EGFR-targeted immunoliposomes as a selective delivery system of simvastatin, with potential use in treatment of triple-negative breast cancers.

A promising strategy for treatment of EGFR-dependent tumours is EGFR signal transduction suppression via inhibition of HMG-CoA reductase using high doses of statins, popular cholesterol-lowering drugs. The main purpose of this study was to obtain targeted long circulating immunoliposomes containing simvastatin (tLCLS) with anti-EGFR antibody attached to their surface and to test whether they can be effective in treatment of TNBC. The designed tLCLS were characterized in terms of physicochemical properties and long-term stability. In vitro experiments conducted on MDA-MB-231 cells demonstrated that tLCLS induced apoptosis and are characterized by IC50 of 7.5 µM. Treatment of studied cells with tLCLS led to a decrease in membrane order and inhibited PI3K/Akt signalling. Analyses of efficacy of the tLCLS in in vivo experiments in model animals indicate that immunoliposomes were effectively delivered to tumours. Our results showed that regardless of whether tLCLS were administered before or after tumour formation, at the tested dose they inhibited tumour growth by an average of 25% in comparison to the control. However, the results were not statistically significant. The experiments described above allowed us to test the possibility of using immunoliposomes as simvastatin carriers delivering increased amounts of the drug to tumour cells.

The microdomain-organizing protein MPP1 is required for insulin-stimulated activation of H-Ras.

Signaling complexes are localized to distinct plasma-membrane domains which undergo precise spatiotemporal regulation. A crucial link between membrane dynamics and the small GTPase, H-Ras, has been suggested, connecting membrane localization, clustering and scaffolding with its activity and signal transduction. Results of this study suggest a relationship between MPP1 and/or MPP1-dependent plasma-membrane organization and H-Ras activation. Namely, we show here that in HEL cells, MPP1 knock-down lead to the disruption of signaling cascade(s) from the activated insulin receptor. The signal inhibition occurred at the level of H-Ras, as it showed impaired GDP-to-GTP exchange and further interaction with its effector molecule, Raf. Moreover, in these cells H-Ras detergent-resistant membrane localization was not sensitive to insulin treatment which may imply molecular mechanism via which MPP1 affects functions of other proteins which may be connected with functional domain formation. Understanding the link between MPP1 and activation of H-Ras, may provide an important insight into the complexity of Ras related signaling pathways which may become a potential target for associated cancer therapies.

A novel regulatory function of CDKN1A/p21 in TNFα-induced matrix metalloproteinase 9-dependent migration and invasion of triple-negative breast cancer cells.

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.

The role of stearoyl-CoA desaturase in the regulation of cardiac metabolism.

The uptake and utilization of energetic substrates in the myocardium are under strict control, any disturbances of which may lead to myocardial dysfunction, such as in the case of ischemia and heart failure. Stearoyl-CoA desaturase (SCD) is an enzyme that converts saturated fatty acids to monounsaturated fatty acids. It is an important player in the regulation of heart metabolism. Our previous studies showed that SCD1 affects substrate utilization by the heart, with a preference for glucose. Large cohort studies established a positive correlation between the plasma fatty acid desaturation index and cardiovascular disease mortality. Therefore, SCD1 might serve as a potential target for future therapies. We review recent findings on the role of SCD1 in the heart, with a focus on cardiac metabolism reprogramming and its involvement in heart dysfunction.

MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC.

Protein palmitoylation: Palmitoyltransferases and their specificity.

A plethora of novel information has emerged over the past decade regarding protein lipidation. The reversible attachment of palmitic acid to cysteine residues, termed S-palmitoylation, has focused a special attention. This is mainly due to the unique role of this modification in the regulation of protein trafficking and function. A large family of protein acyltransferases (PATs) containing a conserved aspartate-histidine-histidine-cysteine motif use ping-pong kinetic mechanism to catalyze S-palmitoylation of a substrate protein. Here, we discuss the topology of PAT proteins and their cellular localization. We will also give an overview of the mechanism of protein palmitoylation and how it is regulated. New information concerning the recent discovery of depalmitoylating enzymes belonging to the family of α/ß-hydrolase domain-containing protein 17 (ABHD17A) is included. Considering the recent advances that have occurred in understanding the mechanisms underlying the interplay between palmitoylation and depalmitoylation, it is clear that we are beginning to understand the fundamental nature of how cellular signal-transduction mediates membrane-level organization in health and disease. Impact statement Protein palmitoylation is one of most important reversible post-translational modifications of protein function in cell-signaling systems. This review gathers the latest information on the molecular mechanism of protein palmitoyl transferase action. It also discusses the issue of substrate specificity of palmitoyl transferases. Another important question is the role of depalmitoylation enzymes. This review should help to formulate questions concerning the regulation of activity of particular PATs as well as of depalmitoylating enzymes (APT).

Investigation of oxidative stress parameters in different lifespan erythrocyte fractions in young untrained men after acute exercise.

NEW FINDINGS: What is the central question of this study? What is the influence of a single bout of exercise on the properties of erythrocyte fractions at different ages? What is the main finding and its importance? A single bout of exercise in untrained men induced oxidative stress in erythrocytes and had an influence on antioxidant defense in these cells. Old erythrocytes were more sensitive to oxidative damage than young and middle-aged cells. Higher levels of glutathione in old erythrocyte fractions did not protect them against oxidative stress. It seems that exercise may promote the removal of old erythrocytes from the circulation. The objective of this study was to establish the role of exercise-induced oxidative stress in the erythrocyte fractions [young (YF), middle-aged (MAF) and old (OF)] of young untrained men after acute exercise. Blood samples were collected before exercise, immediately after and 1 h after exercise. The maximal power generated was 292 ± 27 W, and exercise duration was 8.73 ± 0.9 min. Different optical properties and oxidative stress parameters were found in each erythrocyte fraction. Total thiols in YF and MAF after exercise and after 1 h rest were similar to values before exercise; however, in OF {32.7 ± 9.8 nmol [mg haemoglobin (Hb)]-1 } the concentration was lower in comparison to YF [55.5 ± 3.2 nmol (mg Hb)-1 ] and MAF [56.8 ± 7.7 nmol (mg Hb)-1 ] and increased 1 h later (P < 0.0002). The glutathione concentration was higher in OF [8.4 ± 0.4 nmol (mg Hb)-1 ] than in YF [4.5 ± 0.6 nmol (mg Hb)-1 ] and MAF [4.8 ± 0.5 nmol (mg Hb)-1 ; P < 0.0002] and did not change after exercise or 1 h later. In OF, the peroxide level was higher after exercise [1.2 ± 0.2 nmol (mg Hb)-1 ] and 1 h later [1.1 ± 0.2 nmol (mg Hb)-1 ], when compared with samples before exercise [0.9 ± 0.1 nmol (mg Hb)-1 ; P < 0.05]. Similar results were observed in YF and MAF. The level of thiobarbituric acid reactive substances (TBARS) was ∼2.5-fold higher in OF [0.19 ± 0.04 nmol (mg Hb)-1 ] when compared with YF [0.07 ± 0.01 nmol (mg Hb)-1 ] and MAF [0.08 ± 0.02 nmol (mg Hb)-1 ; P < 0.0002] and was increased after exercise, remaining unchanged 1 h later. In YF and MAF, no difference in the level of TBARS was detected after exercise or 1 h later. No difference in membrane fluidity was observed in all fractions. The erythrocyte OF appeared to be more sensitive to cellular oxidative damage.

Anti-tumor potential of nitroxyl derivative Pirolin in the DMBA-induced rat mammary carcinoma model: A comparison with quercetin.

BACKGROUND: Considering the role of oxidative stress in carcinogenesis, we investigated the effect of synthetic antioxidant Pirolin (3-carbamoyl-2,2,5,5-tetramethylpyrroline-1-oxyl) on breast cancer progression. Since the anticancer drugs may cause cardiotoxicity due to oxidative stress in the heart muscle, we also evaluated Pirolin performance in heart tissue and compared its effect with that of the natural dietary flavonoid quercetin. METHODS: Sprague-Dawley rats were administered with 7,12-dimethylbenz(a)anthracene (DMBA) and then treated ip with an antioxidant (each at a dose of 10mg/kg b.w.) for 14 days. The histopathology of tumors, their size and multiplicity were assesed. The effect of antioxidants on heart tissue was evaluated by the oxidative stress markers and poly (ADP-ribose) polymerase 1 (PARP 1) cleavage. RESULTS: The median number of tumors and their volume, at the end of the study, were considerably smaller in both antioxidant-treated groups. We found a better antioxidative performance of quercetin in the heart, since a restoration of the GSH pool and decreased amount of hydroperoxides were observed. Antioxidants did not prevent cardiomyocytes from apoptosis. CONCLUSION: The attenuation of tumor progression by Pirolin was comparable with the action of quercetin. No negative changes were observed in the heart of animals after Pirolin treatment. Thus, its use in targeting deregulated redox pathways should be further studied.

Quercetin attenuates oxidative stress in the blood plasma of rats bearing DMBA-induced mammary cancer and treated with a combination of doxorubicin and docetaxel.

The development of side-effects during doxorubicin-docetaxel (DOX-DTX) chemotherapy is considered as related to generation of oxidative stress by DOX. The addition of docetaxel potentiates this effect. Thus, antioxidants are assumed as a promising remedy for neutralizing deteriorating effects of reactive oxygen species (ROS) in pathological conditions and polyphenolic antioxidants are suitable candidates for such a therapeutic approach. We evaluated the ability of quercetin to attenuate oxidative stress developed during the process of DMBA carcinogenesis and DOX-DTX chemotherapy in the blood plasma of rats bearing mammary tumors. We have found that quercetin significantly improved the plasma nonenzymatic antioxidant capacity (NEAC) and reduced lipid peroxidation, which suggest the beneficial effect of flavonoid. The inclusion of quercetin to the DOX-DTX chemotherapy was also advantageous. A considerable decrease of carbonyls and lipid peroxidation products (TBARS) and improvement of the endogenous antioxidant defense system (an increase of NEAC, thiols and SOD activity) were observed compared to rats treated with DOX-DTX chemotherapy. These results suggest that quercetin could protect blood plasma constituents against oxidative damage evoked by DOX and DTX.

Alterations in red blood cells and plasma properties after acute single bout of exercise.

The aim of this study was to investigate alterations in haemoglobin conformation and parameters related to oxidative stress in whole erythrocytes, membranes, and plasma after a single bout of exercise in a group of young untrained men. Venous blood samples from eleven healthy young untrained males (age = 22 ± 2 years, BMI = 23 ± 2.5 kg/m(2)) were taken from the antecubital vein before an incremental cycling exercise test, immediately after exercise, and 1 hour after exercise. Individual heart rate response to this exercise was 195 ± 12 beats/min and the maximum wattage was 292 ± 27 W. Immediately after exercise, significant increase in standard parameters (haemoglobin, haematocrit, lactate levels, and plasma volume) of blood was observed as well as plasma antioxidant capacity one hour after exercise. Reversible conformational changes in haemoglobin, measured using a maleimide spin label, were found immediately following exercise. The concentration of ascorbic acid inside erythrocytes significantly decreased after exercise. A significant decline in membrane thiols was observed one hour after exercise, but simultaneously an increase in plasma thiols immediately after and 1 h after exercise was also observed. This study shows that a single bout of exercise can lead to mobilization of defensive antioxidant systems in blood against oxidative stress in young untrained men.

[Nitroxides as antioxidants - possibilities of their application in chemoprevention and radioprotection]. / Nitroksydy jako antyoksydanty ­ mozliwosci ich zastosowania w celach chemioprewencyjnych oraz radioprotekcyjnych.

Nitroxides as stabile organic radicals were used initially as spin labels in spectroscopy of electron paramagnetic resonance (EPR) with respect to parameters such as pH of an intercellular environment, oxygenation of cells and tissues, fluidity of biological membranes, conformational state and topography of proteins. Nitroxides have also been used in biology and medicine as contrast agents in magnetic resonance imaging (MRI). When their antioxidant activities were discovered, an era of research on the potential utility of these agents began. Nitroxides can modulate the redox state of the cell by participation in oxidation/reduction reactions. Therefore, they are extensively examined in various models of oxidative stress. The antioxidant effect of nitroxides is a result of their ability to catalyze dismutation of superoxide radical (superoxide dismutase-like activity), inhibit lipid peroxidation, prevent Fenton and Haber-Weiss reactions by oxidation of transition metal ions to a higher oxidative state, and confer catalase-like activity on heme proteins. In the present paper the antioxidative mechanisms of nitroxides are presented. The relation between structure, function and the rate of nitroxide reduction inside cells and tissues is also presented. The application of nitroxides in chemoprevention and radioprotection is discussed.

[Molecular mechanisms of antitumor activity of taxanes. I. Interaction of docetaxel with microtubules]. / Molekularne mechanizmy aktywnosci przeciwnowotworowej taksanów. I. Oddzialywanie docetakselu na mikrotubule.

Docetaxel (Taxotere), a new semisynthetic taxoid, is a mitotic inhibitor, widely used in monotherapy or in combination with other anticancer drugs against many types of cancer. The structure and dynamics of microtubules as the main target for docetaxel activity inside the cell and the taxane-binding site on ß-tubulin are discussed. Microtubules are highly dynamic assemblies of α- and ß-tubulin. They readily polymerize and depolymerize in cells and these dynamic behaviours are crucial to cell mitosis. Microtubule instability is attributed to their capability to hydrolyze GTP to GDP, which causes their depolymerization. Addition of new α-, ß-tubulin heterodimer bound to GTP leads to tubulin polymerization, which increases the length of the microtubule. Docetaxel alters the polymerization dynamics of microtubules, which causes blockage of cell mitosis, and consequently induces apoptotic and non-apoptotic cell death. Docetaxel specifically acts on the S, M and G2 phases of the cell cycle. This paper reviews the current state of knowledge related to the molecular mechanisms of docetaxel action on the cell cycle and microtubule dynamics. In addition, a brief survey of the present state of research on the new generation (2nd and 3rd) of taxanes is presented.