Biological activity of anti-miR-221 Peptide Nucleic Acids and relative building blocks

The study of new molecules able to selectively and stably interact with DNA and RNA is a field of great interest in consideration all the possible applications in medicine. Our study is related to the biomedical applications of the final product of PNA synthesis (the PNA itself) and the intermediate molecules obtained during the synthetic activity. In all the chemical synthesis approach of any pharmaceutical laboratory several molecules are produced, which are usually not considered for biological assays and technology transfer. We screened a set of C(5) uracil derivatives monomers, that were employed during the PNA synthesis, for activity on differentiated functions in K562 cells. We found that the highest antiproliferative effect and erythroid induction ability was exhibited by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine (compound 2) did not show any effect, suggesting the importance of the linear alkil chain in N(1) position. Compound 9, furthermore, exhibits induce erythroid terminal differentiation without activation of apoptotic pathway. The interest in the context on anti-tumor differentiation therapy is related to the fact that, when compared to known erythroid differentiation antitumor inducers (such as for instance cytosine arabinoside, mithramycin, resveratrol), the lead compound we were able to identify is the most active agent. Therefore these molecules in our opinion deserves further research activity in order to define its possible application, for instance in the control of proliferation/differentiation of CML primary cells resistance to the commonly employed Imatinib (Gleevec®) therapy. As far as the final product of the synthetic strategy (a PNA recognizing miR- 221 and able to be internalized in target tumor cells thanking to a linked Arg-8 peptide), the results here presented allows to conclude that (a) it is internalized at high efficiency into target tumor cells; (b) inhibits the miR-221 hybridization availability and (c) has important effects on biological functions regulated by miR-221 (i.e. expression of the p27Kip1 mRNA/protein). These results are in our opinion of interest, considering on one hand the role of miR-221 in cancer and, on the other hand, the role of p27Kip1. As far as miR-221, it was found to be up-regulated in several tumors; in breast cancer miR-221 is up-regulated in breast cancer cell line (such as MDA-MB-231) and in metastatic tumors. In the contest of breast cancer, it was identified p27kip1 mRNA as possible target of miR-221. The cyclin-kinase inhibitor p27kip1 is a tumorsuppressor protein, involved in the control of cell cycle during the G0/G1 check-point transition: the loss or decrease of p27kip1, together with others, is one of contributory causes of the “proliferating state” of invasive cancer, which remains in this growth phase without arrive to differentiation. It is of great interest since it was found to be down-regulated in several type of tumors. The studies presented in this PhD Thesis teach that even intermediate synthetic molecules deserve attention in respect to possible biological effects on cells relevant to human pathologies. Moreover, we have identify two reagents of possible interest for the development of anti-cancer protocols: N(1)-octyl-thymine for the treatment of CML cells and Rpep-PNA-a221 for possible use in the treatment of breast cancer cells

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 ?-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating ?-globin gene expression, which is responsible for the clinical symptoms of ß-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying ß-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 ß-thalassemia.<br/

Targeting oncomiRNAs and mimicking tumor suppressor miRNAs: Ew trends in the development of miRNA therapeutic strategies in oncology

MicroRNA (miRNA or miR) therapeutics in cancer are based on targeting or mimicking miRNAs involved in cancer onset, progression, angiogenesis, epithelial-mesenchymal transition and metastasis. Several studies conclusively have demonstrated that miRNAs are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This review focuses on the most promising examples potentially leading to the development of anticancer, miRNAbased therapeutic protocols. The inhibition of miRNA activity can be readily achieved by the use of miRNA inhibitors and oligomers, including RNA, DNA and DNA analogues (miRNA antisense therapy), small molecule inhibitors, miRNA sponges or through miRNA masking. On the contrary, the enhancement of miRNA function (miRNA replacement therapy) can be achieved by the use of modified miRNA mimetics, such as plasmid or lentiviral vectors carrying miRNA sequences. Combination strategies have been recently developed based on the observation that i) the combined administration of different antagomiR molecules induces greater antitumor effects and ii) some anti-miR molecules can sensitize drug-resistant tumor cell lines to therapeutic drugs. In this review, we discuss two additional issues: i) the combination of miRNA replacement therapy with drug administration and ii) the combination of antagomiR and miRNA replacement therapy. One of the solid results emerging from different independent studies is that miRNA replacement therapy can enhance the antitumor effects of the antitumor drugs. The second important conclusion of the reviewed studies is that the combination of anti-miRNA and miRNA replacement strategies may lead to excellent results, in terms of antitumor effects

Inhibition of Cancer Cell Proliferation and Antiradical Effects of Decoction, Hydroalcoholic Extract, and Principal Constituents of Hemidesmus indicus R. Br

Indian Sarsaparilla (Hemidesmus indicus R. Br.) is widely used in Indian traditional medicine. In the present work, we explored the effects of decoction, traditional Ayurvedic preparation, and hydroalcoholic extract, a phytocomplex more traditionally studied and commercialized as food supplement in western medicine, from the roots as possible source of chemicals with new functional potential linked to their nutritional uses. The antiproliferative and antioxidant properties were assayed. To test antiproliferative affects, different cancer cell lines, growing both as monolayers (CaCo2, MCF-7, A549, K562, MDA-MB-231, Jurkat, HepG2, and LoVo) and in suspension (K562 and Jurkat) were used. The decoction showed strong activity on HepG2 cells, while the hydroalcoholic extracts were active on HepG2, LoVo, MCF-7, K562, and Jurkat cell lines. Weak inhibition of cancer cell proliferation was observed for the principal constituents of the preparations: 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, and 3-hydroxy-4-methoxybenzaldehyde that were tested alone. The antiradical activity was tested with 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt tests and inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages. Interesting result has also been obtained for hydroalcoholic extract regarding genoprotective potential (58.79% of inhibition at 37.5 µg/mL). Copyright © 2015 John Wiley & Sons, Ltd

Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAsmiRNA Maturation

The involvement of microRNAs in human pathologies is a firmly established fact. Accordingly, the pharmacological modulation of their activity appears to be a very appealing issue in the development of new types of drugs (miRNA therapeutics). One of the most interesting issues is the possible development of miRNA therapeutics for development of anti-cancer molecules. In this respect appealing molecules are based on peptide nucleic acids (PNAs), displaying a pseudo-peptide backbone composed of N-(2-aminoethyl) glycine units and found to be excellent candidates for antisense and antigene therapies. The major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells. The aim of this chapter is to describe methods for determining the activity of PNAs designed to oncomiRNA targets, using as model system miR-221 and its target p27(Kipl) mRNA. The effects of PNAs targeting miR-221 are here presented discussing data obtained using as model system the human breast cancer cell line MDA-MB-231, in which miR-221 is up-regulated and p27(Kipl) down-regulated

In vitro evaluation of the anti-proliferative and geno-protective activity of traditional preparations of ayurvedic crude drugs

The research of new botanicals is an important aspect of the modern research focused on prevention and cure of cancer. This project evaluated the in vitro geno-protective capacity (SOS-Chromotest) and anti-proliferative activity (MCF7, A549, MDA-MB-231, LoVo, HepG2, K562, Jurkat and IB3-1 cell lines) of two traditional preparations, decoction and mother tincture, of two ayurvedic crude drugs: Hemidesmus indicus roots and Azadirachta indica leaves. Our previous results demonstrated an interesting antileukemic effect1,2 of H. indicus decoction. In present study both H. indicus preparations possess anti-proliferative activity against all the cell lines considered. Among phytochemical markers, the most active, 2-hydroxy-4-methoxybenzaldehyde, showed an IC50 of 12.09±2.78 μg/ml against K562 and an IC50 of 13.15±5.57 μg/ml against Jurkat cells. The only relevant data for A. indica were against: MDA-MB-231 cell (IC50= 381.57±43.62 μg/ml) for decoction and K562 (IC50= 276.05±26.60 μg/ml) and Jurkat cells (IC50= 207.18±29.97 μg/ml) for mother tincture. Further analyses of anti-proliferative bioactivity for A. indica are still in progress. In conclusion: H. indicus preparations were more effective than those of A. indica; mother tincture of both crude drugs showed a general wider anti-proliferative activity than decoction, in particular for H. indicus, it evidenced promising data against Jurkat (63.79±7.97 μg/ml), Hep-G2 (34.50±0.14) and LoVo (29.84±0.24); H. indicus mother tincture and decoction were more effective than 2-hydroxy-4-methoxybenzaldehyde against Hep-G2 cell, pointing out possible synergistic (agonistic) activity of minor compounds

Author&apos;s personal copy Induction of erythroid differentiation and increased globin mRNA production with furocoumarins and their photoproducts

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. 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 realtime 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 0 -Dimethylpsoralen photoproducts induced an important increase in c-globin gene transcription in K562 cells

trans-Resveratrol in Nutraceuticals: Issues in Retail Quality and Effectiveness

Fourteen brands of resveratrol-containing nutraceuticals were evaluated in order to verify their actual resveratrol content and to control if their health-promoting properties are related to manufacturing quality. Products included pure resveratrol capsules or multi-ingredient formulations with standardized amounts of resveratrol and other phytochemicals. Samples were analyzed for total trans-resveratrol, flavonoids, procyanidin, polyphenol content and the results were compared with the content declared on-label. Only five out of 14 brands had near label values, compliant with Good Manufacturing Practices (GMP) requirements (95–105% content of active constituent), four products were slightly out of this range (83–111%) and three were in the 8–64% range. Two samples were below the limit of detection. The greater the difference between actual and labeled resveratrol content, the lower was the antioxidant and antiproliferative activity strength. Dietary supplements containing pure trans-resveratrol exhibited a greater induction of differentiation towards human leukemic K562 cells when compared to multicomponent products. Great differences currently exist among resveratrol food supplements commercially available and GMP-grade quality should not be taken for granted. On the other side, dosages suggested by most “pure”, “high-dosage” supplements may allow a supplementation level adequate to obtain some of the purported health benefits

Induction of erythroid differentiation and increased globin mRNA production with furocoumarins and their photoproducts

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 \u3b3-globin gene transcription in K562 cells