Enhanced Ca(2+) response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines.

Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1ß enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.

Antagonism of bradykinin B2 receptor prevents inflammatory responses in human endothelial cells by quenching the NF-kB pathway activation.

BACKGROUND: Bradykinin (BK) induces angiogenesis by promoting vessel permeability, growth and remodeling. This study aimed to demonstrate that the B2R antagonist, fasitibant, inhibits the BK pro-angiogenic effects. METHODOLOGY: We assesed the ability of fasibitant to antagonize the BK stimulation of cultured human cells (HUVEC) and circulating pro-angiogenic cells (PACs), in producing cell permeability (paracellular flux), migration and pseocapillary formation. The latter parameter was studied in vitro (matrigel assay) and in vivo in mice (matrigel plug) and in rat model of experimental osteoarthritis (OA). We also evaluated NF-κB activation in cultured cells by measuring its nuclear translocation and its downstream effectors such as the proangiogenic ciclooxygenase-2 (COX-2), prostaglandin E-2 and vascular endothelial growth factor (VEGF). PRINCIPAL FINDINGS: HUVEC, exposed to BK (1-10 µM), showed increased permeability, disassembly of adherens and tight-junction, increased cell migration, and pseudocapillaries formation. We observed a significant increase of vessel density in the matrigel assay in mice and in rats OA model. Importantly, B2R stimulation elicited, both in HUVEC and PACs, NF-κB activation, leading to COX-2 overexpression, enhanced prostaglandin E-2 production. and VEGF output. The BK/NF-κB axis, and the ensuing amplification of inflammatory/angiogenic responses were fully prevented by fasitibant as well as by IKK VII, an NF-κB. Inhibitor. CONCLUSION: This work illustrates the role of the endothelium in the inflammation provoked by the BK/NF-κB axis. It also demonstates that B2R blockade by the antaogonist fasibitant, abolishes both the initial stimulus and its amplification, strongly attenuating the propagation of inflammation.

MicroRNA-130b promotes tumor development and is associated with poor prognosis in colorectal cancer.

MicroRNA-130b (miR-130b) is involved in several biologic processes; its role in colorectal tumorigenesis has not been addressed so far. Herein, we demonstrate that miR-130b up-regulation exhibits clinical relevance as it is linked to advanced colorectal cancers (CRCs), poor patients' prognosis, and molecular features of enhanced epithelial-mesenchymal transition (EMT) and angiogenesis. miR-130b high-expressing cells develop large, dedifferentiated, and vascularized tumors in mouse xenografts, features that are reverted by intratumor injection of a specific antisense RNA. In contrast, injection of the corresponding mimic in mouse xenografts from miR-130b low-expressing cells increases tumor growth and angiogenic potential while reduces the epithelial hallmarks. These biologic effects are reproduced in human CRC cell lines. We identify peroxisome proliferator-activated receptor γ (PPARγ) as an miR-130b direct target in CRC in vitro and in vivo. Notably, the effects of PPARγ gain- and loss-of-function phenocopy those due to miR-130b down-regulation or up-regulation, respectively, underscoring their biologic relevance. Furthermore, we provide mechanistic evidences that most of the miR-130b-dependent effects are due to PPARγ suppression that in turn deregulates PTEN, E-cadherin, Snail, and vascular endothelial growth factor, key mediators of cell proliferation, EMT, and angiogenesis. Since higher levels of miR-130b are found in advanced tumor stages (III-IV), we propose a novel role of the miR-130b-PPARγ axis in fostering the progression toward more invasive CRCs. Detection of onco-miR-130b and its association with PPARγ may be useful as a prognostic biomarker. Its targeting in vivo should be evaluated as a novel effective therapeutic tool against CRC.

Comparison between oral and intra-articular antinociceptive effect of dexketoprofen and tramadol combination in monosodium iodoacetate-induced osteoarthritis in rats.

Dexketoprofen and tramadol, alone or in combination, were evaluated after oral or intra-articular administration on knee osteoarthritis nociception induced by intra-articular (i.ar.) monosodium iodoacetate (MIA, 1 mg/25 µl) in the rat right knee while the left knee received saline (25 µl). Seven days after MIA treatment, dexketoprofen, tramadol, their combination or the vehicle were administered. Nociception was evaluated as alteration in hind limb weight distribution with Incapacitance tester at different time-points after drug administration. Oral dexketoprofen (0.1-1 mg/kg) or tramadol (0.5-5 mg/kg) induced maximal antinociception at 1 and 5 mg/kg, respectively. Their combination dose-dependently increased the intensity and duration of antinociception, that was additive and lasted up to 3 days. Also the intra-articular administration of dexketoprofen or tramadol (10-100 µg/25 µl) inhibited MIA-induced nociception, and the combination of the lower doses (10 µg/25 µl) produced a long lasting more than additive antinociceptive effect indicating a synergistic interaction between the two drugs. This effect was significantly reduced by naloxone (10 µg/25 µl, i.ar.) co-administered with both compounds. The intra-articular administration of both drugs at 10 µg/25 µl in the contralateral control knee joint provoked a marked synergistic antinociceptive effect indicating significant systemic diffusion through synovial membrane. The oral or intra-articular combination of dexketoprofen and tramadol produced additive or synergistic antinociceptive effects, respectively, in the model of MIA-induced osteoarthritis in rats, that might allow to obtain therapeutic advantages with lower side effects.

Characterization of a novel proinflammatory effect mediated by BK and the kinin B2 receptor in human preadipocytes.

Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca²âº](i) mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B1 receptor agonist, Lys-[des-Arg9]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B2 receptor antagonist, fasitibant chloride, but not by the B1 selective antagonist, Lys-[Leu8][des-Arg9]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B2 receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B2 receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride.

Abagovomab as maintenance therapy in patients with epithelial ovarian cancer: a phase III trial of the AGO OVAR, COGI, GINECO, and GEICO--the MIMOSA study.

PURPOSE: To determine whether abagovomab maintenance therapy prolongs recurrence-free (RFS) and overall survival (OS) in patients with ovarian cancer in first clinical remission. PATIENTS AND METHODS: Patients with International Federation of Gynecology and Obstetrics stage III to IV ovarian cancer in complete clinical remission after primary surgery and platinum- and taxane-based chemotherapy were randomly assigned at a ratio of 2:1 in a phase III, double-blind, placebo-controlled, multicenter study. Abagovomab 2 mg or placebo was administered as 1-mL suspension once every 2 weeks for 6 weeks (induction phase) and then once every 4 weeks (maintenance phase) until recurrence or up to 21 months after random assignment of the last patient. The primary end point was RFS; secondary end points were OS and immunologic response. RESULTS: Characteristics of the 888 patients included: mean age, 56.3 years; Eastern Cooperative Oncology Group performance status, ≤ 1 in > 99% of patients; serous papillary subtype, 81.5%; stage III, 85.9%; and cancer antigen 125 ≤ 35 U/mL after third cycle, 80.9%. Mean exposure to study treatment (± standard deviation) was 449.7 ± 333.08 days. Hazard ratio (HR) of RFS for the treatment group using tumor size categorization (≤ 1 cm, > 1 cm) was 1.099 (95% CI, 0.919 to 1.315; P = .301). HR of OS using tumor size categorization (≤ 1 cm, > 1 cm) was 1.150 (95% CI, 0.872 to 1.518; P = .322). The most frequently reported type of adverse event was an injection site reaction in 445 patients (50.2%), followed by injection site erythema and fatigue in 227 (25.6%) and 212 patients (23.9%), respectively. By the final visit, median anti-anti-idiotypic antibody level was 493,000.0 ng/mL, indicating a robust response. CONCLUSION: Abagovomab administered as repeated monthly injections is safe and induces a measurable immune response. Administration as maintenance therapy for patients with ovarian cancer in first remission does not prolong RFS or OS.

New insight into the binding mode of peptides at urotensin-II receptor by Trp-constrained analogues of P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-D-Trp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp(7) residue was replaced by the highly constrained L-Tpi and D-Tpi residues. The replacement of the Trp(7) by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation-activity relationships previously reported on UT receptor ligands.

Characterization of ibodutant at NK(2) receptor in human colon.

We have characterized the pharmacological profile of the nonpeptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) through radioligand binding and contractility assays in the human colon smooth muscle. The antagonist affinity of ibodutant was evaluated through concentration-dependent inhibition curves at the [(125)I]NKA specific binding by using membranes prepared from human colon smooth muscle. In this assay the affinity of ibodutant (pKi 9.9) was compared to that of other two selective NK2 receptor antagonists, nepadutant (pKi 8.4) and saredutant (pKi 9.2). The antagonist potency of ibodutant was evaluated towards the [ßAla(8)]NKA(4-10)-mediated contractions of human colon smooth muscle strips. In this assay ibodutant (3, 10, 30 and 100 nM) induced a concentration-dependent rightward shift of the [ßAla(8)]NKA(4-10) concentration-response curves without depressing the maximal contractile effect. The analysis of the curves yielded a Schild-plot linear regression with a slope not different from unity (1.02), thus indicating a surmountable antagonist behavior. The calculated apparent antagonist potency as pKB value was 9.1. No sex related differences were observed in NK2 receptor pharmacology for [ßAla(8)]NKA(4-10) or ibodutant in colonic strips obtained from male or female patients. Reversibility experiments of tachykinin NK2 receptor blockade indicated that the inhibition of the agonist-induced contractions in preparations pre-exposed to ibodutant, and afterwards subjected to repeated washing cycles remained almost constant showing no sign of recovery during the 3h observation period. Overall, the present study indicates ibodutant as a potent tachykinin NK2 receptor antagonist in the human colon tissue, also endowed with a persistent duration of action.

OC125, M11 and OV197 epitopes are not uniformly distributed in the tandem-repeat region of CA125 and require the entire SEA domain.

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments in E. coli and Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

Antagonist profile of ibodutant at the tachykinin NK(2) receptor in guinea pig isolated bronchi.

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK(2) receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK(2) receptor agonist [ßAla8]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300 nM) induced a concentration-dependent rightward shift of the [ßAla8]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pK(B) value was 8.31 ± 0.05. Ibodutant (0.3-100 nM) produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100 nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95 ± 4% inhibition), the calculated IC(50) value was 2.98 nM (95% c.l. 1.73-5.16 nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100 nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK(2) receptor antagonist in guinea pig airways.

Set-up of a new series of HDAC inhibitors: the 5,11-dihydrodibenzo[b,e]azepin-6-ones as privileged structures.

We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC) inhibitors. A series of 5,11-dihydrodibenzo[b,e]azepine-6-ones alkylated on the amide nitrogen with an alkyl chain bearing an hydroxamic acids moiety at the end, has been designed (based upon the general motif for HDAC inhibitors), synthesized and tested. This allowed us to identify a new series of submicromolar HDAC inhibitors, which showed antiproliferative activity on HCT-116 colon carcinoma cells.

SAHA/Vorinostat induces the expression of the CD137 receptor/ligand system and enhances apoptosis mediated by soluble CD137 receptor in a human breast cancer cell line.

HDAC inhibitors (HDACis) represent a class of anticancer agents including suberoylanilide hydroxamic acid (SAHA, Vorinostat), which has shown a strong antitumor effect, both in vitro and in vivo. Induction of apoptotic genes is an important pathway of SAHA cytotoxic mechanism of action and it has been largely described that SAHA induces sensitization of cell death receptor-resistant breast cancer cells to apoptosis. In this study, we investigated the activation of some apoptotic genes which could be responsible for the in vivo antitumor potency of SAHA in a model of human breast cancer. We found that the apoptotic gene pattern induced by SAHA in the MDA-MB-231 cell line involves the upregulation of some molecules belonging to the TNF superfamily. In particular, we demonstrated that the upregulation of the CD137 receptor/ligand system correlates with a synergistic cytotoxic effect when MDA-MB-231 cells are treated with the combination of SAHA and soluble CD137 receptor. To our knowledge, this is the first study to indicate that this member of the TNF superfamily, CD137, is modulated by SAHA treatment in breast cancer cells, suggesting that the combination of SAHA with this TNF-related receptor could be a new therapeutic approach for the treatment of tumors.

Design and synthesis of novel sulfonamide-containing bradykinin hB(2) receptor antagonists. Synthesis and structure-relationships of α,α-tetrahydropyranylglycine.

A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R'). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.

Fasitibant chloride, a kinin B2 receptor antagonist, and dexamethasone interact to inhibit carrageenan-induced inflammatory arthritis in rats.

BACKGROUND AND PURPOSE: Bradykinin, through the kinin B2 receptor, is involved in inflammatory processes related to arthropathies. B2 receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated. EXPERIMENTAL APPROACH: Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1ß, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h. KEY RESULTS: The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg9-[Leu8]-bradykinin (B2 receptor, leukotriene, catecholamine and B1 receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS: Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B2 receptor, or through the indirect effects mediated by release of eicosanoids and cytokines.

hNK2 receptor antagonists. The use of intramolecular hydrogen bonding to increase solubility and membrane permeability.

Starting from in-house capped tripeptide libraries, we have developed two series of compounds as potent antagonists of the hNK(2) receptor with a reduced peptide character. These two series maintained a crucial amide bond, which could not be methylated or substituted with classical isostere without a dramatic loss in binding affinity, very likely due conformational changes. We report here the planning, synthesis and evaluation of molecules belonging to the selected chemical series, which contain a strategically placed hydrogen bond acceptor. The aim of the work was to improve membrane permeability via the formation of an intramolecular hydrogen bonding, and at the same time to maintain the structural characteristics geometry and polarity of the amide linkage so as to retain a relevant binding affinity for the biological target.

Maintenance therapy in ovarian cancer: Molecular basis and therapeutic approach.

Ovarian cancer has the highest mortality rate among gynaecological tumours despite the fact that the majority of patients with advanced disease achieve complete remission after first-line surgery and chemotherapy. Unfortunately, disease recurrence occurs in the majority of patients and second-line treatments are not curative. Clearly, the persistence of dormant and drug-resistant cells after front-line treatments results in the inability to cure the disease. The identification of cancer-initiating cells or cancer stem cells as key players in the development of recurrence has opened up a novel field of research aimed at identifying additional innovative therapeutic approaches. Strategies of maintenance therapy to extend the survival of patients have been studied, but to date no overall survival benefit has been detected. Currently, numerous clinical trials have just been completed or are ongoing involving patients achieving a complete clinical response after first-line chemotherapy in order to evaluate the efficacy of different therapeutic approaches in terms of disease-free survival and overall survival. At the 2010 ASCO meeting, the first positive results of a phase III clinical trial in this setting were presented: bevacizumab (15 mg/kg i.v. every 21 days) added to first-line chemotherapy and continued for an additional 15 cycles was found to prolong progression-free survival of 3.8 months in comparison to 6 cycles of chemotherapy alone or only 6 cycles of chemotherapy plus bevacizumab. In addition, positive results were announced for a second phase III trial testing bevacizumab in the same setting, but at half dose. The final assessment of the overall clinical benefit and the approval of bevacizumab in maintenance therapy by regulatory agencies is expected to be positive, as are the final results of abagovomab phase III trial MIMOSA, another antibody-based therapy tested as a maintenance treatment for advanced ovarian cancer patients. Encouraging preliminary results confirming the safety profile and the immunogenic activity of abagovomab were presented at the last ASCO meeting. The final results are expected to be released in the first half of 2011.

Differences between zofenopril and ramipril, two ACE inhibitors, on cough induced by citric acid in guinea pigs: role of bradykinin and PGE2.

Dry and persistent cough is one of the commonest side effects experienced by patients treated with angiotensin-converting enzyme (ACE) inhibitors for the therapy of hypertension and congestive heart failure. The present study investigated the effect of zofenopril and ramipril on cough induced by citric acid in guinea pig and the involvement of bradykinin (BK) and prostaglandin E2 (PGE2) in mediating the responses of these drugs. Zofenopril (10 mg/kg) or ramipril (3-10 mg/kg), which is threefold more potent than zofenopril, on a mg basis, in lowering blood pressure, was orally administered daily in drinking water for 2 weeks. At the end of this period, aerosol of citric acid solution (0.1 M) was performed and the number of cough counted for 10 min. The role of the kinin B(2) receptor was also investigated. BK and PGE2 levels in the bronchoalveolar lavage (BAL) fluid were measured after repeated oral treatment with zofenopril or ramipril (10 mg/kg). Ramipril (3-10 mg/kg) increased citric acid-induced cough by 40% and 60%, respectively, as compared to the vehicle control group (15.0 ± 1.8), while zofenopril (10 mg/kg) was without effect. The enhancement of citric acid-induced cough caused by ramipril (10 mg/kg) was reduced by the kinin B(2) receptor antagonist MEN16132 (0.25 mg/kg ip). BK and PGE2 levels in the BAL fluid were increased, in comparison to the control group, after ramipril treatment, while they were unchanged after zofenopril administration. Zofenopril, contrary to ramipril, did not affect either citric acid-induced cough in the guinea pigs or BK and PGE2 production in the airways.

Induction of a less aggressive phenotype in human colon carcinoma HCT116 cells by chronic exposure to HDAC inhibitor SAHA.

Histone deacetylase inhibitors (HDACis) are anticancer molecules that epigenetically modulate cell functions. Chronic exposure of HCT116 colon cancer cells to SAHA has been investigated for a better understanding of resistance mechanisms but, surprisingly, a less aggressive tumor phenotype both in vitro and in vivo was obtained after exposure to increasing concentrations of SAHA. Indeed, HCT116/SAHA cells when injected into nude mice showed a reduced engraftment and growth with respect to HCT116 cells. This difference was not observed inoculating the cells into NOD/SCID mice that, differently from nude mice, lack NK activity, thus suggesting the involvement of the native immune response in impairment of HCT116/SAHA cell growth. In agreement with this result, a growing induction of NKG2D ligand expression, MICA and MICB, that are molecular mediators of NK cell killing, was confirmed in HCT116/SAHA chronically exposed to SAHA. A reduced clonogenic efficiency was also observed in HCT116/SAHA with respect to HCT116 cells. Interestingly, even after chronic exposure to SAHA, HCT116/SAHA cells developed only a moderate resistance to SAHA both in vitro and in vivo and they acquired a collateral sensitivity to anthracyclines. These results are of note and probably rely on the fact that, having simultaneously many different targets, HDACis would require many different mutations to display high resistance index. Moreover, to understand the molecular basis of HCT116/SAHA cell phenotype a gene expression profile of cancer genes was evaluated in HCT116 incubated with SAHA for 24 h and in HCT116/SAHA cells to identify selectively regulated genes.

Radioligand binding characterization of the bradykinin B(2) receptor in the rabbit and pig ileal smooth muscle.

Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and

Antiproliferative and differentiating activities of a novel series of histone deacetylase inhibitors.

Histone deacetylases are promising molecular targets for the development of antitumor agents. A novel series of histone deacetylase inhibitors of the hydroxamic acid type were synthesized for structure-activity studies. Thirteen tricyclic dibenzo-diazepine, -oxazepine, and -thiazepine analogues were studied and shown to induce variable degrees of histone H3/H4 and tubulin acetylation in a cellular model of myeloid leukemia sensitive to all-trans retinoic acid (ATRA). Multiparametric correlations between acetylation of the three substrates, tumor cell growth inhibition, and ATRA-dependent cytodifferentiation were performed, providing information on the chemical functionalities governing these activities. For two analogues, antitumor activity in the animal was demonstrated.