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MicroRNAs (miRNAs) are small non-coding RNA molecules capable of silencing mRNA targets. miRNA dysregulation has been linked to cancer development, cardiovascular and neurological diseases, lipid metabolism, and impaired immunity. Therefore, miRNAs are gaining interest as putative novel disease biomarkers and therapeutic targets. Recent studies have shown that purinergic surface receptors activated by extracellular nucleotides (ATP, ADP, UTP, UDP), and by nucleosides such as adenosine (ADO), are subject to miRNA regulation. This opens a new and previously unrecognized opportunity to modulate the purinergic network with the aim of avoiding abnormal activation of specific receptor subtypes. miRNA technology will hopefully contribute strategies to prevent purinergic-mediated tissue damage in conditions of neurodegeneration, atherosclerosis, transplantation, and even neoplasia. [ABSTRACT FROM AUTHOR]

Several investigations have demonstrated a mild clinical status in patients with β-globin disorders and congenital high persistence of foetal haemoglobin. This can be mimicked by a pharmacological increase of foetal γ-globin genes expression and foetal haemoglobin production. Our goal was to apply a multistep assay including few screening methods (benzidine staining, RT-PCR and HPLC analyses) and erythroid cellular model systems (the K562 cell line and erythroid precursors collected from peripheral blood) to select erythroid differentiation agents with foetal haemoglobin inducing potential. With this methodology, we have identified a butyric acid derivative, namely the 4174 cyclopropanecarboxylic acid compound, able to induce erythroid differentiation without antiproliferative effect in K562 cells and increase of γ-globin gene expression in erythroid precursor cells. The results are relevant for pharmacological treatments of haemoglobinopathies, including β-thalassaemia and sickle cell anaemia. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we compared some bile acid derivatives and their platinum(II) complexes with respect to their ability to induce erythroid differentiation of human leukemic K562 cells. The complexes analyzed were cis-[(3-dehydrocholanoyliden-l-tartrate)-diammineplatinum(II)] (compound 1) and cis-[di(dehydrocholanoate)-bis(triphenylphosphine)-platinum(II)] (compound 3), together with their free ligands, respectively, 3-dehydrocholanoyliden-l-tartaric acid (compound 2) and dehydrocholanoic acid (4), and their parent compounds, respectively, cisplatin and cis-[dichloride-bis(triphenylphosphine)-platinum(II)] (5). We found that compound 1 stimulates erythroid differentiation of K562 cells and an increase of fetal hemoglobin (HbF) production in erythroid precursor cells isolated from peripheral blood of human subjects. This increase is similar to that obtained by hydroxyurea, a potent inducer of HbF production both in vitro and in vivo. Another important conclusion of this study is related to the evaluation of the effects of compound 1 on production of γ-globin mRNA in human erythroid precursors grown in the two-stage liquid culture system. We demonstrated that compound 1 induces preferential accumulation of γ-globin mRNA. The results presented in this manuscript could have practical impact, since it is well known that an increase in HbF production could ameliorate the clinical status of patients with β-thalassemia and sickle cell anemia. [Copyright &y& Elsevier]

Synthesis, DNA binding properties and biological activity of a series of bis-benzoheterocycle derivatives 5–11, structurally related to the natural dipyrrole antitumor agent netropsin, and tethered to a benzoyl nitrogen mustard (BAM) as alkylating moiety is reported and structure–activity relationships determined. These compounds 5–11 have been evaluated for sequence selective alkylating properties and cytotoxicity against murine L1210 and human K562 leukaemia cells. Using as target sequence a portion of the long terminal repeat of the type-1 human immunodeficiency virus, we found that these compounds induce similar patterns of DNA fragmentation. In addition, the results obtained indicate that all synthesized compounds retain a good antiproliferative activity in the submicromolar range, and generally are more active against L1210 than K562 cells. With respect to both these cell lines, compounds 6, 7, 10 and 11 showed the greatest potency, ranging from 0.3 to 1 μM, while compounds 8 and 9 exhibit the lowest activity (IC50=2–12 μM). Among compounds 5–11, the derivative 11 was found to be the most potent member of this class and it is 5 and 10-fold less active than the bis-pyrrole counterpart 2 against K562 and L1210 cell lines, respectively. For compound 11, the substitution of the C-terminus benzofurane with N-methylindole and indole (to give the compounds 5 and 6, respectively) led to a decrease in cytotoxicity, which is more evident against the K562 cell line. Finally, differences were found among compounds 5–11 in induction of K562 differentiation. Some of them (compounds 7, 8 and 9) are potent inducers of erythroid differentiation of K562 cells, and could be proposed for differentiation anti-cancer therapy. [Copyright &y& Elsevier]

Abstract: Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose), a gallotannin identified in several plants, including Phyllanthus urinaria, has been shown to exhibit versatile medicinal activities. As far as possible anti-inflammatory effects of corilagin, only few reports are available, and the potential use of corilagin as possible therapeutic molecule for cystic fibrosis has not been evaluated. In the present paper we report experiments aimed at determining the activity of corilagin on nuclear factor kappaB (NF-kappaB) binding to DNA target and on the expression of the major pro-inflammatory gene involved in cystic fibrosis, interleukin-8 (IL-8). Both IL-8 mRNA content and IL-8 protein secretion were analyzed in cystic fibrosis bronchial IB3-1 cells stimulated by tumor necrosis factor-alpha (TNF-alpha), one of the most potent pro-inflammatory agents. The data obtained demonstrate that corilagin binds to NF-kappaB, inhibits NF-kappaB/DNA interactions and affects IL-8 gene expression in TNF-alpha treated IB3-1 cells. In addition, corilagin inhibits TNF-alpha induced secretion of MCP-1 and RANTES, exhibiting low or no effect on the release of G-CSF, IL-6 and VEGF. Therefore, corilagin might be of interest for experimental anti-inflammatory therapy of cystic fibrosis. [Copyright &y& Elsevier]

Abstract: The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo. [Copyright &y& Elsevier]

Abstract: Nuclear Factor kappaB (NF-κB) plays a very important role in the control of gene expression and is deeply involved in several human pathologies. Accordingly, molecules targeting NF-κB dependent biological functions are considered of great interest. Virtual screening of furocoumarin libraries against NF-κB p50 allowed to rank compounds in respect to their expected ability to bind NF-κB and the identified compound might be considered for the development of analogs to be tested for biological activity on inhibition of NF-κB/DNA complex formation. The data reported in the present paper suggest that, following this approach, the best ranked compounds identified by virtual screening (a) strongly bind in silico to NF-κB and (b) efficiently inhibit the molecular interactions between 32P-labeled NF-κB double stranded DNA and p50 or p50/p65 complex. These data allowed to develop a novel lead of great interest for inhibiting NF-κB dependent biological functions. This novel molecule (compound 2), bearing a methyl group in the 9 position of the psoralen nucleus, exhibits high efficiency in inhibiting NF-κB/DNA interactions. In addition, we found that compound 2 is a potent inhibitor of IL-8 gene expression in TNF-α treated IB3-1 cystic fibrosis cells. Taken together, our data indicate that compound 2 might find an important place in the set of molecules of interest for the development of pharmaceutical strategies against the inflammatory phenotype of cystic fibrosis. [Copyright &y& Elsevier]

Abstract: In the present study, a structured-based virtual screening (VS) of differently substituted furocoumarins and analogues has been carried out against nuclear factor kappa B (NF-κB), with the objective of selecting molecules able to inhibit the binding of this transcription factor to the DNA. The focus library was developed starting from chemical structures obtained from the literature, as well as retrieving compounds from available commercial databases. A two dimensional substructure searching method based on four different chemical scaffolds was used for this purpose. Among the 10 highest-scored ligands selected from the docking studies, five commercially available molecules were investigated in biological assays. Four furocoumarin derivatives showed IC50 values in the range of 40–100μM in inhibiting NF-κB/DNA interactions studied by electrophoretic mobility shift assay (EMSA). Three compounds significantly inhibited NF-κB dependent biological functions (expression of IL-8) in cellular analysis based on Pseudomonas aeruginosa infection of cystic fibrosis IB3-1 cells. These findings validated the virtual screening approach here presented and reinforce the successful results of our previously computational studies aimed at the identification of molecules targeting NF-κB. The discovered novel compounds could be of relevance to identify more potent inhibitors of NF-κB dependent biological functions beneficial to control lung inflammation occurring in patients affected by cystic fibrosis. [ABSTRACT FROM AUTHOR]

Abstract: Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PCBs are characterized by low production costs and large-scale development, both essential elements in the frame of disposable applications. LOAC platforms have been employed not only for diagnostic and/or analytical purposes, but also for identification and isolation of eukaryotic cells, including cancer and stem cells. Accordingly, the compatibility of the employed materials with the biological system under analysis is critical for the development of LOAC devices to be proposed for efficient and safe cell isolation. In this study, we analyzed the in-vitro compatibility of a large set of materials and surface treatments used for LOAC development and evaluation with quasi-standard PCB processes. Biocompatibility was analyzed on hippocampal primary cells (a model of attached cell cultures), in comparison with the reference K562 cell line (a model of cells growing in suspension). We demonstrate here that some of the materials under study alter survival, organization, morphology and adhesion capacity of hippocampal cells, and inhibit growth and differentiation of K562 cells. Nonetheless, a subset of the materials tested did not negatively affect these functions, thus demonstrating that PCB technology, with some limitations, is suitable for the realization of LOAC devices well compatible at least with these preparations. [Copyright &y& Elsevier]

Abstract: Persistent recruitment of neutrophils in the bronchi of cystic fibrosis patients contributes to airway tissue damage, suggesting the importance of intervening on the expression of the neutrophil chemokine IL-8. Extracts from plants have been investigated to select components able to reduce IL-8 expression in bronchial epithelial cells challenged with Pseudomonas aeruginosa. Extracts and purified components have been added to cells 24h before pro-inflammatory challenge with P. aeruginosa and IL-8 transcription was quantified in the IB3-1 CF cells in vitro. P. aeruginosa-dependent IL-8 mRNA induction was increased by Argemone mexicana and Vernonia anthelmintica whereas no significant modification of transcription was observed with Aphanamixis polystachya, Lagerstroemia speciosa and Hemidesmus indicus. Finally, inhibition of IL-8 was observed with Polyalthia longifolia (IC50 =200μg/ml) and Aegle marmelos (IC50 =20μg/ml). Compounds from A. marmelos were isolated and identified by GC–MS. No significant effect was observed with butyl-p-tolyl sulphate, whereas the inhibition obtained with 6-methyl-4-chromanone concentration was accompanied by an anti-proliferative effect. On the contrary, 5-methoxypsoralen resulted in IL-8 inhibition at 10μM concentration, without effects on cell proliferation. In synthesis, 5-methoxypsoralen can be taken into consideration to investigate mechanisms of neutrophil chemotactic signalling and for its potential application in modulating the excessive CF lung inflammation. [Copyright &y& Elsevier]

Abstract: A series of carbamides derived from 1,2:5,6-di-O-isopropylidene-d-gluco- (1) and d-allofuranose (3) as well as their 5,6-O-deprotected analogues (2 and 4) and methyl 3,4-O-isopropylidene-α- and β-d-galactopyranosides (5 and 6) have been prepared in order to evaluate their ability to induce erythroid differentiation of human erythroleukemic K562 cells. Twenty out of 51 carbamides tested exhibit an appreciable activity as inducers of erythroid differentiation and have been fully characterized and described. [Copyright &y& Elsevier]

Abstract: The most relevant cause of morbidity and mortality in cystic fibrosis (CF) patients is the lung pathology characterized by chronic infection and inflammation sustained mainly by Pseudomonas aeruginosa (P. aeruginosa). Innovative pharmacological approaches to control the excessive inflammatory process in the lung of CF patients are thought to be beneficial to reduce the extensive airway tissue damage. Medicinal plants from the so-called traditional Asian medicine are attracting a growing interest because of their potential efficacy and safety. Due to the presence of different active compounds in each plant extract, understanding the effect of each component is important to pursue selective and reproducible applications. Extracts from Emblica officinalis (EO) were tested in IB3-1 CF bronchial epithelial cells exposed to the P. aeruginosa laboratory strain PAO1. EO strongly inhibited the PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-α, GRO-γ, of the adhesion molecule ICAM-1 and of the pro-inflammatory cytokine IL-6. Pyrogallol, one of the compounds extracted from EO, inhibited the P. aeruginosa-dependent expression of these pro-inflammatory genes similarly to the whole EO extract, whereas a second compound purified from EO, namely 5-hydroxy-isoquinoline, had no effect. These results identify Pyrogallol as an active compound responsible for the anti-inflammatory effect of EO and suggest to extend the investigation in pre-clinical studies in airway animal models in vivo, to test the efficacy and safety of this molecule in CF chronic lung inflammatory disease. [Copyright &y& Elsevier]

Abstract: Psoralens, also known as furocoumarins, are a class of photosensitizers largely used in the therapy of various skin diseases. In this study we have evaluated the combined effects of UVA irradiation and furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemia K562 cells and (b) globin gene expression in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a series of linear and angular furocoumarins derivatives, we employed the human leukemia K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. Quantitative real-time reverse transcription polymerase-chain assay (Q-RT-PCR) was employed for quantification of the accumulation of globin mRNAs. The results obtained demonstrate that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we have selected two derivatives, 5-methoxypsoralen (5-MOP) and trimethylangelicin (TMA), for which we have investigated their mechanism of action. The cell cycle analysis showed that these derivatives induce, after irradiation, a cell cycle arrest in the G2/M phase, followed by apoptosis. Mitochondrial depolarisation and caspases activation seem to be involved in the mechanism of cell death. In erythroid precursor cells, psoralens in combination with UVA irradiation, stimulate at very low concentrations a preferential increase of γ-globin mRNA. Altogether, these data suggest that psoralen derivatives warrant further evaluation as potential therapeutic drugs in β-thalassemia and sickle cell anemia. [Copyright &y& Elsevier]

Abstract: The synthesis and biological evaluation of some new glycose esters bearing the 1,2-O-isopropylidene-d-hexofuranose functionality and belonging to the 3-O-acyl-d-allose and 6-O-acyl-d-glucose series are reported. When the results concerning cell growth inhibition are compared, it appears that the 6-O-acyl-d-glucose derivatives are more active than the 3-O-acyl-d-allose compounds. Within both 6-O-acyl-d-glucose and 3-O-acyl-d-allose derivatives, butyric esters displayed the highest inhibitory effects. Inhibition of cell growth is not associated with high induction levels of erythroid differentiation, despite the fact that pivaloates induce erythroid differentiation to an extent similar to that exhibited by previously reported molecules [Bioorg. Med. Chem. Lett. 1999, 9, 3153–3158]. [Copyright &y& Elsevier]

The binding properties of a set of four hybrids, prepared combining from one to four polypyrrole minor groove binders and pyrrolo [2,1-c][1,4]benzodiazepine (PBD), have been studied using as target molecule the HIV-1 TAR-RNA. We found that these hybrids bind to TAR-RNA and inhibit TAR/protein(s) interactions. The anti-proliferative activity of the hybrids has been tested in vitro on HL3T1 cells and compared to the anti-proliferative effects of the natural product distamycin A and PBD. The effects on HIV-1 LTR directed transcription were studied using the chloramphenicol-acetyltransferase gene reporter system, and structure–activity relationships are discussed. The results obtained demonstrate that the hybrids 22–25 exhibit different TAR-RNA binding activity with respect to both distamycin A and PBD. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22–25 and anti-proliferative effects. It was found that increased length of the polypyrrole backbone leads to an increased in vitro anti-proliferative effect, i.e. the hybrid 25, containing the four pyrroles distamycin analogous, is more active than 22, 23 and 24 against cell proliferation. With respect to inhibition of HIV-1 LTR-driven transcription, it was found that the hybrids 23–25 containing two–four pyrroles are active. Therefore, when anti-proliferative effects are considered together with the inhibitory effects of HIV-1 LTR driven transcription, our results suggest that the hybrid 23 is the more interesting, since it exhibits low anti-proliferative activity and inhibits HIV-1 LTR driven transcription both in vitro and in ex vivo experiments. [Copyright &y& Elsevier]

The design, synthesis and in vitro activities of novel α-bromoacryloyl pyrazole, imidazole and benzoheterocyclic derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure–activity relationships are discussed. In spite of the relevance of these modifications on the distamycin frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of α-bromoacrylic derivatives of distamycin. [Copyright &y& Elsevier]

Determines whether double-stranded molecules based on peptide nucleic acids (PNA) and PNA-DNA-PNA chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family. Ability to act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA; Potential applications in gene therapy.

Peptide nucleic acids (PNAs) have been recently proposed as useful molecules in pharmacogenetic therapy, especially due to the fact that they show a very high stability with respect to DNA and RNA. However, PNAs are not efficient decoy molecules, are characterized by negligible cell internalization and low solubility and are not suitable to be delivered by liposomes. With respect to the biological activity of PNA-based molecules, PDP deserve great consideration, due to the fact that they exhibit high levels of solubility, and are expected to be resistant to proteinases and exonucleases. In this manuscript we determined whether double-stranded molecules based on PNA–DNA chimeras containing NF-κB binding sites, exhibit decoy activity against NF-κB transcription factors. In addition, we determined whether they can be complexed by cationic liposomes. The results obtained demonstrated that hybrids based on PNA–DNA chimeras are powerful decoy molecules against NF-κB p52 transcription factor. In addition, we found that cationic liposomes can be proposed for in vitro delivery to target cells of these decoy molecules. The results presented in this paper are thus of practical importance, since the simplicity and the versatility of the cationic liposome technology have made cationic liposomes useful nonviral gene delivery systems for human gene therapy. [Copyright &y& Elsevier]

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. [ABSTRACT FROM AUTHOR]