Michael W. Kilgore

Signal cross-talk between estrogen receptor alpha and beta and the peroxisome proliferator-activated receptor gamma1 in MDA-MB-231 and MCF-7 breast cancer cells

We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed and transcriptionally responsive to both synthetic and natural ligands in a variety of human breast cancer cells. We also observed significant differences in basal and ligand-mediated transactivation of PPARgamma in cells with variable expression of the estrogen receptor. While previous reports indicate that PPARgamma can mediate the expression of estrogen target genes, no data have suggested that estrogen receptor (ER) expression can alter the transcriptional regulation of PPARgamma target gene expression. Here we have demonstrated that the expression of either ERalpha or beta, but not the androgen or aryl hydrocarbon receptors, lowers both basal and stimulated PPARgamma-mediated reporter activity. Interestingly, the presence of an ER antagonist does not inhibit this response while estradiol treatment further inhibits the ligand-stimulated transactivation of PPARgamma in cells expressing ERalpha but not ERbeta. Cells transfected with ERalpha deletion mutants demonstrate that the DNA binding domain of the ER is required to repress PPAR transactivation in these cells. Finally, using RNase protection assays we show that the inhibition of PPAR function is not due to a decrease in the expression of PPARgamma. These data suggest that signal cross talk exists bidirectionally between PPARgamma and ER in breast cancer cells.

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat

Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

MCF-7 and T47D human breast cancer cells contain a functional peroxisomal response

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors that regulate transcription of target genes. Since attempts have been made to correlate the ingestion of high-fat diets, itself a peroxisome proliferator, with the occurrence of breast cancer, we set about to determine if human breast cancer cells contained a functional PPAR. In this report we demonstrate the presence of an mRNA in two breast cancer cell lines (MCF-7 and T47D) which is specifically recognized by a mouse PPARgamma2 probe. Furthermore, in gel shift assays a consensus PPAR response element (PPRE) was specifically bound by nuclear extracts from MCF-7 cells and was further retarded by antibodies raised to mouse PPARgamma. Finally, when transfected with a PPRE-luciferase transcriptional reporter construct, transcription was increased in response to activators of PPAR and its dimmeric partner the retinoic acid X receptor (RXR). These data indicate that peroxisomal proliferators are capable of mediating transcription in human breast cells and suggest the possibility of a physiological role in the breast.

Selective activation of PPARγ in breast, colon, and lung cancer cell lines

Abstract Peroxisome proliferator-activated receptor gamma (PPARγ) 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 PPARγ 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-Δ12,14-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 PPARγ within a single cell line by different ligands. Interestingly, GW, a PPARγ 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 PPARγ ligands. For instance, Ros, Cig, and PGJ2 were all potent agonist of PPARγ 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 PPARγ and retinoid X receptor alpha (RXRα) in the individual cell lines were quantitated by real time-polymerase chain reaction (RT-PCR). The ratio of PPARγ to RXRα was predictive of how cells responded to co-treatment of Ros and 9-cis-retinoic acid, an RXRα agonist, in two out of three cell lines tested. These data indicate that PPARγ can be selectively modulated and suggests that it may be used as a therapeutic target for individual tumors.

Differential transcriptional activation of peroxisome proliferator-activated receptor gamma by omega-3 and omega-6 fatty acids in MCF-7 cells.

While the role of dietary fats in breast cancer remains controversial, the recent cloning of peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor, from human breast cancer cells lines provides a potential molecular link. Several fatty acids from four classes of dietary fats were tested for their ability to mediate the transcriptional activity of PPARgamma in MCF-7 and MDA-MB-231 cells using growth media with minimal serum. Whereas omega-3 fatty acids inhibit transactivation of PPARgamma to levels below control, omega-6, monounsaturated and saturated fatty acids stimulate the activity of the transcriptional reporter. These studies indicate that individual fatty acids differentially regulate the transcriptional activity of PPARgamma by selectively acting as agonists or antagonists. Furthermore, the transcriptional activation of PPARgamma correlates with cell proliferation in MCF-7 cells. Understanding the effects of individual fats on breast cancer cells and PPARgamma transactivation could provide important new insights into the epidemiology of breast cancer and the role of dietary fat.

Distinct modulation of voltage-gated and ligand-gated Ca2+ currents by PPAR-γ agonists in cultured hippocampal neurons

Type 2 diabetes mellitus is a metabolic disorder characterized by hyperglycemia and is especially prevalent in the elderly. Because aging is a risk factor for type 2 diabetes mellitus, and insulin resistance may contribute to the pathogenesis of Alzheimer’s disease (AD), anti‐diabetic agents (thiazolidinediones‐TZDs) are being studied for the treatment of cognitive decline associated with AD. These agents normalize insulin sensitivity in the periphery and can improve cognition and verbal memory in AD patients. Based on evidence that Ca2+ dysregulation is a pathogenic factor of brain aging/AD, we tested the hypothesis that TZDs could impact Ca2+ signaling/homeostasis in neurons. We assessed the effects of pioglitazone and rosiglitazone (TZDs) on two major sources of Ca2+ influx in primary hippocampal cultured neurons, voltage‐gated Ca2+ channel (VGCC) and the NMDA receptor (NMDAR). VGCC‐ and NMDAR‐mediated Ca2+ currents were recorded using patch‐clamp techniques, and Ca2+ intracellular levels were monitored with Ca2+ imaging techniques. Rosiglitazone, but not pioglitazone reduced VGCC currents. In contrast, NMDAR‐mediated currents were significantly reduced by pioglitazone but not rosiglitazone. These results show that TZDs modulate Ca2+‐dependent pathways in the brain and have different inhibitory profiles on two major Ca2+ sources, potentially conferring neuroprotection to an area of the brain that is particularly vulnerable to the effects of aging and/or AD.

Down-regulation of PPARgamma1 suppresses cell growth and induces apoptosis in MCF-7 breast cancer cells

BackgroundPeroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily and is highly expressed in many human tumors including breast cancer. PPARγ has been identified as a potential target for breast cancer therapy based on the fact that its activation by synthetic ligands affects the differentiation, proliferation, and apoptosis of cancer cells. However, the controversial nature of current studies and disappointing results from clinical trials raise questions about the contribution of PPARγ signaling in breast cancer development in the absence of stimulation by exogenous ligands. Recent reports from both in vitro and in vivo studies are inconsistent and suggest that endogenous activation of PPARγ plays a much more complex role in initiation and progression of cancer than previously thought.ResultsWe have previously demonstrated that an increase in expression of PPARγ1 in MCF-7 breast cancer cells is driven by a tumor-specific promoter. Myc-associated zinc finger protein (MAZ) was identified as a transcriptional mediator of PPARγ1 expression in these cells. In this study, using RNA interference (RNAi) to inhibit PPARγ1 expression directly or via down-regulation of MAZ, we report for the first time that a decrease in PPARγ1 expression results in reduced cellular proliferation in MCF-7 breast cancer cells. Furthermore, we demonstrate that these changes in proliferation are associated with a significant decrease in cell transition from G1 to the S phase. Using a dominant-negative mutant of PPARγ1, Δ462, we confirmed that PPARγ1 acts as a pro-survival factor and showed that this phenomenon is not limited to MCF-7 cells. Finally, we demonstrate that down-regulation of PPARγ1 expression leads to an induction of apoptosis in MCF-7 cells, confirmed by analyzing Bcl-2 expression and PARP-1 cleavage.ConclusionThus, these findings suggest that an increase in PPARγ1 signaling observed in breast cancer contributes to an imbalance between proliferation and apoptosis, and may be an important hallmark of breast tumorigenesis. The results presented here also warrant further investigation regarding the use of PPARγ ligands in patients who are predisposed or already diagnosed with breast cancer.

Specific Thiazolidinediones Inhibit Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARγ Independent Manner

Background Peroxisome Proliferator Activated Receptor gamma (PPARγ) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs—Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)—on ovarian cancer cell proliferation, PPARγ expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARγ dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARγ activity. Principal Findings Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G0/G1 stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARγ mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARγ activity. Overexpression of wild type PPARγ increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. To determine whether PPARγ mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARγ construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARγ independent. Conclusions CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARγ independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARγ did not affect the changes in cell proliferation and cell cycle.

The Increased Expression of Peroxisome Proliferator-Activated Receptor-γ1 in Human Breast Cancer Is Mediated by Selective Promoter Usage

Peroxisome proliferator-activated receptor-γ1 (PPARγ1) is transactivated by a wide range of ligands in normal human mammary epithelial and breast cancer cells. Although transactivation of PPARγ mediates the expression of genes that are markers of differentiation, its overexpression in cancers of the breast, thyroid, colon, and lung suggests its dysregulation may play a role in oncogenesis, cancer progression, or both. We report the overexpression of PPARγ is caused by the use of a tumor-specific promoter in breast cancer cells that is distinct from the promoter used in normal epithelia. Thus, the increase in PPARγ expression seen in breast cancer cells results from promoter recruitment, providing new insights into the expression and actions of PPARγ in breast cancer.

Halo-Enediynes: Probing the Electronic and Stereoelectronic Contributions to the Bergman Cycloaromatization

Efficient routes to three classes of 10-membered oxa-enediynes are presented. The electronic and stereoelectronic contributions to half-lives are supported by density functional theory calculations. One member of this class cyclizes to give an isochroman which binds to and degrades the aryl hydrocarbon receptor (AhR).