GO Nanosheets: Promising Nano Carrier for the S29, 1-(2-Chloro-2-(4-chlorophenyl-ethyl)-N-(4-fluorobenzyl)-1H-pyrazolo[3,4-d] pyrimidin-4-amine, Therapeutic Agent in Neuroblastoma.

Graphene oxide (GO) derivatives are reported as a valid alternative to conventional carriers of therapeutic agents, because they have a large surface area, an excellent electrical and thermal conductivity and a great capacity for selective binding of drugs and therapeutics, due to the functionalization of their surfaces, edges and sides. In this work GO nanosheets, synthesized by electrochemical exfoliation of graphite (patent N 102015000023739, Tor Vergata University), were investigated as possible carriers of an anticancer drug, the S29, an inhibitor of a cytoplasmic tyrosine kinase (c-SRC) on a neuroblastoma cell line (SK N BE 2 cells). Neuroblastoma is a heterogenous tumor whose characteristics range from spontaneous regression to aggressive phenotypes that are due to different mutations that often occur in SRC family kinases. Inhibitors of tyrosine kinases are currently investigated for their anti-tumoral effects on aggressive neuroblastomas, but their uptake in cells and pharmacokinetics needs to be improved. In this work S29 was stably conjugated with highly water-dispersible GO nanoparticles. S29/GO complex formation was induced by 1h sonication and its stability was analyzed by chromatography coupled with spectrophotometry and mass spectrometry. The synthesized composite (GO-S29) was delivered into SK N BE 2 cells and its effects on cell viability, production of reactive oxygen species (ROS) and migration were studied. The results show that the compound GO-S29 exerts anti-tumoral effects on the neuroblastoma cell line, higher than both GO and S29 do alone and that GO has an additive effect on S29.

Adaptation to intermittent hypoxia restricts nitric oxide overproduction and prevents beta-amyloid toxicity in rat brain.

This study tested the hypothesis that adaptation to intermittent hypoxia (AIH) can prevent overproduction of nitric oxide (NO) in brain and neurodegeneration induced by beta-amyloid (Aß) toxicity. Rats were injected with a Aß protein fragment (25-35) into the nucleus basalis magnocellularis. AIH (simulated altitude of 4000 m, 14 days, 4h daily) was produced prior to the Aß injection. A passive, shock-avoidance, conditioned response test was used to evaluate memory function. Degenerating neurons were visualized in stained cortical sections. NO production was evaluated in brain tissue by the content of nitrite and nitrate. Expression of nNOS, iNOS, and eNOS was measured in the cortex and the hippocampus using Western blot analysis. 3-Nitrotyrosine formation, a marker of protein nitration, was quantified by slot blot analysis. Aß injection impaired memory of rats; AIH significantly alleviated this disorder. Histological examination confirmed the protective effect of AIH. Degenerating neurons, which were numerous in the cortex of Aß-injected, unadapted rats, were essentially absent in the brain of hypoxia-adapted rats. Injections of Aß resulted in significant increases in NOx and in expression of all NOS isoforms in brain; AIH blunted these increases. NO overproduction was associated with increased amounts of 3-nitrotyrosine in the cortex and hippocampus. AIH alone did not significantly influence tissue 3-nitrotyrosine, but significantly restricted its increase after the Aß injection. Therefore, AIH affords significant protection against experimental Alzheimer's disease, and this protection correlates with restricted NO overproduction.

Cancer immunotherapy through the prism of adaptation: Will Achilles catch the tortoise?

There is no secret that despite the rapid development of new methods of cancer therapy, we still are not able to completely destroy the tumor. Every time we attack the tumor, the tumor neutralizes our attempts. Carcinogenesis can be presented as a tree whose branches are different pro-tumor mechanisms and whose trunk is a biological phenomenon that "feeds" those branches. A tree can be destroyed in two ways: either by cutting a branch for a branch without a guarantee that new branches will not grow, or cutting down the trunk and letting the branches wither away. To cut down the trunk, it is necessary to understand the nature of the biological phenomenon, which helps the tumor to avoid attack by the immune system, drugs and immunotherapy. The clue is that the pro-tumor mechanisms are united by one goal - to increase the resistance of the tumor cell to immune factors and drugs. A phenomenon that improves cell resistance is well known in biology - adaptation. If the immunity does not immediately destroy the tumor cell, the cell begins to adapt to it. Our hypothesis is that short range adaptation to immune factors plays a role in the formation of tumor tolerance for immunity and immunotherapy. This gives rise to the idea of reducing the survival of tumor cells by disrupting adaptation mechanisms. Indeed, "turning off" the immune system for a period of time before therapy and applying immunotherapy only to tumor cells that have lost their increased resistance could be a new approach to increase the effectiveness of immunotherapy.

M3 Macrophages Stop Division of Tumor Cells In Vitro and Extend Survival of Mice with Ehrlich Ascites Carcinoma.

BACKGROUND M1 macrophages target tumor cells. However, many tumors produce anti-inflammatory cytokines, which reprogram the anti-tumor M1 macrophages into the pro-tumor M2 macrophages. We have hypothesized that the problem of pro-tumor macrophage reprogramming could be solved by using a special M3 switch phenotype. The M3 macrophages, in contrast to the M1 macrophages, should respond to anti-inflammatory cytokines by increasing production of pro-inflammatory cytokines to retain its anti-tumor properties. Objectives of the study were to form an M3 switch phenotype in vitro and to evaluate the effect of M3 macrophages on growth of Ehrlich ascites carcinoma (EAC) in vitro and in vivo. MATERIAL AND METHODS Tumor growth was initiated by an intraperitoneal injection of EAC cells into C57BL/6J mice. RESULTS 1) The M3 switch phenotype can be programed by activation of M1-reprogramming pathways with simultaneous inhibition of the M2 phenotype transcription factors, STAT3, STAT6, and/or SMAD3. 2) M3 macrophages exerted an anti-tumor effect both in vitro and in vivo, which was superior to anti-tumor effects of cisplatin or M1 macrophages. 3) The anti-tumor effect of M3 macrophages was due to their anti-proliferative effect. CONCLUSIONS Development of new biotechnologies for restriction of tumor growth using in vitro reprogrammed M3 macrophages is very promising.

Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage "Switch" Phenotype.

Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-ß-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity.

C57BL/6N Mice Are More Resistant to Ehrlich Ascites Tumors Than C57BL/6J Mice: The Role of Macrophage Nitric Oxide.

BACKGROUND Effectiveness of the immune defense formed by the genotype often determines the predisposition to cancer. Nitric oxide (NO) produced by macrophages is an important element in this defense. MATERIAL AND METHODS We hypothesized that genetic characteristics of NO generation systems can predetermine the vulnerability to tumor development. The study was conducted on mice of 2 genetic substrains - C57BL/6J and C57BL/6N - with Ehrlich ascites carcinoma (EAC). NO production in the tumor was changed using ITU, an iNOS inhibitor; c-PTIO, a NO scavenger; and SNP, a NO donor. Macrophage NO production was estimated by nitrite concentration in the culture medium. iNOS content was measured by Western blot analysis. Macrophage phenotype was determined by changes in NO production, iNOS level, and CD markers of the phenotype. RESULTS The lifespan of C57BL/6N mice (n=10) with EAC was 25% longer (p<0.01) than in C57BL/6J mice (n=10). Decreased NO production 23% reduced the survival duration of C57BL/6N mice (p<0.05), which were more resistant to tumors. Elevated NO production 26% increased the survival duration of C57BL/6J mice (p<0.05), which were more susceptible to EAC. Both the NO production and the iNOS level were 1.5 times higher in C57BL/6N than in C57BL/6J mice (p<0.01). CD markers confirmed that C57BL/6N macrophages had the M1 and C57BL/6J macrophages had the M2 phenotype. CONCLUSIONS The vulnerability to the tumor development can be predetermined by genetic characteristics of the NO generation system in macrophages. The important role of NO in anti-EAC immunity should be taken into account in elaboration of new antitumor therapies.

Macrophages Reprogrammed In Vitro Towards the M1 Phenotype and Activated with LPS Extend Lifespan of Mice with Ehrlich Ascites Carcinoma.

BACKGROUND The majority of tumors trigger macrophage reprogramming from an anti-tumor M1 phenotype towards a pro-tumor M2 phenotype. The M2 phenotype promotes tumor growth. We hypothesized that increasing the number of M1 macrophages in a tumor would limit carcinogenesis and extend the lifespan of the tumor host. The aim of this study was to verify this hypothesis in Ehrlich ascites carcinoma (EAC). The objectives were to evaluate effects of 1) EAC on a macrophage phenotype and NO-producing macrophage activity in vivo; 2) ascitic fluid from mice with EAC on a macrophage phenotype and NO-producing macrophage activity in vitro; and 3) in vitro reprogrammed M1 macrophages on lifespan of mice with EAC. MATERIAL AND METHODS The study was conducted using C57BL/6J mice. RESULTS Concentration of nitrite, a stable NO metabolite and an index of NO production, was measured spectrophotometrically. Shifts of macrophage phenotype were assessed by changes in NO production as well as by amounts of CD80, a marker of M1 phenotype, and CD206, a marker of M2 phenotype. The CD markers were measured by flow cytometry. Macrophages were reprogrammed towards the M1 phenotype using two reprogramming factors: 0% FBS and 20 ng/ml IFN-γ. The study results showed that 1) EAC inhibited the macrophage NO production in vivo and reprogrammed macrophages towards the M2 phenotype; 2) ascitic fluid of mice with EAC inhibited the macrophage NO production in vitro and reprogrammed macrophages towards the M2 phenotype; and 3) injection of in vitro reprogrammed M1 macrophages into mice with EAC significantly increased the lifespan of mice. CONCLUSIONS These findings suggest that promising biotechnologies for restriction of tumor growth could be developed based on the in vitro macrophage reprogramming.

Physiological organization of immune response based on the homeostatic mechanism of matrix reprogramming: implication in tumor and biotechnology.

It is accepted that the immune system responds to pathogens with activation of antigen-independent innate and antigen-dependent adaptive immunity. However many immune events do not fit or are even inconsistent with this notion. We developed a new homeostatic model of the immune response. This model consists of four units: a sensor, a regulator, an effector and a rehabilitator. The sensor, macrophages or lymphocytes, recognize pathogenic cells and generate alarm signals. The regulator, antigen-presenting cells, Тregs and myeloid-derived suppressor cells, evaluate the signals and together with sensor cells program the effector. The effector, programmed macrophages and lymphocytes, eliminate the pathogenic cells. The rehabilitator, M2 macrophages, restrict inflammation, provide angiogenesis and reparation of tissue damage, and restore the homeostasis. We suggest the terms "immune matrix" for a biological template of immune responses to pathogens and "matrix reprogramming" for the interdependent reprogramming of different cells in the matrix. In an adequate immune response, the matrix forms a negative feedback mechanism to support the homeostasis. We defined the cellular and phenotypic composition of a tumor immune matrix. A tumor reprograms the homeostatic negative feedback mechanism of matrix into a pathogenic positive feedback mechanism. M2 macrophages play a key role in this transformation. Therefore, macrophages are an attractive target for biotechnology. Based on our hypotheses, we are developing a cell biotechnology method for creation of macrophages with a stable antitumor phenotype. We have shown that such macrophages almost doubled the survival time of mice with tumor.

Possible use of adaptation to hypoxia in Alzheimer's disease: a hypothesis.

Disorders in memory and other cognitive functions in Alzheimer's disease (AD) may result from an exhaustion of adaptive reserves in the brain. Therefore it is a challenge to find methods to increase the adaptive reserve of the organism to combat AD. Excitotoxicity, Ca2+ homeostasis disruptions, oxidative stress, disturbed synthesis of NO, and impaired cerebral circulation are suggested as key pathogenic factors of AD. At present it appears that stimulation of the self-defense systems in neural cells is a promising strategy in restricting the progression of AD. These systems include those of antioxidants, heat shock proteins (HSPs), NO, and other so-called stress-limiting systems. Non-drug activation of these systems can be achieved most efficiently by adaptation of the organism to environmental challenges, such as hypoxia. In this paper the potential of methods used in adaptive medicine is explored. The protective mechanisms of adaptation to hypoxia may be related to restriction of oxidative stress in the hippocampus, the limitation of a decrease in NO production induced by beta-amyloid, and increased density of the vascular network in the brain. In this review we selectively present data that support the idea that adaptation to hypoxia is a possible non-drug means in the prevention of AD. In our opinion this strategy may provide a break-through in the clinical approach of this disease.

Application of the nitric oxide donor SNAP to cardiomyocytes in culture provides protection against oxidative stress.

Multiple data indicates that nitric oxide (NO) donors retain immediate protective effects against different disturbances in cardiovascular system. The aim of the present study was to investigate delayed effects of nitric oxide donor S-nitroso-N-acetyl-l,l-penicillamine (SNAP) application in cardiac H9c2 cell line. Cardiomyocytes were treated with SNAP for 2h followed by 24h wash with fresh growth medium. The concentration curve was constructed in range from 0.5 to 2mM, toxicity was observed at 2mM concentration of SNAP. For the study of SNAP-induced protection against t-butyl hydroperoxide-induced oxidative injury 1mM concentration was used. Cell viability was assessed by MTT reductase activity assay; mitochondrial transmembrane potential (mdeltapsi) was measured by flow cytometry with fluorescent dye DiOC(6). Synthesis of heat-shock proteins (hsps) was analyzed by Western blot. Analysis of the cell viability and mdeltapsi reflected delayed protective effect of 1mM SNAP application against oxidative injury. SNAP in 1mM concentration caused 70% induction of hsp75 synthesis in cardiomyocytes. However, the other analyzed hsps (hsp70, hsp27, hsp60, hsp10, and CyP A) did not display any significant induction after incubation with SNAP. Present work demonstrates that the NO donor SNAP causes delayed protection against oxidative stress in H9c2 cardiomyocyte cell line, reflected in cell viability increase and preservation of the mdeltapsi. We suppose the major pathway for the development of SNAP-induced protection is through mitochondria. Induction of hsp75 expression following SNAP pretreatment is one possible way to explanation the mechanisms of this protection.

Nitric oxide production and intensity of free radical processes in young men with high normal and hypertensive blood pressure.

BACKGROUND: Young individuals with high normal blood pressure (HNBP) are at risk for hypertension. The aim of our study was to compare NO production and the intensity of free radical processes in young males with different BP levels. MATERIAL/METHODS: Male subjects aged 18-45 years with normal BP, HNBP, and hypertension underwent physical and cardiological examination. NO production was evaluated by measuring plasma nitrite and nitrate (NOx) and 24-hour urinary excretion of NOx. Lipid peroxidation (LP) intensity and serum antioxidant activity (AOA) were measured using the biochemiluminescence method. RESULTS: HNBP was associated with increased 24-hour systolic BP (SBP), diastolic BP (DBP), SBP and DBP variability, plasma and 24-hour urinary NOx and LP intensity, and decreased total AOA as compared to normotensive controls. We observed a direct nonlinear correlation between plasma NOx and SBP and between 24-hour urinary NOx and DBP, and a close inverse correlation between LP intensity and AOA in patients with HNBP. In the presence of two cardiovascular risk factors (smoking and obesity), patients with HNBP displayed higher LP intensity and lower levels of NOx than in both nonsmokers with normal body weight and control subjects. In hypertensive patients, SBP, DBP and LP intensity inversely correlated with plasma and urinary NOx. CONCLUSIONS: Activation of LP processes and depression of AOA proceed in parallel with declining NO production and severity of hypertension. Early correction of the revealed disorders before the appearance of clinical symptoms may be promising in terms of prevention and treatment of cardiovascular disease.