Endothelin-1 stimulates cyclooxygenase-2 expression in ovarian cancer cells through multiple signaling pathways: evidence for involvement of transactivation of the epidermal growth factor receptor

Ovarian carcinoma cells release high amounts of endothelin-1 and exhibit increased expression of endothelin-A receptor. Engagement of the endothelin-A receptor triggers tumor growth, survival, neoangiogenesis and invasion. Cyclooxygenase-1 and cyclooxygenase-2 are enzymes involved in the production of prostaglandins and play a role in the regulation of tumor progression in several malignancies, including ovarian carcinomas. Endothelin-1 significantly increases the expression of cyclooxygenase-1 and cyclooxygenase-2 mRNA and protein, the activity of the cyclooxygenase- 2 promoter, and the release of prostaglandin E2 from two ovarian carcinoma cell lines, HEY and OVCA 433. The cyclooxygenase- 2 inhibitor, NS-398 drastically decreased the endothelin- 1-induced prostaglandin E2 production and vascular endothelial growth factor upregulation, indicating a role for cyclooxygenase-2 in endothelin-1-induced vascular endothelial growth factor-mediated angiogenesis. In this study we demonstrated that endothelin-1-induced cyclooxygenase-2 and related prostaglandin E2 release were dependent upon the activation of endothelin-A receptor and of multiple mitogen-activated protein kinase signal pathways, including extracellular signal-regulated kinase 1/2 kinase, p38 mitogen-activated protein kinase and the transactivation of the epidermal growth factor receptor. In human ovarian xenografts, the levels of cyclooxygenase-2 protein expression were significantly reduced following treatment with the endothelin-A receptor selective antagonist, atrasentan, compared with untreated mice. These results suggest that the pharmacological blocking of endothelin-A receptor is an attractive strategy to control the cyclooxygenase-2 protein expression in ovarian carcinoma.     

Ethanol and nitric oxide modulate expression of glucocorticoid receptor in the rat adrenal cortex

BackgroundThis study was performed to investigate expression and distribution of glucocorticoid receptor (GR) in the rat adrenal cortex, acute effect of ethanol on its expression and possible role of endogenous nitric oxide (NO) in this phenomenon.MethodsAdult female Wistar rats showing diestrus day 1 were treated with: a) ethanol (2 or 4 g/kg body weight (b.w.), ip), b) N^ω-nitro-L-argmine methyl ester (L-NAME), well-known competitive inhibitor of all isoforms of NO synthase (NOS), (30 mg/kg b.w., sc) followed by ethanol (4 g/kg, ip) 3 h later and c) L-NAME (30 mg/kg b.w., sc) followed by saline (ip) 3 h later. Untreated rats were used as controls. Adrenocortical expression of GR was estimated by immunohistochemistry.ResultsStrong nuclear GR staining was observed throughout the cortex of control rats. Acute ethanol treatment significantly decreased the expression of GR in the zona fasciculata and zona reticularis. Blockade of NO formation had no influence on this effect of ethanol, whereas L-NAME itself induced significant decline in GR immunoreactivity.ConclusionsObtained findings are the first to demonstrate localization and distribution of the GR throughout the rat adrenal cortex and to suggest that ethanol as well as endogenous NO may modulate adrenocortical expression of this steroid receptor.     

Lovastatin suppresses erythropoietin receptor surface expression through dual inhibition of glycosylation and geranylgeranylation

Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64 kDa and the mature 66 kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and geranylgeranylation.     

Synthesis and characterization of nonsteroidal glucocorticoid receptor modulators for multiple myeloma

Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.     

大鼠糖皮质激素受体基因腺病毒载体的构建及在肾系膜细胞中的表达

目的 利用AdEasy系统构建大鼠糖皮质激素受体(GR)基因重组腺病毒载体,转染大鼠肾小球系膜细胞(GMC),并通过Western Blot法检测GR蛋白的表达.方法 将质粒pcDNA1 Neo-Rat GR扩增、凝胶回收获得的Rat GR cDNA双酶切片段,插入腺病毒穿梭载体质粒pShuttle-CMV的巨细胞病毒(CMV)启动子下游,构建重组穿梭载体pShuttle-CMV-Rat GR,线性化后与骨架载体AdEasy-1在细菌BJ5183内经同源重组得到腺病毒重组质粒pAd-Rat GR,经人胚肾293A细胞包装后得到复制缺陷型重组腺病毒Ad-Rat GR;用包装后的病毒上清转染大鼠GMC,提取细胞中总蛋白,通过Western Blot方法 检测GR蛋白的表达.结果 连接、重组后通过酶切和测序法筛选出pAd-Rat GR重组质粒,经人胚肾293细胞包装,48 h后观察到增强型绿色荧光蛋白(EGFP)明显表达,氯化铯梯度离心纯化最终获得1×109 PFU /ml 滴度的重组病毒;用该滴度的Ad-Rat GR,转染大鼠GMC 48 h后提取细胞总蛋白,Western Blot检测GR蛋白有明显表达.结论 构建的大鼠GR基因腺病毒载体能够在大鼠GMC 中表达GR蛋白,并有上调作用.     

Binding of umespirone to the sigma receptor: evidence for multiple affinity states

Umespirone (3,7-diazabicyclo[3.3.1]nonane-2,4,6,8-tetraon; KC9172) has been recently described as a potential antipsychotic/anxiolytic. In the present study the interaction of umespirone and several other typical and atypical psychotropics with the sigma and phencyclidine (PCP) receptor subtypes, in rat brain membranes, was investigated. Umespirone, haloperidol, chlorpromazine and buspirone, but not clozapine and diazepam, compete for the binding of the sigma-ligand, (+)[3H]-3-(3-hydroxy-phenyl)-N-1-(propyl) piperidine [(+)]3H-3-PPP]. The compounds do not compete for the binding of PCP-receptor ligands. Umespirone, buspirone and (+)-3-PPP, unlike haloperidol and chlorpromazine, inhibit (+)[3H]-3-PPP binding in a manner indicating the existence of high and low affinity states of the sigma receptor. 5'-Guanylylimidodiphosphate (Gpp(NH)p; 0.1mM) abolishes the high affinity binding component associated with the binding of umespirone, buspirone and (+)-3-PPP, but does not affect the affinities of haloperidol and chlorpromazine for the sigma receptor. These findings suggest that different coupling states of the sigma receptor may be associated with the binding of umespirone and buspirone compared to the binding of haloperidol and chlorpromazine.     

Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization

Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFκB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFκB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFκB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNβ in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFκB or IRF3 induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFκB activation. Further, cinnamaldehyde inhibited ligand-independent NFκB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.     

Further evidence for the involvement of kinin in anaphylactic shock in the rat

1. Sodium phenylbutazone, soya bean trypsin inhibitor, or the concomitant administration of ascorbic acid and mepyramine protected rats against anaphylactic shock at 10 days after sensitization but gave no protection against anaphylaxis at 20 days.2. During anaphylactic shock in rats at 10 days after sensitization, the plasma bradykinin and bradykininogen levels, as well as those in the intestinal lumen and peritoneal cavity, were markedly raised.3. The results support the hypothesis that there are two phases in anaphylaxis in the rat-an early phase in which bradykinin is a mediator and against which phenylbutazone or soya bean trypsin inhibitor or the mixture of ascorbic acid and mepyramine, give protection, and a late phase which does not involve bradykinin.     

Functional evidence for multiple receptor activation by kappa-ligands in the inhibition of spinal nociceptive reflexes in the rat.

1. The evidence for kappa-receptor heterogeneity is equivocal. We have now investigated this question by comparing the effects of five putatively selective kappa-agonists. The parameters examined were: the relative potencies in depressing hindlimb flexor muscle reflexes to noxious pinch stimuli in both spinalized and sham-spinalized rats; the reversibility of these effects by naloxone; and the effects on blood pressure. 2. Two types of drug effect was discriminated. One drug group, represented by U-50,488, U-69,593 and PD-117,302, had a potency ratio between sham and spinalized rats approximately 10 fold lower than the other group, which comprised GR103545 and CI-977. 3. Under sham-spinalized conditions, CI-977 and GR103545 at high doses caused only sub-maximal reductions of spinal reflexes. U-50,488 was still active when superimposed on these high doses of GR103545. 4. Naloxone reversed all effects, but different doses were required between compounds, with GR103545 taking some 20 times higher doses of naloxone to cause reversal than did U-50,488. 5. The effects on mean arterial pressure were opposite between groups. 6. The results imply that more than one type of naloxone-sensitive non-mu opioid receptor must be involved in mediating these complex actions of ligands that have been claimed to be selective for kappa-receptors.     

On the involvement of multiple muscarinic receptor subtypes in the activation of phosphoinositide metabolism in rat cerebral cortex

We have examined the activation of phosphoinositide metabolism by muscarinic agonists in rat cerebral cortex, in an attempt to delineate the mechanisms by means of which some selective antagonists inhibit this response in a manner that deviates from simple mass action law. The accumulation of [3H]inositol phosphates induced by the full agonist carbamylcholine in cell aggregates preparations was inhibited by muscarinic antagonists with the following order of potency: telenzepine greater than atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than pirenzepine greater than hexahydro-sila-difenidol greater than AF-DX 116. The same order of potency was found for the competition of these antagonists with [3H]telenzepine binding to M1 muscarinic receptors. The inhibition of the formation of [3H]inositol phosphates activated by acetylcholine, carbamylcholine, and oxotremorine-M by pirenzepine and telenzepine showed biphasic curves, with 62-73% of the response being inhibited with high affinity. Atropine, AF-DX 116, and pirenzepine shifted the concentration-response curves of oxotremorine-M to the right in a parallel manner. However, pirenzepine at micromolar concentrations showed deviation from linearity of the Schild regression. The blockade by high concentrations of pirenzepine and telenzepine showed less than additive dose ratios when assayed in the presence of atropine, suggesting deviation of their antagonism from simple competition. However, after alkylation with propylbenzilylcholine mustard in the presence of low concentrations of pirenzepine, the response to carbamylcholine and oxotremorine-M showed monophasic inhibition curves by pirenzepine and linear Schild regression for this antagonist. These results support the interpretation that the formation of [3H]inositol phosphates is activated by multiple muscarinic receptor subtypes in rat cerebral cortex. The profile of affinities of muscarinic antagonists indicates that a major component of the response is activated by an M1 receptor subtype and a minor component is probably mediated by M3 muscarinic receptors when acetylcholine, carbamylcholine, or oxotremorine-M are used to stimulate the response. Conversely, pirenzepine inhibited the response induced by methacholine and bethanechol in a monophasic manner with high affinity (Ki = 13 nM), suggesting that these agonists can selectively stimulate phosphoinositide metabolism through activation of M1 muscarinic receptors in rat cerebral cortex.     

Effect of phenobarbital on the glucocorticoid receptor in rat hepatoma cells

Phenobarbital is a potent inducer of several liver-specific genes such as those encoding detoxication enzymes, including cytochromes P450. However, the mechanisms of action of the barbiturate are poorly understood. Since both, phenobarbital and glucocorticoids, are capable of inducing the same cytochrome P450 species, we asked whether the glucocorticoid receptor could participate to the phenobarbital induced responses. The results presented here show that phenobarbital was able to induce a two-fold increase in the affinity of the glucocorticoid receptor for the binding of dexamethasone, as well as a 30% increase of the receptor number in Reuber rat hepatoma cells of the Fao line. These effects may have a biological significance since they were paralleled by an enhancement of the dexamethasone-induced tyrosine aminotransferase activity, a glucocorticoid inducible function in rat hepatoma cells and in rat liver. To our knowledge, phenobarbital is the first compound shown to be able to induce, in intact cells, an increase in the affinity of the glucocorticoid receptor for the binding of its ligand.     

Effects of SKF-10047 in the phencyclidine-dependent rat: evidence for common receptor mechanisms

The repeated administration of phencyclidine (PCP, 72 mg/kg/day) to rats led to physical dependence, as evidenced by a withdrawal syndrome exhibited approx. 24-48 h following suspension of drug. All components of the withdrawal syndrome were suppressed by s.c. injections of PCP (16 mg/kg), (+/-)-N-allylnormetazocine (SKF-10047, 16 mg/kg) and (+)-SKF-10047 (16 mg/kg), but not by injections of saline or (-)-SKF-10047. Moreover, tolerance to the behavioral effects of PCP, as well as cross-tolerance to (+/-)-SKF-10047 and (+)-SKF-10047 were observed. These data indicate that PCP and the sigma opiate SKF-10047 share mechanisms of action, which are mediated by the (+)-isomer of the sigma agonist.     

Chronic methamphetamine exposure suppresses the striatal expression of members of multiple families of immediate early genes (IEGs) in the rat: normalization by an acute methamphetamine injection

Rationale  

Repeated injections of cocaine cause blunted responses to acute cocaine challenge-induced increases in the expression of immediate early genes (IEGs).