Biophysical insights into the antitumoral activity of crotalicidin against breast cancer model membranes

Bioactive peptides have emerged as promising therapeutic agents with antimicrobial, antifungal, antiparasitic, and, recently, antitumoral properties with a mechanism of action based on membrane destabilization and cell death, often involving a conformational change in the peptide. This biophysical study aims to provide preliminary insights into the membrane-level antitumoral mode of action of crotalicidin, a cationic host defense peptide from rattlesnake venom, toward breast cancer cell lines. The lipid composition of breast cancer cell lines was obtained after lipid extraction and quantification to prepare representative cell membrane models. Membrane–peptide interaction studies were performed using differential scanning calorimetry and Fourier-transform infrared spectroscopy. The outcome evidences the potential antitumoral activity and selectivity of crotalicidin toward breast cancer cell lines and suggests a mechanism initiated by the electrostatic interaction of the peptide with the lipid bilayer surface and posterior conformation change with membrane intercalation between the acyl chains in negatively charged lipid systems. This research provides valuable information that clears up the antitumoral mode of action of crotalicidin

Configuration of polyisoprenoids affects the permeability and thermotropic properties of phospholipid/polyisoprenoid model membranes.

The influence of α-cis- and α-trans-polyprenols on the structure and properties of model membranes was analyzed. The interaction of Ficaprenol-12 (α-cis-Prenol-12, α-Z-Prenol-12) and Alloprenol-12 (α-trans-Prenol-12, α-E-Prenol-12) with model membranes was compared using high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC) and fluorescent methods. L-α-phosphatidylcholine from egg yolk (EYPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as the main lipid components of unilamellar (SUVs) and multilamellar (MLVs) vesicles were used. The two-step extraction procedure (n-pentane and hexane, respectively) allowed to separately analyze the fractions of polyprenol as non-incorporated (PrenolNonInc) and incorporated (PrenolInc) into liposomes. Consequently, distribution coefficients, P', describing the equilibrium of prenol content between phospholipid (EYPC) membrane and the aqueous phase gave different logP' for α-cis- and α-trans-Prenol-12, indicating that the configuration of the α-terminal residue significantly alters the hydrophobicity of the polyisoprenoid molecule and consequently the affinity of polyprenols for EYPC membrane. In fluorescence experiments α-trans-Pren-12 increased up to 1.7-fold the permeability of EYPC bilayer for glucose while the effect of α-cis-Pren-12 was almost negligible. Considerable changes of thermotropic behavior of DPPC membranes in the presence of both prenol isomers were observed. α-trans-Pren-12 completely abolished the pretransition while in the case of α-cis-Pren-12 it was noticeably reduced. Furthermore, for both prenol isomers, the temperature of the main phase transition (Tm) was shifted by about 1ºC to lower values and the height of the peak was significantly reduced. The DSC analysis profiles also showed a new peak at 38.7°C, which may suggest the concomitant presence of more that one phase within the membrane. Results of these experiments and the concomitant occurrence of alloprenols and ficaprenols in plant tissues suggest that cis/trans isomerization of the α-residue of polyisoprenoid molecule might comprise a putative mechanism responsible for modulation of the permeability of cellular membranes

An in vitro study on the antioxidant capacity of usnic acid on human erythrocytes and molecular models of its membrane

AbstractUsnic acid (UA) has been associated with chronic diseases through its antioxidant action. Its main target is the cell membrane; however, its effect on that of human erythrocytes has been scarcely investigated. To gain insight into the molecular mechanisms of the interaction between UA and cell membranes human erythrocytes and molecular models of its membrane have been utilized. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Results by X-ray diffraction showed that UA produced structural perturbations on DMPC and DMPE bilayers. DSC studies have indicated that thermotropic behavior of DMPE was most strongly distorted by UA than DMPC, whereas the latter is mainly affected on the pretransition. Scanning electron (SEM) and defocusing microscopy (DM) showed that UA induced alterations to erythrocytes from the normal discoid shape to echinocytes. These results imply that UA molecules were located in the outer monolayer of the erythrocyte membrane. Results of its antioxidant properties showed that UA neutralized the oxidative capacity of HClO on DMPC and DMPE bilayers; SEM, DM and hemolysis assays demonstrated the protective effect of UA against the deleterious oxidant effects of HClO upon human erythrocytes

Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate

AbstractResearch on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50μM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25–1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations