Rubén Martínez-Romero

Inhibition of Poly(ADP-Ribose) Polymerase Modulates Tumor-Related Gene Expression, Including Hypoxia-Inducible Factor-1 Activation, during Skin Carcinogenesis

Poly(ADP-ribose) polymerase (PARP)-1, an enzyme that catalyzes the attachment of ADP ribose to target proteins, acts as a component of enhancer/promoter regulatory complexes. In the present study, we show that pharmacologic inhibition of PARP-1 with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) results in a strong delay in tumor formation and in a dramatic reduction in tumor size and multiplicity during 7,12-dimethylbenz(a)anthracene plus 12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis. This observation was parallel with a reduction in the skin inflammatory infiltrate in DPQ-treated mice and tumor vasculogenesis. Inhibition of PARP also affected activator protein-1 (AP-1) activation but not nuclear factor-kappaB (NF-kappaB). Using cDNA expression array analysis, a substantial difference in key tumor-related gene expression was found between chemically induced mice treated or not with PARP inhibitor and also between wild-type and parp-1 knockout mice. Most important differences were found in gene expression for Nfkbiz, S100a9, Hif-1alpha, and other genes involved in carcinogenesis and inflammation. These results were corroborated by real-time PCR. Moreover, the transcriptional activity of hypoxia-inducible factor-1alpha (HIF-1alpha) was compromised by PARP inhibition or in PARP-1-deficient cells, as measured by gene reporter assays and the expression of key target genes for HIF-1alpha. Tumor vasculature was also strongly inhibited in PARP-1-deficient mice and by DPQ. In summary, this study shows that inhibition of PARP on itself is able to control tumor growth, and PARP inhibition or genetic deletion of PARP-1 prevents from tumor promotion through their ability to cooperate with the activation AP-1, NF-kappaB, and HIF-1alpha.

PARP-1 modulates deferoxamine-induced HIF-1α accumulation through the regulation of nitric oxide and oxidative stress

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a nuclear protein that, once activated by genotoxic agents, modulates the activity of several nuclear proteins including itself. Previous studies have established that PARP‐1 inhibition may provide benefit in the treatment of different diseases, particularly those involving a hypoxic situation, in which an increased oxidative and nitrosative stress occurs. One of the most important transcription factors involved in the response to the hypoxic situation is the hypoxia‐inducible factor‐1 (HIF‐1). The activity of HIF‐1 is determined by the accumulation of its α subunit which is regulated, in part, by oxidative stress (ROS) and nitric oxide (NO), both of them highly dependent on PARP‐1. Besides, HIF‐1α can be induced by iron chelators such as deferoxamine (DFO). In this sense, the therapeutical use of DFO to strengthen the post‐hypoxic response has recently been proposed. Taking into account the increasing interest and potential clinical applications of PARP inhibition and DFO treatment, we have evaluated the impact of PARP‐1 on HIF‐1α accumulation induced by treatment with DFO. Our results show that, in DFO treated cells, PARP‐1 gene deletion or inhibition decreases HIF‐1α accumulation. This lower HIF‐1α stabilization is parallel to a decreased inducible NO synthase induction and NO production, a higher response of some antioxidant enzymes (particularly glutathione peroxidase and glutathione reductase) and a lower ROS level. Taken together, these results suggest that the absence of PARP‐1 modulates HIF‐1 accumulation by reducing both NO and oxidative stress. J. Cell. Biochem. 104: 2248–2260, 2008.

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.

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.

Combined data mining strategy for the systematic identification of sport drug metabolites in urine by liquid chromatography time-of-flight mass spectrometry

The development of comprehensive methods able to tackle with the systematic identification of drug metabolites in an automated fashion is of great interest. In this article, a strategy based on the combined use of two complementary data mining tools is proposed for the screening and systematic detection and identification of urinary drug metabolites by liquid chromatography full-scan high resolution mass spectrometry. The proposed methodology is based on the use of accurate mass extraction of diagnostic ions (compound-dependent information) from in-source CID fragmentation without precursor ion isolation along with the use of automated mass extraction of accurate-mass shifts corresponding to typical biotransformations (non compound-dependent information) that xenobiotics usually undergo when metabolized. The combined strategy was evaluated using LC-TOFMS with a suite of nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol, ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline), after single doses administered to rats. The metabolite identification coverage rate obtained with the systematic method (compared to existing literature) was satisfactory, and provided the identification of several non-previously reported metabolites. In addition, the combined information obtained helps to minimize the number of false positives. As an example, the systematic identification of urinary metabolites of propranolol enabled the identification of up to 24 metabolites, 15 of them non previously described in literature, which is a valuable indicator of the usefulness of the proposed systematic procedure.

The hypoxic preconditioning agent deferoxamine induces poly(ADP-ribose) polymerase-1-dependent inhibition of the mitochondrial respiratory chain

We previously reported that treatment with a single dose of deferoxamine (DFO), which acts as a hypoxic-mimetic agent, only induces reactive oxygen species (ROS) production in the presence of poly(ADP-ribose) polymerase (PARP-1). Given that mitochondria are one of the main sources of ROS, the present study was designed to assess the effect of DFO treatment on the activity of mitochondrial respiratory chain complexes, and more importantly, to determine whether this effect is modulated by PARP-1. We found that DFO treatment induced a progressive decline in complex II and IV activity, but that this activity was preserved in PARP-1 knock-out cells, demonstrating that this decrease is mediated by PARP-1. We also confirmed that complex II inhibition after DFO treatment occurs in parallel with poly-ADP ribosylation. Consequently, we recommend that PARP-1 activation be taken into account when using DFO as a hypoxia-mimetic agent, because it mediates alteration of the mitochondrial respiratory chain.

Cardiotrophin-1 decreases liver apoptosis through calpastatin induction

BACKGROUND Cardiotrophin-1 (CT1) has been used to prevent cell death in different models of liver injury in rats. D-galactosamine induces cell death in culture rat and human hepatocytes. The present study evaluated the cytoprotective effects of CT1 in an experimental model of apoptosis induced by D-galactosamine in hepatocytes. METHODS DNA fragmentation, calpain activity and Western blots of caspase-3, calpastatin and Stat3, and Akt phosphorylation were measured. Stat3 and Akt inhibitors were used to analyze the mechanisms of action of CT1. RESULTS CT1 caused an increase in Stat3 and Akt phosphorylation and a decrease of DNA fragmentation, calpain activity, and caspase-3 induced by D-galactosamine. The reduction of calpain activity by CT1 was associated with an increase of calpastatin (its endogenous inhibitor). The effects of CT1 were also dependent on the activation of Sta3 or Akt. CONCLUSIONS CT1 decreases cell death through a mechanism related to Stat3 and Akt phosphorylation and activation of calpastatin in D-galactosamine-treated hepatocytes.

Plasmatic nitric oxide correlates with weight and red cell distribution width in exercised rats supplemented with quercetin.

Abstract Quercetin is suggested as a nitric oxide regulator which may in turn influence blood parameters and weight gain. Wistar rats were classified as: quercetin-exercise training, QT; placebo-exercise training, PT; quercetin-sedentary, QS; and placebo sedentary, PS. After 6 weeks of treatment with quercetin and/or exercise, an incremental test was run to measure oxygen consumption. QT had lower levels of NO compared with PS (p = 0.029) and QS (p = 0.002). Red cell distribution width increased in both exercised groups, especially in the QT group (p < 0.001). Pearson correlation analysis showed that nitric oxide levels were associated with weight (r = 0.675) and red distribution width (r = −0814) in the QT group. Quercetin effect on NO production seems to be more powerful when it is supplemented during exercise training. Moreover, RDW relationship with NO production need to be further investigated in regards to health.