Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G + C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.
Raptor, regulatory associated protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; m-TORC2, mTOR complex 2; Sp1, specific protein 1; MTH, mithramycin; RAPA, rapamycin; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS, tris-buffered saline; HbF, fetal hemoglobin; ODN, oligonucleotide; Raptor; mTOR; Sp1; Mithramycin; Erythroid induction; Fetal hemoglobin
In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies.
peptide nucleic acids; sickle-cell anemia; β-globin; hemoglobin; erythroid differentiation
•Low expression of miR501-5p correlates with good prognosis for patients with ccRCC.•miRNA501-5p downregulation stimulates apoptosis by p53 activation.•miR501-5p upregulation promotes cell proliferation and survival.•Increased cell growth occurs by activation of mTOR kinase and MDM2 expression.•This miRNA modulates apoptosis/cell growth, making it a prognostic biomarker for ccRCC.
Renal cell carcinoma is a common neoplasia of the adult kidney that accounts for about 3% of adult malignancies. Clear cell renal carcinoma is the most frequent subtype of kidney cancer and 20–40% of patients develop metastases. The absence of appropriate biomarkers complicates diagnosis and prognosis of this disease. In this regard, small noncoding RNAs (microRNAs), which are mutated in several neoplastic diseases including kidney carcinoma, may be optimal candidates as biomarkers for diagnosis and prognosis of this kind of cancer. Here we show that patients with clear cell kidney carcinoma that express low levels of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. Consistently, in kidney carcinoma cells the downregulation of miR501-5p induced an increased caspase-3 activity, p53 expression as well as decreased mTOR activation, leading to stimulation of the apoptotic pathway. Conversely, miR501-5p upregulation enhanced the activity of mTOR and promoted both cell proliferation and survival. These biological processes occurred through p53 inactivation by proteasome degradation in a mechanism involving MDM2-mediated p53 ubiquitination. Our results support a role for miR501-5p in balancing apoptosis and cell survival in clear cell renal carcinoma. In particular, the downregulation of microRNA501-5p promotes a good prognosis, while its upregulation contributes to a poor prognosis, in particular, if associated with p53 and MDM2 overexpression and mTOR activation. Thus, the expression of miR501-5p is a possible biomarker for the prognosis of clear cell renal carcinoma.
ccRCC, clear cell renal cell carcinoma; MDM2, mouse double minute 2 homolog; mTOR, mammalian target of rapamycin; pRCC, papillary renal cell carcinoma; MicroRNA501-5p; mTOR signaling; p53; Apoptosis; Cell survival
Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2.
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
, 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
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
peptide nucleic acid
microRNA replacement therapy
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.
► 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.
Psoralen; Angelicin; Photoproducts; K562 cells; Erythroid differentiation; Fetal hemoglobin
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.
aminoglycoside antibiotics; K562 cell; locus control region; nonsense mutation; thalassaemia
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.
Breast cancer and its metastatic progression is mainly directed by epithelial to mesenchymal transition (EMT), a phenomenon supported by specific transcription factors and miRNAs.
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.
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.
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.
Slug; miR-221; Epithelial mesenchymal transition; Breast cancer
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.
FACS; fluorescence; miR-210; PNA; pre-miR; thiazole orange
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.
NF-kappaB; transcription factor decoy; inflammation; Peptide Nucleic Acids; PNA-DNA chimeras
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.
β-thalassemia; Gene therapy; Lentiviral vectors; HbF induction; Erythroid progenitor cells
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.
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.
microfluidics; lab-on-a-chip; design of experiments; erythroid differentiation; human erythroid precursor cells
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.
Erythroid differentiation; Tumor growth; Isoorotic acid derivative; Chronic myelogenous leukemia; Beta-thalassemia
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.
Benzo[b]thiophene; Benzo[b]furan; Tumor cell growth; Antiproliferative agents
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.
β-thalassemia; β-globin; gene therapy; lentiviral vectors; hemoglobin; erythroid progenitor 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.
Biomaterials; Encapsulation; Alginate; Cystic fibrosis
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.
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.
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.
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.
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.
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.
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.
Pyrazino[1,2-a]indole; Human leukemia K562 cell line; Anticancer agents
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.
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.
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".
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.