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The role of the microRNA miR-93-5p on the secretome profile and the expression levels of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) was investigated in the neuroblastoma SK-N-AS cell line by Bio-Plex analysis and RT-qPCR. The results indicate that VEGF and IL-8 are the major miR-93-5p molecular targets. This conclusion was based on in vitro transfection with pre-miR-93-5p and anti-miR-93-5p; these treatments inversely modulated both VEGF and IL-8 gene expression and protein release in the neuroblastoma SK-N-AS cell line. Computational analysis showed the presence of miR-93-5p consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93-5p and confirming a potential regulatory role of this microRNA.

The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR‑221 and miR‑222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR‑221 and anti-miR‑222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222.

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.

The activity of a peptide nucleic acid (PNA) targeting cancer-associated microRNA-221 is described. PNAs against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in the cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-PNA-a221) showed both very high affinity for RNA and efficient cellular uptake without the addition of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only Rpep-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by PNA resulted in i) lowering of the hybridization levels of miR-221 measured by RT-qPCR, ii) upregulation of p27Kip1 gene expression, measured by RT-qPCR and western blot analysis. The major conclusion of this study is that efficient delivery of anti‑miR PNA through a suitable peptide carrier (Rpep‑PNA-a221) leads to inhibition of miR-221 activity, altering the expression of miR-221-regulated functions in breast cancer cells.

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and β-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.

Derivatives of distamycin A modified at the C-terminal amidine moiety and tethered to bis-epoxyethyl moieties at the N-terminal position were tested for their ability to induce erythroid differentiation in the human erythroleukemic cell line K562. None of the compounds without bis-epoxyethyl moiety were active. A comparison of the biological activity of diepoxy compounds containing different non-basic amidine-modified moieties, showed low activity of amidoxime, carbamoyl and N-methyl carbamoyl derivatives as differentiation agents. In contrast, a cyanamidine derivative, compound 3, was able to induce erythroid differentiation of K562 cells. In addition, the cyanamidine derivative 3 was able to induce HbF accumulation following treatment of cultures of erythroid precursor cells isolated from the peripheral blood of normal subjects.

The isolation of rare cells, such as fetal nucleated red blood cells and trophoblasts, from maternal blood for non-invasive prenatal diagnosis is a new field of research exhibiting several difficulties since this strategy requires unresolved basic technological protocols for a successful outcome. However, several achievements in the field of Laboratory-on-a-chip (Lab-on-a-chip) technology have provided clear advancements in projects aimed at the isolation of rare cells from biological fluids. Among the most interesting approaches are those based on dielectrophoresis (DEP). DEP-based Lab-on-a-chip platforms have been demonstrated to be suitable for several applications in biotechnology and biomedicine. DEP-based arrays are able to manipulate single cells, which can be identified and moved throughout the DEP chip to recovery places. DEP buffers are compatible with molecular interactions between monoclonal antibodies and target cells, allowing integration of these devices with magnetic cell sorting (MACS). DEP treatment does not alter the viability of manipulated cells.

Vanadium complexes are known to possess potent insulin-mimetic effects, high affinity for several enzymes and anticancer activity, which deserve increasing attention for application to biomedical sciences. Different vanadium complexes have been found to be more effective than the simple vanadium-(IV) and -(V) salts in experiments performed both in vitro and in vivo. Application of polyoxometalates as potential drugs against Herpes Simplex Virus and AIDS have also increased the interest to study the association between vanadium containing species and proteins. The aim of our research was to investigate the in vitro antiproliferative activity of a variety of vanadium-containing compounds, and study their ability to interfere with the molecular interactions between GATA-1 and NF-kappaB transcription factors and target DNA elements, employing electrophoretic mobility shift assays. All of the used vanadium compounds were found to exhibit antiproliferative activity, despite with differences in efficacy. Inhibition of K562 cell growth was not associated with differentiation, but with activation of apoptosis. Vanadium complexes with a +5 oxidation state and their discrete anionic units appear essential for the respective effects on K562 cells; a +4 oxidation state appears to be important in inhibiting transcription factors/DNA interactions.

In this study we determined the activity of extracts from Bangladeshi medicinal plants (Emblica officinalis, Aegle marmelos, Vernonia anthelmintica, Oroxylum indicum, Argemone mexicana) on human breast tumor cell lines. Extracts from E. officinalis and O. indicum displayed anti-proliferative activity on MCF7 and MDA-MB-231 breast cancer cell lines, while extracts from A. mexicana were active on MCF7 cells, exhibiting on the contrary low antiproliferative effects on MDA-MB-231 cells. Extracts from A. marmelos and V. anthelmintica were antiproliferative on both cell lines, but at higher concentrations. The accumulation of estrogen receptor alpha (ERalpha) mRNA, a marker of neoplastic status, was analysed by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The data obtained demonstrated that only extracts from E. officinalis induce an increase of ERalpha mRNA in MCF7 cells. When MDA-MB-231 cell line was employed, extracts from E. officinalis, V. anthelmintica and A. mexicana were found to be inducers of the increase of ERalpha mRNA accumulation. Since activation of ERalpha gene expression could have clinical impact, our results suggest a possible use of extracts from medicinal plants to identify compounds of possible interest in the treatment of breast cancer.

The class II products of the major histocompatibility complex have a distribution restricted to certain tissues and cells. For instance, they are constitutively expressed by B lymphocytes, but not by resting T lymphocytes. In this study, we report the identification of a novel DNase I hypersensitive site within a putative regulatory region of the human HLA-DRA gene, the so-called far upstream region. This hypersensitive site was present in the genome of the DRalpha-positive human B-lymphoid Raji cell line, and absent in the DRalpha-negative T-lymphoid Jurkat cell line. In addition, this hypersensitive site was also present in transgenic B lymphocytes isolated from the murine transgenic line TG 53, carrying a single integrated copy of the human HLA-DRA gene per haploid genome. The correlation between DRA expression and the presence of this far upstream hypersensitive site suggests novel long distance chromatin remodeling mechanisms possibly shared by human and murine class II genes.

In this study we compared the in vitro antiproliferative activity of extracts from medicinal plants toward human tumor cell lines, including human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat, erythroleukemic HEL cell lines. Extracts from Emblica officinalis were the most active in inhibiting in vitro cell proliferation, after comparison to those from Terminalia arjuna, Aphanamixis polystachya, Oroxylum indicum, Cuscuta reflexa, Aegle marmelos, Saraca asoka, Rumex maritimus, Lagerstroemia speciosa, Red Sandalwood. Emblica officinalis extracts have been studied previously, due to their hepatoprotective, antioxidant, antifungal, antimicrobial and anti-inflammatory medicinal activities. Gas chromatography/mass spectrometry analyses allowed to identify pyrogallol as the common compound present both in unfractionated and n-butanol fraction of Emblica officinalis extracts. Antiproliferative effects of pyrogallol were therefore determined on human tumor cell lines thus identifying pyrogallol as an active component of Emblica officinalis extracts.

The inhibition of gene transcription mediated by peptide nucleic acids (PNAs) has been described as mainly due to a process that involves strand invasion of target DNA containing homopurine stretches with generation of PNA/DNA/PNA triplexes. Scarce information is available on DNA strand invasion of mixed purine-pyrimidine stretches and theirs possible use as molecules able to interact with transcription factors. With this aim, we compared the effects of DNA/DNA, DNA/PNA or PNA/PNA hybrid molecules mimicking the NF-kappaB binding sites of HIV-1 on LTR-driven in vitro transcription. The results allowed us to conclude that PNA/PNA molecules and DNA/PNA hybrids are capable of inhibiting transcription, like the DNA/DNA. We investigated the mechanisms by which PNA/PNA and DNA/PNA hybrids affect the transcription. The results suggest that DNA/PNA and PNA/PNA molecules inhibit transcription in distinct manner; the inhibitory effects are due to strand invasion in the case of PNA/PNA hybrids and decoy effects on transcription factors belonging the NF-kappaB/Rel family in the case of DNA/PNA hybrids. It is noteworthy that DNA/PNA hybrids are more resistant to nucleases than DNA/DNA hybrids.