Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine

Conventional anticancer therapies, such as chemo- and/or radio-therapy are often unable to completely eradicate cancers due to abnormal tumor microenvironment, as well as increased drug/radiation resistance. More effective therapeutic strategies for overcoming these obstacles are urgently in demand. Aptamers, as chemical antibodies that bind to targets with high affinity and specificity, are a promising new and novel agent for both cancer diagnostic and therapeutic applications. Aptamer-based cancer cell targeting facilitates the development of active targeting in which aptamer-mediated drug delivery could provide promising anticancer outcomes. This review is to update the current progress of aptamer-based cancer diagnosis and aptamer-mediated active targeting for cancer therapy in vivo, exploring the potential of this novel form of targeted cancer therapy.

Glutaredoxin1 protects neuronal cells from copper-induced toxicity

Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.

Aptamer-mediated cancer gene therapy

Cancer as a genetic disorder is one of the leading causes of death worldwide. Conventional anticancer options such as chemo- and/or radio-therapy have their own drawbacks and could not provide a cure in most cases at present. More effective therapeutic strategies with less side effects are urgently needed. Aptamers, also known as chemical antibodies, are single strand DNA or RNA molecules that can bind to their target molecules with high affinity and specificity. Such site-specific binding ability of aptamers facilitates the delivery and interaction of exogenous nucleic acids with diseased genes. Thus, aptamer-guided gene therapy has emerged as a promising anticancer strategy in addition to the classic treatment regimen. Aptamers can directly deliver anti-cancer nucleic acids, e.g. small interfering RNA, micro RNA, antimicroRNA and small hairpin RNA, to cancer cells or function as a targeting ligand to guide nanoparticles containing therapeutic nucleic acids. This review focuses on recent progress in aptamer-mediated gene therapy for the treatment of hepatocellular carcinoma and other types of cancers, shedding light on the potential of this novel approach of targeted cancer gene therapy.

Redox sulfur chemistry of the copper chaperone Atox1 is regulated by the enzyme glutaredoxin 1, the reduction potential of the glutathione couple GSSG/2GSH and the availability of Cu(I)

Glutaredoxins have been characterised as enzymes regulating the redox status of protein thiols via cofactors GSSG/GSH. However, such a function has not been demonstrated with physiologically relevant protein substrates in in vitro experiments. Their active sites frequently feature a Cys-xx-Cys motif that is predicted not to bind metal ions. Such motifs are also present in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone. In this work, we present the first demonstration that: (i) human glutaredoxin 1 (hGrx1) efficiently catalyses interchange of the dithiol and disulfide forms of the Cys(12)-xx-Cys(15) fragment in Atox1 but does not act upon the isolated single residue Cys(41); (ii) the direction of catalysis is regulated by the GSSG/2GSH ratio and the availability of Cu(I); (iii) the active site Cys(23)-xx-Cys(26) in hGrx1 can bind Cu(I) tightly with femtomolar affinity (K(D) = 10(-15.5) M) and possesses a reduction potential of E(o)' = -118 mV at pH 7.0. In contrast, the Cys(12)-xx-Cys(15) motif in Atox1 has a higher affinity for Cu(I) (K(D) = 10(-17.4) M) and a more negative potential (E(o)' = -188 mV). These differences may be attributed primarily to the very low pKa of Cys23 in hGrx1 and allow rationalisation of conclusion (ii) above: hGrx1 may catalyse the oxidation of Atox1(dithiol) by GSSG, but not the complementary reduction of the oxidised Atox1(disulfide) by GSH unless Cu(aq)(+) is present at a concentration that allows binding of Cu(I) to reduced Atox1 but not to hGrx1. In fact, in the latter case, the catalytic preferences are reversed. Both Cys residues in the active site of hGrx1 are essential for the high affinity Cu(I) binding but the single Cys(23) residue only is required for the redox catalytic function. The molecular properties of both Atox1 and hGrx1 are consistent with a correlation between copper homeostasis and redox sulfur chemistry, as suggested by recent cell experiments. These proteins appear to have evolved the features necessary to fill multiple roles in redox regulation, Cu(I) buffering and Cu(I) transport.

Locked nucleic acid modified bi-specific aptamer-targeted nanoparticles carrying survivin antagonist towards effective colon cancer therapy

We investigated the anti-cancer activity of alginate coated chitosan nanoparticles (CHNP) encapsulating cell-permeable dominant negative survivin (SR9) with locked nucleic acid (LNA) aptamers targeting EpCAM and nucleolin (termed as "nanobullets") in vitro (2D and 3D cell culture models) and in vivo (colon cancer mouse xenograft model). We incorporated three LNA modifications in each sequence in order to enhance the stability of these aptamers. Confocal microscopy revealed binding of the LNA-aptamers to their specific markers with high affinity. The muco-adhesive nanobullets showed 6-fold higher internalization in cancer cells when compared to non-cancerous cells, suggesting a tumour specific uptake. A higher intensity of nanobullets was observed in both the periphery and the core of the multicellular tumour spheroids compared to non-targeted CHNP-SR9. The nanobullets were found to be the highly effective as they led to a 2.26 fold (p < 0.05) reduction at 24 h and a 4.95 fold reduction (p ≤ 0.001) in the spheroid size at 72 h. The tumour regression was 4 fold higher in mice fed on a nanobullet diet when compared to a control diet. The nanobullets were able to show a significantly high apoptotic (p ≤ 0.0005) and necrotic index in the tumour cell population (p ≤ 0.005) when compared to void NPs. Therefore, our nanoparticles have shown highly promising results and therefore deliver a new conduit towards the approach of cancer-targeted nanodelivery. This journal is

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions. with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following Michaelian kinetics with a specific activity of 36.7 U/mg and K-0.5 = 88 mu M. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited Michaclian kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed. K-m values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K-0.5 = 40 and 90 mu M, for magnesium and calcium, respectively), high-affinity sites were also observed with K-0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 mu M), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed. (C) 1998 Published by Elsevier B.V. Ltd. All rights reserved.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative transcriptome analysis of Paracoccidioides brasiliensis during in vitro adhesion to type I collagen and fibronectin: identification of potential adhesins

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T-o and T-x, as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.

Crotoxin-induced behavioral effects in rats

Crotoxin is the major component of Crotalus durissus terrificus venom. In view of the presence of high-affinity specific binding sites for crotoxin in the brain, the objective of this work was to investigate whether crotoxin induces behavioral effects in the open-field and hole-board tests. Adult male Wistar rats (180-220 g) treated with crotoxin, 100, 250 and 500 mu g/kg, ip, administered 2 h before the test, presented statistically significant behavioral alterations (ANOVA for one-way classification complemented with Dunnet test, P<0.05). In the open-field test, 250 and 500 mu g/kg of crotoxin increased freezing (from 3.22 sec to 10.75 sec and 11.2 sec) and grooming (from 13.44 sec to 22.75 sec and 21.22 sec) and decreased ambulation (from 64.8 to 39.38 and 45.8). The dose of 500 mu g/kg also decreased rearing (from 24.9 to 17.5). In the hole-board test, 500 mu g/kg of crotoxin decreased head-dip count (from 6.33 to 4.00). All the crotoxin-induced behavioral effects were antagonized by an anxiolytic dose of diazepam (1.5 mg/kg, ip, 30 min before the tests). These results show that crotoxin reduced open-field activity and exploratory behavior as well. We suggest that these effects express an increased emotional state induced by this toxin.

2-Mercaptoimidazole covalently bonded to a silica gel surface for the selective separation of mercury(II) from an aqueous solution

Silica gel with a specific surface area of 365 m(2).g(-1) and an average pore diameter of 60 Angstrom was chemically modified with 2-mercaptoimidazole. The degree of functionalization of the covalently attached molecule, (drop SiO)(3)(CH2)(3) - MI, where MI is the 2-mercaptoimidazole bound to the silica surface by a propyl group, was 0.58 mmolg.(-1). In individual metal adsorption experiments from aqueous solutions by the batch procedure, the affinity order was Hg(II)much greater than Cd-II > Cu-II approximate to Zn-II approximate to Pb-II > Mn-II at solution pHs between 4 and 7. Due to the high affinity by the sulfur atom, Hg-II is strongly bound to the functional groups. When solution containing a mixture of Hg-II, Cd-II, Cu-II, Zn-II, Pb-II, and Mn-II ions was passed through a column packed with the adsorbent, Hg-II was the only one whose adsorption and elution was not affected by the presence of other ions.

The role of hydration on the mechanism of allosteric regulation: In situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98%, relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

Effects of unilateral decortication on β-adrenergic receptors in the remaining cortex and in the hypothalamus of female rats

Many experiments have been performed to evaluate the physiological role of catecholaminergic mechanisms of gonadotropin release. The purpose of the present study was to determine the concentration of β-adrenoreceptors in the remaining (right) cerebral cortex and in right and left hypothalamic halves of hemi-decorticated female rats which exhibited elevated plasma gonadotropin levels as observed previously. The density of β-receptors was measured using a high-affinity β-adrenergic ligand, iodocyanopindolol (ICYP). Scatchard estimates were obtained for maximum binding (B(max) fmol/mg of tissues) from pooled cerebral cortical and hypothalamic tissue of animals under several experimental conditions after hemi-decortication and sham operation. There was an increase in β-adrenoreceptor density in the remaining (right) cerebral cortex at all times examined in hemi-decorticate in comparison with the sham-operated animals (7 days, +10.9%; 21 days, +8.4%; 90 days, +22%; and 90 days plus ovariectomy, +34.8%). The number of β-adrenoreceptors in the right hypothalamic half in hemi-decorticates decreased at 21 days (-42.20%) and then increased at 90 days (+76.63%) and 90 days plus ovariectomy (+51.75%) when compared with the left hypothalamic half. At the same time there were no significant changes in the sham-operated animals when comparing the receptor density in the right and left hypothalamic halves, respectively. Thus, our results suggest a direct adrenergic pathway by which the left cortex can influence the right cortex and a crossed pathway to the contralateral hypothalamus changing adrenergic activity which can alter the β-adrenergic receptor binding capacity in the hypothalamus.

Nickel chloride: potential accumulation from the airway. Toxicokinetics in rats.

The rate removal of nickel from the airway was measured in vivo. Removal in vivo was studied by intratracheal injection of nickel chloride solutions. Regardless of time after injection, the lungs and heart retained the greatest concentration of nickel and 40 days after 1.68 mumol administration they were the organs where nickel was still significantly measurable. The slow removal of nickel may indicate the presence of high affinity binding sites in the lung. Nickel can interact with others metals, such as copper and zinc, so that nickel exposure may have public health implications.