Simon N S Louis

Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/MTUS1 in hepatocellular carcinoma.

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein (ATIP)/mitochondrial tumor suppressor gene (MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.

Functional angiotensin II type 2 receptors inhibit growth factor signaling in LNCaP and PC3 prostate cancer cell lines.

There is clear evidence of a tissue‐based renin‐angiotensin system in the prostate and studies to date suggest that AT1‐receptor blocking drugs inhibit the growth of some prostate cancer cell lines and delay the development of prostate cancer. The present studies examine the action of Ang II in two prostate cancer cell lines and report the presence of functional AT2‐receptors that regulate the actions of growth factors.

Role of β-adrenergic receptor subtypes in Lipolysis

In vitro lipolysis stimulated by low (-)-isopre-naline concentrations (≤30 nM) in epididymal white adipo-cytes from Sprague-Dawley rats was inhibited at least 60–80% by the specific β1-antagonists LK 204-545 and CGP 20712A (1 μM), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via β1-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for β1-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, β3-adrenergic receptors were fully activated and were the dominant β-adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the β1-antagonists, demonstrating that the β3-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively β2-selective agonist formoterol in the presence of β1-blockade (1 μM CGP 20712A) demonstrated the inability of the β2-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 ≤ 1 μM. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (∼500-fold lower than its β2-adrenergic receptor pA2, 7.80 ± 0.21), suggesting that formoterol was not acting via β2-adrenergic receptors. These data are consistent with β1-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that β2-adrenergic receptors play no obvious direct role in mediating β-adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a β3-selective antagonist), was found to be a non-selective antagonist at the three β-adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.

Expression and function of ATIP/MTUS1 in human prostate cancer cell lines

We have previously demonstrated Ang II type 2 (AT2‐) receptor‐mediated inhibition of EGF‐induced prostate cancer cell growth in androgen‐dependent (LNCaP) and independent (PC3) prostate cancer cell lines.

Role of the renin-angiotensin system in prostate cancer.

Prostate cancer is highly prevalent in Western society, and its early stages can be controlled by androgen ablation therapy. However, the cancer eventually regresses to an androgen-independent state for which there is no effective treatment. The renin-angiotensin system (RAS), in particular the octapeptide angiotensin II, is now recognised to have important effects on growth factor signalling and cell growth in addition to its well known actions on blood pressure, fluid homeostasis and electrolyte balance. All components of the RAS have been recently identified in the prostate, consistent with the expression of a local RAS system in this tissue. This review focuses on the role of the RAS in the prostate, and the possibility that this pathway may be a potential therapeutic target for the treatment of prostate cancer and other prostatic diseases.

LK 204–545, a highly selective β1-adrenoceptor antagonist at human β-adrenoceptors

LK 204-545 ((+/-)-1-(2-(3-(2-cyano-4-(2-cyclopropyl-methoxy-ethoxy)phenoxy)-2-hydro xy-propyl-amino)-ethyl)-3-(4-hydrxy-phenyl) urea), an antagonist that possesses high beta1-/beta2-selectivity in the rat, and a range of cardio-selective and non-selective beta-adrenoceptor antagonists were examined to compare their radioligand binding affinities for human beta1-, beta2- and beta3-adrenoceptors transfected into CHO cells. LK 204-545 and CGP 20712A displayed the highest beta1-/beta2- (approximately 1800 and approximately 650, respectively) and beta1-/beta3-selectivity (approximately 17000 and approximately 2200, respectively) at human beta-adrenoceptors with LK 204-545 being approximately 2.75-fold more beta1-/beta2-selective and approximately 8-fold beta1-/beta3-selective than CGP 20712A. The high potency of LK 204-545 at transfected human beta1-adrenoceptors and in functional models of rat beta1-adrenoceptors together with its high selectivity, identify it as a useful ligand for studying beta1-adrenoceptors and suggest that it may be the preferred ligand for human beta-adrenoceptor studies.

Partial agonist activity of celiprolol.

Celiprolol given intravenously (i.v.) to pithed rats in the dose range of 0.1–10,000 μg/g produced a dose-dependent increase in heart rate (HR) which was greatest (123 beats/min) at 1,000–3,000 μg/kg. This partial agonist effect was blocked by the selective β1-adrenoceptor antagonist CGP 20712A. Celiprolol also produced a vasodepressor effect in this dose range which was abolished by the relatively selective β1-adrenoceptor antagonist ICI 118551 but not CGP 20712A. The magnitude of this intrinsic sympathomimetic activity (ISA) response was not significantly altered by reserpine pretreatment. Celiprolol also antagonised the effects of isoprenaline 0.05 μg/kg on HR and blood pressure (BP). The β1 selectivity of celiprolol as an antagonist in pithed rats (β1/β2 = 340:1) was similar to that observed in studies with isolated guinea pig atria and trachea (β1/β2 = 63:1), both being considerably greater than that observed with atenolol. Celiprolol, however, like atenolol, potentiated the bronchoconstrictor responses to histamine (3 μg/kg). Metabolic studies of rats and human urine failed to show significant amounts ++of potentially vasoactive metabolites. These data are consistent with celiprolol acting as both a β1- and β2-adrenoceptor partial agonist.

Synthesis, β-adrenoceptor pharmacology and toxicology of S-(−)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride, a short acting β1-specific antagonist

The synthesis of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride (D140S.HCl 6), a novel short acting beta(1)-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S.HCl 6 has been compared with esmolol, another short acting agent, and other well known beta-adrenoceptor antagonists in isolated rat tissue preparations. The pharmacokinetics of D140S.HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced heart rate increase and the pharmacodynamic half-life of D140S.HCl 6 has been compared with esmolol in a conscious rat model. In addition, the results of a range of toxicological studies are presented. The results indicate that D140S.HCl 6 is a highly specific beta(1)-adrenoceptor antagonist (pA(2) = 8.15+/-0.22, beta(1)/beta(2) selectivity > 4400). The in vitro studies suggest D140S.HCl is ca. ten times more potent and 60 times more beta(1)-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day intravenous infusion at toxicological doses above 10 mg kg(-1) h(-1) in the rat and 2.5 mg kg(-1) h(-1) in the dog. Both D140S.HCl 6 and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is < 15 min for D140S.HCl and 10 min for esmolol). The toxicological tests indicate that D140S.HCl 6 shows no unexpected toxicity and none of the tissue irritancy problems reported for esmolol formulations.

β1- and β2-Adrenoceptor antagonist activity of a series of para-substituted N-isopropylphenoxypropanolamines

Abstract To further explore the structure-activity relationships of β-adrenoceptor (β-AR) antagonists, a series of 25 para -substituted N -isopropylphenoxy-propanolamines were synthesised, nine of which are new compounds. All have been examined for their ability to antagonise β 1 -ARs in rat atria and β 2 -ARs in rat trachea. Substitution in the para -position of the phenyl ring is thought to confer β 3 -specificity and the selectivity of these compounds for the β 1 -AR ranges from 1.5–234. None of the compounds tested were selective for the β 2 -AR. Of the 25 compounds studied, 22 had reasonable (pA 2 > 7) potencies for the rat β 1 -AR. Only compound 1 displayed reasonable (pA 2 > 7) potency for the rat β 2 -AR. Twenty two compounds were used as the training set for comparative molecular field analysis (CoMFA) of antagonist potency (pA 2 ) at the rat β 1 - and β 2 -ARs. The inclusion of a number of additional physical characteristics improved the QSAR analysis over models derived solely using the CoMFA electrostatic and steric fields. The final models predicted the β 1 - and β 2 -AR potency of the compounds in the training set with high accuracy (r 2 = 0.93 and 0.86 respectively). The final β 1 -AR model predicted the β 1 -potencies of two out of the three test compounds, not included in the training set, with residual pA 2 values 2 -AR model (residual pA 2 values

The Expression of MTUS1/ATIP and Its Major Isoforms, ATIP1 and ATIP3, in Human Prostate Cancer

Angiotensin II (Ang II), the main effector of the renin angiotensin system, acts upon two distinct transmembrane receptors, the Ang II type 1 and the type 2 (AT2-) receptor, to induce promotion and inhibition of ERK2 phosphorylation. The AT2-receptor, through an interaction with its putative signaling partner MTUS1/ATIP (AT2-receptor interacting protein), inhibits the mitogenic effects of EGF in prostate cancer cell lines representing both early and late stage disease. This is the first report on the expression of ATIP in normal and malignant human prostatic biopsies. The expression of ATIP and its major isoforms, ATIP1 and ATIP3, in normal prostatic cells and three prostate cancer cell lines was examined using QPCR and immunohistochemistry. Human biopsies containing benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and well, moderately and poorly differentiated prostate cancer were also examined. Overall, ATIP1 and ATIP3 mRNA expression was increased in malignant compared to normal tissues and cell lines. ATIP immunostaining was low or absent in both the basal and columnar epithelial cell layers surrounding BPH acini; however, it was observed in high concentration in neoplastic epithelial cells of HGPIN and was clearly evident in cytoplasms of malignant cells in all prostate cancer grades. ATIP immunostaining was also identified in the cytoplasms of LNCaP and PC3 prostate cancer cells. As the AT2-receptor/ATIP inhibitory signaling pathway exists in malignant cells in all grades of prostate cancer, enhancement of this pathway may be a therapeutic target even after the development of androgen-independence.