Activation of the PKC pathway stimulates ovarian cancer cell proliferation, migration, and expression of MMP7 and MMP10.

Postmenopausal women are at a higher risk of ovarian cancer due, in part, to increased levels of gonadotropins such as luteinizing hormone (LH). Gonadotropins and other stimuli are capable of activating two pathways, PKA and PKC, that are altered in ovarian cancer. To determine the role of LH on ovarian cancer, we explored the effects of human chorionic gonadotropin (hCG), an LH mimic, and an activator of the PKC pathway, phorbol-12-myristate 13-acetate (PMA), on ovarian cancer cell-cycle kinetics and apoptosis in Ovcar3 cells. PMA treatment increased cells in the S phase of the cell cycle and initially increased apoptosis after 4 h before diminishing apoptosis after 8 h. Treatment of ovarian cancer cells with hCG had no effect on these parameters. The PKC pathway is known to differentially regulate matrix metalloproteinase (MMP) expression. Results showed that ovarian cancer cells treated with PMA increased MMP7 and MMP10 mRNA levels after 8 h of treatment, and expression remained high after 12 h before decreasing at 24 h. The mRNA expression of extracellular matrix metalloproteinase inducer (BSG), an activator of MMPs, was unaffected by PMA. Due to the role that MMPs play in migration, we investigated the effect of PMA activation of MMPs on ovarian cancer cell migration. The use of the MMP inhibitor GM6001 blocked the increased migratory effects of PMA on ovarian cancer cells. Together, these studies show that activating the PKC pathway causes significant changes in cell cycle kinetics and selective expression of MMPs that are involved in enhancing ovarian cancer cell proliferation and migration.

Distinct modulation of voltage-gated and ligand-gated Ca2+ currents by PPAR-gamma 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 Ca(2+) dysregulation is a pathogenic factor of brain aging/AD, we tested the hypothesis that TZDs could impact Ca(2+) signaling/homeostasis in neurons. We assessed the effects of pioglitazone and rosiglitazone (TZDs) on two major sources of Ca(2+) influx in primary hippocampal cultured neurons, voltage-gated Ca(2+) channel (VGCC) and the NMDA receptor (NMDAR). VGCC- and NMDAR-mediated Ca(2+) currents were recorded using patch-clamp techniques, and Ca(2+) intracellular levels were monitored with Ca(2+) 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 Ca(2+)-dependent pathways in the brain and have different inhibitory profiles on two major Ca(2+) 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.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily and is highly expressed in many human tumors including breast cancer. PPARgamma 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 PPARgamma 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 PPARgamma plays a much more complex role in initiation and progression of cancer than previously thought. RESULTS: We have previously demonstrated that an increase in expression of PPARgamma1 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 PPARgamma1 expression in these cells. In this study, using RNA interference (RNAi) to inhibit PPARgamma1 expression directly or via down-regulation of MAZ, we report for the first time that a decrease in PPARgamma1 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 PPARgamma1, Delta462, we confirmed that PPARgamma1 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 PPARgamma1 expression leads to an induction of apoptosis in MCF-7 cells, confirmed by analyzing Bcl-2 expression and PARP-1 cleavage. CONCLUSION: Thus, these findings suggest that an increase in PPARgamma1 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 PPARgamma ligands in patients who are predisposed or already diagnosed with breast cancer.

PPARgamma1 as a molecular target of eicosapentaenoic acid in human colon cancer (HT-29) cells.

Diets high in (n-3) PUFA decrease colon cancer development and suppress colon tumor growth, but the molecular mechanism through which these compounds act is largely unknown. We sought to determine whether PPARgamma1 serves as a molecular link between the physiological actions of eicosapentaenoic acid (EPA) in human colon cancer cells (HT-29). At nutritionally relevant concentrations, EPA stimulated a PPAR response element (PPRE) reporter assay in a dose-responsive manner in HT-29 cells. Cotreatment with GW9662 (GW), a PPARgamma antagonist, significantly inhibited this effect, whereas overexpressing the receptor enhanced it. EPA also stimulated the PPRE reporter in a PPARgamma negative cancer cell line (22Rv1) when the cells were cotransfected with a PPARgamma1 expression plasmid and this effect was again inhibited by GW. Furthermore, in vitro incubation of EPA with PPARgamma1 enhanced binding of the protein to DNA containing a PPRE. Next, we sought to determine whether EPA or a prostaglandin formed from EPA is the functional ligand of PPARgamma. Cotreatment in HT-29 and 22Rv1 cells with EPA and acetyl salicylic acid, an inhibitor of cyclooxygenase activity, activated the PPRE reporter at levels similar to EPA alone, suggesting that EPA itself is a ligand of PPARgamma. Finally, EPA suppressed HT-29 cell growth and this effect was significantly reversed by the addition of GW, suggesting that in part the physiological actions of EPA are the result of PPARgamma activation. These studies identify PPARgamma as a molecular mediator of (n-3) PUFA actions in colon cancer cells.

PPARalpha ligands reduce PCB-induced endothelial activation: possible interactions in inflammation and atherosclerosis.

Exposure to polychlorinated biphenyls (PCBs) can activate inflammatory responses in vascular endothelial cells. Activation of peroxisome proliferator-activated receptors (PPARs) by nutrients or synthetic agonists has been shown to block pro-inflammatory responses both in vitro and in vivo. Here we demonstrate that activation of PPARalpha by synthetic agonists can reduce 3,3'4,4'-tetrachlorobiphenyl (PCB77)-induced endothelial cell activation. Primary vascular endothelial cells were pretreated with the PPARalpha ligands fenofibrate or WY14643 followed by exposure to PCB77. PPARalpha activation protected endothelial cells against PCB77-induced expression of the pro-inflammatory proteins vascular cell adhesion molecule-1 (VCAM-1), cycloxygenase-2 (COX-2), and PCB77-induced expression and activity of the aryl hydrocarbon receptor (AHR) responsive cytochrome P450 1A1 (CYP1A1). Furthermore, basal AHR expression was downregulated by fenofibrate and WY14643. We also investigated the possible interactions between PCBs, and basal PPAR activity and protein expression. Treatment with PCB77 significantly reduced basal mRNA expression of PPARalpha and the PPAR responsive gene CYP4A1, as well as PPARalpha protein expression. Also, PCB77 exposure caused a significant decrease in basal PPAR-dependent reporter gene expression in MCF-7 cells. Overall, these findings suggest that PPARalpha agonists can reduce PCB77 induction of endothelial cell activation by inhibition of the AHR pathway, and that coplanar PCB induced pro-inflammatory effects could be mediated, in part, by inhibition of PPARalpha expression and function.

The increased expression of peroxisome proliferator-activated receptor-gamma1 in human breast cancer is mediated by selective promoter usage.

Peroxisome proliferator-activated receptor-gamma1 (PPARgamma1) is transactivated by a wide range of ligands in normal human mammary epithelial and breast cancer cells. Although transactivation of PPARgamma 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 PPARgamma 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 PPARgamma expression seen in breast cancer cells results from promoter recruitment, providing new insights into the expression and actions of PPARgamma in breast cancer.

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

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.

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.

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 G(0)/G(1) 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 PPARgamma antagonist T0070907 suppresses breast cancer cell proliferation and motility via both PPARgamma-dependent and -independent mechanisms.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) is overexpressed in many types of cancer, including breast cancer, and it is regulated by ligand binding and post-translational modifications. It was previously demonstrated that endogenous transactivation promotes an aggressive phenotype of malignant breast cells. This study examines whether selective antagonism of PPARγ with T0070907 is a potential strategy for breast cancer therapy. MATERIALS AND METHODS: PPARγ activation was inhibited using both pharmacological and molecular approaches and proliferation, apoptosis, migration and invasion were measured in MDA-MB-231 and MCF-7 breast cancer cells. RESULTS: T0070907 treatment inhibited proliferation, invasion and migration but did not significantly affect apoptosis. Molecular inhibition using a dominant negative (Δ462) receptor yielded similar results. T007 also mediated a dose-dependent decrease in phosphorylation of PPARγ, and its ability to bind to DNA, and may directly affect mitogen-activated protein kinase signaling. CONCLUSION: These data indicate that inhibiting endogenous PPARγ signaling may be a promising new approach to breast cancer therapy.

MAZ drives tumor-specific expression of PPAR gamma 1 in breast cancer cells.

The peroxisome proliferator-activated receptor gamma 1 (PPARgamma1) is a nuclear receptor that plays a pivotal role in breast cancer and is highly over-expressed relative to normal epithelia. We have previously reported that the expression of PPARgamma1 is mediated by at least six distinct promoters and expression in breast cancer is driven by a tumor-specific promoter (pA1). Deletional analysis of this promoter fragment revealed that the GC-rich, 263 bp sequence proximal to the start of exon A1, is sufficient to drive expression in breast cancer cells but not in normal, human mammary epithelial cells (HMEC). By combining the disparate technologies of microarray and computer-based transcription factor binding site analyses on this promoter sequence the myc-associated zinc finger protein (MAZ) was identified as a candidate transcription factor mediating tumor-specific expression. Western blot analysis and chromatin immunoprecipitation assays verify that MAZ is overexpressed in MCF-7 cells and is capable of binding to the 263 bp promoter fragment, respectively. Furthermore, the over-expression of MAZ in HMEC is sufficient to drive the expression of PPARgamma1 and does so by recruiting the tumor-specific promoter. This results in an increase in the amount of PPARgamma1 capable of binding to its DNA response element. These findings help to define the molecular mechanism driving the high expression of PPARgamma1 in breast cancer and raise new questions regarding the role of MAZ in cancer progression.

Transactivation of ERalpha by Rosiglitazone induces proliferation in breast cancer cells.

In the present study, we demonstrate that Rosiglitazone (Rosi), a thiazolidinedione and PPARgamma agonist, induces ERE (Estrogen Receptor Response Element) reporter activity, pS2 (an endogenous ER gene target) expression, and proliferation of ER positive breast cancer (MCF-7) cells. By performing a dose-response assay, we determined that high concentrations of Rosi inhibit proliferation, while low concentrations of Rosi induce proliferation. Using the anti-estrogen ICI, ER negative breast cancer (MDA-MB-231) cells, and a prostate cancer cell line (22Rv1) deficient in both ERalpha and PPARgamma, we determined that Rosiglitazone-induced ERE reporter activation and proliferation is through an ERalpha dependent mechanism. Rosiglitazone-induced ERE activation is also dependent on activation of the Extracellular Signal-Regulated Kinase-Mitogen Activated Protein Kinase (ERK-MAPK) pathway, since it is inhibited by co-treatment with U0126, a specific inhibitor of this pathway. We also demonstrate that when ERalpha and PPARgamma are both present, they compete for Rosi, inhibiting each others transactivation. To begin to unravel the pharmacological mechanism of Rosi-induced ER activation, sub-maximally effective concentrations of E(2) were used in combination with increasing concentrations of Rosi in luciferase reporter assays. From these assays it appears that E(2) and Rosi both activate ERalpha via similar pharmacological mechanisms. Furthermore sub-maximally effective concentrations of E(2) and Rosi additively increase both ERE reporter activity and MCF-7 cell proliferation. The results of this study may have clinical relevancy for Rosi's use both as an anti-diabetic in post-menopausal women and as an anti-cancer drug in women with ER positive breast cancer.

Oxa-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).