A novel leptin antagonist peptide inhibits breast cancer growth in vitro and in vivo

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I–III. Site I is crucial for the formation of an active leptin–leptin receptor complex and in its subsequent activation. Amino acids 39‐42 (Leu‐Asp‐Phe‐Ile‐ LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild‐type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin‐induced anchorage‐dependent and ‐independent growth as well as the migration of ERα‐positive (MCF‐7) and ‐negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin‐mediated up‐regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women.     

A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies

The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging.     

A novel RGDS-analog inhibits angiogenesis in vitro and in vivo

In this study the anti-angiogenic action of a novel non-peptide RGDS-analog named RAM was tested in vitro and in vivo. RAM inhibited FGF-2-induced chemotaxis by 80% in an adhesion-independent way. Further, it induced HUVEC-apoptosis in collagen-seeded HUVEC, indicating that such pro-apoptotic effect was adhesion-independent. In vivo studies revealed that RAM inhibited FGF-2 induced angiogenesis by 60% in the mouse Matrigel-assay and in the chicken-egg chorion-allantoic membrane assay. Finally, RAM was markedly more stable in serum as compared to the template RGDS and after 24 h incubation in 100% serum was significantly more active than RGDS. Taken together these results show that RAM exerts anti-chemotactic and pro-apoptotic effects, by an unexpected adhesion-independent mechanism, as we have recently shown for the template RGDS molecule [Blood 103 (2004) 4180], and has in vivo relevant anti-angiogenic properties, with marked stability in serum; therefore, RAM represents a novel promising anti-angiogenic molecule.     

Development of a potent bombesin receptor antagonist with prolonged in vivo inhibitory activity on bombesin-stimulated amylase and protein release in the rat.

Of the various types of potent bombesin(Bn)/gastrin releasing peptide receptor antagonists that have been discovered, the desMet14-methyl ester peptides are devoid of residual agonist activity and are among the most potent in terms of in vitro receptor blockade and also in terms of their prolonged inhibition of bombesin-stimulated amylase and protein release in the rat. We have now examined the in vitro and in vivo properties of a new series of methyl ester analogues, [D-Phe6]Bn(6-13)OMe, [D-Phe6,D-Ala11]Bn(6-13)OMe, N alpha-propionyl-[D-Ala24]GRP(20-26)OMe, and [D-pentafluoro-Phe6,D-Ala11]Bn(6-13)OMe, which have an additional D-amino acid substituent and some highly lipophilic moieties at the N-terminus. All analogues were able to potently antagonize the ability of Bn to stimulate amylase release from rat acinar cells, with IC50 values of 2.4, 2.5, 0.6, and 1.3 nM, respectively. The four peptides were found to have binding affinities for these cells comparable to Bn itself, with K(i)s of 10.3, 2.8, 5.5, and 3.6 nM, respectively, but all had little or no affinity for neuromedin B receptors on murine C6 cells. Single bolus IV injections of these peptides were found to potently inhibit amylase and protein release caused by IV infusion of bombesin into the rat. Generally the peptides containing the D-Ala substituent were longer acting than [D-Phe6]Bn(6-13)OMe, so that [D-Phe6,D-Ala11]Bn(6-13)OMe and N alpha-propionyl-[D-Ala24]GRP(20-26)OMe displayed significant inhibitory effects for up to 1.5 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bombesin inhibits insulin release from isolated pancreatic islets of rats in vitro.

The effect of bombesin on insulin release from isolated pancreatic islets of rats was examined in vitro. Bombesin, at the doses ranging from 10 ng/ml to 1 microgram/ml, significantly inhibited 16.7 mM glucose-induced insulin release, while bombesin had no inhibitory effect on insulin release at 8.3 mM and 3.3 mM glucose. Moreover, bombesin also suppressed insulin release elicited by 10 mM arginine at the doses of 100 ng/ml and 1 microgram/ml. These results indicate that bombesin has a direct inhibitory action on insulin release.     

In vitro and in vivo pharmacological characterization of the novel NK(1) receptor selective antagonist Netupitant

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

Periovulatory changes in the local release of vasoactive peptides, prostaglandin f(2alpha), and steroid hormones from bovine mature follicles in vivo

We previously proposed that an endothelin-angiotensin-atrial natriuretic peptide system may contribute to inducing ovulation of mature bovine follicles by modulating follicular secretion of steroids and prostaglandins (PGs). Thus, this study aimed to determine the real-time changes in the local release of angiotensin II (Ang II), endothelin (ET), atrial natriuretic peptide (ANP), PGF(2alpha), and steroid hormones from bovine mature follicles during the periovulatory period in vivo. Seven cows were treated for superovulation using FSH and PGF(2alpha) injections. Two dialysis capillary membranes per follicle were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system (MDS). Fractions of the perfusate were collected from Day -1 (Day 0 = LH surge) to Day 3. Five out of seven treated cows were normally ovulated, and the newly formed corpora lutea were observed at the end of the experiment. In these five ovulated cows, the release of estradiol, androstenedione, and progesterone in the theca layer increased (P < 0.05) synchronously with the LH surge. Acute increases in PGF(2alpha) and Ang II concentrations in the ovarian venous plasma (OVP) were observed at 24-48 h after the peak of the LH surge, when multiple ovulations were expected to occur. The follicular Ang II release was low during the pre-LH surge period and rose (P < 0.05) at the beginning of the increase in the LH surge. On the other hand, ET-1 release dropped (P < 0.05) when plasma LH started to increase. However, no clear changes in ANP concentration in the MDS perfusate and plasma were observed. The above local changes in Ang II, PGF(2alpha), as well as steroid hormones were not observed in cows (n = 2) that did not show an LH surge and ovulation. The present results demonstrate for the first time the local release of Ang II, ET-1, and ANP from the bovine mature follicle in real-time in vivo and show that Ang II and PGF(2alpha) concentrations in the OVP acutely increase around the time of ovulation. The overall results support the concept of a local functional ET-Ang-ANP system in the bovine mature follicle that may be involved in the ovulatory process.     

In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031

Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC(50)=3.0nM), ligand-induced Ca(2+) flux (IC(50)=5.4nM), and the chemotaxis of human CCR3-expressing cells (IC(50)=19.9nM). YM-344031 (1-10mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.     

Ketanserin, an antagonist of 5-HT2A receptor of serotonin, inhibits testosterone secretion by rat Leydig cells in vitro.

The effect of ketanserin, an antagonist of 5-HT2A receptor of serotonin, added to the culture medium, on basal and LH-stimulated testosterone secretion was studied in primary cultures of adolescent rat Leydig cells. Ketanserin decreased the basal secretion of testosterone but showed an insignificant influence on the LH-stimulated process. It can be concluded that ketanserin may affect the testosterone-secreting cells by an indirect action at the vascular level as well as directly at the level of Leydig cells, at least in adolescent rats, leading to down-regulation of the basal testosterone secretion.     

A novel labdane-type trialdehyde from myoga (Zingiber mioga Roscoe) that potently inhibits human platelet aggregation and human 5-lipoxygenase

We screened myoga extracts for inhibitors of human platelet aggregation and human 5-lipoxygenase. We identified a novel labdane type of diterpene, together with three known diterpenes (miogadial and galanals A and B) from the flower buds of myoga. Spectroscopic data indicated the structure of the new compound to be 12(E)-labdene-15,16,(8beta)17-trial (miogatrial). Miogatrial and miogadial were potent inhibitors of human platelet aggregation and human 5-lipoxygenase (5-LOX). The sesquiterpene, polygodial, also exhibited strong inhibitory activity against human platelet aggregation and 5-LOX. On the other hand, galanals A and B did not have inhibitory activity in either experimental system. It thus appears that a 3-formyl-3-butenal structure was essential for the potent inhibition of human platelet aggregation and human 5-LOX.     

Mouse IL-1 receptor antagonist protein. Molecular characterization, gene mapping, and expression of mRNA in vitro and in vivo.

The IL-1R antagonist protein (IL-1RN/IL-1rn) is a member of the IL-1 family of inflammatory mediators. We have isolated and analyzed a mouse IL-1rn cDNA clone and established that the derived mouse IL-1rn protein sequence is highly homologous to the human counterpart molecule. Mouse IL-1rn mRNA may be induced in P388D1 monocytic cells with PMA and in mouse liver in vivo by development of an experimental inflammation via s.c. injection of azocasein. This latter observation implies the existence of an autocrine hepatic negative feedback loop that down-regulates the acute phase response and is itself induced at the same time as the major acute-phase proteins. The mouse Il 1rn gene was mapped to the proximal region of chromosome 2 between the centromere and Spna2; the other known members of the mouse IL-1 gene family, Il 1a and Il 1b, both map to the same chromosome, although not in close linkage.     

Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity

Each peptide bond CONH group in the most important COOH-terminal octapeptide region of [Leu14]bombesin was replaced by a CH2NH group using recently developed rapid solid-phase methods. The resulting analogues were then examined for amylase releasing activity in guinea pig pancreatic acini and for their ability to inhibit binding of [125I-Tyr4]bombesin to acinar cells. Replacement of the Trp8-Ala9, Gly11-His12, and His12-Leu13 peptide bonds resulted in about 1000-, 200-, and 300-fold losses in both amylase releasing activity and binding affinity. The Val10-Gly11 replacement, however, retained 30% potency relative to the parent peptide. Ala9-Val10 and Leu13-Leu14 bond replacement analogues exhibited no detectable amylase releasing activity but were still able to bind to acini with Kd values of 1060 and 60 nM, respectively (compared to 15 nM for [Leu14]bombesin itself). Subsequently, both analogues were demonstrated to be competitive inhibitors of bombesin-stimulated amylase release with IC50 values of 937 and 35 nM, respectively. [Leu14-psi-CH2NH-Leu13]Bombesin exhibits a 100-fold improvement in binding affinity compared to previously reported bombesin receptor antagonists and showed no affinity for substance P receptors. It was also a potent inhibitor of bombesin-stimulated growth of murine Swiss 3T3 cells with an IC50 of 18 nM. In terms of a bombesin receptor-binding conformation, these results may aid in the delineation of intramolecular hydrogen-bonding points and the eventual design of improved, conformationally restricted analogues.     

Atrial natriuretic peptide inhibits neurohypophysial hormones' release in the rat (in vitro and in vivo studies).

Intracerebroventricular hANP (50 nmol) inhibits release of vasopressin and oxytocin following dehydration as well as after haemorrhage. 10 nmol/L hANP markedly inhibits vasopressin and oxytocin release in vitro from the neurointermediate lobes both under basal condition as well as during stimulation with excess (56 mM) potassium. It is suggested that ANP may serve as a modulator of vasopressin and oxytocin release. The respective processes are localized, at least in part, at the neurohypophysial level.     

In vivo characterization of a novel GnRH (gonadotropin-releasing hormone) antagonist, LXT-101, in normal male rats

LXT-101 is a newly developed GnRH (gonadotropin-releasing hormone) analogue. In this study, the in vivo pharmacological profile in intact male rats and binding characters of LXT-101 were illustrated, and regulation of mRNA of hormone receptors related to the pituitary-gonadal axis during and after administration was observed to reveal its molecular mechanism of potent effect and reversibility. After single subcutaneous injections, LXT-101 produced a dose- and time-dependent suppression of serum testosterone level. Multiple administrations and osmotic pump implantation revealed that the time of onset and dose needed to maintain the effect of chemical castration decreased as the frequency of injection increased and gave direct proof that depot formulation could significantly improve the duration of antagonist delivery and pharmacological activities compared to the injectable formulation. And LXT-101 showed excellent character of regulating the pituitary-gonadal axis quickly and reversibly. Competitive binding assay showed that LXT-101 could specifically bind a pituitary GnRH receptor with high affinity. These results indicated that LXT-101 is fit for sustained-release formulation and it might possibly be developed as an ideal candidate for treating sex hormone-sensitive tumors and other disorders.     

A novel peptide derived from human pancreatitis-associated protein inhibits inflammation in vivo and in vitro and blocks NF-kappa B signaling pathway

Background

Pancreatitis-associated protein (PAP) is a pancreatic secretory protein belongs to the group VII of C-type lectin family. Emerging evidence suggests that PAP plays a protective effect in inflammatory diseases. In the present study, we newly identified a 16-amino-acid peptide (named PAPep) derived from C-type lectin-like domain (CTLD) of human PAP with potent anti-inflammatory activity using both in vivo and in vitro assays.

Methodology/Principal Findings

We assessed the anti-inflammatory effect of PAPep on endotoxin-induced uveitis (EIU) in rats and demonstrated that intravitreal pretreatment of PAPep concentration-dependently attenuated clinical manifestation of EIU rats, reduced protein leakage and cell infiltration into the aqueous humor (AqH), suppressed tumor necrosis factor (TNF)-α, interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein (MCP)-1 production in ocular tissues, and improved histopathologic manifestation of EIU. Furthermore, PAPep suppressed the LPS-induced mRNA expression of TNF-α and IL-6 in RAW 264.7 cells, inhibited protein expression of ICAM-1 in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) as well as U937 cells adhesion to HUVECs. Western blot analysis in ocular tissues and different cell lines revealed that the possible mechanism for this anti-inflammatory effect of PAPep may depend on its ability to inhibit the activation of NF-kB signaling pathway.

Conclusions/Significance

Our studies provide the first evidence that the sequence of PAPep is within the critically active region for the anti-inflammatory function of PAP and the peptide may be a promising candidate for the management of ocular inflammatory diseases.     

Interleukin-4 (IL-4) enhances and soluble interleukin-4 receptor (sIL-4R) inhibits histamine release from peripheral blood basophils and mast cells in vitro and in vivo

The aim of the study was to analyse the effect of interleukin-4 (IL-4) on allergen and anti-IgE mediated histamine release from basophils and human skin mast cells and to assess whether soluble recombinant interleukin-4 receptor (sIL4R) can inhibit these effects. Anti-IgE stimulated histamine release from peripheral blood basophils and mast cells of atopic donors was enhanced after preincubation with IL-4, whereas after preincubation with sIL-4R it was inhibited. These effects were even more pronounced when samples were stimulated with a clinically relevant allergen. In IL-4 preincubated skin mast cells, there was a similar enhancement of anti-IgE stimulated histamine release, which could again be inhibited by sIL-4R. The effects of IL-4 and sIL4R were dose- and time-dependent. Mice sensitized to ovalbumin and treated with soluble recombinant murine sIL-4R showed significantly reduced immediate-type cutaneous hypersensitivity responses compared with untreated mice. These in vivo effects were IgE independent, since there were no significant differences in total and allergen specific IgE/IgG1 antibody titres between treated and untreated mice. This indicates that IL4 exerts priming effects on histamine release by effector cells of the allergic response and that these effects are potently antagonized by soluble IL-4R both in vitro and in vivo.