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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]

Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa-dependent IL-8 transcription in bronchial epithelial cell lines, with an IC50 of 10 μM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411-1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4′-trimethylangelicin (TMA), which inhibits P. aeruginosa-dependent IL-8 transcription at nanomolar concentration in IB3-1, CuFi-1, CFBE41o-, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa-dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function. [ABSTRACT FROM AUTHOR]

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]

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of gamma-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the gamma-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and beta-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human gamma-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including beta-thalassemia and sickle cell anemia. [ABSTRACT FROM AUTHOR]

In this review we summarize the structure and biological effects of linear and angular psoralens. These compounds exhibit interesting biological effects on the cell cycle, apoptosis and differentiation. These molecules should be considered promising drugs in the therapy of several diseases, including psoriasis, mycosis fungoides and cancer. Also, preclinical data demonstrate a possible use of these molecules for the treatment of β-thalassemia and other hematological disorders. [ABSTRACT FROM AUTHOR]

The objective of this review is to present examples of lead compounds identified from biological material (fungi, plant extracts and agro-industry material) and of possible interest in the field of a pharmacological approach to the therapy of b-thalassemia using molecules able to stimulate production of fetal hemoglobin (HbF) in adults. Concerning the employment of HbF inducers as potential drugs for pharmacological treatment of b-thalassemia, the following conclusions can be reached: (i) this therapeutic approach is reasonable, on the basis of the clinical parameters exhibited by hereditary persistence of fetal hemoglobin patients, (ii) clinical trials (even if still limited) employing HbF inducers were effective in ameliorating the symptoms of b-thalassemia patients, (iii) good correlation of in vivo and in vitro results of HbF synthesis and g-globin mRNA accumulation indicates that in vitro testing might be predictive of in vivo responses and (iv) combined use of different inducers might be useful to maximize HbF, both in vitro and in vivo. In this review, we present three examples of HbF inducers from the natural world: (i) angelicin and linear psoralens, contained in plant extracts from Angelica arcangelica and Aegle marmelos, (ii) resveratrol, a polyphenol found in grapes and several plant extracts and (iii) rapamycin, isolated from Streptomyces hygroscopicus. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to determine whether angelicin is able to increase the expression of γ -globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and γ -globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of γ -globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically-mediated regulation of the expression of human γ -globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including β -thalassemia and sickle cell anemia. [ABSTRACT FROM AUTHOR]

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: Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in β-thalassaemia and sickle cell anaemia. [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]