RESUMO
Metabolic alterations shared between the nervous system and skin fibroblasts have emerged in amyotrophic lateral sclerosis (ALS). Recently, we found that a subgroup of sporadic ALS (sALS) fibroblasts (sALS1) is characterized by metabolic profiles distinct from other sALS cases (sALS2) and controls, suggesting that metabolic therapies could be effective in sALS. The metabolic modulators nicotinamide riboside and pterostilbene (EH301) are under clinical development for the treatment of ALS. Here, we studied the transcriptome and metabolome of sALS cells to understand the molecular bases of sALS metabotypes and the impact of EH301. Metabolomics and transcriptomics were investigated at baseline and after EH301 treatment. Moreover, weighted gene coexpression network analysis (WGCNA) was used to investigate the association of the metabolic and clinical features. We found that the sALS1 transcriptome is distinct from sALS2 and that EH301 modifies gene expression differently in sALS1, sALS2 and the controls. Furthermore, EH301 had strong protective effects against metabolic stress, an effect linked to the antiinflammatory and antioxidant pathways. WGCNA revealed that the ALS functional rating scale and metabotypes are associated with gene modules enriched for the cell cycle, immunity, autophagy and metabolic genes, which are modified by EH301. The meta-analysis of publicly available transcriptomic data from induced motor neurons by Answer ALS confirmed the functional associations of genes correlated with disease traits. A subset of genes differentially expressed in sALS fibroblasts was used in a machine learning model to predict disease progression. In conclusion, multiomic analyses highlighted the differential metabolic and transcriptomic profiles in patient-derived fibroblast sALS, which translate into differential responses to the investigational drug EH301.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/metabolismo , Antioxidantes/metabolismo , Drogas em Investigação/metabolismo , Drogas em Investigação/uso terapêutico , Fibroblastos/metabolismo , Humanos , Transcriptoma/genéticaRESUMO
BACKGROUND AND AIMS: The prevalence of NAFLD is increasing globally and on a path to becoming the most frequent cause of chronic liver disease. Strategies for the prevention and treatment of NAFLD are urgently needed. APPROACH AND RESULTS: A 6-month prospective, randomized, double-blind, placebo-controlled clinical trial was conducted to assess the efficacy of daily NRPT (commercially known as Basis, a combination of nicotinamide riboside and pterostilbene) supplementation in 111 adults with NAFLD. The study consisted of three arms: placebo, recommended daily dose of NRPT (NRPT 1×), and a double dose of NRPT (NRPT 2×). NRPT appeared safe and well tolerated. At the end of the study, no significant change was seen in the primary endpoint of hepatic fat fraction with respect to placebo. However, among prespecified secondary outcomes, a time-dependent decrease in the circulating levels of the liver enzymes alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) was observed in the NRPT 1× group, and this decrease was significant with respect to placebo. Furthermore, a significant decrease in the circulating levels of the toxic lipid ceramide 14:0 was also observed in the NRPT 1× group versus placebo, and this decrease was associated with a decrease in ALT in individuals of this group. A dose-dependent effect was not observed with respect to ALT, GGT, or ceramide 14:0 in the NRPT 2× group. CONCLUSIONS: This study demonstrates that NRPT at the recommended dose is safe and may hold promise in lowering markers of hepatic inflammation in patients with NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Adulto , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/complicações , Estudos Prospectivos , gama-Glutamiltransferase , Inflamação/tratamento farmacológico , Inflamação/complicações , Método Duplo-Cego , Alanina TransaminaseRESUMO
BACKGROUND: Environmental ultraviolet radiation has deleterious effects on humans, including sunburn and immune perturbations. These immune changes are involved in skin carcinogenesis. OBJECTIVES: To determine whether nicotinamide riboside and/or pterostilbene administered systemically inhibits inflammatory and immune effects of exposure to mid-range ultraviolet radiation. METHODS: To examine UVB radiation-induced inflammatory effects, mice were fed standard chow/water, 0.04% pterostilbene in chow and 0.2% nicotinamide riboside in drinking water, diet with nicotinamide riboside alone, or diet with pterostilbene alone. After 4 weeks, mice were exposed to UVB radiation (3500 J/m2), and 24-/48-h ear swelling was assessed. We also asked if each agent or the combination inhibits UVB radiation suppression of contact hypersensitivity in two models. Mice were fed standard diet/water or chow containing 0.08% pterostilbene, water with 0.4% nicotinamide riboside, or both for 4 weeks. Low-dose: Half the mice in each group were exposed on the depilated dorsum to UVB radiation (1700 J/m2) daily for 4 days, whereas half were mock-irradiated. Mice were immunized on the exposed dorsum to dinitrofluorobenzene 4 h after the last irradiation, challenged 7 days later on the ears with dinitrofluorobenzene, and 24-h ear swelling assessed. High dose: Mice were treated similarly except that a single dose of 10,000 J/m2 of radiation was administered and immunization was performed on the unirradiated shaved abdomen 3 days later. RESULTS: Nicotinamide riboside and pterostilbene together inhibited UVB-induced skin swelling more than either alone. Pterostilbene alone and both given together could inhibit UVB-induced immune suppression in both the low-dose and high-dose models while nicotinamide riboside alone was more effective in the low-dose model than the high-dose model. CONCLUSION: Nicotinamide riboside and pterostilbene have protective effects against UVB radiation-induced tissue swelling and immune suppression.
Assuntos
Niacinamida , Niacinamida/análogos & derivados , Compostos de Piridínio , Estilbenos , Raios Ultravioleta , Animais , Niacinamida/farmacologia , Compostos de Piridínio/farmacologia , Camundongos , Raios Ultravioleta/efeitos adversos , Estilbenos/farmacologia , Feminino , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Dermatite de Contato/etiologiaRESUMO
Nicotinamide riboside (NR) is a naturally occurring form of vitamin B3 shown to preferentially elevate the nicotinamide adenine dinucleotide (NAD+) metabolome compared to other vitamin B3 forms (nicotinic acid and nicotinamide). Although daily requirements of vitamin B3 are typically met through the diet, recent studies have shown that additional supplementation with NR may be an effective method to counter the age-related decline in NAD+ levels as NR bypasses the rate-limiting step in NAD+ biosynthesis. Furthermore, pharmaceutical applications of NR for age-related disorders have been proposed. In this study, the safety of a high-purity, nature-identical, synthetic NR (NR-E), manufactured under the guidelines of good manufacturing practices for dietary supplements (21 CFR 111) as well as for drugs (21 CFR 210), was investigated in a 90-day oral toxicity study in Sprague Dawley rats at 300, 500, and 1,200 mg/kg/d. There were no mortality or clinical observations attributable to the test substance at any dose. A small but statistically significant decrease in body weight was observed at day 92 in the 1,200 mg/kg/d NR-treated male rats only. In contrast to a previously published safety assessment using a different synthetic NR (NIAGEN), whose no-observed-adverse-effect-level (NOAEL) was reported to be 300 mg/kg/d, there were no adverse changes in clinical pathology parameters and no notable macroscopic or microscopic findings or treatment-related effects at similar doses. In the current study, the NOAEL for systemic toxicity of NR-E in Sprague-Dawley rats was conservatively determined to be 500 mg/kg/d for males (solely based on body weight) and 1,200 mg/kg/d for females.
Assuntos
Suplementos Nutricionais/toxicidade , Niacinamida/análogos & derivados , Compostos de Piridínio/toxicidade , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Masculino , Niacinamida/toxicidade , Nível de Efeito Adverso não Observado , Ratos Sprague-Dawley , Caracteres Sexuais , Testes de Toxicidade SubcrônicaRESUMO
The high number of somatic mutations in the melanoma genome associated with cumulative ultra violet (UV) exposure has rendered it one of the most difficult of cancers to treat. With new treatment approaches based on targeted and immune therapies, drug resistance has appeared as a consistent problem. Redox biology, including reactive oxygen and nitrogen species (ROS and RNS), plays a central role in all aspects of melanoma pathophysiology, from initiation to progression and to metastatic cells. The involvement of melanin production and UV radiation in ROS/RNS generation has rendered the melanocytic lineage a unique system for studying redox biology. Overall, an elevated oxidative status has been associated with melanoma, thus much effort has been expended to prevent or treat melanoma using antioxidants which are expected to counteract oxidative stress. The consequence of this redox-rebalance seems to be two-fold: on the one hand, cells may behave less aggressively or even undergo apoptosis; on the other hand, cells may survive better after being disseminated into the circulating system or after drug treatment, thus resulting in metastasis promotion or further drug resistance. In this review we summarize the current understanding of redox signaling in melanoma at cellular and systemic levels and discuss the experimental and potential clinic use of antioxidants and new epigenetic redox modifiers.
Assuntos
Antioxidantes/metabolismo , Melanoma/metabolismo , Melanoma/fisiopatologia , Estresse Oxidativo , Animais , Humanos , Melanoma/genética , Oxirredução , Transdução de Sinais/genéticaRESUMO
The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. CONCLUSION: NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630).
Assuntos
Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/fisiologia , Fígado/patologia , NAD/biossíntese , Niacinamida/análogos & derivados , Animais , Modelos Animais de Doenças , Imunofluorescência , Hepatectomia/métodos , Immunoblotting , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NAD/metabolismo , Niacinamida/farmacologia , Compostos de Piridínio , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
Elevated plasma total homocysteine (tHcy) is associated with the development of Alzheimer's disease and other forms of dementia. In this study, we report the relationship between tHcy and epigenetic age in older adults with mild cognitive impairment from the VITACOG study. Epigenetic age and rate of aging (ROA) were assessed using various epigenetic clocks, including those developed by Horvath and Hannum, DNAmPhenoAge, and with a focus on Index, a new principal component-based epigenetic clock that, like DNAmPhenoAge, is trained to predict an individual's "PhenoAge." We identified significant associations between tHcy levels and ROA, suggesting that hyperhomocysteinemic individuals were aging at a faster rate. Moreover, Index revealed a normalization of accelerated epigenetic aging in these individuals following treatment with tHcy-lowering B-vitamins. Our results indicate that elevated tHcy is a risk factor for accelerated epigenetic aging, and this can be ameliorated with B-vitamins. These findings have broad relevance for the sizable proportion of the worldwide population with elevated tHcy.
RESUMO
Oxidative stress and neuroinflammation are major contributors to the pathophysiology of ALS. Nicotinamide riboside (a NAD+ precursor) and pterostilbene (a natural antioxidant) were efficacious in a human pilot study of ALS patients and in ALS SOD1G93A transgenic mice. Ibudilast targets different phosphodiesterases and the macrophage migration inhibitory factor, reduces neuroinflammation, and in early-phase studies improved survival and slowed progression in ALS patients. Using two ALS murine models (SOD1G93A, FUSR521C) the effects of nicotinamide riboside, pterostilbene, and ibudilast on disease onset, progression and survival were studied. In both models ibudilast enhanced the effects of nicotinamide riboside and pterostilbene on survival and neuromotor functions. The triple combination reduced microgliosis and astrogliosis, and the levels of different proinflammatory cytokines in the CSF. TNFα, IFNγ and IL1ß increased H2O2 and NO generation by motor neurons, astrocytes, microglia and endothelial cells isolated from ALS mice. Nicotinamide riboside and pterostilbene decreased H2O2 and NO generation in all these cells. Ibudilast specifically decreased TNFα levels and H2O2 generation by microglia and endothelial cells. Unexpectedly, pathophysiological concentrations of H2O2 or NO caused minimal motor neuron cytotoxicity. H2O2-induced cytotoxicity was increased by NO via a trace metal-dependent formation of potent oxidants (i.e. OH and -OONO radicals). In conclusion, our results show that the combination of nicotinamide riboside, pterostilbene and ibudilast improve neuromotor functions and survival in ALS murine models. Studies on the underlying mechanisms show that motor neuron protection involves the decrease of oxidative and nitrosative stress, the combination of which is highly damaging to motor neurons.
Assuntos
Esclerose Lateral Amiotrófica , Indolizinas , Niacinamida/análogos & derivados , Pirazóis , Compostos de Piridínio , Camundongos , Animais , Humanos , Superóxido Dismutase-1 , Esclerose Lateral Amiotrófica/tratamento farmacológico , Doenças Neuroinflamatórias , Fator de Necrose Tumoral alfa , Células Endoteliais , Peróxido de Hidrogênio , Projetos Piloto , Neurônios Motores , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Camundongos Transgênicos , Modelos Animais de Doenças , Superóxido Dismutase , Medula EspinalAssuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/análise , Carcinoma de Células Escamosas/química , Ceratose Actínica/metabolismo , Neoplasias Cutâneas/química , Biópsia , Carcinoma de Células Escamosas/patologia , Celecoxib/farmacologia , Celecoxib/uso terapêutico , Quimioprevenção , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Glucuronosiltransferase/metabolismo , Humanos , Ceratose Actínica/patologia , Inibidores de Lipoxigenase/farmacologia , Inibidores de Lipoxigenase/uso terapêutico , Neoplasias Induzidas por Radiação/química , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controleRESUMO
Dietary vitamin B3 components, such as nicotinamide and nicotinic acid, are precursors to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+). NAD+ levels are thought to decline with age and disease. While the drivers of this decline remain under intense investigation, strategies have emerged seeking to functionally maintain NAD+ levels through supplementation with NAD+ biosynthetic intermediates. These include marketed products, such as nicotinamide riboside (NR) and its phosphorylated form (NMN). More recent developments have shown that NRH (the reduced form of NR) and its phosphorylated form NMNH also increases NAD+ levels upon administration, although they initially generate NADH (the reduced form of NAD+). Other means to increase the combined levels of NAD+ and NADH, NAD(H), include the inhibition of NAD+-consuming enzymes or activation of biosynthetic pathways. Multiple studies have shown that supplementation with an NAD(H) precursor changes the profile of NAD(H) catabolism. Yet, the pharmacological significance of NAD(H) catabolites is rarely considered although the distribution and abundance of these catabolites differ depending on the NAD(H) precursor used, the species in which the study is conducted, and the tissues used for the quantification. Significantly, some of these metabolites have emerged as biomarkers in physiological disorders and might not be innocuous. Herein, we review the known and emerging catabolites of the NAD(H) metabolome and highlight their biochemical and physiological function as well as key chemical and biochemical reactions leading to their formation. Furthermore, we emphasize the need for analytical methods that inform on the full NAD(H) metabolome since the relative abundance of NAD(H) catabolites informs how NAD(H) precursors are used, recycled, and eliminated.
Assuntos
NAD , Niacina , NAD/metabolismo , Niacinamida/metabolismo , Metaboloma , Oxirredução , Biomarcadores/metabolismoRESUMO
INTRODUCTION: Effective agents that could confer long-term protection against ionizing radiation in vivo would have applications in medicine, biotechnology, and in air and space travel. However, at present, drugs that can effectively protect against lethal ionizing radiations are still an unmet need. OBJECTIVE: To investigate if combinations of natural polyphenols, known for their antioxidant potential, could protect against ionizing radiations. METHODS: Plant-derived polyphenols were screened for their potential ability to confer radioprotection to mice given a lethal whole-body γ radiation (137Cs) dose expected to kill 50% of the animals in 30 days. Telomere and centromere staining, Q-FISH and comet assays were used to investigate chromosomal aberration, micronuclei formation and DNA breaks. Molecular oxidations were investigated by enzyme immunoassays and UPLC-MS/MS. RT-PCR, western blotting and siRNA-induced gene silencing were used to study signaling mechanisms and molecular interactions. RESULTS: The combination of pterostilbene (PT) and silibinin (SIL) was the most effective against γ-irradiation, resulting in 100% of the mice surviving at 30 days and 20% survival at one year. Treatment post γ-irradiation with two potential radiomitigators nicotinamide riboside (NR, a vitamin B3 derivative), and/or fibroblast-stimulating lipoprotein 1 (FSL1, a toll-like receptor 2/6 agonist), did not extend survival. However, the combination of PT, SIL, NR and FSL1 achieved a 90% survival one year post γ-irradiation. The mechanism involves induction of the Nrf2-dependent cellular antioxidant defense, reduction of NF-kB signaling, upregulation of the PGC-1α/sirtuins 1 and 3 axis, PARP1-dependent DNA repair, and stimulation of hematopoietic cell recovery. The pathway linking Nrf2, sirtuin 3 and SOD2 is key to radioprotection. Importantly, this combination did not interfere with X-ray mediated killing of different tumor cells in vivo. CONCLUSION: The combination of the radioprotectors PT and SIL with the radiomitigators NR and FSL1 confer effective, long-term protection against γ radiation in vivo. This strategy is potentially capable of protecting mammals against ionizing radiations.
Assuntos
NAD , Protetores contra Radiação , Camundongos , Animais , Raios gama , Antioxidantes , Receptor 2 Toll-Like/agonistas , Lipopeptídeos , Protetores contra Radiação/farmacologia , Protetores contra Radiação/uso terapêutico , Polifenóis/farmacologia , Fator 2 Relacionado a NF-E2 , Cromatografia Líquida , Ligantes , Espectrometria de Massas em Tandem , MamíferosRESUMO
In clinical trials, oral supplementation with nicotinamide riboside (NR) fails to increase muscle mitochondrial respiratory capacity and insulin sensitivity but also does not increase muscle NAD+ levels. This study tests the feasibility of chronically elevating skeletal muscle NAD+ in mice and investigates the putative effects on mitochondrial respiratory capacity, insulin sensitivity, and gene expression. Accordingly, to improve bioavailability to skeletal muscle, we developed an experimental model for administering NR repeatedly through a jugular vein catheter. Mice on a Western diet were treated with various combinations of NR, pterostilbene (PT), and voluntary wheel running, but the metabolic effects of NR and PT treatment were modest. We conclude that the chronic elevation of skeletal muscle NAD+ by the intravenous injection of NR is possible but does not affect muscle respiratory capacity or insulin sensitivity in either sedentary or physically active mice. Our data have implications for NAD+ precursor supplementation regimens.
RESUMO
BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.
Assuntos
Doenças Musculares , Cadeias Pesadas de Miosina , Idoso , Creatina Quinase Forma MM , Suplementos Nutricionais , Humanos , Músculo Esquelético , Mioglobina/farmacologia , Niacinamida/análogos & derivados , Compostos de Piridínio , EstilbenosRESUMO
Charcot first described amyotrophic lateral sclerosis (ALS) between 1865 and 1874 as a sporadic adult disease resulting from the idiopathic progressive degeneration of the motor neuronal system, resulting in rapid, progressive, and generalized muscle weakness and atrophy. There is no cure for ALS and no proven therapy to prevent it or reverse its course. There are two drugs specifically approved for the treatment of ALS, riluzol and edaravone, and many others have already been tested or are following clinical trials. However, at the present moment, we still cannot glimpse a true breakthrough in the treatment of this devastating disease. Nevertheless, our understanding of the pathophysiology of ALS is constantly growing. Based on this background, we know that oxidative stress, alterations in the NAD+-dependent metabolism and redox status, and abnormal mitochondrial dynamics and function in the motor neurons are at the core of the problem. Thus, different antioxidant molecules or NAD+ generators have been proposed for the therapy of ALS. This review analyzes these options not only in light of their use as individual molecules, but with special emphasis on their potential association, and even as part of broader combined multi-therapies.
RESUMO
Natural polyphenols are organic chemicals which contain phenol units in their structures and possess antitumor properties. However, a key problem is their short half-life and low bioavailability under in vivo conditions. Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene; PT) is a phytoalexin originally isolated from the heartwood of red sandalwood. As recently reported by our group, PT was shown to be effective in the treatment of melanoma. Counterintuitively, PT is not effective (cytotoxic) against melanoma in vitro, and only under in vivo conditions does PT display its anticancer activity. This study elucidated that PT can be effective against melanoma through the inhibition of adrenocorticotropic hormone production in the brain of a mouse, which weakens the Nrf2-dependent antioxidant defenses of melanoma and also pancreatic cancers. This results in both the inhibition of tumor growth and sensitization of the tumor to oxidative stress. Moreover, PT can promote cancer cell death via a mechanism involving lysosomal membrane permeabilization. Different grades of susceptibility were observed among the different cancer cells depending on their lysosomal heat shock protein 70 content, a known stabilizer of lysosomal membranes. In addition, the safety of PT administered i.v. has been evaluated in mice. PT was found to be pharmacologically safe because it showed no organ-specific or systemic toxicity (including tissue histopathologic examination and regular hematology and clinical chemistry data) even when administered i.v. at a high dose (30 mg/kg per day × 23 days). Moreover, new pharmacological advances are being developed to increase its bioavailability and, thereby, its bioefficacy. Therefore, although applications of PT in cancer therapy are just beginning to be explored, it represents a potential (and effective) adjuvant/sensitizing therapy which may improve the results of various oncotherapies. The aim of this review is to present and discuss the results that in our opinion best support the usefulness of PT in cancer therapy, making special emphasis on the in vivo evidence.
RESUMO
OBJECTIVE: Exemestane is a third-generation aromatase inhibitor used in the treatment of breast cancer in postmenopausal women. Reduction to form 17-dihydroexemestane and subsequent glucuronidation to exemestane-17-O-glucuronide is a major pathway for exemestane metabolism. The goal of this study was to analyze 17-dihydroexemestane anti-aromatase activity, characterize the 17-dihydroexemestane glucuronidation pathway, and determine whether the functional polymorphisms in active UGTs could play a role in altered 17-dihydroexemestane glucuronidation. METHODS: Homogenates from a HEK293 aromatase-overexpressing cell line (HEK293-aro) were used to examine exemestane versus 17-dihydroexemestane anti-aromatase activities. UGT-overexpressing cell lines and a panel (n=110) of human liver microsome (HLM) were screened for glucuronidation activity against 17-dihydroexemestane. UGT2B17 genotyping and liver mRNA expression were performed by real-time PCR. RESULTS: The inhibition of estrone formation from androst-4-ene-3,17-dione in HEK293-aro cell homogenates was similar for 17-dihydroexemestane (IC(50)=2.3±0.83 µmol/l) and exemestane (IC(50)=1.4±0.42 µmol/l). UGTs 2B17 and 1A4 were high-expression hepatic UGTs that exhibited activity against 17-dihydroexemestane, with UGT2B17 exhibiting a 17-fold higher V(max)/K(M) than UGT1A4. The rate of exemestane-17-O-glucuronide formation was shown to be significantly (P<0.001) decreased (14-fold) in HLMs exhibiting the UGT2B17(*2/*2) deletion genotype versus wild-type UGT2B17(*1/*1) HLMs; a 36-fold lower V(max)/K(M) (P=0.023) was observed in UGT2B17(*2/*2) versus UGT2B17(*1/*1) HLMs. A significant (P<0.0001, R(2)=0.72) correlation was observed between HLM exemestane-17-O-glucuronide formation and liver UGT2B17 expression. CONCLUSION: These data suggest that 17-dihydroexemestane is an active metabolite of exemestane and that the UGT2B17 deletion polymorphism could play an important role in determining levels of excretion of 17-dihydroexemestane and overall exemestane metabolism.
Assuntos
Androstadienos/metabolismo , Androstadienos/farmacologia , Deleção de Genes , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Androstadienos/química , Aromatase/metabolismo , Inibidores da Aromatase/química , Inibidores da Aromatase/metabolismo , Inibidores da Aromatase/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Glicosilação/efeitos dos fármacos , Humanos , Cinética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Antígenos de Histocompatibilidade MenorRESUMO
The extrahepatic UDP-glucuronosyltransferase 1A10 (UGT1A10) is a phase II metabolizing enzyme that is active against a number of potent carcinogens. In the present study, UGT1A10 was examined for activity against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the major procarcinogenic metabolite of the potent tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and the promoter region of UGT1A10 was examined for variants that could lead to altered UGT1A10 expression. UGT1A10-overexpressing cell homogenates exhibited high O-glucuronidation activity against NNAL (K(M) = 5.95 mM). A 2000-base pair (bp) product corresponding to the UGT1A10 proximal promoter region was polymerase chain reaction (PCR)-amplified using genomic DNA from 97 white subjects, and 42 of these were sequenced. In addition to a previously reported C/G single-nucleotide polymorphism at -1271 bp (rs2741032), a novel 1664-bp deletion located between nucleotides -190 to -1856 relative to the UGT1A10 translation start site was identified. Using real-time multiplex PCR, this deletion exhibited a prevalence of 0.022 in whites (n = 156) and 0.056 in blacks (n = 133). To determine whether either polymorphism altered gene expression, in vitro assays were performed using luciferase constructs containing up to 2000 bp of the proximal UGT1A10 promoter. Constructs containing the 1664-bp deletion exhibited a significant (p = 0.009) 3-fold increase in luciferase activity compared with constructs containing the wild-type UGT1A10 promoter. No effect on luciferase activity was observed for the UGT1A10(-1271G) promoter variant. These data are consistent with previous studies that indicate the presence of a transcriptional repressor element within the newly identified deletion and that this deletion polymorphism may contribute to altered UGT1A10 expression and altered carcinogen detoxification between individuals.
Assuntos
Carcinógenos/metabolismo , Carcinoma Hepatocelular/genética , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Nitrosaminas/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas , Piridinas/metabolismo , Células CACO-2 , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/cirurgia , Regulação Neoplásica da Expressão Gênica/genética , Frequência do Gene , Genes Reporter , Estudos de Associação Genética , Predisposição Genética para Doença/etnologia , Variação Estrutural do Genoma , Glucuronosiltransferase/biossíntese , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirurgia , Desintoxicação Metabólica Fase II/genética , Antígenos de Histocompatibilidade Menor , Polimorfismo de Nucleotídeo Único , Deleção de Sequência/genéticaRESUMO
The UDP-glucuronosyltransferase (UGT) 1A9 has been shown to play an important role in the detoxification of several carcinogens and clearance of anticancer and pain medications. The goal of the present study was to identify novel polymorphisms in UGT1A9 and characterize their effect on glucuronidation activity. The UGT1A9 gene was analyzed by direct sequencing of buccal cell genomic DNA from 90 healthy subjects. In addition to a previously identified single nucleotide polymorphism (SNP) at codon 33 resulting in an amino acid substitution (Met>Thr), two low-prevalence (<0.02) novel missense SNPs at codons 167 (Val>Ala) and 183 (Cys>Gly) were identified and are present in both white and African-American subjects. Glucuronidation activity assays using HEK293 cell lines overexpressing wild-type or variant UGT1A9 demonstrated that the UGT1A9(33Thr) and UGT1A9(183Gly) variants exhibited differential glucuronidation activities compared with wild-type UGT1A9, but this was substrate-dependent. The UGT1A9(167Ala) variant exhibited levels of activity similar to those of wild-type UGT1A9 for all substrates tested. Whereas the wild-type and UGT1A9(33Thr) and UGT1A9(167Ala) variants formed homodimers as determined by Western blot analysis of native polyacrylamide gels, the UGT1A9(183Gly) variant was incapable of homodimerization. These results suggest that several low-prevalence missense polymorphisms exist for UGT1A9 and that two of these (M33T and C183G) are functional. These results also suggest that although Cys183 is necessary for UGT1A9 homodimerization, the lack of capacity for UGT1A9 homodimerization is not sufficient to eliminate UGT1A9 activity.
Assuntos
Predisposição Genética para Doença/epidemiologia , Glucuronosiltransferase/genética , Polimorfismo Genético , Prevalência , Negro ou Afro-Americano/genética , Alelos , Substituição de Aminoácidos , Linhagem Celular , Frequência do Gene , Genótipo , Glucuronosiltransferase/fisiologia , Humanos , Microssomos Hepáticos , Mutação , UDP-Glucuronosiltransferase 1A , População Branca/genéticaRESUMO
The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/fisiologia , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina , DNA , Ativação Enzimática , Fase G1 , Humanos , Mamíferos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Distribuição Tecidual , Células Tumorais Cultivadas , XenopusRESUMO
Nicotine, the major addicting agent in tobacco and tobacco smoke, undergoes a complex metabolic pathway, with approximately 22% of nicotine urinary metabolites in the form of phase II N-glucuronidated compounds. Recent studies have shown that UGT2B10 is a major enzyme involved in the N-glucuronidation of several tobacco-specific nitrosamines. In the present study, microsomes of UGT2B10-overexpressing HEK293 cells exhibited high N-glucuronidation activity against both nicotine and cotinine with apparent KM's that were 37- and 3-fold lower than that observed for microsomes of UGT1A4-overexpressing cells against nicotine and cotinine, respectively. The KM of microsomes from wild-type (WT) UGT2B10-overexpressing cells for nicotine and cotinine was similar to that observed for human liver microsomes (HLM) against both substrates. The level of glucuronidated nicotine or cotinine in 112 HLM samples was correlated with UGT2B10 genotype; the levels of nicotine- and cotinine-glucuronide were 21% to 30% lower in specimens from subjects with the UGT2B10 (*1/*2) genotype compared with specimens from subjects with the WT UGT2B10 (*1/*1) genotype; a 5- and 16-fold lower level of nicotine- and cotinine-glucuronide formation, respectively, was observed in HLM from subjects with the UGT2B10 (*2/*2) genotype. In contrast to the relatively high activity observed for cells overexpressing WT UGT2B10 in vitro, little or no glucuronidation was observed for microsomes from cells overexpressing the UGT2B10*2 variant against either nicotine or cotinine. These data suggest that UGT2B10 is the major hepatic enzyme involved in nicotine/cotinine glucuronidation and that the UGT2B10*2 variant significantly reduces nicotine- and cotinine-N-glucuronidation formation and plays an important role in nicotine metabolism and elimination.