Balazs Sumegi

Direct effect of Taxol on free radical formation and mitochondrial permeability transition.

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.

Novel phenanthridinone inhibitors of poly(adenosine 5'-diphosphate-ribose) synthetase: Potent cytoprotective and antishock agents

ObjectiveTo synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5′-diphosphate [ADP]-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock. DesignRandomized, prospective laboratory study. SettingResearch laboratory. SubjectsMurine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats. InterventionsMacrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis. Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity. Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1–10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor. The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability. Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally). PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment. Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured. In addition, survival rates and vascular contractile and relaxant responses were recorded. Measurements and Main ResultsAppropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents. The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM. PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34. The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized). The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages. PJ34 was selected for the subsequent in vivo studies. PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse. PJ34 reduced peak plasma concentrations of tumor necrosis factor-&agr;, interleukin-1&bgr;, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats. PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut. Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34. Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment). ConclusionsThe novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

Cardioprotection by resveratrol: A human clinical trial in patients with stable coronary artery disease

Several beneficial effects of resveratrol (RES), a natural antioxidant present in red wine have already been described. The aim of our study was to investigate if RES had a clinically measurable cardioprotective effect in patients after myocardial infarction. In this double-blind, placebo controlled trial 40 post-infarction Caucasian patients were randomized into two groups. One group received 10 mg RES capsule daily for 3 months. Systolic and diastolic left ventricular function, flow-mediated vasodilation (FMD), several laboratory and hemorheological parameters were measured before and after the treatment. Left ventricular ejection fraction showed an increasing tendency (ns) by RES treatment. However, left ventricular diastolic function was improved significantly (p < 0.01) by RES. A significant improvement in endothelial function measured by FMD was also observed (p < 0.05). Low-density lipoprotein (LDL) level significantly decreased (p < 0.05) in the RES treated group. Red blood cell deformability decreased and platelet aggregation increased significantly in the placebo group (p < 0.05), while resveratrol treatment has prevented these unfavourable changes. Concerning other measured parameters no significant changes were observed neither in placebo nor in RES group. Our results show that resveratrol improved left ventricle diastolic function, endothelial function, lowered LDL-cholesterol level and protected against unfavourable hemorheological changes measured in patients with coronary artery disease (CAD).

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.

BGP-15 — a novel poly(ADP-ribose) polymerase inhibitor — protects against nephrotoxicity of cisplatin without compromising its antitumor activity

Nephrotoxicity is one of the major dose limiting side effects of cisplatin chemotherapy. The antitumor and toxic effects are mediated in part by different mechanisms, thus, permitting a selective inhibition of certain side effects. The influence of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) - a poly(ADP-ribose) polymerase (PARP) inhibitor - on the nephrotoxicity and antitumor efficacy of cisplatin has been evaluated in experimental models. BGP-15 either blocked or significantly reduced (60-90% in 100-200 mg/kg oral dose) cisplatin induced increase in serum urea and creatinine level in mice and rats and prevented the structural degeneration of the kidney, as well. The nephroprotective effect of BGP-15 treatment was revealed also in living mice by MRI analysis manifesting in the lack of oedema which otherwise developed as a result of cisplatin treatment. The protective effect was accompanied by inhibition of cisplatin-induced poly-ADP-ribosylation and by the restoration of the disturbed energy metabolism. The preservation of ATP level in the kidney was demonstrated in vivo by localized NMR spectroscopy. BGP-15 decreased cisplatin-induced ROS production in rat kidney mitochondria and improved the antioxidant status of the kidney in mice with cisplatin-induced nephropathy. In rat kidney, cisplatin caused a decrease in the level of Bcl-x, a mitochondrial protective protein, and this was normalized by BGP-15 treatment. On the other hand, BGP-15 did not inhibit the antitumor efficacy of cisplatin in cell culture and in transplantable solid tumors of mice. Treatment with BGP-15 increased the mean survival time of cisplatin-treated P-388 leukemia bearing mice from 13 to 19 days. PARP inhibitors have been demonstrated to diminish the consequences of free radical-induced damage, and this is related to the chemoprotective effect of BGP-15, a novel PARP inhibitor. Based on these results, we propose that BGP-15 represents a novel, non-thiol chemoprotective agent.

Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin, a Major Red Wine Polyphenol

Background Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. Methods & Findings The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. Conclusions These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease.

Inhibiting poly(ADP-ribose) polymerase: a potential therapy against oligodendrocyte death

Oligodendrocyte loss and demyelination are major pathological hallmarks of multiple sclerosis. In pattern III lesions, inflammation is minor in the early stages, and oligodendrocyte apoptosis prevails, which appears to be mediated at least in part through mitochondrial injury. Here, we demonstrate poly(ADP-ribose) polymerase activation and apoptosis inducing factor nuclear translocation within apoptotic oligodendrocytes in such multiple sclerosis lesions. The same morphological and molecular pathology was observed in an experimental model of primary demyelination, induced by the mitochondrial toxin cuprizone. Inhibition of poly(ADP-ribose) polymerase in this model attenuated oligodendrocyte depletion and decreased demyelination. Poly(ADP-ribose) polymerase inhibition suppressed c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation, increased the activation of the cytoprotective phosphatidylinositol-3 kinase-Akt pathway and prevented caspase-independent apoptosis inducing factor-mediated apoptosis. Our data indicate that poly(ADP-ribose) polymerase activation plays a crucial role in the pathogenesis of pattern III multiple sclerosis lesions. Since poly(ADP-ribose) polymerase inhibition was also effective in the inflammatory model of multiple sclerosis, it may target all subtypes of multiple sclerosis, either by preventing oligodendrocyte death or attenuating inflammation.

PARP inhibition delays transition of hypertensive cardiopathy to heart failure in spontaneously hypertensive rats

AIMS Oxidative stress followed by abnormal signalling can play a critical role in the development of long-term, high blood pressure-induced cardiac remodelling in heart failure (HF). Since oxidative stress-induced poly(ADP-ribose)polymerase (PARP) activation and cell death have been observed in several experimental models, we investigated the possibility that inhibition of nuclear PARP improves cardiac performance and delays transition from hypertensive cardiopathy to HF in a spontaneously hypertensive rat (SHR) model of HF. METHODS AND RESULTS SHRs were divided into two groups: one received no treatment (SHR-C) and the other (SHR-L) received 5 mg/kg/day L-2286 (PARP-inhibitor) orally for 46 weeks. A third group was a normotensive age-matched control group (CFY) and a fourth was a normotensive age-matched group receiving L-2286 treatment 5 mg/kg/day (CFY+L). At the beginning of the study, systolic function was similar in both CFY and SHR groups. In the SHR-C group at the end of the study, eccentric hypertrophy with poor left ventricular (LV) systolic function was observed, while PARP inhibitor treatment preserved systolic LV function. Due to these favourable changes, the survival rate of SHRs was significantly improved (P < 0.01) by the administration of the PARP inhibitor (L-2286). The PARP inhibitor used did not affect the elevated blood pressure of SHR rats, but moderated the level of plasma-BNP (P < 0.01) and favourably influenced all the measured gravimetric parameters (P < 0.05) and the extent of myocardial fibrosis (P < 0.05). The inhibition of PARP increased the phosporylation of Akt-1/GSK-3beta (P < 0.01), ERK 1/2 (P < 0.01), and PKC epsilon (P < 0.01), and decreased the phosphorylation of JNK (P < 0.05), p-38 MAPK (P < 0.01), PKC pan betaII and PKC zeta/lambda (P < 0.01), and PKC alpha/betaII and delta (P < 0.05). CONCLUSION These data demonstrate that chronic inhibition of PARP induces long-term favourable changes in the most important signalling pathways related to oxidative stress. PARP inhibition also prevents remodelling, preserves systolic function, and delays transition of hypertensive cardiopathy to HF in SHRs.

Concentration dependent mitochondrial effect of amiodarone.

Although, the antiarrhythmic effect of amiodarone is well characterized, its effect on post-ischemic heart and cardiomyocytes, as well as the mechanism of its toxicity on extracardiac tissues is still poorly understood. In this study, we analyzed energy metabolism in situ during ischemia-reperfusion in Langendorff-perfused heart model by measuring the high-energy phosphate metabolites using 31P NMR spectroscopy. The toxicity of amiodarone on cardiomyocytes and cell lines of extracardiac origin, as well as direct effect of the drug on mitochondrial functions in isolated mitochondria was also analyzed. Amiodarone, when was present at low concentrations and predominantly in membrane bound form, protected heart and mitochondrial energy metabolism from ischemia-reperfusion-induced damages in Langendorff-perfused heart model. Toxicity of the drug was significantly higher on hepatocytes and pancreatic cells than on cardiomyocytes. In isolated mitochondria, amiodarone did not induce reactive oxygen species formation, while it affected mitochondrial permeability transition in a concentration dependent way. Up to the concentration of 10 microM, the drug considerably inhibited Ca(2+)-induced permeability transition, while at higher concentrations it induced a cyclosporin A independent permeability transition of its own. At concentrations where it inhibited the Ca(2+)-induced permeability transition (IC(50)=3.9+/-0.8 microM), it did not affect, between 6 and 30 microM it uncoupled, while, at higher concentrations it inhibited the respiratory chain. Thus, the concentration dependent nature of amiodarones effect on permeability transition together with the different sensitivities of the tissues toward amiodarone can be involved in the beneficial cardiac and the simultaneous toxic extracardiac effects of the drug.

Differential expression of Akt/protein kinase B, Bcl-2 and Bax proteins in human leiomyoma and myometrium

The expression and activation of serine/threonine protein kinase, Akt, in leiomyoma and in adjacent myometrium of human uteri was studied parallel with the changes of Bcl-2, Bax proteins, estrogen and progesterone receptors during menstrual cycle and early stage of the menopause. Abundant expression of Akt protein was detected in the studied tissues during menstrual cycle, the rate of increase was higher in leiomyoma than in corresponding myometrium. The expression of estrogen receptor alpha, progesterone receptor and of Bcl-2 protein changed parallel with that of Akt protein. The level of phosphorylated Akt (pAkt(473)) was seen only in leiomyoma samples from the growing period of tumors. At early stage of menopause levels of all studied proteins were lower than that in the menstrual cycle with the exception of Bax protein expression, which was high in leiomyoma. Our data suggest the involvement of phosphatidylinositol 3-kinase/Akt signaling in the pathomechanism of leiomyoma.