Ebru Bodur

A Nanomedicine Transports a Peptide Caspase-3 Inhibitor across the Blood–Brain Barrier and Provides Neuroprotection

Caspases play an important role as mediators of cell death in acute and chronic neurological disorders. Although peptide inhibitors of caspases provide neuroprotection, they have to be administered intracerebroventricularly because they cannot cross the blood–brain barrier (BBB). Herein, we present a nanocarrier system that can transfer chitosan nanospheres loaded with N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK), a relatively specific caspase-3 inhibitor, across BBB. Caspase-3 was chosen as a pharmacological target because of its central role in cell death. Polyethylene glycol-coated nanospheres were conjugated to an anti-mouse transferrin receptor monoclonal antibody (TfRMAb) that selectively recognizes the TfR type 1 on the cerebral vasculature. We demonstrate with intravital microscopy that this nanomedicine is rapidly transported across the BBB without being measurably taken up by liver and spleen. Pre- or post-treatment (2 h) with intravenously injected Z-DEVD-FMK-loaded nanospheres dose dependently decreased the infarct volume, neurological deficit, and ischemia-induced caspase-3 activity in mice subjected to 2 h of MCA occlusion and 24 h of reperfusion, suggesting that they released an amount of peptide sufficient to inhibit caspase activity. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17 after closure of the BBB. Neither nanospheres functionalized with TfRMAb but not loaded with Z-DEVD-FMK nor nanospheres lacking TfRMAb but loaded with Z-DEVD-FMK had any effect on either paradigm, suggesting that inhibition of caspase activity and subsequent neuroprotection were due to efficient penetration of the peptide into brain. Thus, chitosan nanospheres open new and exciting opportunities for brain delivery of biologically active peptides that are useful for the treatment of CNS disorders.

Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells

Statins show antiproliferative activity in various cancer cells. The aim of this study was to evaluate the effects of rosuvastatin treatment on papillary thyroid carcinoma. The papillary thyroid carcinoma (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines were treated with rosuvastatin at 12.5, 18.5, 25, 50, 100, and 200 μM concentrations. After 48 and 72 h of rosuvastatin treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Ki-67 immunolabeling, FACS analysis, electron microscopy, caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis were performed. Decreased cell viability and G1 phase arrest were detected in papillary thyroid cell line treated with rosuvastatin. Positive immunoreactivity of Ki-67 and dose-dependent increase in S phase on Nthy-ori 3-1 cells were also detected. B-CPAP cells showed intense vacuolisation and autophagosomes with low concentrations and 48 h incubations, while Nthy-ori 3-1 cells showed these changes at higher concentrations. A decrease in the percentage of cells showing autophagy was determined with increasing concentrations of rosuvastatin in B-CPAP cells. Rosuvastatin treatment also caused a dose- and time-dependent increase in caspase-3 activity and apoptotic index by TUNEL assay in B-CPAP cells compared with the Nthy-ori 3-1 cells. Apoptotic cells with nuclear condensation and fragmentation were observed in B-CPAP cell line. Rosuvastatin induced autophagic changes in B-CPAP papillary thyroid cancer cells in lower doses and caused a shift from autophagy to apoptosis. Rosuvastatin may be an alternative treatment for refractory papillary thyroid cancer. Further in vivo studies are necessary to clarify the effects of rosuvastatin in papillary thyroid carcinoma and the clinical implications of rosuvastatin treatment.

A comparative study of treatment for brain edema: Magnesium sulphate versus dexamethasone sodium phosphate

Treatments for brain edema are important and one of the major options is corticosteroids. Cell membrane stabilization and prevention of formation of free radicals are the main mechanisms of action of steroids in edema treatment. As an alternative therapeutic agent, magnesium sulphate has been used for its neuroprotective effect in various injury models. In our animal model of brain injury, cold has been used in Sprague-Dawley rats. After brain injury, magnesium sulphate (600 mg/kg) or dexamethasone sodium phosphate (0.2 mg/kg) were administered to experimental groups. The degree of brain edema and lipid peroxidation was evaluated using the wet-dry weight method, the determination of malondialdehyde (MDA) levels and an ultrastructural grading system. Magnesium sulphate treatment was found to be the most effective choice due to the absence of side effects and comparable efficacy to corticosteroids.

Atorvastatin efficiency after traumatic brain injury in rats

BACKGROUND The neuroprotective effects of statins possibly depend on their pleiotropic effect such as antioxidative and anti-inflammatory properties. In this study, we have evaluated the efficiency of atorvastatin on brain edema, lipid peroxidation, and ultrastructural changes in TBI animal model. METHODS Modified Feeney method has been used for the trauma model in rats. Only craniectomy for group A and trauma after craniectomy for group B was the procedure for animals. For the trauma, rods weighing 24 g were dropped on a foot plate just over the dura. Atorvastatin (1 mg/kg, IP) was administered to the animals in group C after craniectomy and trauma; but on the other hand, animals in group D received only 0.5 mL PEG as the vehicle. Brains were harvested 24 hours after the trauma for the assays of wet-dry weight, lipid peroxidation level, and ultrastructural investigations. Lipid peroxidation levels, TEM, and UNGS were the investigated parameters. The statistical comparisons between the groups were investigated by 1-way ANOVA and post hoc analysis by Duncan and Dunnett T3 test within the groups at the significance level P = .05. RESULTS Trauma increased water contents of the brain tissues and lipid peroxidation levels in groups B and D. When compared with the results of group B (brain edema, 84.694% +/- 1.510%; lipid peroxidation, 74.932 +/- 2.491 nmol/g tissue), atorvastatin (1 mg/kg) significantly decreased brain edema (77.362% +/- 1.448%), lipid peroxidation level (58.335 +/- 3.980 nmol/g tissue), and UNGS scores in group C (P < 0.05). CONCLUSION In this descriptive study, the remarkable improvements of atorvastatin on brain edema, lipid peroxidation, and ultrastructural investigations encouraged us for a further dose optimization study.

Transport of a caspase inhibitor across the blood-brain barrier by chitosan nanoparticles.

The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Several approaches taking advantage of molecular and physicochemical characteristics of the BBB have been developed recently to improve drug delivery to the brain. Here, we introduce a nanomedicine that can efficiently transport BBB-impermeable peptides to the brain. This nanomedicine is made of chitosan nanoparticles into which considerable amounts of a peptide can be incorporated. The nanoparticle surface is modified with polyethylene glycol to enhance the plasma residence time by preventing their capture by the reticuloendothelial system. Monoclonal antibodies against the transferrin receptor (TfR), which is highly expressed on the brain capillary endothelium, are conjugated to nanoparticles via biotin-streptavidin bonds. The activation of TfR by the nanoparticle-antibody complex induces transcytosis and thus delivers the loaded drug to the brain. Penetration of nanoparticles to the brain can be illustrated in vivo by intravital microscopy as well as ex vivo by fluorescence or electron microscopy. N-Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK)-loaded nanoparticles rapidly release their contents within brain parenchyma, inhibit ischemia-induced caspase-3 activity, and thereby provide neuroprotection.

Effects of conjugated linoleic acid supplementation and exercise on post-heparin lipoprotein lipase, butyrylcholinesterase, blood lipid profile and glucose metabolism in young men

This study was designed to investigate the effects of conjugated linoleic acid (CLA) supplementation and endurance exercise training-induced changes on post-heparin lipoprotein lipase (PH-LPL) and butyrylcholinesterase (BChE) activities along with leptin, insulin and lipid levels in plasma by a randomized double blind experiment. Eighteen sedentary male volunteers were randomly divided into CLA and Placebo (PLC) supplementation groups. Both groups underwent daily supplementation of either 3g CLA or 3g placebo for 30 days, respectively, and performed exercise on a bicycle ergometer 3 times per week for 30-40 min at 50% VO2 peak workload. For plasma glucose, insulin and leptin levels and BChE activity fasting blood was used. For PH-LPL measurements, blood was collected 15 min after 50 IU/kg iv heparin injection. In all groups, there is a statistically significant decrease in BChE (p = 0.03, p = 0.02) and leptin (p = 0.002), insulin and HOMA-IR levels (p = 0.02). Exercise with or without CLA supplementation decreased insulin levels and increased insulin sensitivity. PH-LPL activity was increased significantly in both groups, displaying increased fatty acid mobilization. We conclude that though CLA supplementation and exercise can affect these parameters, CLA is not more effective than exercise alone. Hence, a prolonged supplementation regime may be more effective. Taken together in our small study group, our findings display that BChE is a potential marker for synthetic function of liver, fat metabolism, an obesity marker, a function long overlooked.

Plasma 3‐nitrotyrosine estimates the reperfusion‐induced cerebrovascular stress, whereas matrix metalloproteinases mainly reflect plasma activity: a study in patients treated with thrombolysis or endovascular recanalization

Cerebral reperfusion injury may account for complications of thrombolysis and endovascular recanalization. Experimental studies have shown that brain matrix metalloproteinase (MMP) activity increases during reperfusion and is correlated with oxidative/nitrative stress. Increased plasma MMP levels have been reported in stroke, but no information is available for reperfusion‐induced plasma MMP and 3‐nitrotyrosine (3‐NT, a marker of oxidative/nitrative stress) changes immediately after recanalization. We obtained plasma from 29 patients undergoing endovascular recanalization, 12 patients treated with thrombolysis, and six control patients having diagnostic angiogram before and 1,3, and 24 h after treatment to investigate the effect of cerebral reperfusion on plasma MMP gelatinolytic activity and 3‐NT level. Hypoperfusion was shown distal to the stenotic artery in endovascular treatment patients. Presence of an occluded artery and recanalization was documented in thrombolysis patients. A significant increase was detected in plasma 3‐NT levels 3 and 24 h after stenting/angioplasty. Plasma MMP‐9 gelatinolytic activity rose more than 50% of the pre‐treatment level in 12 of 29 patients. However, this was not statistically significant and not correlated with any of the clinical or radiological correlates of reperfusion injury (e.g., hyperperfusion and hemorrhage). After thrombolysis, a significant increase in plasma MMP‐9 gelatinolytic activity at 3 and 24 h and the cleaved form of MMP‐9 were detected. 3‐NT levels increased by 44% and 62% at 3 and 24 h, which did not achieve statistical significance, but was highly correlated with admission NIH Stroke Scale (r = 0.930 p < 0.001). No change was detected in MMP‐2 in all groups. In conclusion, these data suggest that the increased plasma MMP‐9 levels is not a direct measure of MMP‐9 activity in the reperfused brain but rather a consequence of tissue plasminogen activator infusion, whereas plasma 3‐NT levels appear to originate from the reperfused brain vasculature. The changes in 3‐NT levels may therefore be useful to monitor oxygen/nitrogen radical formation during reperfusion with serial measurements.

Counter-regulation of cholinesterases: differential activation of PKC and ERK signaling in retinal cells through BChE knockdown.

The ubiquitous cholinesterase (ChE) enzymes, functioning in the termination of acetylcholine mediated neural transmission, are also reported to have additional functions. Through application of siRNAs against butyrylcholinesterase (BChE) in R28 cells, a retinal cell line with pluripotent properties, a counter-regulation between ChEs was revealed. BChE knock down resulted in an up-regulation of not only acetylcholinesterase (AChE), but also altered the signaling status of PKC and ERK. Knockdown of BChE modified ERK signaling most notably through ERK1/2 proteins, together with the transcription activator P90RSK1 and c-fos. Stimulation of the R28 cell line by forskolin revealed that ChEs are involved in an intricate cross talk between different signaling pathways. Forskolin-stimulated R28 cells displayed a robust cholinergic response, as detected by both electrophysiology and ChE expression, and changed the activation status of PKC/ERK signaling pathways. The findings in R28 cells show that ChE expressions are inversely co-regulated and act through the transcription factors c-fos and P90RSK1. Since R28 cells have the capacity to differentiate into different cell types through stimulation of signaling pathways, ChEs are likely to be associated with cell fate determination, rather than just terminating cholinergic responses.

Human serum butyrylcholinesterase interactions with cisplatin and cyclophosphamide

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 +/- 50.4 microM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 x 10(-5) M(-1) min(-1), 0.063 min(-1), and 9.83 x 10(-4) M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 +/- 71.2 microM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.

The effect of HDL-bound and free PON1 on copper-induced LDL oxidation.

Oxidative modification of LDL plays an important role in the development of atherosclerosis. High-density lipoprotein (HDL) confers protection against atherosclerosis and the antioxidative properties of paraoxonase 1 (PON1) has been suggested to contribute to this effect of HDL. The PON1 exist in two major polymorphic forms (Q and R), which regulate the concentration and activity of the enzyme and alter its ability to prevent lipid oxidation. However, the association of Q192R polymorphism with PON1s capacity to protect against LDL lipoperoxidation is controversial. The aim of this study was to evaluate the effects of the purified PON1 Q192R and the partially purified HDL-bound PON1 Q192R isoenzymes (HDL-PON1 Q192R) on LDL oxidation, with respect to their arylesterase/homocysteine thiolactonase (HTLase) activities. Cupric ion-induced LDL oxidation was reduced up to 48% by purified PON1 Q192, but only 33% by an equivalent activity of PON1 R192. HDL-PON1 Q192 isoenzyme caused a 65% reduction, whereas HDL-PON1 R192 isoenzyme caused only 46% reduction in copper ion-induced LDL oxidation. These findings reflect the fact that PON1 Q and PON1 R allozymes may have different protective characteristics against LDL oxidation. The protection against LDL oxidation provided by HDL-PON1 Q192R isoenzymes is more prominent than the purified soluble enzymes. Inhibition of the Ca(+2)-dependent PON1 Q192R arylesterase/HTLase by the metal chelator EDTA, did not alter PON1s ability to inhibit LDL oxidation. These studies indicate that the active site involvement of the purified enzyme is not similar to the HDL-bound one, in terms of both PON1 arylesterase/HTLase activity and the protection of LDL from copper ion-induced oxidation. Moreover, PON1s ability to protect LDL from oxidation does not seem to require calcium.