Ana Cañuelo

PARP inhibition sensitizes p53-deficient breast cancer cells to doxorubicin-induced apoptosis

p53 deficiency confers resistance to doxo (doxorubicin), a clinically active and widely used antitumour anthracycline antibiotic. The purpose of the present study was to investigate the reversal mechanism of doxo resistance by the potent PARP [poly(ADP-ribose) polymerase] inhibitor ANI (4-amino-1,8-naphthalimide) in the p53-deficient breast cancer cell lines EVSA-T and MDA-MB-231. The effects of ANI, in comparison with doxo alone, on doxo-induced apoptosis, were investigated in matched pairs of EVSA-T or MDA-MB-231 with or without ANI co-treatment. Doxo elicited PARP activation as determined by Western blotting and immunofluorescence of poly(ADP-ribose), and ANI enhanced the cytotoxic activity of doxo 2.3 times and in a caspase-dependent manner. The long-term cytotoxic effect was studied by a colony-forming assay. Using this assay, ANI also significantly potentiates the long-term cytotoxic effect with respect to treatment with doxo alone. Decrease in mitochondrial potential together with an increase in cytochrome c release, association of Bax with the mitochondria and caspase 3 activation were also observed in the presence of ANI. Therefore PARP inhibition may represent a novel way of selectively targeting p53-deficient breast cancer cells. The underlying mechanism is probably a potentiation of unrepaired DNA damage, shifting from DNA repair to apoptosis due to the effective inhibition of PARP activity.

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.

Tyrosol, a main phenol present in extra virgin olive oil, increases lifespan and stress resistance in Caenorhabditis elegans.

Extra virgin olive oil (EVOO) consumption has been traditionally related to a higher longevity in the human population. EVOO effects on health are often attributed to its unique mixture of phenolic compounds with tyrosol and hydroxityrosol being the most biologically active. Although these compounds have been extensively studied in terms of their antioxidant potential and its role in different pathologies, their actual connection with longevity remains unexplored. This study utilized the nematode Caenorhabditis elegans to investigate the possible effects of tyrosol in metazoan longevity. Significant lifespan extension was observed at one specific tyrosol concentration, which also induced a higher resistance to thermal and oxidative stress and delayed the appearance of a biomarker of ageing. We also report that, although tyrosol was efficiently taken up by these nematodes, it did not induce changes in development, body length or reproduction. In addition, lifespan experiments with several mutant strains revealed that components of the heat shock response (HSF-1) and the insulin pathway (DAF-2 and DAF-16) might be implicated in mediating tyrosol effects in lifespan, while caloric restriction and sirtuins do not seem to mediate its effects. Together, our results point to hormesis as a possible mechanism to explain the effects of tyrosol on longevity in C. elegans.

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Aging affects but does not eliminate the enzymatic antioxidative response to hypoxia/reoxygenation in cerebral cortex.

The effect of aging on basal and hypoxia/reoxygenation levels of both oxidative stress (protein carbonyl and TBARS) and antioxidative-enzyme activity (Cu/Zn-SOD; Mn-SOD; Catalase, CAT; Se-independent and Se-dependent glutathione peroxidase, GPX; glutathione transferase, GST and glutathione reductase, GR) has been studied in the cerebral cortex of adult and old rats. Oxidative stress markers increased with aging and show an age-dependent post-hypoxic response. Moreover, aging caused either no change (GST, GR and CAT) or an increase (Se-GPX, Cu/Zn-SOD, Mn-SOD) in the basal activity of the enzymes analysed. Only Se-independent GPX activity decreases. However, we detected an age-dependent response of SODs to the hypoxic injury. The early and sustained Cu/Zn-SOD activity rise in adult animals became late and weak in aged animals. Meanwhile, aging slowed the Mn-SOD post-hypoxic response although this activity was consistently higher in aged rats. Aging eliminated the post-hypoxic CAT response, but, perhaps offset by increased GPX activity, did not affect the GST response and slightly reduced post-hypoxic GR activity. In conclusion, aging rise basal ROS production, does not diminish or even increase the antioxidative-enzyme activity, and may slow but does not usually eliminate the enzymatic antioxidant response to the increased post-hypoxic ROS generation.

Poly(ADP-ribose) polymerase-1 modulation of in vivo response of brain hypoxia-inducible factor-1 to hypoxia/reoxygenation is mediated by nitric oxide and factor inhibiting HIF

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a nuclear protein that once activated by genotoxic agents, modulates its own activity and that of several other nuclear proteins. The absence or pharmacological inhibition of this protein has been proven to be beneficial in the treatment of different diseases involving a hypoxic situation. We previously reported that PARP‐1 modulates the hypoxia‐inducible factor‐1 (HIF‐1) response in vitro, but this effect has not yet been demonstrated in vivo. The brain is especially susceptible to hypoxic injury, and the present study demonstrates that PARP‐1 plays a major role in the post‐hypoxic response of HIF‐1α in the cerebral cortex. Immediate post‐hypoxic HIF‐1α accumulation was higher in the presence of PARP‐1, and this differential response was mediated by nitric oxide and to a lesser extent, reactive oxygen species. PARP‐1 was also found to induce a more rapid but less sustained HIF‐1 transcriptional activity by up‐regulating the factor inhibiting HIF. The implication of PARP‐1 in these results was further demonstrated by pharmacologically inhibiting PARP in wild‐type mice. In conclusion, our data suggest that PARP‐1 has an important regulatory role in the in vivo response of brain HIF‐1 to hypoxia/reoxygenation.

The nitric oxide system response to hypoxia/reoxygenation in the aged cerebral cortex.

Aged individuals are more susceptible to hypoxic insults, but little is known about the response of the nitric oxide (NO) system to hypoxia in the senescent brain. We have analysed the effect of aging on the hypobaric hypoxia/reoxygenation NO synthase (NOS) expression and activity in the cerebral cortex. In aged animals, the absence of significant changes in NOx and activity indicates a weaker response of the systems involving NO production in this pathological situation. The nNOS protein levels remained invariable and similar in both age groups after hypoxia, although in aged animals the mRNA did not change and was consistently lower than in adults. Both eNOS mRNA and protein increased shortly after hypoxia. However, although eNOS protein levels were quite similar in both age groups, the increase appeared later and was less persistent in aged animals. Real-time RT-PCR revealed a similar basal inducible NOS (iNOS) mRNA expression that responded late in reoxygenation, mainly in aged rats. However, neither iNOS protein nor activity was detected in any age group. Altogether our results indicate that aging attenuates the response of the NO system to a hypoxic injury, particularly at eNOS level, the activity of which is crucial for maintaining vascular homeostasis.

Upregulation of endothelial nitric oxide synthase maintains nitric oxide production in the cerebellum of thioacetamide cirrhotic rats

This study examines the expression and cellular distribution pattern of nitric oxide synthase (NOS) isoforms, nitrotyrosine-derived complexes, and the nitric oxide (NO) production in the cerebellum of rats with cirrhosis induced by thioacetamide (TAA). The results showed local changes in the tissue distribution pattern of the NOS isoforms and nitrated proteins in the cerebellum of these animals. Particularly, eNOS immunoreactivity in perivascular glial cells of the white matter was detected only in TAA-treated animals. In addition, although neither neuronal NOS (nNOS) nor inducible NOS (iNOS) cerebellar protein levels appeared to be affected, the endothelial NOS (eNOS) isoform significantly increased its expression, and NO production slightly augmented in TAA-treated rats. These NOS/NO changes may contribute differently to the evolution of the hepatic disease either by maintaining the guanosine monophosphate-NO signal transduction pathways and the physiological cerebellar functions or by inducing oxidative stress and cell damage. This model gives rise to the hypothesis that the upregulation of the eNOS maintains the physiological production of NO, while the iNOS is silenced and the nNOS remains unchanged. The differential NOS-distribution and expression pattern may be one of the mechanisms involved to balance cerebellar NO production in order to minimize TAA toxic injury. These data help elucidate the role of the NOS/NO system in the development and progress of hepatic encephalopathy associated with TAA cirrhosis.

Nitric oxide modulates hypoxia-inducible factor-1 and poly(ADP-ribose) polymerase-1 cross talk in response to hypobaric hypoxia

The physiological response to hypobaric hypoxia represents a complex network of biochemical pathways in which the nitrergic system plays an important role. Previous studies have provided evidence for an interplay between the hypoxia-inducible factor-1 (HIF-1) and poly(ADP-ribose) polymerase-1 (PARP-1) under hypoxia. Here, we evaluate the potential involvement of nitric oxide (NO) in the cross talk between these two proteins. With this aim, we studied comparatively the effect of pharmacological inhibitors of NO production or PARP activity in the response of the mouse cerebral cortex to 4 h of exposure to a simulated altitude of 31,000 ft. Particularly, we analyzed the NO and reactive oxygen species production, the expression of NO synthase (NOS) isoforms, PARP-1 activity, HIF-1α expression and HIF-1 transcriptional activity, the protein level of the factor inhibiting HIF, and, finally, beclin-1 and fractin expression, as markers of cellular damage. Our results demonstrate that the reduction of NO level did not affect reactive oxygen species production but significantly 1) dampened the posthypoxic increase in neuronal NOS and inducible NOS expression without altering endothelial NOS protein level; 2) prevented PARP activation; 3) decreased HIF-1α response to hypoxia; 4) achieved a higher long-term HIF-1 transcriptional activity by reducing factor inhibiting HIF expression; and 5) reduced hypoxic damage. The pharmacological inhibition of PARP reproduced the NOS expression pattern and the HIF-1α response observed in NOS-inhibited mice, supporting its involvement in the NO-dependent regulation of hypoxia. As a whole, these results provide new data about the molecular mechanism underlying the beneficial effects of controlling NO production under hypobaric hypoxic conditions.

Oral quercetin supplementation hampers skeletal muscle adaptations in response to exercise training

We aimed to test exercise‐induced adaptations on skeletal muscle when quercetin is supplemented. Four groups of rats were tested: quercetin sedentary, quercetin exercised, placebo sedentary, and placebo exercised. Treadmill exercise training took place 5 days a week for 6 weeks. Quercetin groups were supplemented with quercetin, via gavage, on alternate days throughout the experimental period. Sirtuin 1 (SIRT1), peroxisome proliferator‐activated receptor γ coactivator‐1α mRNA levels, mitochondrial DNA (mtDNA) content, and citrate synthase (CS) activity were measured on quadriceps muscle. Redox status was also quantified by measuring muscle antioxidant enzymatic activity and oxidative damage product, such as protein carbonyl content (PCC). Quercetin supplementation increased oxidative damage in both exercised and sedentary rats by inducing higher amounts of PCC (P < 0.001). Quercetin supplementation caused higher catalase (P < 0.001) and superoxide dismutase (P < 0.05) activity in the non‐exercised animals, but not when quercetin is supplemented during exercise. Quercetin supplementation increased SIRT1 expression, but when quercetin is supplemented during exercise, this effect is abolished (P < 0.001). The combination of exercise and quercetin supplementation caused lower (P < 0.05) mtDNA content and CS activity when compared with exercise alone. Quercetin supplementation during exercise provides a disadvantage to exercise‐induced muscle adaptations.