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1.  From microRNA functions to microRNA therapeutics: Novel targets and novel drugs in breast cancer research and treatment  
International Journal of Oncology  2013;43(4): 985 - 994 .
MicroRNAs (miRNAs or miRs) are a family of small non-coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences. miRNAs play a crucial role in cancer. In the case of breast tumors, several studies have demonstrated a correlation between: i) the expression profile of oncogenic miRNAs (oncomiRs) or tumor suppressor miRNAs and ii) the tumorigenic potential of triple-negative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] primary breast cancers. Among the miRNAs involved in breast cancer, miR-221 plays a crucial role for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancers; ii) the oncosuppressor p27 Kip1 , a validated miR-221 target, is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network has been suggested, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27 Kip1 downregulation. Interference with this process can be achieved using antisense miRNA (antagomiR) molecules targeting miR-221, inducing the down-regulation of Slug and the upregulation of p27 Kip1 .
doi: 10.3892/ijo.2013.2059
PMCID: PMC3829774  PMID: 23939688
microRNAs ;  breast cancer ;  miR-221 ;  Slug ;  p27 Kip1 ;  peptide nucleic acid ;  microRNA replacement therapy ;  antagomiR
2.  Modulation of the Expression of the Proinflammatory IL-8 Gene in Cystic Fibrosis Cells by Extracts Deriving from Olive Mill Waste Water 
A persistent recruitment of neutrophils in the bronchi of cystic fibrosis (CF) patients contributes to aggravate the airway tissue damage, suggesting the importance of modulating the expression of chemokines, including IL-8 during the management of the CF patients. Polyphenols rich extracts derived from waste water from olive mill, obtained by a molecular imprinting approach, have been investigated in order to discover compounds able to reduce IL-8 expression in human bronchial epithelial cells (IB3-1 cells), derived from a CF patient with a ΔF508/W1282X mutant genotype and stimulated with TNF-alpha. Initially, electrophoretic mobility shift assays (EMSAs) were performed to determine whether the different active principles were able to inhibit the binding between transcription factor (TF) NF-kappaB and DNA consensus sequences. Among different representative active principles present in the extract, three compounds were selected, apigenin, oleuropein, and cyanidin chloride, which displayed remarkable activity in inhibiting NF-kappaB/DNA complexes. Utilizing TNF-alpha-treated IB3-1 cells as experimental model system, we demonstrated that apigenin and cyanidin chloride are able to modulate the expression of the NF-kappaB-regulated IL-8 gene, while oleuropein showed no effect in regulating the expression of the gene IL-8.
doi:10.1155/2013/960603
PMCID: PMC3723063  PMID: 23935691
3.  Induction of erythroid differentiation and increased globin mRNA production with furocoumarins and their photoproducts 
Highlights
► Furocoumarins photoinduce erythroid differentiation in K562 cells with UVA. ► Furocoumarins photoinduce high increase in α-, ζ-, ε-, and γ-globin mRNA sequences. ► Furocoumarin erythrodifferentiation is connected with DNA photodamage. ► 5,5′-Dimethylpsoralen photoproducts present differentiating properties.
Differentiation-therapy is an important approach in the treatment of cancer, as in the case of erythroid induction in chronic myelogenous leukemia. Moreover, an important therapeutic strategy for treating beta-thalassemia and sickle-cell anemia could be the use of drugs able to induce erythroid differentiation and fetal hemoglobin (HbF) accumulation: in fact, the increased production of this type of hemoglobin can reduce the clinical symptoms and the frequency of transfusions. An important class of erythroid differentiating compounds and HbF inducers is composed by DNA-binding chemotherapeutics: however, they are not used in most instances considering their possible devastating side effects. In this contest, we approached the study of erythrodifferentiating properties of furocoumarins. In fact, upon UV-A irradiation, they are able to covalently bind DNA. Thus, the erythrodifferentiation activity of some linear and angular furocoumarins was evaluated in the experimental K562 cellular model system. Quantitative real-time reverse transcription polymerase-chain reaction assay was employed to evaluate the accumulation of different globin mRNAs. The results demonstrated that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we selected the most active compounds and investigated the role of DNA photodamage in their erythroid inducing activity and mechanism of action. Moreover, some cytofluorimetric experiments were carried out to better study cell cycle modifications and the mitochondrial involvement. A further development of the work was carried out studying the erythroid differentiation of photolysis products of these molecules. 5,5′-Dimethylpsoralen photoproducts induced an important increase in γ-globin gene transcription in K562 cells.
doi:10.1016/j.jphotobiol.2013.02.011
PMCID: PMC3625112  PMID: 23518160
Psoralen; Angelicin; Photoproducts; K562 cells; Erythroid differentiation; Fetal hemoglobin
4.  Development of K562 cell clones expressing β-globin mRNA carrying the β039 thalassaemia mutation for the screening of correctors of stop-codon mutations 
Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30 % of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039-thalassaemia. In this context, we started the development of a cellular model of the β039-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing β-thalassaemia.
doi:10.1042/BA20080266
PMCID: PMC3582994  PMID: 19216718
aminoglycoside antibiotics; K562 cell; locus control region; nonsense mutation; thalassaemia
5.  Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients 
American journal of hematology  2009;84(11):720-728.
In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations.
doi:10.1002/ajh.21539
PMCID: PMC3572903  PMID: 19810011
6.  Correlation between Slug transcription factor and miR-221 in MDA-MB-231 breast cancer cells 
BMC Cancer  2012;12:445.
Background
Breast cancer and its metastatic progression is mainly directed by epithelial to mesenchymal transition (EMT), a phenomenon supported by specific transcription factors and miRNAs.
Methods
In order to investigate a possible correlation between Slug transcription factor and miR-221, we performed Slug gene silencing in MDA-MB-231 breast cancer cells and evaluated the expression of genes involved in supporting the breast cancer phenotype, using qRT-PCR and Western blot analysis. Chromatin immunoprecipitation and wound healing assays were employed to determine a functional link between these two molecules.
Results
We showed that Slug silencing significantly decreased the level of miR-221 and vimentin, reactivated Estrogen Receptor α and increased E-cadherin and TRPS1 expression. We demonstrated that miR-221 is a Slug target gene, and identified a specific region of miR-221 promoter that is transcriptionally active and binds the transcription factor Slug “in vivo”. In addition, we showed that in Slug-silenced cells, wich retained residual miR-221 (about 38%), cell migration was strongly inhibited. Cell migration was inhibited, but to a less degree, following complete knockdown of miR-221 expression by transfection with antagomiR-221.
Conclusions
We report for the first time evidence of a correlation between Slug transcription factor and miR-221 in breast cancer cells. These studies suggest that miR-221 expression is, in part, dependent on Slug in breast cancer cells, and that Slug plays a more important role than miR-221 in cell migration and invasion.
doi:10.1186/1471-2407-12-445
PMCID: PMC3534407  PMID: 23031797
Slug; miR-221; Epithelial mesenchymal transition; Breast cancer
7.  Targeting pre-miRNA by Peptide Nucleic Acids 
Artificial DNA, PNA & XNA  2012;3(2):88-96.
PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs.
doi:10.4161/adna.20911
PMCID: PMC3429535  PMID: 22699795
FACS; fluorescence; miR-210; PNA; pre-miR; thiazole orange
8.  Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3–1 cells 
Artificial DNA, PNA & XNA  2012;3(2):97-296.
One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3–1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.
doi:10.4161/adna.21061
PMCID: PMC3429536  PMID: 22772035
NF-kappaB; transcription factor decoy; inflammation; Peptide Nucleic Acids; PNA-DNA chimeras
9.  A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction 
Annals of Hematology  2012;91(8):1201-1213.
Gene therapy might fall short in achieving a complete reversion of the β-thalassemic phenotype due to current limitations in vector design and myeloablative regimen. Following gene transfer, all or a large proportion of erythroid cells might express suboptimal levels of β-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the β-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual α-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from β039-thalassemia patients were employed. Gene therapy was performed with the lentiviral vector T9W. Induction of fetal hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on K562 transgenic cell lines harboring different copies of a lentiviral vector carrying the human β-globin gene, showing that γ-globin mRNA expression and HbF production can be induced in the presence of high levels of β-globin gene expression and HbA accumulation. We then treated erythroid progenitor cells from β-thalassemic patients with T9W, which expresses the human β-globin gene and mithramycin separately or in combination. When transduction with our lentiviral vector is insufficient to completely eliminate the unpaired α-globin chains, combination of β-globin gene transfer therapy together with fetal hemoglobin induction might be very efficacious to remove the excess of α-globin proteins in thalassemic erythroid progenitor cells.
doi:10.1007/s00277-012-1430-5
PMCID: PMC3389244  PMID: 22460946
β-thalassemia; Gene therapy; Lentiviral vectors; HbF induction; Erythroid progenitor cells
10.  Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients 
PLoS ONE  2012;7(3):e32345.
Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.
We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).
Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.
doi:10.1371/journal.pone.0032345
PMCID: PMC3314006  PMID: 22479321
11.  Mithramycin encapsulated in polymeric micelles by microfluidic technology as novel therapeutic protocol for beta-thalassemia 
This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic® block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially γ-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating α-globin gene expression, which is responsible for the clinical symptoms of β-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying β-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for β-thalassemia.
doi:10.2147/IJN.S25657
PMCID: PMC3265999  PMID: 22287841
microfluidics; lab-on-a-chip; design of experiments; erythroid differentiation; human erythroid precursor cells
12.  C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells 
European Journal of Pharmacology  2011;672(1-3):30-37.
The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects.
doi:10.1016/j.ejphar.2011.09.024
PMCID: PMC3271358  PMID: 21958870
Erythroid differentiation; Tumor growth; Isoorotic acid derivative; Chronic myelogenous leukemia; Beta-thalassemia
13.  Synthesis and Evaluation of Haloacetyl, α-Bromoacryloyl and Nitrooxyacetyl Benzo[b]furan and Benzo[b]thiophene Derivatives as Potent Antiproliferative Agents Against Leukemia L1210 and K562 Cells 
Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. In search of new compounds with strong antiproliferative activity and simple molecular structure, we have synthesized three different series of compounds in which different substituents were linked to the 3-amino position of the 2-(3′, 4′, 5′-trimethoxybenzoyl)-benzo[b]furan or benzo[b]thiophene ring system. These substituents, corresponding to acetyl/haloacetyl, α-bromoacryloyl and nitrooxyacetyl moieties had different electrophilic properties. The benzoheterocycle parent structures were selected because of their reported bioactivities. Compounds bearing a methoxy group at the 6-position of the benzo[b]furan skeleton, were identified as potent antiproliferative agents against the human chronic myelogenous K562 and murine L1210 leukemia cell lines. Comparison of positional isomers indicated that moving the methoxy group from the 6- to the 5- or 7-position yielded inactive compounds. The effects of a selected series of compounds on cell cycle progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. The analysis of structure-activity relationships observed in the series of compounds described here may represent a platform for the design of more active molecules.
PMCID: PMC2911241  PMID: 20676361
Benzo[b]thiophene; Benzo[b]furan; Tumor cell growth; Antiproliferative agents
14.  A preclinical approach for gene therapy of β-thalassemia 
Lentiviral-mediated β-globin gene transfer successfully treated β-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different β-globin mutations found in patients. Most mutations can be classified as β0 or β+, based on the amount of β-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human β-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley’s anemia patients.
doi:10.1111/j.1749-6632.2010.05594.x
PMCID: PMC3068625  PMID: 20712784
β-thalassemia; β-globin; gene therapy; lentiviral vectors; hemoglobin; erythroid progenitor cells
15.  Encapsulation of eukaryotic cells in alginate microparticles: cell signaling by TNF-alpha through capsular structure of cystic fibrosis cells 
Entrapment of mammalian cells in natural or synthetic biomaterials represents an important tool for both basic and applied research in tissue engineering. For instance, the encapsulation procedures allow to physically isolate cells from the surrounding environment, after their transplantation maintaining the normal cellular physiology. The first part of the current paper describes different microencapsulation techniques including bulk emulsion technique, vibrating-nozzle procedure, gas driven mono-jet device protocol and microfluidic based approach. In the second part, the application of a microencapsulation procedure to the embedding of IB3-1 cells is also described. IB3-1 is a bronchial epithelial cell line, derived from a cystic fibrosis (CF) patient. Different experimental parameters of the encapsulation process were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of protein secretion, analysing the culture medium by Bio-Plex strategy. The analyzed factors include members of the interleukin family (IL-6), chemokines (IL-8 and MCP-1) and growth factors (G-CSF). The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent.
doi:10.1007/s12079-010-0105-z
PMCID: PMC3088797  PMID: 21484183
Biomaterials; Encapsulation; Alginate; Cystic fibrosis
16.  Bergamot (Citrus bergamia Risso) fruit extracts and identified components alter expression of interleukin 8 gene in cystic fibrosis bronchial epithelial cell lines 
BMC Biochemistry  2011;12:15.
Background
Cystic fibrosis (CF) airway pathology is a fatal, autosomal, recessive genetic disease characterized by extensive lung inflammation. After induction by TNF-α, elevated concentrations of several pro-inflammatory cytokines (i.e. IL-6, IL-1β) and chemokines (i.e. IL-8) are released from airway epithelial cells. In order to reduce the excessive inflammatory response in the airways of CF patients, new therapies have been developed and in this respect, medicinal plant extracts have been studied. In this article we have investigated the possible use of bergamot extracts (Citrus bergamia Risso) and their identified components to alter the expression of IL-8 associated with the cystic fibrosis airway pathology.
Methods
The extracts were chemically characterized by 1H-NMR (nuclear magnetic resonance), GC-FID (gas chromatography-flame ionization detector), GC-MS (gas chromatography-mass spectrometry) and HPLC (high pressure liquid chromatography). Both bergamot extracts and main detected chemical constituents were assayed for their biological activity measuring (a) cytokines and chemokines in culture supernatants released from cystic fibrosis IB3-1 cells treated with TNF-α by Bio-Plex cytokine assay; (b) accumulation of IL-8 mRNA by real-time PCR.
Results
The extracts obtained from bergamot (Citrus bergamia Risso) epicarps contain components displaying an inhibitory activity on IL-8. Particularly, the most active molecules were bergapten and citropten. These effects have been confirmed by analyzing mRNA levels and protein release in the CF cellular models IB3-1 and CuFi-1 induced with TNF-α or exposed to heat-inactivated Pseudomonas aeruginosa.
Conclusions
These obtained results clearly indicate that bergapten and citropten are strong inhibitors of IL-8 expression and could be proposed for further studies to verify possible anti-inflammatory properties to reduce lung inflammation in CF patients.
doi:10.1186/1471-2091-12-15
PMCID: PMC3095539  PMID: 21496221
17.  Predictive Analyses of Biological Effects of Natural Products: From Plant Extracts to Biomolecular Laboratory and Computer Modeling 
Year by year, the characterization of the biological activity of natural products is becoming more competitive and complex, with the involvement in this research area of experts belonging to different scientific fields, including chemistry, biochemistry, molecular biology, immunology and bioinformatics. These fields are becoming of great interest for several high-impact scientific journals, including eCAM. The available literature in general, and a survey of reviews and original articles recently published, establishes that natural products, including extracts from medicinal plants and essential oils, retain interesting therapeutic activities, including antitumor, antiviral, anti-inflammatory, pro-apoptotic and differentiating properties. In this commentary, we focus attention on interest in networks based on complementary activation and comparative evaluation of different experimental strategies applied to the discovery and characterization of bioactive natural products. A representative flow chart is shown in the paper.
doi:10.1093/ecam/nep096
PMCID: PMC3135393  PMID: 19752166
18.  Induction by TNF-α of IL-6 and IL-8 in Cystic Fibrosis Bronchial IB3-1 Epithelial Cells Encapsulated in Alginate Microbeads 
We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-α induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells.
doi:10.1155/2010/907964
PMCID: PMC2946646  PMID: 20936184
19.  Discovery of 8-methoxypyrazino[1,2-a]indole as a New Potent Antiproliferative Agent Against Human Leukemia K562 Cells. A Structure-Activity Relationship Study 
Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. The indole nucleus, frequently encountered as a molecular fragment in natural products and pharmaceutically active compounds, was employed as the initial building block for the synthesis of a series of pyrazino[1,2-a]indoles 1a–k, variably substituted at the 6, 7, 8 and 9-positions. Compound 1e, bearing the methoxy group at the 8-position of the pyrazino[1,2-a]indole nucleus was identified as a novel potent antiproliferative agent against the human chronic myelogenous leukemia K562 cell line, but it was much less active against several other cancer cell lines. Comparison of positional isomers indicated that moving the methoxy group from the 8- to the 7- or 6-position, to furnish compounds 1f and 1g, respectively, yielded inactive compounds. The analysis of structure-activity relationships observed in the series of investigated compounds may represent the basis for the design of more active molecules.
doi:10.2174/157018009788452519
PMCID: PMC2744412  PMID: 20161090
Pyrazino[1,2-a]indole; Human leukemia K562 cell line; Anticancer agents
20.  Hybrid α-bromoacryloylamido chalcones. Design, synthesis and biological evaluation 
Research into the anti-tumor properties of chalcones has received significant attention over the last few years Two novel large series of α-bromoacryloylamido chalcones 1a–m and 2a–k containing a pair of Michael acceptors in their structures, corresponding to the α-bromoacryloyl moiety and the α,β-unsaturated ketone system of the chalcone framework, were synthesized and evaluated for antiproliferative activity against five cancer cell lines. Such hybrid derivatives demonstrated significantly increased anti-tumor activity compared with the corresponding amino chalcones. The most promising lead molecules were 1k, 1m and 2j, which had the highest activity toward the five cell lines. Flow cytometry with K562 cells showed that the most active compounds resulted in a large proportion of the cells entering in the apoptotic sub-G0–G1 peak. Moreover, compound 1k induced apoptosis through the mitochondrial pathway and activated caspase-3.
doi:10.1016/j.bmcl.2009.02.038
PMCID: PMC2714055  PMID: 19250822
21.  SLUG: a new target of lymphoid enhancer factor-1 in human osteoblasts 
BMC Molecular Biology  2010;11:13.
Background
Lymphoid Enhancer Factor-1 (Lef-1) is a member of a transcription factor family that acts as downstream mediator of the Wnt/β-catenin signalling pathway which plays a critical role in osteoblast proliferation and differentiation. In a search for Lef-1 responsive genes in human osteoblasts, we focused on the transcriptional regulation of the SLUG, a zinc finger transcription factor belonging to the Snail family of developmental proteins. Although the role of SLUG in epithelial-mesenchymal transition and cell motility during embryogenesis is well documented, the functions of this factor in most normal adult human tissues are largely unknown. In this study we investigated SLUG expression in normal human osteoblasts and their mesenchymal precursors, and its possible correlation with Lef-1 and Wnt/β-catenin signalling.
Results
The experiments were performed on normal human primary osteoblasts obtained from bone fragments, cultured in osteogenic conditions in presence of Lef-1 expression vector or GSK-3β inhibitor, SB216763. We demonstrated that the transcription factor SLUG is present in osteoblasts as well as in their mesenchymal precursors obtained from Wharton's Jelly of human umbilical cord and induced to osteoblastic differentiation. We found that SLUG is positively correlated with RUNX2 expression and deposition of mineralized matrix, and is regulated by Lef-1 and β-catenin. Consistently, Chromatin Immunoprecipitation (ChIP) assay, used to detect the direct Lef/Tcf factors that are responsible for the promoter activity of SLUG gene, demonstrated that Lef-1, TCF-1 and TCF4 are recruited to the SLUG gene promoter "in vivo".
Conclusion
These studies provide, for the first time, the evidence that SLUG expression is correlated with osteogenic commitment, and is positively regulated by Lef-1 signal in normal human osteoblasts. These findings will help to further understand the regulation of the human SLUG gene and reveal the biological functions of SLUG in the context of bone tissue.
doi:10.1186/1471-2199-11-13
PMCID: PMC2834684  PMID: 20128911
22.  Foetal haemoglobin inducers and thalassaemia: novel achievements 
Blood Transfusion  2010;8(1):5-7.
doi:10.2450/2009.0137-09
PMCID: PMC2809505  PMID: 20104272
23.  Synthesis and biological evaluation of 2-(3′,4′,5′-trimethoxybenzoyl)-3-N,N-dimethylamino benzo[b]furan derivatives as inhibitors of tubulin polymerization 
Bioorganic & medicinal chemistry  2008;16(18):8419-8426.
Molecules that target microtubules have an important role in the treatment of cancer. A new class of inhibitors of tubulin polymerization based on the 2-(3,4,5-trimethoxybenzoyl)-2-dimethylamino-benzo[b]furan molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. The most promising compound in this series was 2-(3,4,5-trimethoxybenzoyl)-3-dimethylamino-6-methoxy-benzo[b]furan, which inhibits cancer cell growth at nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.
doi:10.1016/j.bmc.2008.08.029
PMCID: PMC2574800  PMID: 18755591
Microtubules; Tubulin polymerization; Colchicine binding site; Combretastatin A-4; Bioisosteric replacement
24.  Gene Therapy in Thalassemia and Hemoglobinopathies 
Sickle cell disease (SCD) and ß-thalassemia represent the most common hemoglobinopathies caused, respectively, by the alteration of structural features or deficient production of the ß-chain of the Hb molecule. Other hemoglobinopathies are characterized by different mutations in the α- or ß-globin genes and are associated with anemia and might require periodic or chronic blood transfusions. Therefore, ß-thalassemia, SCD and other hemoglobinopathies are excellent candidates for genetic approaches since they are monogenic disorders and, potentially, could be cured by introducing or correcting a single gene into the hematopoietic compartment or a single stem cell. Initial attempts at gene transfer of these hemoglobinopathies have proved unsuccessful due to limitations of available gene transfer vectors. With the advent of lentiviral vectors many of the initial limitations have been overcame. New approaches have also focused on targeting the specific mutation in the ß-globin genes, correcting the DNA sequence or manipulating the fate of RNA translation and splicing to restore ß-globin chain synthesis. These techniques have the potential to correct the defect into hematopoietic stem cells or be utilized to modify stem cells generated from patients affected by these disorders. This review discusses gene therapy strategies for the hemoglobinopathies, including the use of lentiviral vectors, generation of induced pluripotent stem cells (iPS) cells, gene targeting, splice-switching and stop codon readthrough.
doi:10.4084/MJHID.2009.008
PMCID: PMC3033156  PMID: 21415990
25.  Release of sICAM-1 in Oocytes and In Vitro Fertilized Human Embryos 
PLoS ONE  2008;3(12):e3970.
Background
During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.
Methodology/Principal Findings
The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.
Conclusions/Significance
The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.
doi:10.1371/journal.pone.0003970
PMCID: PMC2599884  PMID: 19092999

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