Edgar J. Paredes-Gamero

Characterization of dual effects induced by antimicrobial peptides: Regulated cell death or membrane disruption

BACKGROUND Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. In this study, we compare the cell death-inducing activities of four β-hairpin AMPs (gomesin, protegrin, tachyplesin and polyphemusin II) along with their linear analogs in the human erythroleukemia K562 cell line to investigate the relationship between their structure and activity. METHODS K562 cells were exposed to AMPs. Morphological and biochemistry alterations were evaluated using light microscopy, confocal microscopy and flow cytometry. RESULTS Gomesin and protegrin displayed cytotoxic properties that their linear counterparts did not. Tachyplesin and polyphemusin II and also their linear analogs induced cell death. We were able to distinguish two ways in which these AMPs induced cell death. Lower concentrations of AMPs induced controlled cell death mechanisms. Gomesin, tachyplesin and linear-tachyplesin promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation, but was also inhibited by necrostatin-1. Gomesin and protegrin induced cell death was dependent on intracellular Ca2+ mechanisms and the participation of free radicals was observed in protegrin induced cell death. Polyphemusin II and its linear analog mainly induced necrosis. Conversely, treatment with higher concentrations of AMPs primarily resulted in cell membrane disruption, but with clearly different patterns of action for each AMP tested. CONCLUSION Different actions by β-hairpin AMPs were observed at low concentrations and at higher concentrations despite the structure similarity. GENERAL SIGNIFICANCE Controlled intracellular mechanism and direct membrane disruption were clearly distinguished helping to understand the real action of AMPs in mammalian cells.

Antimicrobial, antioxidant and cytotoxic activities of propolis from Melipona orbignyi (Hymenoptera, Apidae)

Propolis from stingless bees is well known for its biologic properties; however, few studies have demonstrated these effects. Therefore, this study aimed to investigate the chemical composition and antimicrobial, antioxidant and cytotoxic activities of propolis from the stingless bee Melipona orbignyi, found in Mato Grosso do Sul, Brazil. The chemical composition of the ethanol extract of propolis (EEP) indicated the presence of aromatic acids, phenolic compounds, alcohols, terpenes and sugars. The EEP was active against the bacterium Staphylococcus aureus and the fungus Candida albicans. The EEP showed antioxidant activity by scavenging free radicals and inhibiting hemolysis and lipid peroxidation in human erythrocytes incubated with an oxidizing agent. Additionally, EEP promoted cytotoxic activity and primarily necrotic death in K562 erythroleukemia cells. Taken together, these results indicate that propolis from M. orbignyi has therapeutic potential for the treatment and/or prevention of diseases related to microorganism activity, oxidative stress and tumor cell proliferation.

Phosphoproteome Reveals an Atlas of Protein Signaling Networks During Osteoblast Adhesion

Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion‐related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3β during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell‐cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems‐oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell–substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering. J. Cell. Biochem. 109: 957–966, 2010.

Retinyl palmitate flexible polymeric nanocapsules: characterization and permeation studies.

Polymeric nanocapsules with elastic characteristics were prepared by the pre-formed polymer interfacial deposition method. The system consists of an oily core of retinyl palmitate with Span 60 and a polymeric wall of poly(D,L-lactide) (PLA). A narrow size distribution (215 nm, P.D.I. 0.10) was showed by dynamic light scattering (DLS) analyses. Particle deformability was observed by transmission electron microscopy (TEM) images and permeation of the particles through two superposed membranes of smaller pore diameters. Permeation studies were achieved using plastic surgery abdominal human skin by Franz diffusion cell. Retinyl palmitate permeates into deep skin layers. Besides, a PLA fluorescent derivative conjugated with Nile blue dye by an amide covalent bound was additionally obtained. Permeation profile of the nanocapsules with the fluorescent polymer was evaluated by confocal laser scanning microscopy (CLSM). The CLSM showed that nanocapsules were distributed uniformly, suggesting that the permeation mechanism through skin is intercellular. Thus, the use of these nanocapsules may be a feasible strategy to enhance the permeation of actives into the skin when delivery to deep layers is aimed.

Influence of protein corona on the transport of molecules into cells by mesoporous silica nanoparticles.

Although there are several studies reporting the promising biological efficiency of mesoporous silica nanoparticles (loaded with antitumoral drugs) against cancer cells and tumors, there are no reports on the influence of the bio-nano interface interactions on the molecular diffusion process occurring along their pores. In this context, we show here that the protein coating formed on multifunctionalized colloidal mesoporous silica nanoparticles (MSNs) dispersed in a cell culture medium decreases the release of camptothecin (CPT, a hydrophobic antitumoral drug) from the pores of MSNs. This effect is related to the adsorption of biomolecules on the nanoparticle surface, which partially blocks the pores. Parallely, the hydrophobic functionalization inside the pores can offer suitable sites for the adsorption of other molecules present in the cell culture medium depending on the hydrophobicity, size, and conformation aspects of these molecules and adsorption sites of MSNs. Thus, the molecular cargo loaded in the pores (i.e. CPT) can be replaced by specific molecules present in the dispersion medium. As a consequence, we show that a non-permeable cellular staining molecule such as SYTOX green can be incorporated in MSNs through this mechanism and internalized by cells in an artificial fashion. By extrapolating this phenomenon for applications in vivo, one has to consider now the possible manifestation of unpredicted biological effects from the use of porous silica nanoparticles and others with similar structure due to these internalization aspects.

Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis.

Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process.

Changes in Intracellular Ca2+ Levels Induced by Cytokines and P2 Agonists Differentially Modulate Proliferation or Commitment with Macrophage Differentiation in Murine Hematopoietic Cells

The role of intracellular Ca2+ (Ca2+i) on hematopoiesis was investigated in long term bone marrow cultures using cytokines and agonists of P2 receptors. Cytokines interleukin 3 and granulocyte/macrophage colony stimulator factor promoted a modest increase in Ca2+i concentration ([Ca2+]i) with activation of phospholipase Cγ, MEK1/2, and Ca2+/calmodulin kinase II. Involvement of protein kinase C was restricted to stimulation with interleukin 3. In addition, these cytokines promoted proliferation (20 times) and an increase in the Gr-1-Mac-1+ population with participation of gap junctions (GJ). Nevertheless ATP, ADP, and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), a reduction in the primitive Gr-1-Mac-1-c-Kit+ population, and differentiation into macrophages without participation of GJ. It is likely that Ca2+i participates as a regulator of hematopoietic signaling: moderate increases in [Ca2+]i would be related to cytokine-dependent proliferation with participation of GJ, whereas high increases in [Ca2+]i would be related to macrophage differentiation without maintenance of the primitive population.

Cell-Permeable Gomesin Peptide Promotes Cell Death by Intracellular Ca2+ Overload

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.

Antimicrobial, Antioxidant, Anti-Inflammatory, and Cytotoxic Activities of Propolis from the Stingless Bee Tetragonisca fiebrigi (Jataí)

Propolis from stingless bees Tetragonisca fiebrigi found in Brazil is used in folk medicine by their nutritional and therapeutic properties. However, there are no scientific records evidencing such properties. The present study was designed to investigate the chemical composition and the biological properties of propolis from T. fiebrigi. For this, the chemical composition of the ethanol extract of propolis (EEP) was determined by GC-MS and presented phenolic compounds, alcohol, and terpenes as its major class compounds. The antimicrobial activity was accessed in gram-positive and gram-negative bacteria and in fungi, isolated from different biological fluids and reference strains. The EEP was active against all microorganisms and showed antioxidant activity by scavenging free radicals, inhibiting hemolysis and lipid peroxidation in human erythrocytes incubated with an oxidizing agent. The anti-inflammatory potential of the EEP was confirmed by inhibition of the hyaluronidase enzyme. The cytotoxic activity was concentration-dependent against K562 cells, with a predominance of death by necrosis. Taken together, these results show that propolis from T. fiebrigi has important therapeutic activities, which suggest its potential application in the pharmaceutical industry, as well as in health foods, beverages, and nutritional supplements.

Pre-clinical antitumour evaluation of Biphosphinic Palladacycle Complex in human leukaemia cells

Previous studies reported by our group have introduced a new antitumoural drug called Biphosphinic Palladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60 and Jurkat), but not in normal human lymphocytes. IC(50) values obtained for both cell lines using the MTT and trypan blue exclusion assays 5h after BPC treatment were lower than 8.0 microM. Using metachromatic fluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Furthermore, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatin condensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lysosomal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation, which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We conclude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptotic signalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no toxic effects for human lymphocytes were observed, we suggest that BPC is more selective for transformed cells, mainly due to their exacerbated lysosome expression.