determined by fluorescence microscopy and flow cytometry using annexin Vpropidium iodide (PI) staining. ER expression was determined by Western blotting. Intracellular calcium was stained with Fluo4 AM while intracellular caspase activities were detected with CaspaseFLICA® and measured by flow cytometry. We showed that 15dPGJ2 caused a significant increase in apoptosis in MCF7 and MDAMB231 cells. ERα protein expression was reduced in treated MCF7 cells but preincubation with the ERα inhibitor' ICI 182 780' did not affect the percentage of apoptotic cells. The expression of ERβ was unchanged in both cell lines. In addition, 15dPGJ2 increased intracellular calcium (Ca²+) staining and caspase 8, 9 and3/7 activities. We therefore conclude that 15dPGJ2 induces caspasedependent apoptosis that is associated with an influx of intracellular Ca²+ with no involvement of ER signaling.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Calcium; Caspases; Cell Proliferation; Enzyme Activation; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Prostaglandin D2; Tumor Cells, Cultured

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ2 on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ2 dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ2 suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ2 blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ2 exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ2 substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ2 prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ2 may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.

Topics: Anilides; Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Disease Models, Animal; Estrogens; Female; Humans; Male; Mice; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Ovariectomy; Parathyroid Hormone-Related Protein; PPAR gamma; Prostaglandin D2; RANK Ligand

Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the sites in the proteome that are most sensitive to electrophilic modification requires more quantitative methods. Here we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against >1,000 cysteines in parallel in the human proteome. Using this approach, we identified a select set of proteins that constitute 'hot spots' for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxy-2-nonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine and that the resulting enzyme inhibition creates a negative feedback mechanism that can suppress the activation of JNK pathways normally induced by oxidative stress.

Topics: Amino Acid Sequence; Breast Neoplasms; Catalytic Domain; Cell Line, Tumor; Cysteine; Dose-Response Relationship, Drug; Electrochemistry; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Inhibitory Concentration 50; Lipids; MAP Kinase Kinase Kinases; Mass Spectrometry; Molecular Sequence Data; Oxidative Stress; Prostaglandin D2; Protein Kinases; Protein Processing, Post-Translational; Proteome; Proteomics; Sequence Homology, Amino Acid

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that has antioxidant and cytoprotective functions. However, HO-1 has oncogenic functions in cancerous or transformed cells. In the present work, we investigated the effects of HO-1 on the expression of p53 induced by 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) in human breast cancer (MCF-7) cells. Treatment of MCF-7 cells with 15d-PGJ2 led to time-dependent increases in the expression of p53 as well as HO-1. Upregulation of p53 expression by 15d-PGJ2 was abrogated by si-RNA knock-down of HO-1. In MCF-7 cells transfected with HO-1 si-RNA, 15d-PGJ2 failed to induce expression of p53 as well as HO-1. In addition, HO-1 inducers enhanced the p53 expression. We speculated that iron, a by-product of HO-1-catalyzed reactions, could mediate 15d-PGJ2-induced p53 expression. Upregulation of p53 expression by 15d-PGJ2 was abrogated by the iron chelator desferrioxamine in MCF-7 cells. Iron released from heme by HO-1 activity is mostly in the Fe(2+) form. When MCF-7 cells were treated with the Fe(2+)-specific chelator phenanthroline, 15d-PGJ2-induced p53 expression was attenuated. In addition, levels of the Fe-sequestering protein H-ferritin were elevated in 15d-PGJ2-treated MCF-7 cells. In conclusion, upregulation of p53 and p21 via HO-1 induction and subsequent release of iron with accumulation of H-ferritin may confer resistance to oxidative damage in cancer cells frequently challenged by redox-cycling anticancer drugs.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Heme Oxygenase-1; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; NF-E2-Related Factor 2; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Suppressor Protein p53; Up-Regulation

Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is maintained not only by its biosynthesis, but also by inactivation/degradation. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the conversion of oncogenic PGE2 to a biologically inactive keto metabolite. In the present study, we demonstrate that 15-deoxy-Δ(12,14)-prostaglandin J2 (15 d-PGJ2), one of the terminal products of cyclooxygenase-2, updregulates the expression and the activity of 15-PGDH in human breast cancer MDA-MB-231 cells. By using deletion constructs of the 15-PGDH promoter, we have found that E-twenty six (Ets) is the most essential determinant for 15-PGDH induction. 15 d-PGJ2 induced phosphorylation of Elk-1, one of Ets transcription factor family members, in the nucleus. Knockdown of Elk-1 abolished the ability of 15 d-PGJ2 to upregulate 15-PGDH expression. Furthermore, 15 d-PGJ2-mediated activation of Elk-1 was found to be dependent on activation of extracellular-signal related kinase (ERK) 1/2. Treatment of U0126, a pharmacological inhibitor of MEK1/2-ERK, abolished phosphorylation and DNA binding of Elk-1 as well as 15-PGDH induction in 15 d-PGJ2-treated MDA-MB-231 cells. Moreover, 15 d-PGJ2 generated reactive oxygen species (ROS), which contribute to the expression of 15-PGDH as well as phosphorylation of ERK1/2 and Elk-1. 15 d-PGJ2 inhibited the migration of MDA-MB-231 cells, which was attenuated by transient transfection with 15-PGDH siRNA. Taken together, these findings suggest that 15 d-PGJ2 induces the expression of 15-PGDH through ROS-mediated activation of ERK1/2 and subsequently Elk-1 in the MDA-MB-231 cells, which may contribute to tumor suppressive activity of this cyclopentenone prostaglandin.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; ets-Domain Protein Elk-1; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxyprostaglandin Dehydrogenases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Prostaglandin D2; Reactive Oxygen Species

6-Shogaol has been shown to possess many antitumor properties including inhibition of cancer cell growth, inhibition of cancer metastasis, induction of apoptosis in cancer cells and induction of cancer cell differentiation. Despite its prominent antitumor effects, the direct molecular target of 6-shogaol has remained elusive. To identify the direct targets of 6-shogaol, a comprehensive antitumor profile of 6-shogaol (NSC752389) was tested in the NCI-60 cell line in an in vitro screen. The results show that 6-shogaol is COMPARE negative suggesting that it functions via a mechanism of action distinct from existing classes of therapeutic agents. Further analysis using microarray gene profiling and Connectivity Map analysis showed that MCF-7 cells treated with 6-shogaol display gene expression signatures characteristic of peroxisome proliferator activated receptor γ (PPARγ) agonists, suggesting that 6-shogaol may activate the PPARγ signaling pathway for its antitumor effects. Indeed, treatment of MCF-7 and HT29 cells with 6-shogaol induced PPARγ transcriptional activity, suppressed NFκB activity, and induced apoptosis in breast and colon cancer cells in a PPARγ-dependent manner. Furthermore, 6-shogaol is capable of binding to PPARγ with a binding affinity comparable to 15-delta prostaglandin J2, a natural ligand for PPARγ. Together, our findings suggest that the antitumor effects of 6-shogaol are mediated through activation of PPARγ and imply that activation of PPARγ might be beneficial for breast and colon cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Catechols; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Ligands; MCF-7 Cells; NF-kappa B; Oligonucleotide Array Sequence Analysis; PPAR gamma; Prostaglandin D2; Signal Transduction; Transcription, Genetic

The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), is expressed in various cancer cells including breast, prostate, colorectal and cervical examples. An endogenous ligand of PPARγ, 15-deoxy-Δ12,14 prostaglandin J2 (PGJ2), is emerging as a potent anticancer agent but the exact mechanism has not been fully elucidated, especially in breast cancer. The present study compared the anticancer effects of PGJ2 on estrogen receptor alpha (ERα)-positive (MCF-7) and ERα-negative (MDA-MB-231) human breast cancer cells. Based on the reported signalling cross-talk between PPARγ and ERα, the effect of the ERα ligand, 17β-estradiol (E2) on the anticancer activities of PGJ2 in both types of cells was also explored. Here we report that PGJ2 inhibited proliferation of both MCF-7 and MDA-MB-231 cells by inducing apoptotic cell death with active involvement of mitochondria. The presence of E2 potentiated PGJ2-induced apoptosis in MCF-7, but not in MDA-MB-231 cells. The PPARγ antagonist, GW9662, failed to block PGJ2-induced activities but potentiated its effects in MCF-7 cells, instead. Interestingly, GW9662 also proved capable of inducing apoptotic cell death. It can be concluded that E2 enhances PPARγ-independent anticancer effects of PGJ2 in the presence of its receptor.

Topics: Anilides; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Drug Synergism; Estradiol; Estrogens; Female; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; PPAR gamma; Prostaglandin D2; Tumor Cells, Cultured

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor and plays important roles in breast cancer cell proliferation. The complexity of the underlying biochemical and molecular mechanisms of breast cancer and the involvement of PPARγ in breast cancer pathophysiology are unclear. In this study, we carried out prediction of the peroxisome proliferator response element (PPRE) motifs in 2332 genes reported to be involved in breast cancer in literature. A total of 178 genes were found to have PPRE (DR1/DR2) and/or PPAR-associated conserved motif (PACM) motifs. We further constructed protein-protein interaction network, disease gene network and gene ontology (GO) analyses to identify novel key genes for experimental validation. We identified two genes in the glycolytic pathway (phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M2 (PKM2)) at the ATP production steps and experimentally validated their repression by PPARγ in two breast cancer cell lines MDA-MB-231 and MCF-7. Further analysis suggested that this repression leads to decrease in ATP levels and apoptosis. These investigations will help us in understanding the molecular mechanisms by which PPARγ regulates the cellular energy pathway and the use of its ligands in human breast cancer therapeutics.

Topics: Adenosine Triphosphate; Apoptosis; Breast Neoplasms; Carrier Proteins; Databases, Genetic; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Ligands; MCF-7 Cells; Membrane Proteins; Phosphoglycerate Kinase; PPAR gamma; Prostaglandin D2; Protein Interaction Domains and Motifs; Response Elements; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcriptional Activation

Our aim was to get new information about the Peroxisome Proliferator Activated Receptor gamma (PPARγ)-independent pathway involved in the antiproliferative action of PPARγ ligands in breast cancer cells. We investigated the effects of Troglitazone (TGZ), Ciglitazone (CGZ), Rosiglitazone (RGZ) and, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ(2)) on the hormone-dependent breast cancer cell line MCF7. The early transcription factor EGR1 (Early Growth Response gene 1) mRNA and protein levels peaked after 3h of incubation with 25μM TGZ, CGZ or 15d-PGJ(2) and then gradually decreased. RGZ, the most potent activator of PPARγ, did not show this effect. The PPARγ antagonist GW 9662 did not block EGR1 mRNA induction which also still occurred in case of PPARγ silencing as well as in case of treatment with the PPARγ-inactive compound Δ2-TGZ. EGR1 mRNA induction required ERK1/2 phosphorylation which was not blocked by EGF Receptor (EGFR) inhibition. The ERK1/2 pathway was also involved in Δ2-TGZ-induced EGR1 mRNA expression in the hormone-independent breast cancer cell line MDA-MB-231. Using the fluorescent dye Fura2, we showed in MCF7 that TGZ or Δ2-TGZ induced an immediate increase in cytosolic calcium which was required for ERK1/2 phosphorylation and EGR1 mRNA induction as demonstrated by calcium chelation experiments. Furthermore, in MCF7 transfected with siRNA targeting EGR1, Δ2-TGZ inhibited less efficiently cell proliferation.

Topics: Base Sequence; Breast Neoplasms; Calcium; Cell Line, Tumor; Cell Proliferation; DNA Primers; Early Growth Response Protein 1; Female; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; PPAR gamma; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction; Thiazolidinediones

15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a representative J-series cyclopentenone prostaglandin bearing an electrophilic α,β-unsaturated carbonyl group. In the present study, treatment of human breast cancer MCF-7 cells with 15d-PGJ(2) caused the up-regulation of the glutamate cysteine ligase catalytic (GCLC) subunit, the rate-limiting enzyme in glutathione (GSH) synthesis. 15d-PGJ(2) treatment caused nuclear translocation and transactivation of Nrf2, a redox-sensitive transcription factor responsible for induced expression of antioxidant and other cytoprotective genes. siRNA knockdown of Nrf2 abrogated 15d-PGJ(2)-induced GCLC expression. Following 15d-PGJ(2) treatment, the intracellular GSH level was initially diminished but eventually enhanced even above the basal level. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) abolished the 15d-PGJ2-induced Nrf2 activation and GCLC expression. Pharmacologic inhibition or siRNA knockdown of Akt, the target of phosphoinositide 3-kinase (PI3-K), attenuated 15d-PGJ(2)-induced Nrf2 activation and GCLC expression, and NAC treatment inhibited phosphorylation of Akt, and subsequently Nrf2 activation and GCLC upregulation. 9,10-Dihydro-15-PGJ2, a nonelectrophilic analogue of 15d-PGJ(2) that lacks the ability to form a conjugate with GSH, failed to induce activation of Akt and Nrf2 as well as ROS generation. These findings, taken all together, suggest that intracellular accumulation of ROS formed as a consequence of initial depletion of GSH can activate Akt, which in turn induces Nrf2 activation and subsequently the expression of GCLC, leading to the restoration of GSH. Interestingly, the extracellular GSH level was increased, concomitantly with the depletion of the intracellular GSH following 15d-PGJ(2) treatment. However, 15d-PGJ(2) was unable to influence both intra- and extra-cellular GSH levels when multidrug resistance-associated protein 1 (MRP1), the efflux pump for GSH conjugates, was blocked by its antagonist, MK571. Moreover, 15d-PGJ(2)-induced GCLC expression was attenuated by the MK571 and also by siRNA knockdown of MRP1, suggesting that MRP1 contributes to 15d-PGJ(2)-mediated up-regulation of GCLC by pumping out the 15d-PGJ(2)-GSH conjugate. It is speculated that 15d-PGJ(2), once effluxed through MRP, liberates from the GSH conjugate, and the free 15d-PGJ(2) re-enters the cell and forms the GSH conjugate again. In conclusion, MRP1 mediates Nrf2-dependent up-regulation of GCLC in

Topics: Acetylcysteine; Breast Neoplasms; Cell Line, Tumor; Female; Glutamate-Cysteine Ligase; Glutathione; Humans; Multidrug Resistance-Associated Proteins; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; Propionates; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Quinolines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Signal Transduction; Up-Regulation

Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(4)-androstenedione to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)).

Topics: 20-alpha-Dihydroprogesterone; 3-Hydroxysteroid Dehydrogenases; 5-alpha-Dihydroprogesterone; Aldo-Keto Reductase Family 1 Member C3; Androstenedione; Androsterone; Biocatalysis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dihydrotestosterone; Dinoprost; Estradiol; Estrone; Etiocholanolone; Female; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Hydroxyprostaglandin Dehydrogenases; Ketosteroids; Kinetics; Progesterone; Prostaglandin D2; Prostaglandins; Recombinant Proteins; Testosterone; Transfection; Up-Regulation

Heme oxygenase-1 (HO-1) has recently been found to be involved in angiogenesis and metastasis. In this study, we investigated whether HO-1 could potentiate the metastatic potential of human breast cancer cells. Treatment of MCF-7 and MDA-MB-231 cells with 30 microM of 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) increased the expression of HO-1, which preceded the induction of matrix metalloproteinases (MMPs). The 15d-PGJ2-induced upregulation of MMP-1 was abrogated by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) as well as introduction of HO-1 short interfering RNA. In addition, HO-1 inducers, such as cobalt protoporphyrin IX and hemin, upregulated the expression of MMP-1. Overexpression of HO-1 in the MCF-7 cells caused the induction of MMP-1 expression. Treatment with the HO-1 inhibitor ZnPP abolished the migrative phenotype of 15d-PGJ2-treated MCF-7 cells. MCF-7 cells treated with 15d-PGJ2 exhibited intracellular accumulation of reactive oxygen species (ROS) which was abolished by ZnPP. We hypothesize that excess iron, released as a consequence HO-1 activity induced by 15d-PGJ2, is transiently available for the stimulation of intracellular ROS generation and subsequently MMP-1 expression. 15d-PGJ2-mediated upregulation of MMP-1 expression was blocked by the iron chelator desferrioxamine and the Fe2+-specific chelator 1,10-phenanthroline. The iron chelators as well as the antioxidant N-acetyl-L-cysteine abrogated ROS formation by 15d-PGJ2. In conclusion, 15d-PGJ2 upregulates MMP-1 expression via induction of HO-1 and subsequent production of iron capable of generating ROS, which may contribute to increased metastasis and invasiveness of the human breast cancer cells.

Topics: Breast Neoplasms; Cell Movement; Heme Oxygenase-1; Humans; Immunologic Factors; Iron; Iron Chelating Agents; Luciferases; Matrix Metalloproteinase 1; Matrix Metalloproteinase Inhibitors; NF-E2-Related Factor 2; Phenanthrolines; Prostaglandin D2; Protoporphyrins; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Up-Regulation; Wound Healing

Cyclopentenone prostaglandins (cyPGs) exert diverse cellular functions, such as anti-inflammatory and cytoprotective effects, via multiple mechanisms. CyPGs, especially those of the A and J series, are characterized by the presence of a chemically reactive alpha,beta-unsaturated carbonyl group. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyPG of the J series, has been reported to directly inhibit the activity of redox-sensitive transcription factors, such as activator protein-1 and nuclear factor-kappaB. In the present study, we examined the effects of 15d-PGJ(2) on activation of p53 tumor suppressor in human breast cancer (MCF-7) cells. MCF-7 cells treated with 15d-PGJ(2) exhibited elevated p53 protein expression in time- and concentration-related manners, whereas prostaglandin A(2) (PGA(2)) and the nonprostaglandin derivative 2-cyclopenten-1-one exerted an effect to a lesser extent than did 15d-PGJ(2). In addition, MCF-7 cells exposed to 15d-PGJ(2) significantly accumulated p53 in both cytosolic and nuclear fractions. Despite the elevated levels of p53, its DNA-binding activity was reduced in 15d-PGJ(2)-treated MCF-7 cells. Moreover, isolated MCF-7 nuclear extracts directly treated with 15d-PGJ(2) exhibite diminished DNA-binding ability of p53, while the same concentration of PGA(2) or 2-cyclopenten-1-one was much less inhibitory. Thus, the electrophilic carbon center located in the alpha,beta-unsaturated carbonyl moiety of the cyclopentenone ring might be critical for the control of DNA-binding activity as well as cellular levels of p53 by 15d-PGJ(2).

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell-Free System; Cyclopentanes; Cytosol; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Female; Humans; Immunohistochemistry; Microscopy, Confocal; Molecular Structure; Prostaglandin D2; Prostaglandins A; Protein Binding; Time Factors; Tumor Suppressor Protein p53

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative J-series cyclopentenone prostaglandin, exerts cytoprotective effects that are mainly mediated by Nrf2. Nrf2 is a major transcription factor involved in the transactivation of genes encoding many phase 2 detoxifying and antioxidant enzymes via interaction with the antioxidant response element (ARE). Recently it has been reported that expression of phase 3 efflux transporters, such as multidrug resistance-associated proteins (MRPs), is also regulated by Nrf2. It is well known that cancer cells overexpressing MRPs are more resistant to anticancer drugs. In the present study we have found that 15d-PGJ(2) induces the expression of MRP1, one of the phase 3 efflux transporters, in human breast cancer cells (MCF-7). In addition, treatment of MCF-7 cells with 15d-PGJ(2) resulted in nuclear translocation and DNA binding of Nrf2. In contrast to 15d-PGJ(2), 9,10-dihydro-15d-PGJ(2), an analogue of 15d-PGJ(2) that lacks an electrophilic cyclopentenone ring moiety, failed to induce not only Nrf2 activation but also MRP1 upregulation in MCF-7 cells. 15d-PGJ(2)-induced MRP1 overexpression was abrogated by Nrf2 gene knockdown, using RNA interference. These results, taken together, suggest that 15d-PGJ(2) induces MRP1 upregulation via Nrf2-ARE signaling.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Structure; Multidrug Resistance-Associated Proteins; NF-E2-Related Factor 2; Oligonucleotides; Prostaglandin D2; Protein Binding; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection; Up-Regulation

Recent studies suggest that inflammation is causally linked to carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandins, is inappropriately expressed in various cancers and hence recognized as one of the hallmarks of chronic inflammation-associated malignancies. However, the mechanistic role of COX-2 as a link between inflammation and cancer remains undefined. Here, we report that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), one of the final products of COX-mediated arachidonic acid metabolism, upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line. 15d-PGJ(2)-induced COX-2 expression was mediated by activation of Akt and subsequently activator protein-1 (AP-1). Furthermore, 15d-PGJ(2) formed reactive oxygen species, which led to increased phosphorylation of Akt, DNA binding of AP-1 and expression of COX-2. In contrast to 15d-PGJ(2), 9,10-dihydro-15d-PGJ(2) did not elicit any of effects induced by 15d-PGJ(2) in this study, suggesting that an electrophilic carbon center present in 15d-PGJ(2) is critical for COX-2 expression as well activation of upstream signal transduction induced by this cyclopentenone prostaglandin. Taken together, these observations suggest that 15d-PGJ(2) produced by COX-2 overexpression may function as a positive regulator of COX-2 in human breast cancer MCF-7 cells.

Topics: Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2; DNA Probes; DNA Replication; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Transcription Factor AP-1; Transfection

BAG-1 is a pleiotropic protein that exists as multiple isoforms. BAG-1 overexpression in breast cancer is associated with outcome. BAG-1 modulates the function of various nuclear hormone receptors, including the oestrogen receptor, and BAG-1 can influence the in vitro action of anti-hormonal therapies such as cyproterone acetate in prostate cancer. Activation of PPARgamma, a nuclear hormone receptor important for lipid and glucose homeostasis, may present a new therapeutic approach for breast cancer, since PPARgamma agonists promote cell cycle arrest, differentiation and apoptosis in breast cancer cells. Here we determined whether BAG-1 also modulated PPARgamma function in MCF7 cells. 15-deoxy-Delta12, 14-prostaglandin J(2) (15dPGJ2), an agonistic ligand for PPARgamma, induced expression of HSP70, a BAG-1 binding partner, but did not alter BAG-1 isoform expression. Overexpression of BAG-1 isoforms did not alter PPARgamma-dependent transcription or interfere with 15dPGJ2-induced cell cycle arrest or differentiation. However, overexpression of BAG-1 isoforms did interfere with induction of cell death by 15dPGJ2. Thus, BAG-1 is unlikely to directly modulate PPARgamma function, but the overexpression of BAG-1 in some breast cancers may limit the efficacy of PPARgamma agonists as cancer therapies, by suppression of PPARgamma-induced cell death pathways.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Line, Tumor; DNA-Binding Proteins; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; PPAR gamma; Prostaglandin D2; Protein Isoforms; Transcription Factors; Transcription, Genetic

The cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-gamma (PPARgamma)-dependent and PPARgamma-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-alpha (ERalpha) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ(2) inhibits both 17beta-estradiol (E(2))-dependent and E(2)-independent ERalpha transcriptional activity by PPARgamma-independent mechanism. In addition, 15d-PGJ(2) directly modifies ERalpha protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ(2) into ERalpha, both in vitro and in vivo. Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys(227) and Cys(240)) within the COOH-terminal zinc finger of ERalpha DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ(2). Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ(2) inhibits DNA binding of ERalpha and subsequent repression of ERalpha target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ(2) can block ERalpha function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERalpha DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERalpha transcriptional activity.

Topics: Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Cysteine; DNA, Neoplasm; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Humans; Molecular Sequence Data; PPAR gamma; Prostaglandin D2; Protein Structure, Tertiary; Transcriptional Activation; Transfection; Zinc Fingers

The PPAR-gamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J(2) and ciglitazone, and the PPAR-alpha ligand, WY-14643, were examined for their effects on proliferation and apoptosis of A375 melanoma, DU145 and PC3 prostate cancer, and MB-MDA-231 breast cancer. While 15-deoxy-Delta(12,14)-prostaglandin J(2) inhibited proliferation of A375 melanoma, ciglitazone was inactive against this and the other cell lines. Restriction of specific amino acids known to inhibit proliferation and induce apoptosis sensitized all cell lines to ciglitazone, and the combined effects were greater than the individual effects of either treatment. WY-14643 alone or in combination with amino acid deprivation was inactive. Normal fibroblasts were resistant to the treatments.

Topics: Amino Acids; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Hypoglycemic Agents; Immunologic Factors; Ligands; Male; Melanoma; Methionine; Phenylalanine; PPAR gamma; Prostaglandin D2; Prostatic Neoplasms; Thiazolidinediones; Tyrosine

It has been reported that agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibit proliferation of breast carcinoma cells, but the biological significance of PPARgamma remains undetermined in human breast carcinomas. Therefore, we immunolocalized PPARgamma in 238 human breast carcinoma tissues. PPARgamma immunoreactivity was detected in 42% of carcinomas, and was significantly associated with the status of estrogen receptor (ER) alpha, ERbeta, progesterone receptor, retinoic X receptors, p21 or p27, and negatively correlated with histological grade or cyclooxygenase-2 status. PPARgamma immunoreactivity was significantly associated with an improved clinical outcome of breast carcinoma patients by univariate analysis, and multivariate analysis demonstrated that PPARgamma immunoreactivity was an independent prognostic factor for overall survival in ERalpha-positive patients. We then examined possible mechanisms of modulation by PPARgamma on estrogenic actions in MCF-7 breast carcinoma cells. A PPARgamma activator, 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2)), significantly inhibited estrogen-responsive element-dependent transactivation by estradiol in MCF-7 cells, which was blocked by addition of a PPARgamma antagonist GW9662. Subsequent study, employing a custom-made microarray focused on estrogen-responsive genes, revealed that mRNA expression was significantly regulated by estradiol in 49 genes, but this significance vanished on addition of 15d-PGJ(2) in 16 out of 49 (33%) genes. These findings were confirmed by real-time PCR in 11 genes. 15d-PGJ(2) significantly inhibited estrogen-mediated proliferation of MCF-7 cells, and caused accumulation of p21 and p27 protein. These results suggest that PPARgamma is mainly expressed in well-differentiated and ER-positive breast carcinomas, and modulates estrogenic actions.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Proliferation; Chemotherapy, Adjuvant; Cyclin-Dependent Kinase Inhibitor p21; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Immunologic Factors; Middle Aged; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; PPAR gamma; Prostaglandin D2; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; Transcriptional Activation; Tumor Cells, Cultured

Nuclear factor-kappaB (NF-kappaB), a transcription factor with a critical role in promoting inflammation and cell survival, is constitutively activated in estrogen-receptor (ER)-negative breast cancer and is considered a potential therapeutic target for this type of neoplasia. We have previously demonstrated that cyclopentenone prostaglandins are potent inhibitors of NF-kappaB activation by inflammatory cytokines, mitogens, and viral infection, via direct binding and modification of the beta subunit of the IkappaB kinase complex (IKK). Herein, we describe the NF-kappaB-dependent anticancer activity of natural and synthetic cyclopentenone IKK inhibitors. We demonstrate that the natural cyclopentenone 15-deoxy-Delta(12,14)prostaglandin J(2) (15d-PGJ(2)) is a potent inhibitor of constitutive IkappaB-kinase and NF-kappaB activities in chemotherapy-resistant ER-negative breast cancer cells. 15d-PGJ(2)-induced inhibition of NF-kappaB function is rapidly followed by down-regulation of NF-kappaB-dependent antiapoptotic proteins cIAPs 1/2, Bcl-X(L), and cellular FLICE-inhibitory protein, leading to caspase activation and induction of apoptosis in breast cancer cells resistant to treatment with paclitaxel and doxorubicin. We then demonstrate that the cyclopentenone ring structure is responsible for these activities, and we identify a new synthetic cyclopentenone derivative, 3-tert-butyldimethylsilyloxy-5-(E)-iso-propylmethylenecyclopent-2-enone (CTC-35), as a potent NF-kappaB inhibitor with proapoptotic activity in ER-negative breast cancer cells. The results open new perspectives in the search for novel proapoptotic molecules effective in the treatment of cancers presenting aberrant NF-kappaB regulation.

Topics: Antineoplastic Agents; Apoptosis; Arachidonic Acid; Breast Neoplasms; Caspases; Cyclopentanes; Down-Regulation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; I-kappa B Kinase; Inhibitor of Apoptosis Proteins; NF-kappa B; Prostaglandin D2; Receptors, Estrogen; Tumor Cells, Cultured

The S14 (spot 14) gene encodes a protein that is predominantly expressed in lipogenic tissues, such as the liver, white and brown adipose tissues and the lactating mammary glands. Accumulated evidence suggests that S14 could play an important role in the induction of lipogenic enzymes. In humans, the S14 locus resides in the chromosome region 11q13, which is frequently amplified in breast tumours, and as a result, it has been suggested that this protein could play a role in the metabolism and growth of these kinds of tumours. In the present study, we have examined the effects of S14 overexpression in MCF-7 human breast cancer cells. We found that S14 causes (i) an inhibition of cell proliferation and of anchorage-independent growth, (ii) a marked reduction in the number of viable cells and (iii) the induction of differentiation and cell death of these cells. The inhibition of cell growth was associated with a decrease in the expression of cyclin D1 and a reduction of cyclin D1 promoter activity. Increased expression of S14 also caused the accumulation of cytochrome c in the cytosol and loss of mitochondrial membrane potential. These findings suggest that S14 may function as an important modulator of tumorigenesis in human breast by decreasing cell growth and inducing cell death and differentiation.

Topics: Breast Neoplasms; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Cyclin D1; Gene Expression; Humans; Nuclear Proteins; Promoter Regions, Genetic; Prostaglandin D2; Proteins; Time Factors; Transcription Factors; Tretinoin

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical albeit poorly defined role in the development and progression of several cancer types including those of the breast, colon, and lung. A PPAR response element (PPRE) reporter assay was utilized to evaluate the selective transactivation of PPARgamma in 10 different cell lines including normal mammary epithelial, breast, lung, and colon cancer cells. Cells were treated with one of four compounds including rosglitizone (Ros), ciglitizone (Cig), 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2), or GW 9662 (GW). We observed differences in transactivation between cell lines from different tissue origin, across cell lines from a single tissue type, and selective modulation of PPARgamma within a single cell line by different ligands. Interestingly, GW, a PPARgamma antagonist in adipocytes, enhanced PPRE reporter activation in normal mammary epithelial cells while it had virtually no effect in any of the cancer cell lines tested. Within each cancer type, individual cell lines were found to respond differently to distinct PPARgamma ligands. For instance, Ros, Cig, and PGJ2 were all potent agonist of PPARgamma transactivation in lung adenocarcinoma cell lines while these same ligands had no effect in squamous cell or large cell carcinomas of the lung. Message levels of PPARgamma and retinoid X receptor alpha (RXRalpha) in the individual cell lines were quantitated by real time-polymerase chain reaction (RT-PCR). The ratio of PPARgamma to RXRalpha was predictive of how cells responded to co-treatment of Ros and 9-cis-retinoic acid, an RXRalpha agonist, in two out of three cell lines tested. These data indicate that PPARgamma can be selectively modulated and suggests that it may be used as a therapeutic target for individual tumors.

Topics: Alitretinoin; Anilides; Breast Neoplasms; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; HT29 Cells; Humans; Ligands; Lung Neoplasms; PPAR gamma; Prostaglandin D2; Retinoid X Receptor alpha; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Transfection; Tretinoin

1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF(3)) and several p-substituted phenyl analogues have been investigated as a new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Structure-activity studies in PPARgamma-dependent transactivation assays in MCF-7 breast cancer cells show that 5-20 micro M concentrations of compounds containing p-trifluoromethyl, t-butyl, cyano, dimethylamino, and phenyl groups were active, whereas p-methyl, hydrogen, methoxy, hydroxyl, or halogen groups were inactive as PPARgamma agonists. Induction of PPARgamma-dependent transactivation by 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2) and DIM-C-pPhCF(3) was inhibited in MCF-7 cells cotreated with the PPARgamma-specific antagonist N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide. In mammalian two-hybrid assays, DIM-C-pPhCF(3) and PGJ2 (5-20 micro M) induced interactions of PPARgamma with steroid receptor coactivator (SRC) 1, SRC2 (TIFII), and thyroid hormone receptor-associated protein 220 but not with SRC3 (AIB1). In contrast, DIM-C-pPhCF(3), but not PGJ2, induced interactions of PPARgamma with PPARgamma coactivator-1. C-substituted diindolylmethanes inhibit carcinogen-induced rat mammary tumor growth, induce differentiation in 3T3-L1 preadipocytes, inhibit MCF-7 cell growth and G(0)/G(1)-S phase progression, induce apoptosis, and down-regulate cyclin D1 protein and estrogen receptor alpha in breast cancer cells. These compounds are a novel class of synthetic PPARgamma agonists that induce responses in MCF-7 cells similar to those observed for PGJ2.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Apoptosis; Breast Neoplasms; Carcinogens; Cell Cycle; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Separation; Cloning, Molecular; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Estrogen Receptor alpha; Female; Flow Cytometry; G1 Phase; Humans; Indoles; Ligands; Luciferases; Methane; Mice; Plasmids; Prostaglandin D2; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Resting Phase, Cell Cycle; Structure-Activity Relationship; Transcription Factors; Transcriptional Activation; Transfection; Two-Hybrid System Techniques

Cyclooxygenase-2 (COX-2) expression and peroxisome proliferator-activated receptor-gamma (PPARgamma) inactivation are linked to increased risk of human breast cancer. The purpose of our study was to examine the relationship between COX-2 (with the resulting prostaglandins E(2), PGE(2)) and PPARgamma (and its natural endogenous ligand 15-Deoxy-Delta(12,14)-prostaglandin J(2), 15d-PGJ(2)) at various stages during the development of human breast cancer and its progression to metastasis. Human breast tissue specimens were collected from normal breasts or from individuals with fibrocystic disease and served as controls (n = 22). Tissues were also collected from uninvolved (n = 25), tumor (n = 25) and lymph node metastasis (n = 15) regions from breast cancer patients. COX-2 and PPARgamma mRNA expression were increased and downregulated, respectively, in tissues from cancer patients compared to controls. Metastatic tissues tended to have higher alterations compared to non-metastatic tissues (p < 0.05). These altered expressions in COX-2 and PPARgamma were paralleled by increases in the tissue levels of PGE(2) and decreases in 15d-PGJ(2). A significant inverse correlation was found between PGE(2) and 15-d-PGJ(2) (r = -0.51, p < 0.05). Significant correlations (p < 0.05) were also obtained between COX-2 and PPARgamma mRNA (inverse, r = -0.72) and between COX-2 and PGE(2) (direct, r = 0.68). Increases in COX-2 mRNA expression and levels of PGE(2) and down-regulation of PPARgamma mRNA expression and 15d-PGJ(2) levels were characterized as predictors of breast cancer risk (p < 0.05). Our results suggest that the altered expression of COX-2 and PPARgamma and the subsequent modulation in the tissue levels of PGE(2) and 15-d-PGJ(2) may influence the development of human breast cancer and its progression to metastasis.

Topics: Adenocarcinoma; Breast Neoplasms; Case-Control Studies; Cyclooxygenase 2; Dinoprostone; DNA Primers; Female; Humans; Isoenzymes; Lymphatic Metastasis; Membrane Proteins; Middle Aged; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transcription Factors

The mechanisms by which prostaglandin (PG)J(2) metabolites inhibit tumorigenicity are poorly understood but may involve thiol reactivity or peroxisome proliferator-activated receptor (PPAR)-dependent pathways. Because aromatase is an important therapeutic target in breast cancer treatment, we have investigated the effect of PGJ(2) metabolites on aromatase activity and evaluated a potential role for redox status during PGJ(2) metabolite action. 15-deoxy-Delta(12,14)PGJ(2) (15d-PGJ(2)) and 9-deoxy-Delta(9,12)13,14-dihydroPGD(2) (Delta(12)PGJ(2)) caused dose-dependent inhibition of both pre-induced aromatase activity in human breast fibroblasts and MDA MB 231 breast cancer cells and of constitutive aromatase activity in JEG-3 choriocarcinoma cells. Structure-activity studies showed that this inhibition was mimicked by 4-cyclopentene-1,3-dione but not by the PPARgamma agonist troglitazone nor the eicosanoids PGE(2) or arachidonic acid. The thiol oxidants diamide and H(2)O(2) simulated the inhibitory action of 15d-PGJ(2) on aromatase activity, whereas the glutathione (GSH) repletor and antioxidant N-acetyl-cysteine (NAC) reversed these actions of 15d-PGJ(2) and H(2)O(2) on aromatase. 15d-PGJ(2) also caused a direct dose-dependent inhibition of aromatase activity in JEG-3 cell sonicates, which was also reversed in the presence of GSH. Kinetic analysis of this 15d-PGJ(2)-induced inhibition of cell-free aromatase indicated the involvement of a non-competitive mechanism possibly resulting from direct thiol-targeted alkylation of the enzyme. These redox-sensitive, PPARgamma-independent actions of 15d-PGJ(2) on aromatase activity demonstrate a novel therapeutic potential for such cyclopentenone PGs in breast cancer treatment.

Topics: Acetylcysteine; Antioxidants; Aromatase; Aromatase Inhibitors; Breast; Breast Neoplasms; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fibroblasts; Glutathione; Humans; Hydrogen Peroxide; Oxidation-Reduction; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Sulfhydryl Reagents; Transcription Factors; Tumor Cells, Cultured

Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Cysteine Endopeptidases; Down-Regulation; Estrogen Receptor alpha; G1 Phase; Humans; Multienzyme Complexes; Prostaglandin D2; Proteasome Endopeptidase Complex; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; RNA, Messenger; S Phase; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Ubiquitin

One of the most potent cyclopentenone prostaglandins, 15-deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), has been shown to be cytotoxic in some tumor cells and, as a ligand of peroxisome proliferator activated receptor gamma (PPARgamma), to influence the transcriptional regulation of several genes. We examined whether a glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, is formed and if the glutathione conjugate efflux pumps, MRP1 and MRP3, could transport this conjugate, thereby attenuating the cytotoxicity and transactivating activity of 15-d-PGJ(2) in MCF7 breast cancer cells. Formation of 15-d-PGJ(2)-SG was demonstrated both in vitro and in cells, and its structure was determined by ESI/MS and NMR. Expression of MRP1 and MRP3 was achieved by stable transduction of parental MCF7 cells. Membrane vesicles derived from these cells supported efficient, ATP-dependent transport of 15-d-PGJ(2)-SG (K(M) 1.4 and 2.9 microM for MRP1 and MRP3, respectively). When compared with parental, MRP-minus MCF7 cells, expression of MRP1 and MRP3 conferred approximately 2-fold protection from 15-d-PGJ(2) cytotoxicity. 15-d-PGJ(2)-mediated transcriptional activation was evaluated in cells transiently transfected with a reporter gene under the transcriptional control of a PPAR responsive element. Treatment of parental MCF7 cells with 15-d-PGJ(2) resulted in a time-dependent induction of reporter gene activity-induction that was measurable with concentrations of added 15-d-PGJ(2) as low as 100 nM. In contrast, expression of MRP1 or MRP3 abolished 15-d-PGJ(2)-dependent reporter gene induction. Depletion of intracellular glutathione reversed MRP1- and MRP3-mediated attenuation of 15-d-PGJ(2) cytotoxicity and transactivation. These data indicate that MRP1 and MRP3 can modulate the biological effects of 15-d-PGJ(2), and likely other cyclopentenone prostaglandins, in a glutathione-dependent manner. The results are consistent with a mechanism for the attenuation of the biological activities of 15-d-PGJ(2) that involves the formation and active efflux of its glutathione conjugate, 15-d-PGJ(2)-SG.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Breast Neoplasms; Cell Membrane; Cell Survival; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Immunologic Factors; Inhibitory Concentration 50; Kinetics; Luciferases; Magnetic Resonance Spectroscopy; Methotrexate; Multidrug Resistance-Associated Proteins; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Spectrometry, Mass, Electrospray Ionization; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Invasion and metastasis are the main causes of death in breast cancer patients. Increased expression of matrix metalloproteinases (MMPs), especially gelatinases (MMP-2 and -9), has been closely associated with tumor progression. One of the nuclear hormone receptors (NHR), peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-activated transcriptional factor that regulates cell proliferation, differentiation and apoptosis in both normal and cancer cells. Recent data indicate that PPARgamma activation by its ligands can also lead to the inhibition of gelatinase B (MMP-9) and the blockage of migration in macrophages and muscle cells, implying the possibility that PPARgamma ligands may possess anti-invasive activities on tumor cells. In this study, we showed that treatment of the highly aggressive human breast cancer cell line MDA-MB-231 with the synthetic PPARgamma ligands pioglitazone (PGZ), rosiglitazone (RGZ), GW7845 or its natural ligand 15-deoxy-delta 12, 14-prostaglandin J2(15d-PGJ2), at concentrations at which no obvious cytotoxicity was observed in vitro, led to a significant inhibition of the invasive capacities of this cell line through a reconstituted basement membrane (Matrigel) in a Transwell chamber model. All-trans-retinoic acid (ATRA), a ligand for retinoic acid receptor (RAR), was also studied and showed a similar inhibitory effect on invasion. Although no change was observed in the expression of MMP-9 after challenge with PPARgamma ligands and/or ATRA on this cell line, the natural tissue inhibitor of gelatinases, namely the tissue inhibitor of MMP 1 (TIMP-1) was upregulated by these treatments and the gelatinolytic activities of gelatinases in the conditioned media were decreased. Since MMP-2 was not detectable in the conditioned media of MDA-MB-231 cells, and the gelatinolytic activities of the conditioned media were reduced only by MMP-9 neutralizing antibodies, it is most likely that the reduction of gelatinolytic activities by PPARgamma ligands and/or ATRA was due to the decrease of MMP-9 activities. Because MMP-9 was absolutely required in the transmigration of this cell line through Matrigel in our in vitro model as demonstrated by neutralizing antibodies against MMP-2 and -9, we concluded that down-regulation of gelatinase activities is, at least in part, responsible for the reduction of the invasive capacities of MDA-MB-231 cell line in vitro. Our results, for the first time, indicate that PPARgamma ligands may

Topics: Breast Neoplasms; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nuclear Proteins; Pioglitazone; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazolidinediones; Tissue Inhibitor of Metalloproteinases; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo. PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer. Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation. Caveolin-1 has growth-inhibitory activities and its disruption is sufficient to induce transformation in fibroblasts. Herein we have tested the hypothesis that caveolins are transcriptional target genes for PPARgamma. In human HT-29 colon carcinoma cells, thiazolidinedione PPARgamma ligands increased the levels of caveolin-1 and caveolin-2 proteins two to fivefold in a concentration-dependent manner within 24 h. In human MCF-7 breast adenocarcinoma cells, nonthiazolidinedione PPARgamma ligands elevated caveolin-2 protein three to fourfold, while the thiazoli-dinediones were less effective. Caveolin-1 mRNA levels were found to be upregulated by PPARgamma ligands already after 3 h in both the cell lines. Ectopic expression of a dominant-negative PPARgamma construct attenuated ligand-induced upregulation of caveolins in both HT-29 and HEK-293T cells, indicating that ligand action is mediated by PPARgamma. Ligand-treated MCF-7 cells exhibited a differentiated phenotype, as evinced by analysis of cell-specific differentiation markers: protein levels of maspin were elevated and perinuclear lipid droplets accumulated. In contrast, in HT-29 cells, caveolin expression was not correlated with differentiation. Interestingly, PPARgamma partially cofractionated in lipid rafts and could be coimmunoprecipitated from cell lysates with caveolin-1, indicating that PPARgamma and caveolin-1 may coexist in a complex. Our data indicate that PPARgamma participates in the regulation of caveolin gene expression in human carcinoma cells and suggest that caveolin-1 may mediate some of the phenotypic changes induced by this nuclear receptor in cancer cells. These findings may have potentially important functional implications in the context of cancer differentiation therapy and multidrug resistance.

Topics: Adenocarcinoma; Antigens, Differentiation; Antigens, Neoplasm; Breast Neoplasms; Caveolin 1; Caveolin 2; Caveolins; Cell Differentiation; Cell Line; Chromans; Colonic Neoplasms; Dimerization; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Humans; Kidney; Ligands; Macromolecular Substances; Membrane Microdomains; Neoplasm Proteins; Phenotype; Phenylacetates; Prostaglandin D2; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; RNA, Neoplasm; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Troglitazone; Tumor Cells, Cultured

Naturally occurring derivatives of arachidonic acid are potent agonists for the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and block cancer cell proliferation through the induction of apoptosis. We have previously reported that induction of apoptosis using cyclopentenone prostaglandins of the J series, including 15deoxydelta(12,14)PGJ(2) (15dPGJ(2)), is associated with a high degree of PPAR-response element (PPRE) activity and requires early de novo gene expression in breast cancer cells. In the current study, we used pharmacologic and genetic approaches to test the hypothesis that PPARgamma is required for 15dPGJ(2)-induced apoptosis. The PPARgamma agonists 15dPGJ(2), trogliltazone (TGZ), and GW7845, a synthetic and highly selective tyrosine-based PPARgamma agonist, all increased transcriptional activity of PPARgamma, and expression of CD36, a PPARgamma-dependent gene. Transcriptional activity and CD36 expression was reduced by GW9662, a selective and irreversible PPARgamma antagonist, but GW9662 did not block apoptosis induced by 15dPGJ(2). Moreover, dominant negative expression of PPARgamma blocked PPRE transcriptional activity, but did not block 15dPGJ(2)-induced apoptosis. These studies show that while 15dPGJ(2) activates PPRE-mediated transcription, PPARgamma is not required for 15dPGJ(2)-induced apoptosis in breast cancer cells. Other likely mechanisms through which cyclopentenone prostaglandins induce apoptosis of cancer cells are discussed.

Topics: Apoptosis; Breast Neoplasms; Cell Nucleus; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Prostaglandin D2; Protein Transport; Receptors, Cytoplasmic and Nuclear; Response Elements; Time Factors; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has become a potential target for the prevention and treatment of breast cancer. However, recent in vitro and in vivo studies have raised the question of whether activation of PPARgamma leads to the promotion or reduction of tumor formation. Studies using several cancer cell lines, animal models, and a variety of PPARgamma agonists have shown discordant results, including changes in cellular proliferation, differentiation, and apoptosis of cancer cells and tumors.. We studied the effects of low-, moderate-, and high-dose treatment of the PPARgamma ligands 15-deoxy-delta1214 prostaglandin J2 (15dPGJ2) and troglitazone (TGZ) on parameters of cell growth, differentiation, and apoptosis in the epithelial breast cancer cell line MDA-MB-231.. The biologic effects of these compounds depend largely on ligand concentration and the degree of PPARgamma activation. For example, low concentrations of 15dPGJ2 (<2.5 microM) and TGZ (<5 microM) increased cellular proliferation, but concentrations of 15dPGJ2 > or = 10 microM and of TGZ at 100 microM blocked cell growth. TGZ (100 microM) slowed cell cycle progression, and 15dPGJ2 (10 microM) caused an S-phase arrest in the cell cycle and induced morphological characteristics consistent with apoptosis. Expression of CD36, a marker of differentiation in these cells, was induced by 2.5 microM 15dPGJ2 or 5 to 100 microM TGZ. However, higher concentrations of 15dPGJ2 did not alter CD36 expression. Transcriptional activation studies demonstrated that 15dPGJ2 is a more potent PPARgamma ligand than TGZ. Regardless of the ligand used, though, low transcriptional activation correlated with an increased cellular proliferation, whereas higher levels of activation correlated with cell cycle arrest and apoptosis.. PPARgamma activation induces several important and seemingly opposite changes in neoplastic cells, depending on the magnitude of PPARgamma activation. These data may explain, at least in part, some of the discordant results previously reported.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Chromans; Female; Humans; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation; Troglitazone; Tumor Cells, Cultured

Cyclopentenone prostaglandin derivatives of arachidonic acid are potent inducers of apoptosis in a variety of cancer cell types. Several investigators have shown that the terminal derivative of prostaglandin J(2) (PGJ(2)) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)), induces apoptosis in breast cancer cells and is a potent activator of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), but 15dPGJ(2) effects can be mediated by PPARgamma-dependent and PPARgamma-independent mechanisms. Here we report that 15dPGJ(2) regulates early gene expression critical to apoptosis. Specifically, 15dPGJ(2) induces potent and irreversible S phase arrest that is correlated with expression of genes critical to cell cycle arrest and apoptosis, including the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Inhibition of RNA or protein synthesis abrogates apoptosis induced by 15dPGJ(2) in breast cancer cells but potentiates apoptosis induced by tumor necrosis factor-alpha or CD95/Fas ligand. Additionally, 15dPGJ(2) induces caspase activation that is blocked by peptide caspase inhibitors. These data show that de novo gene transcription is necessary for 15dPGJ(2)-induced apoptosis in breast cancer cells. Critical candidate genes are likely to be revealed through analysis of differential cDNA array expression.

Topics: Apoptosis; Blotting, Northern; Blotting, Western; Breast Neoplasms; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cycloheximide; Dactinomycin; DNA, Complementary; Down-Regulation; Enzyme Activation; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Immunologic Factors; Ligands; Microscopy, Fluorescence; Nucleic Acid Synthesis Inhibitors; Oligonucleotide Array Sequence Analysis; Prostaglandin D2; Protein Synthesis Inhibitors; Receptors, Cytoplasmic and Nuclear; S Phase; Time Factors; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

One of the most interesting recent developments in the nuclear receptor field has been the identification of natural and synthetic agonists of the peroxisome proliferator-activated receptor (PPAR) family, coupled with a growing recognition that the gamma isoform (PPARgamma) affects pathways important in a variety of human diseases. Here we show that the activation of PPARgamma through the 15-deoxy-Delta-12,14-prostaglandin J(2) (PG-J(2)) ligand causes a dramatic inhibition of ErbB-2 and ErbB-3 tyrosine phosphorylation caused by neuregulin 1 (NRG1) and neuregulin 2 (NRG2) in MCF-7 cells. This effect is accompanied by a very efficient blocking of ErbBs effects upon proliferation, differentiation and cell death in these cells. Preincubation of MCF-7 cells with PG-J(2) before addition of NRG1 and NRG2 had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 compartment of the cell cycle, and a marked increase in apoptosis. NRG1 and NRG2 induce G1 progression, which was associated with stimulation of the phosphatidylinositol-3 kinase (PI 3-K) pathway, whereas survival was dependent on ERK1/ERK2 activation. Both pathways were inhibited by PG-J(2). Furthermore, PG-J(2) can abolish the NRG1 and NRG2-induced increase in anchorage-independent growth of these cells. PG-J(2) also blocks phosphorylation of other receptor tyrosine kinases, such as IGF-IR, in MCF-7 cells, and suppress proliferation of other breast cancer cell lines. In summary, our data show a specific inhibitory action of PG-J(2) on the activity of the ErbB receptors in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Division; Cell Separation; Colony-Forming Units Assay; Culture Media, Serum-Free; Enzyme Inhibitors; ErbB Receptors; Female; Flow Cytometry; Humans; Immunologic Factors; Neuregulins; Oncogene Proteins v-erbB; Phosphorylation; Prostaglandin D2; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, IGF Type 1; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

The relationship between growth and alterations in arachidonic acid (AA) metabolism in human breast (MCF-7) and colon (SW480) cancer cells was studied. Four different fatty acid preparations were evaluated: a mixture of conjugated linoleic acid (CLA) isomers (c9,t11, t10,c12, c11,t13, and minor amounts of other isomers), the pure c9,t11-CLA isomer, the pure t10,c12-CLA isomer, and linoleic acid (LA) (all at a lipid concentration of 16 microg/mL). 14C-AA uptake into the monoglyceride fraction of MCF-7 cells was significantly increased following 24 h incubation with the CLA mixture (P < 0.05) and c9,t11-CLA (P < 0.02). In contrast to the MCF-7 cells, 14C-AA uptake into the triglyceride fraction of the SW480 cells was increased while uptake into the phospholipids was reduced following treatment with the CLA mixture (P < 0.02) and c9,t11-CLA (P < 0.05). Distribution of 14C-AA among phospholipid classes was altered by CLA treatments in both cell lines. The c9,t11-CLA isomer decreased (P < 0.05) uptake of 14C-AA into phosphatidylcholine while increasing (P < 0.05) uptake into phosphatidylethanolamine in both cell lines. Both the CLA mixture and the t10,c12-CLA isomer increased (P < 0.01) uptake of 14C-AA into phosphatidylserine in the SW480 cells but had no effect on this phospholipid in the MCF-7 cells. Release of 14C-AA derivatives was not altered by CLA treatments but was increased (P < 0.05) by LA in the SW480 cell line. The CLA mixture of isomers and c9,t11-CLA isomer inhibited 14C-AA conversion to 14C-prostaglandin E2 (PGE2) by 20-30% (P < 0.05) while increasing 14C-PGF2alpha by 17-44% relative to controls in both cell lines. LA significantly (P < 0.05) increased 14C-PGD2 by 13-19% in both cell lines and increased 14C-PGE2 by 20% in the SW480 cell line only. LA significantly (P < 0.05) increased 5-hydroperoxyeicosatetraenoate by 27% in the MCF-7 cell line. Lipid peroxidation, as determined by increased levels of 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), was observed following treatment with c9,t11-CLA isomer in both cell lines (P < 0.02) and with t10,c12-CLA isomer in the MCF-7 cell line only (P < 0.05). These data indicate that the growth-promoting effects of LA in the SW480 cell line may be associated with enhanced conversion of AA to PGE2 but that the growth-suppressing effects of CLA isomers in both cell lines may be due to changes in AA distribution among cellular lipids and an altered prostaglandin profile.

Topics: Arachidonic Acid; Breast Neoplasms; Carbon Radioisotopes; Cell Survival; Colonic Neoplasms; Dinoprost; Dinoprostone; Humans; Leukotriene B4; Leukotrienes; Linoleic Acid; Prostaglandin D2; Tumor Cells, Cultured

Estrogen biosynthesis is catalyzed by aromatase cytochrome P-450 (the product of the CYP19 gene). Adipose tissue is the major site of estrogen biosynthesis in postmenopausal women, with the local production of estrogen in breast adipose tissue implicated in the development of breast cancer. In human adipose tissue, aromatase is primarily expressed in the mesenchymal stromal cells and is a marker of the undifferentiated preadipocyte phenotype. Aromatase expression in adipose tissue is regulated via the distal promoter I.4, under the control of glucocorticoids and class I cytokines such as oncostatin M, interleukin 6, and interleukin 11, as well as tumor necrosis factor alpha. These cytokines, which are expressed in adipose, also inhibit adipocyte differentiation. Therefore, we hypothesized that factors which stimulate adipocyte differentiation should inhibit aromatase expression. These factors include synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands such as thiazolidinediones, e.g., troglitazone and rosiglitazone (BRL49653) and the endogenous PPARgamma ligand 15-deoxy-delta12,14-prostaglandin J2. We have demonstrated by measurement of aromatase activity and by reverse transcription-PCR/Southern blotting that these PPARgamma ligands inhibit aromatase expression in cultured breast adipose stromal cells stimulated with oncostatin M or tumor necrosis factor alpha plus dexamethasone in a concentration-dependent manner, whereas a metabolite of troglitazone that does not activate PPARgamma has no effect. We have also shown that troglitazone inhibits luciferase activity of reporter constructs containing various lengths of the upstream region of promoter I.4 transfected into mouse 3T3-L1 preadipocyte mesenchymal cells, whereas the troglitazone metabolite does not. Because local estrogen production in breast fat is implicated in breast cancer development in postmenopausal women, the actions of PPARgamma ligands suggest that they may have potential therapeutic benefit in the treatment and management of breast cancer.

Topics: Adipose Tissue; Animals; Aromatase; Aromatase Inhibitors; Binding, Competitive; Breast; Breast Neoplasms; Cell Line; Cells, Cultured; Chromans; Dose-Response Relationship, Drug; Estrogens; Female; Gene Expression Regulation, Enzymologic; Humans; Ligands; Promoter Regions, Genetic; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Rosiglitazone; Stromal Cells; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Troglitazone

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) 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 15dPGJ(2) 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 15dPGJ(2) 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 15dPGJ(2) 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 PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Topics: Animals; Apoptosis; Base Sequence; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; DNA Primers; Humans; Mice; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured

The reduction of mitogen responses of blood lymphocytes which occur after radiation therapy for breast cancer, known to be largely due to inhibitory monocytes, can partly be reverted by indomethacin which is an inhibitor of prostaglandin (PG) synthesis. This may indicate that PG-synthesis by blood monocytes is increased after irradiation or that PG-sensitivity of the lymphocyte population is increased. To test the latter possibility the sensitivity of the PHA-response of blood lymphocytes to varying concentrations of PGE2 and PGD2 was examined in 15 patients with breast cancer before and up to 6 months after local radiation therapy (46 Gy). The results showed that the sensitivity of the PHA-response was not significantly changed after treatment suggesting that the immunosuppression observed after irradiation is partly due to an increased production of PGs rather than an increased PG-sensitivity of lymphocytes.

Topics: Adult; Aged; Breast Neoplasms; Dinoprostone; Female; Humans; Indomethacin; Lymphocyte Activation; Lymphocytes; Middle Aged; Phytohemagglutinins; Prostaglandin D2; Prostaglandins; Prostaglandins D; Prostaglandins E