PI3K pan-inhibition impairs more efficiently proliferation and survival of T-cell acute lymphoblastic leukemia cell lines when compared to isoform-selective PI3K inhibitors.

Class I phosphatidylinositol 3-kinases (PI3Ks) are frequently activated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to the loss of PTEN function. Therefore, targeting PI3Ks is a promising innovative approach for T-ALL treatment, however at present no definitive evidence indicated which is the better therapeutic strategy between pan or selective isoform inhibition, as all the four catalytic subunits might participate in leukemogenesis. Here, we demonstrated that in both PTEN deleted and PTEN non deleted T-ALL cell lines, PI3K pan-inhibition exerted the highest cytotoxic effects when compared to both selective isoform inhibition or dual p110γ/δ inhibition. Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome. PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms. PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis. We further demonstrated that in some T-ALL cell lines, autophagy could exert a protective role against PI3K inhibition. Our findings strongly support clinical application of class I PI3K pan-inhibitors in T-ALL treatment, with the possible exception of ETP-ALL cases.

Therapeutic targeting of Polo-like kinase-1 and Aurora kinases in T-cell acute lymphoblastic leukemia.

Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and dysregulated, thus contributing to uncontrolled cell proliferation and growth. T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy arising in the thymus from T-cell progenitors. Primary chemoresistant and relapsed T-ALL patients have yet a poor outcome, therefore novel therapies, targeting signaling pathways important for leukemic cell proliferation, are required. Here, we demonstrate the potential therapeutic effects of BI6727, MK-5108, and GSK1070916, three selective inhibitors of PLK1, AK-A, and AK-B/C, respectively, in a panel of T-ALL cell lines and primary cells from T-ALL patients. The drugs were both cytostatic and cytotoxic to T-ALL cells by inducing G2/M-phase arrest and apoptosis. The drugs retained part of their pro-apoptotic activity in the presence of MS-5 bone marrow stromal cells. Moreover, we document for the first time that BI6727 perturbed both the PI3K/Akt/mTORC2 and the MEK/ERK/mTORC1 signaling pathways, and that a combination of BI6727 with specific inhibitors of the aforementioned pathways (MK-2206, CCI-779) displayed significantly synergistic cytotoxic effects. Taken together, our findings indicate that PLK1 and AK inhibitors display the potential for being employed in innovative therapeutic strategies for improving T-ALL patient outcome.

Targeting phosphatidylinositol 3-kinase signaling in acute myelogenous leukemia.

INTRODUCTION: Despite continuous advances in our knowledge of the biology of acute myelogenous leukemia (AML), the prognosis of AML patients treated with standard chemotherapy is still poor, especially in the elderly. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. Constitutively active phosphatidylinositol 3-kinase (PI3K) signaling characterizes many types of tumors, including AML, where it negatively influences response to therapeutic treatments. AREAS COVERED: The literature data showed that small inhibitor molecules targeting PI3K signaling induced cell cycle arrest, apoptosis and decreased drug-resistance in AML cells. PI3K inhibitors were also capable of targeting leukemic initiating cells (LICs), the most relevant target for leukemia eradication, whereas they tended to spare healthy hematopoietic stem cells. EXPERT OPINION: Data emerging from pre-clinical settings suggest that the PI3K pathway is critically involved in regulating proliferation, survival and drug-resistance of AML cells. Therefore, we propose that novel drugs targeting this signaling pathway may offer a novel and less toxic treatment option for AML patients, most likely in combination with a lower dosage of traditional chemotherapeutic agents or other innovative therapeutic agents.

Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells.

BACKGROUND: HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7). RESULTS: We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. CONCLUSIONS: The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.

Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist.

PURPOSE: Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action. METHODS: With in vitro studies apoptosis was evaluated in human eosinophils by flow cytometry and western blot of caspase-3 fragments. Eosinophil migration toward platelet-activating factor was evaluated by transwell assays. Interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and the chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation normal T cell expressed, and presumably secreted (RANTES) were measured using a high-throughput multiplex luminex technology. Annexin I and the chemochine receptor C-X-C chemokine receptor 4 (CXCR4) were detected by flow cytometry. With in vivo studies, allergic conjunctivitis was induced in guinea pigs sensitized to ovalbumin by an ocular allergen challenge and evaluated by a clinical score. Conjunctival eosinophils were determined by microscopy or eosinophil peroxidase assay. RESULTS: In cultured human eosinophils, mapracorat showed the same potency as dexamethasone but displayed higher efficacy in increasing spontaneous apoptosis and in counteracting cytokine-sustained eosinophil survival. These effects were prevented by the glucocorticoid receptor antagonist mifepristone. Mapracorat inhibited eosinophil migration and IL-8 release from eosinophils or the release of IL-6, IL-8, CCL5/RANTES, and TNF-α from a human mast cell line with equal potency as dexamethasone, whereas it was clearly less potent than this glucocorticoid in inducing annexin I and CXCR4 expression on the human eosinophil surface; this was taken as a possible sign of glucocorticoid-dependent transactivation. In the guinea pig, mapracorat or dexamethasone eye drops induced an analogous reduction in clinical symptoms of allergic conjunctivitis and conjunctival eosinophil accumulation. CONCLUSIONS: Mapracorat appears to be a promising candidate for the topical treatment of allergic eye disorders. It maintains an anti-allergic profile similar to that of dexamethasone but seems to have fewer transactivation effects in comparison to this classical glucocorticoid. Some of its cellular targets may contribute to eosinophil apoptosis and/or to preventing their recruitment and activation and to inhibiting the release of cytokines and chemokines.

Effect of ΔFosB overexpression on opioid and cannabinoid receptor-mediated signaling in the nucleus accumbens.

The stable transcription factor ΔFosB is induced in the nucleus accumbens (NAc) by chronic exposure to several drugs of abuse, and transgenic expression of ΔFosB in the striatum enhances the rewarding properties of morphine and cocaine. However, the mechanistic basis for these observations is incompletely understood. We used a bitransgenic mouse model with inducible expression of ΔFosB in dopamine D(1) receptor/dynorphin-containing striatal neurons to determine the effect of ΔFosB expression on opioid and cannabinoid receptor signaling in the NAc. Results showed that mu opioid-mediated G-protein activity and inhibition of adenylyl cyclase were enhanced in the NAc of mice that expressed ΔFosB. Similarly, kappa opioid inhibition of adenylyl cyclase was enhanced in the ΔFosB expressing mice. In contrast, cannabinoid receptor-mediated signaling did not differ between mice overexpressing ΔFosB and control mice. These findings suggest that opioid and cannabinoid receptor signaling are differentially modulated by expression of ΔFosB, and indicate that ΔFosB expression might produce some of its effects via enhanced mu and kappa opioid receptor signaling in the NAc.

beta-Arrestin 2-mediated heterologous desensitization of IGF-IR by prolonged exposure of SH-SY5Y neuroblastoma cells to a mu opioid agonist.

Prolonged (12h) exposure of SH-SY5Y neuroblastoma cells to the mu-opioid receptor (MOPr) agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) causes homologous desensitization as well as heterologous desensitization of the extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation induced by insulin-like growth factor (IGF)-I. Brief (15 min) but not prolonged exposure to DAMGO transregulates the insulin-like growth factor-I (IGF-I) receptor, as evidenced by its phosphorylation in the absence of IGF-I. Silencing of beta-arrestin 2 uncouples the crosstalk between the two receptors, thus maintaining IGF-I-mediated receptor phosphorylation and ERK 1/2 activation even after prolonged DAMGO exposure. Furthermore, MOPr-induced activation of IGF-I receptor requires the tyrosine kinase c-Src.

Synthesis and conformational analysis of cyclotetrapeptide mimetic beta-turn templates and validation as 3D scaffolds.

The conformations of all stereoisomers of PMRI cyclotetrapeptide mimetics 1-8 are essentially determined by the predisposition of the diamine to stabilize beta-turns. The peptide mimetics can be regarded as 3D scaffolds for designing molecules with a predictable display of the pharmacophores. We used the models for testing novel RGD analogues as alpha(v)beta(3)-integrin receptor antagonists.

New coumarin-based anti-inflammatory drug: putative antagonist of the integrins alphaLbeta2 and alphaMbeta2.

This study was conducted to investigate putative antagonism of integrin receptors alphaMbeta2 and alphaLbeta2 by a novel coumarin derivative (BOL-303225-A), its efficacy in-vivo after retinal ischaemia-reperfusion injury, and its bioavailability in rat plasma. A cellular adhesion assay in Jurkat and U937 cells, and a flow cytometry assay with an antibody against the beta2 subunit were conducted. BOL-303225-A bioavailability in rat plasma and the retinal levels of myeloperoxidase (MPO) after ischaemia-reperfusion injury were evaluated after oral administration (10 mg kg(-1)). In-vitro cell viability assays revealed no cytotoxicity for BOL-303225-A over a wide dose range, and IC50 values of 32.3 +/- 1.5 muM and 84.95 +/- 2.3 muM were found for Jurkat and U937 cells, respectively. The drug showed specific binding to the alphaMbeta2 and alphaLbeta2 integrin receptors expressed by U937 and Jurkat cells, respectively, producing a fluorescence shift towards lower values in a concentration-dependent manner. The pharmacokinetic profile of BOL-303225-A exhibited rapid absorption following oral administration in the rat. A significant reduction of retinal MPO levels was observed in drug-treated rats. This study demonstrated that BOL-303225-A acts as an antagonist of the integrin alphaLbeta2 and alphaMbeta2 receptors, suggesting that this drug could be used for ocular diseases such as diabetic retinopathy.

Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine.

Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.

Synthesis and biological evaluation of non-peptide alpha(v)beta(3)/alpha(5)beta(1) integrin dual antagonists containing 5,6-dihydropyridin-2-one scaffolds.

Small constrained non-peptidic molecules consisting of a polyfunctionalized rigid core, carrying appendages corresponding to arginine and aspartic acid side chains, have been recently reported to be promising for drug development. In this work, the 5,6-dihydropyridin-2-one was envisaged as a scaffold to turn into potential integrin ligands, introducing a carboxylic acid and a basic appendage. The synthesis and the antiadhesion activity of a small library of peptidomimetics capable to recognize alpha(v)beta(3) and alpha(5)beta(1) integrins has been herein reported.

Inhibition of cancer cell adhesion by heterochiral Pro-containing RGD mimetics.

In this paper, we describe the synthesis of a selected library of heterochiral d-Pro-containing RGD-peptidomimetics (RpD) and we investigate the biological activity as inhibitors of fibronectin adhesion to SK-MEL-24 tumor cells. In particular, peptides 4 and 8 showed an IC(50) in the 10(-8)M range. Despite the linear structure, the peptides tend to adopt a folded conformation in a polar environment.