Repression of NHE1 Expression by PPAR gamma Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells In vitro and In vivo

Ligand-induced activation of peroxisome proliferator-activated receptor gamma (PPAIR gamma) inhibits proliferation in cancer cells in vitro and in vivo; however, the downstream targets remain undefined. We report the identification of a peroxisome proliferator response element in the promoter region of the Na+/ H transporter gene NHE1, the overexpression of which has been associated with carcinogenesis. Exposure of breast cancer cells expressing high levels of PPAR gamma to its natural and synthetic agonists resulted in downregulation of NHE1 transcription as well as protein expression. Furthermore, the inhibitory effect of activated PPAR gamma on tumor colony-forming ability was abrogated on overexpression of NHE1, whereas small interfering RNA-mediated gene silencing of NHE1 significantly increased the sensitivity of cancer cells to growth-inhibitory stimuli. Finally, histopathologic analysis of breast cancer biopsies obtained from patients with type II diabetes treated with the synthetic agonist rosiglitazone showed significant repression of NHE1 in the tumor tissue. These data provide evidence for tumor-selective downregulation of NHE1 by activated PPAR gamma in vitro and in pathologic specimens from breast cancer patients and could have potential implications for the judicious use of low doses of PPAR gamma ligands in combination chemotherapy regimens for an effective therapeutic response. [Cancer Res 2009;69(22):8636-44]

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?), cultures were treated with the PPAR? ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPAR? antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPAR? ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

Therapeutic effects of PPARα agonists on diabetic retinopathy in type 1 diabetes models

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator-activated receptor a (PPARa) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARa dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARa agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-?B in the retinas of OIR and diabetic models. Fenofibrate's beneficial effects were blocked by a specific PPARa antagonist. Furthermore, Ppara knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARa-dependent mechanism.

Fenofibrate and pioglitazone improve endothelial function and reduce arterial stiffness in obese glucose tolerant men

Obesity is a low grade inflammatory state associated with premature cardiovascular morbidity and mortality. Along with traditional risk factors the measurement of endothelial function, insulin resistance, inflammation and arterial stiffness may contribute to the assessment of cardiovascular risk. We conducted a randomised placebo controlled trial to assess the effects of 12 weeks treatment with a PPAR-alpha agonist (fenofibrate) and a PPAR-gamma agonist (pioglitazone) on these parameters in obese glucose tolerant men. Arterial stiffness was measured using augmentation index and pulse wave velocity (PWV). E-selectin, VCAM-1 and ICAM-1 were used as markers of endothelial function. Insulin sensitivity improved with pioglitazone treatment (p=0.001) and, in keeping with this, adiponectin increased by 85.2% (p

Thiazolidinediones: effects on insulin resistance and the cardiovascular system.

Abstract
Thiazolidinediones (TZDs) have been used for the treatment of hyperglycaemia in type 2 diabetes for the past 10 years. They may delay the development of type 2 diabetes in individuals at high risk of developing the condition, and have been shown to have potentially beneficial effects on cardiovascular risk factors. TZDs act as agonists of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) primarily in adipose tissue. PPAR-gamma receptor activation by TZDs improves insulin sensitivity by promoting fatty acid uptake into adipose tissue, increasing production of adiponectin and reducing levels of inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor-1(PAI-1) and interleukin-6 (IL-6). Clinically, TZDs have been shown to reduce measures of atherosclerosis such as carotid intima-media thickness (CIMT). However, in spite of beneficial effects on markers of cardiovascular risk, TZDs have not been definitively shown to reduce cardiovascular events in patients, and the safety of rosiglitazone in this respect has recently been called into question. Dual PPAR-alpha/gamma agonists may offer superior treatment of insulin resistance and cardioprotection, but their safety has not yet been assured

Significance of peroxisome proliferator-activated receptors in the cardiovascular system in health and disease.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that belong to the nuclear receptor superfamily. Three isoforms of PPAR have been identified, alpha, delta and gamma, which play distinct roles in the regulation of key metabolic processes, such as glucose and lipid redistribution. PPARalpha is expressed predominantly in the liver, kidney and heart, and is primarily involved in fatty acid oxidation. PPARgamma is mainly associated with adipose tissue, where it controls adipocyte differentiation and insulin sensitivity. PPARdelta is abundantly and ubiquitously expressed, but as yet its function has not been clearly defined. Activators of PPARalpha (fibrates) and gamma (thiazolidinediones) have been used clinically for a number of years in the treatment of hyperlipidaemia and to improve insulin sensitivity in diabetes. More recently, PPAR activation has been found to confer additional benefits on endothelial function, inflammation and thrombosis, suggesting that PPAR agonists may be good candidates for the treatment of cardiovascular disease. In this regard, it has been demonstrated that PPAR activators are capable of reducing blood pressure and attenuating the development of atherosclerosis and cardiac hypertrophy. This review will provide a detailed discussion of the current understanding of basic PPAR physiology, with particular reference to the cardiovascular system. It will also examine the evidence supporting the involvement of the different PPAR isoforms in cardiovascular disease and discuss the current and potential future clinical applications of PPAR activators.

Routine delirium monitoring in a UK critical care unit

Introduction Delirium in the intensive care unit (ICU) is associated with increased morbidity and mortality. Using an assessment tool has been shown to improve the ability of clinicians in the ICU to detect delirium. The confusion assessment method for the ICU (CAM-ICU) is a validated delirium-screening tool for critically ill intubated patients. The aim of this project was to establish the feasibility of routine delirium screening using the CAM-ICU and to identify the incidence of delirium in a UK critical care unit.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]iodophenyl]2-ethoxy propanoic acid], which is specific for the ? isoform of the peroxisomal proliferator activated receptor (PPAR?), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPAR?1 and to a GST (glutathione S-transferase) fusion protein contg. the ligand binding domain of human PPAR?1 (KD = 70 nM). Using this ligand, the authors characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition expts., rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, resp.). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPAR?1 were comparable with those detd. in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an ?-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPAR?1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPAR? and that the pharmacol. of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

Non-thiazolidinedione antihyperglycaemic agents. Part 3: The effects of stereochemistry on the potency of alpha-methoxy-beta-phenylpropanoic acids.

Rhizopus delemar lipase catalyzed ester hydrolysis of the alpha-methoxy-beta-phenylpropanoate (I) affords the (R)-(+) and (S)-(-) isomers in > 84% enantiomeric excess. Abs. stereochem. was detd. by a single crystal X-ray anal. of a related synthetic analog. The activity of these two enantiomers on glucose transport in vitro and as anti-diabetic agents in vivo is reported and their unexpected equivalence attributed to an enzyme-mediated stereospecific isomerization of the (R)-(+) isomer. Binding studies using recombinant human PPAR-gamma (peroxisomal proliferator activated receptor gamma), now established as a mol. target for this compd. class, indicate a 20-fold higher binding affinity for the (S) antipode relative to the (R) antipode.

Identification of candidate small-molecule therapeutics to cancer by gene-signature perturbation in connectivity mapping.

Connectivity mapping is a recently developed technique for discovering the underlying connections between different biological states based on gene-expression similarities. The sscMap method has been shown to provide enhanced sensitivity in mapping meaningful connections leading to testable biological hypotheses and in identifying drug candidates with particular pharmacological and/or toxicological properties. Challenges remain, however, as to how to prioritise the large number of discovered connections in an unbiased manner such that the success rate of any following-up investigation can be maximised. We introduce a new concept, gene-signature perturbation, which aims to test whether an identified connection is stable enough against systematic minor changes (perturbation) to the gene-signature. We applied the perturbation method to three independent datasets obtained from the GEO database: acute myeloid leukemia (AML), cervical cancer, and breast cancer treated with letrozole. We demonstrate that the perturbation approach helps to identify meaningful biological connections which suggest the most relevant candidate drugs. In the case of AML, we found that the prevalent compounds were retinoic acids and PPAR activators. For cervical cancer, our results suggested that potential drugs are likely to involve the EGFR pathway; and with the breast cancer dataset, we identified candidates that are involved in prostaglandin inhibition. Thus the gene-signature perturbation approach added real values to the whole connectivity mapping process, allowing for increased specificity in the identification of possible therapeutic candidates.

Resource pressure and environmental change on the North African littoral: Epipalaeolithic to Roman gastropods from Cyrenaica, Libya.

This paper discusses the marine and terrestrial shell on Epipalaeolithic to Classical-period sites in the Cyrenaican coastlands, northeast Libya, with particular reference to the Haua Fteah, with parallel studies at a late-Roman farmstead and two small caves. Together they provide evidence for coastal and terrestrial environments and for the continued nutritional importance of gastropods to humans during the Holocene. Land snail evidence is consistent with regional vegetation in coastal Cyrenaica becoming increasingly open through the Holocene, as a result of some combination of climate change and human impact. Marine species suggest that the coastline near the Haua had been rocky throughout the Holocene. At Hagfet al-Gama, changing faunas provide evidence for sand encroachment onto a previously rocky shoreline in Hellenistic times. A biometric study of Osilinus turbinatus shows that in the archaeological sites these shells are systematically smaller than modern specimens, providing evidence for long-term dietary stress in the human populations around the Haua Fteah, with particularly severe stress in parts of the Epipalaeolithic. A biometric study of Patella spp. provided evidence for size selection, but also seems to show evidence for resource pressure. It is unlikely that variations in resource pressure seen in the mollusc biometrics are the result of climatic stress or natural ecological factors and explanations must be sought in society-environment dynamics.

Apo J/clusterin expression and secretion: Evidence for 15-deoxy-Δ12,14-PGJ2-dependent mechanism

Cyclooxygenase-2 (Cox-2) and Apo J/clusterin are involved in inflammatory resolution and have each been reported to inhibit NF-?B signalling. Using a well-validated rat pheochromocytoma (PC12) cell culture model of Cox-2 over-expression the current study investigated inter-dependence between Cox-2 and clusterin with respect to induction of expression and impact on NF-?B signalling. Both gene expression and immunoblot analysis confirmed that intracellular and secreted levels of clusterin were elevated in Cox-2 over-expressing cells (PCXII). Clusterin expression was increased in control (PCMT) cells in a time- and dose-dependent manner by 15-deoxy-? 12,14-prostaglandin J 2 (15d-PGJ 2), but not PGE 2, and inhibited in PCXII cells by pharmacological Cox inhibition. In PCXII cells, inhibition of two transcription factors known to be activated by 15d-PGJ 2, heat shock factor 1 (HSF-1) and peroxisome proliferator activated receptor (PPAR)?, by transcription factor oligonucleotide decoy and antagonist (GW9662) treatment, respectively, reduced clusterin expression. While PCXII cells exhibited reduced TNF-a-induced cell surface ICAM-1 expression, IkB phosphorylation and degradation were similar to control cells. With respect to the impact of Cox-2-dependent clusterin upregulation on NF-?B signalling, basal levels of I?B were similar in control and PCXII cells, and no evidence for a physical association between clusterin and phospho-I?B was obtained. Moreover, while PCXII cells exhibited reduced NF-?B transcriptional activity, this was not restored by clusterin knock-down. These results indicate that Cox-2 induces clusterin in a 15d-PGJ 2-dependent manner, and via activation of HSF-1 and PPAR?. However, the results do not support a model whereby Cox-2/15d-PGJ 2-dependent inhibition of NF-?B signalling involves clusterin.