RESUMEN
The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Paclitaxel , Péptidos , Puntos Cuánticos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HT29 , Humanos , Paclitaxel/química , Paclitaxel/farmacología , Péptidos/química , Péptidos/farmacología , Puntos Cuánticos/química , Puntos Cuánticos/uso terapéuticoRESUMEN
BACKGROUND: Physical exercise plays an important role in bone mineralization as well as factors involved in bone metabolism influence the athletic performance. In European countries, soccer is the most popular sport. The aim of the study was to investigate bone metabolism, bone mass and structural integrity profile in professional male adult football players. METHODS: Sixteen professional male football players from a single team of the Second division Italian League (mean age 22.4±0.7 years) were enrolled. Bone biochemical parameters, including serum calcium, phosphorus, albumin, creatinine, alkaline phosphatase, intact plasma PTH, 25-hydroxy-vitamin D (25-OHD), 24-h urinary calcium and phosphorus, and calcaneal quantitative ultrasound (QUS), were evaluated at the beginning (October 2012) and at the end of the League (May 2013). RESULTS: 25-OHD levels were significantly lower at the end of the League compared to the beginning (27.1±5.9 vs. 36.6±9.5 ng/mL, fold change [FC]=0.25, P=0.008), and the prevalence of 25-OHD deficiency increased from 25% to 73%. Moreover, higher rate of previous bone, cartilage or ligament injuries correlated with 25-OHD deficiencies (P=0.014). T-score and Z-score were at the upper limits of the normality ranges, without significant difference between the beginning and end of the League. Phosphaturia was slightly decreased at the end of the League (691.0±364.5 vs. 934.0±274.3 mg/24h, FC=0.26, P=0.06). A significant correlation was found between phosphaturia and BQI (R2=0.28, P=0.03), and both T-s and Z-s (R2=0.28, P=0.03) at the beginning of the League. CONCLUSIONS: With this pilot study, we demonstrated that vitamin D status significantly worsened at the end of the League. Therefore, vitamin D supplementation might be suggested in adult football players in order to prevent vitamin D deficiency and improve the athletic performance.
Asunto(s)
Huesos/metabolismo , Fútbol/fisiología , Adulto , Rendimiento Atlético , Densidad Ósea , Huesos/química , Calcio/sangre , Calcio/orina , Creatinina/sangre , Humanos , Italia , Masculino , Fósforo/sangre , Fósforo/orina , Proyectos Piloto , Fútbol/lesiones , Vitamina D/análogos & derivados , Vitamina D/sangre , Adulto JovenRESUMEN
BACKGROUND: Young athletes need to consume an appropriate diet in order to maintain health and optimize growth and athletic performance. We evaluated nutritional habits of junior elite skiers. METHODS: Alpine junior elite skiers (N.=68; 42 males and 26 females; age range 16-20 years) coming from 20 countries were recruited during the Alpine Junior World Ski Championship, Roccaraso, Italy. Nutritional habits were assessed using a 3-day food record and the NHANES Food Frequency Questionnaire. Data were compared with nutritional recommendations and Recommended Dietary Allowances (RDAs) for athletes. RESULTS: During the training period, the energy intake in both males and females was significantly lower with respect to estimated energy needs. Carbohydrate intake expressed in terms of grams per kilogram of body weight did not meet the RDAs in both groups (4.19 and 5.15 g/kg in males and females, respectively). Protein and fat consumption exceeded the RDAs with a protein intake of 2.34 g/kg in males and 2.10 g/kg in females, and a fat intake >35% of total daily calories. During competition days, both males and females increased carbohydrate intake to 6.23 and 8.11 g/kg respectively, reaching the RDAs. Protein intake increased to 2.56 and 3.14 g/kg in males and females, respectively, and fat intake slightly decreased, still exceeding the RDAs. CONCLUSIONS: Junior elite skiers reported a low intake of carbohydrates and a high intake of protein and fat. Nutritional counselling should be given to athletes to maintain their health and improve their physical performance.
Asunto(s)
Ingestión de Energía , Conducta Alimentaria , Esquí , Adolescente , Adulto , Carbohidratos de la Dieta/normas , Grasas de la Dieta/normas , Proteínas en la Dieta/normas , Femenino , Humanos , Italia , Masculino , Encuestas Nutricionales , Ingesta Diaria Recomendada , Adulto JovenRESUMEN
Massive poly(ADP-ribose) formation by poly(ADP-ribose) polymerase-1 (PARP-1) triggers NAD depletion and cell death. These events have been invariantly related to cellular energy failure due to ATP shortage. The latter occurs because of both ATP consumption for NAD resynthesis and impairment of mitochondrial ATP formation caused by an increase of the AMP/ADP ratio. ATP depletion is therefore thought to be an inevitable consequence of NAD loss and a hallmark of PARP-1 activation. Here, we challenge this scenario by showing that PARP-1 hyperactivation in cells cultured in the absence of glucose (Glu(-) cells) is followed by NAD depletion and an unexpected PARP-1 activity-dependent ATP increase. We found increased ADP content in resting Glu(-) cells, a condition that counteracts the increase of the AMP/ADP ratio during hyperpoly(ADP-ribosyl)ation and preserves mitochondrial coupling. We also show that the increase of ATP in Glu(-) cells is due to adenylate kinase activity, transforming AMP into ADP which, in turn, is converted into ATP by coupled mitochondria. Interestingly, PARP-1-dependent mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome complex (Cyt c) is reduced in Glu(-) cells, even though cell death eventually occurs. Overall, the present study identifies basal ADP content and adenylate kinase as key determinants of bioenergetics during PARP-1 hyperactivation and unequivocally demonstrates that ATP loss is not metabolically related to NAD depletion.
Asunto(s)
Metabolismo Energético , Glucosa/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células 3T3 , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Animales , Factor Inductor de la Apoptosis/metabolismo , Citocromos c/metabolismo , Activación Enzimática , Células HeLa , Humanos , Ratones , Mitocondrias/metabolismo , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli Adenosina Difosfato Ribosa/metabolismoRESUMEN
Upon massive DNA damage, hyperactivation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 causes severe depletion of intracellular NAD and ATP pools as well as mitochondrial dysfunction. Thus far, the molecular mechanisms contributing to PARP-1-dependent impairment of mitochondrial functioning have not been identified. We found that degradation of the PARP-1 product poly(ADP-ribose) through the concerted actions of poly(ADP-ribose) glycohydrolase and NUDIX (nucleoside diphosphate-X) hydrolases leads to accumulation of AMP. The latter, in turn, inhibits the ADP/ATP translocator, prompting mitochondrial energy failure. For the first time, our findings identify NUDIX hydrolases as key enzymes involved in energy derangement during PARP-1 hyperactivity. Also, these data disclose unanticipated AMP-dependent impairment of mitochondrial exchange of adenine nucleotides, which can be of relevance to organelle functioning and disease pathogenesis.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Adenosina Monofosfato/metabolismo , Metabolismo Energético , Glicósido Hidrolasas/metabolismo , Mitocondrias/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Pirofosfatasas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Western Blotting , Células HeLa , Humanos , Técnicas para Inmunoenzimas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Hidrolasas NudixRESUMEN
Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-kappaB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-kappaB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Inmunofenotipificación , Interleucina-10/antagonistas & inhibidores , Interleucina-10/biosíntesis , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Monocitos/citología , Monocitos/enzimología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Poly(ADP-ribose)polymerase-1 (PARP-1) overactivation is a key event in neurodegeneration but the underlying molecular mechanisms wait to be unequivocally identified. Energy failure, transcriptional derangement and deadly nucleus-mitochondria cross-talk have been proposed as mechanisms responsible for PARP-1 neurotoxicity. In this study, we sought to determine how these mechanisms contributes to PARP-1-dependent neuronal death. We report that the PARP-1 activating agent methyl-nitrosoguanidine (MNNG) caused poly(ADP-ribosyl)ation-dependent death of pure mouse cortical neurons in culture. Upon PARP-1 hyperactivation, NAD and ATP storages only partially decreased, neurons rapidly acquired apoptotic morphology, apoptosis inducing factor and cytochrome c were released from mitochondria and caspase activation occurred. No evidence for p53 activation was found, lactate dehydrogenase release occurred only 18h later, and JNK kinase was constitutively activated and not affected by PARP-1 activation. The PARP-1 inhibitors 6-(5)H-phenanthridinone and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ-34) prevented nucleotide depletion and cell death, whereas the transcription inhibitor actinomycin D did not affect PARP-1-dependent neurotoxicity. Together, our findings provide the first evidence that neither energy collapse nor transcriptional changes are involved in PARP-1-dependent apoptotic neuronal death, and support the existence of a poly(ADP-ribose)-mediated death signaling targeting mitochondria.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Transcripción Genética , Adenosina Trifosfato/metabolismo , Animales , Caspasas/metabolismo , Muerte Celular , Activación Enzimática , Técnicas In Vitro , MAP Quinasa Quinasa 4/metabolismo , Metilnitronitrosoguanidina/farmacología , Ratones , NAD/metabolismoRESUMEN
Poly(ADP-ribose) (PAR) is a polymer synthesized by poly(ADP-ribose) polymerases (PARPs) and metabolized into free adenosine diphosphate (ADP)-ribose units by poly(ADP-ribose) glycohydrolase (PARG). Perturbations in PAR synthesis have been shown to play a key role in brain disorders including postischemic brain damage. A single parg gene but two PARG isoforms (110 and 60 kDa) have been detected in mouse cells. Complete suppression of parg gene causes early embryonic lethality, whereas mice selectively lacking the 110 kDa PARG isoform (PARG(110)(-/-)) develop normally. We used PARG(110)(-/-) mice to evaluate the importance of PAR catabolism to postischemic brain damage. Poly(ADP-ribose) contents were higher in the brain tissue of PARG(110)(-/-) than PARG(110)(+/+) mice, both under basal conditions and after PARP activation. Distal middle cerebral artery occlusion caused higher increase of brain PAR levels and larger infarct volumes in PARG(110)(-/-) mice than in wild-type counterparts. Of note, the brain of PARG(110)(-/-) mice showed reduced heat-shock protein (HSP)-70 and increased cyclooxygenase-2 expression under both control and ischemic conditions. No differences were detected in brain expression/activation of procaspase-3, PARP-1, Akt, HSP-25 and interleukin-1beta. Our findings show that PAR accumulation worsens ischemic brain injury, and highlight the therapeutic potential of strategies capable of maintaining PAR homeostasis.
Asunto(s)
Isquemia Encefálica/patología , Glicósido Hidrolasas/metabolismo , Isoenzimas/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Isquemia Encefálica/metabolismo , Activación Enzimática , Glicósido Hidrolasas/genética , Homeostasis , Técnicas In Vitro , Infarto de la Arteria Cerebral Media , Isoenzimas/genética , Ratones , Ratones Noqueados , NAD/metabolismo , Fármacos Neuroprotectores/metabolismo , Neurotoxinas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.