Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)(2)] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38(MAPK)/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N`]copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

Aims Glycosylation with beta-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Methods and results Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 mu M) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. Conclusion We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.

Neonatal mitochondrial encephaloneuromyopathy due to a defect of mitochondrial protein synthesis

Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). We studied a male infant with severe congenital encephalopathy, peripheral neuropathy, and myopathy. The patient`s lactic acidosis and biochemical defects of respiratory chain complexes I, III, and IV in muscle indicated that he had a mitochondrial disorder while parental consanguinity suggested autosomal recessive inheritance. Cultured fibroblasts from the patient showed a generalized defect of mitochondrial protein synthesis. Fusion of cells from the patient with 143B206 rho(0) cells devoid of mtDNA restored cytochrome c oxidase activity confirming the nDNA origin of the disease. Our studies indicate that the patient has a novel autosomal recessive defect of mitochondrial protein synthesis. (C) 2008 Elsevier B.V. All rights reserved.

Catalase-peroxidase activity is decreased in a Caulobacter crescentus rho mutant

A Caulobacter crescentus rho:Tn5 mutant strain presenting a partially functional transcription termination factor Rho is highly sensitive to hydrogen peroxide in both exponential and stationary phases. The mutant was shown to be permanently under oxidative stress, based on fluorophore oxidation, and also to be sensitive to tert-butyl hydroperoxide and paraquat. However, the results showed that the activities of superoxide dismutases CuZnSOD and FeSOD and the alkylhydroperoxide reductase ahpC mRNA levels in the rho mutant were comparable to the wild-type control in the exponential and stationary phases. In contrast, the KatG catalase activity of the rho mutant strain was drastically decreased and did not show the expected increase in the stationary phase compared with the exponential phase. Transcription of the katG gene was increased in the rho mutant and the levels of the immunoreactive KatG protein do not differ considerably compared with the wild type in the stationary phase, suggesting that KatG activity is affected in a translational or a post-translational step.

Arginine vasopressin controls p27(KiP1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated beta-galactosidase (SA-beta Gal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP`s deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187. (C) 2011 Elsevier B.V. All rights reserved.

The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.