RESUMO
Increasing evidence suggests that kynurenine pathway (KP) dyshomeostasis may promote disease progression in dementia. Studies in Alzheimer's disease (AD) patients confirm KP dyshomeostasis in plasma and cerebrospinal fluid (CSF) which correlates with amyloid-ß and tau pathology. Herein, we performed the first comprehensive study assessing baseline levels of KP metabolites in participants enrolling in the Australian Imaging Biomarkers Flagship Study of Aging. Our purpose was to test the hypothesis that changes in KP metabolites may be biomarkers of dementia processes that are largely silent. We used a cross-sectional analytical approach to assess non-progressors (N = 73); cognitively normal (CN) or mild cognitive impairment (MCI) participants at baseline and throughout the study, and progressors (N = 166); CN or MCI at baseline but progressing to either MCI or AD during the study. Significant KP changes in progressors included increased 3-hydroxyanthranilic acid (3-HAA) and 3-hydroxyanthranilic acid/anthranilic acid (3-HAA/AA) ratio, the latter having the largest effect on the odds of an individual being a progressor (OR 35.3; 95% CI between 14 and 104). 3-HAA levels were hence surprisingly bi-phasic, high in progressors but low in non-progressors or participants who had already transitioned to MCI or dementia. This is a new, unexpected and interesting result, as most studies of the KP in neurodegenerative disease show reduced 3-HAA/AA ratio after diagnosis. The neuroprotective metabolite picolinic acid was also significantly decreased while the neurotoxic metabolite 3-hydroxykynurenine increased in progressors. These results were significant even after adjustment for confounders. Considering the magnitude of the OR to predict change in cognition, it is important that these findings are replicated in other populations. Independent validation of our findings may confirm the utility of 3-HAA/AA ratio to predict change in cognition leading to dementia in clinical settings.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Doenças Neurodegenerativas , Ácido 3-Hidroxiantranílico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Austrália , Biomarcadores , Disfunção Cognitiva/líquido cefalorraquidiano , Estudos Transversais , Progressão da Doença , Humanos , Cinurenina , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidianoRESUMO
BACKGROUND: Immunotherapy has recently been proposed as a promising treatment to stop breast cancer (BrCa) progression and metastasis. However, there has been limited success in the treatment of BrCa with immune checkpoint inhibitors. This implies that BrCa tumors have other mechanisms to escape immune surveillance. While the kynurenine pathway (KP) is known to be a key player mediating tumor immune evasion and while there are several studies on the roles of the KP in cancer, little is known about KP involvement in BrCa. METHODS: To understand how KP is regulated in BrCa, we examined the KP profile in BrCa cell lines and clinical samples (n = 1997) that represent major subtypes of BrCa (luminal, HER2-enriched, and triple-negative (TN)). We carried out qPCR, western blot/immunohistochemistry, and ultra-high pressure liquid chromatography on these samples to quantify the KP enzyme gene, protein, and activity, respectively. RESULTS: We revealed that the KP is highly dysregulated in the HER2-enriched and TN BrCa subtype. Gene, protein expression, and KP metabolomic profiling have shown that the downstream KP enzymes KMO and KYNU are highly upregulated in the HER2-enriched and TN BrCa subtypes, leading to increased production of the potent immunosuppressive metabolites anthranilic acid (AA) and 3-hydroxylanthranilic acid (3HAA). CONCLUSIONS: Our findings suggest that KMO and KYNU inhibitors may represent new promising therapeutic targets for BrCa. We also showed that KP metabolite profiling can be used as an accurate biomarker for BrCa subtyping, as we successfully discriminated TN BrCa from other BrCa subtypes.
Assuntos
Neoplasias da Mama/patologia , Hidrolases/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Quinurenina 3-Mono-Oxigenase/metabolismo , Cinurenina/metabolismo , Redes e Vias Metabólicas , Evasão Tumoral , Adulto , Idoso , Biomarcadores Tumorais/sangue , Neoplasias da Mama/classificação , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de NeoplasiasRESUMO
Intrinsically fluorescent poly(amidoamine) dendrimers (IF-PAMAM) are an emerging class of versatile nanoplatforms for in vitro tracking and bio-imaging. However, limited tissue penetration of their fluorescence and interference due to auto-fluorescence arising from biological tissues limit its application in vivo. Herein, a green IF-PAMAM (FGP) dendrimer is reported and its biocompatibility, circulation, biodistribution and potential role for traceable central nervous system (CNS)-targeted delivery in zebrafish is evaluated, exploring various routes of administration. Key features of FGP include visible light excitation (488 nm), high fluorescence signal intensity, superior photostability and low interference from tissue auto-fluorescence. After intravenous injection, FGP shows excellent imaging and tracking performance in zebrafish. Further conjugating FGP with transferrin (FGP-Tf) significantly increases its penetration through the blood-brain barrier (BBB) and prolongs its circulation in the blood stream. When administering through local intratissue microinjection, including intracranial and intrathecal injection in zebrafish, both FGP and FGP-Tf exhibit excellent tissue diffusion and effective cellular uptake in the brain and spinal cord, respectively. This makes FGP/FGP-Tf attractive for in vivo tracing when transporting to the CNS is desired. The work addresses some of the major shortcomings in IF-PAMAM and provides a promising application of these probes in the development of drug delivery in the CNS.
Assuntos
Sistema Nervoso Central , Dendrímeros , Sistemas de Liberação de Medicamentos , Poliaminas , Animais , Sistema Nervoso Central/diagnóstico por imagem , Dendrímeros/química , Sistemas de Liberação de Medicamentos/métodos , Corantes Fluorescentes/química , Poliaminas/química , Distribuição Tecidual , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Blood markers indicative of neurodegeneration (neurofilament light chain; NFL), Alzheimer's disease amyloid pathology (amyloid-ß; Aß), and neuroinflammation (kynurenine pathway; KP metabolites) have been investigated independently in neurodegenerative diseases. However, the association of these markers of neurodegeneration and AD pathology with neuroinflammation has not been investigated previously. Therefore, the current study examined whether NFL and Aß correlate with KP metabolites in elderly individuals to provide insight on the association between blood indicators of neurodegeneration and neuroinflammation. METHODS: Correlations between KP metabolites, measured using liquid chromatography and gas chromatography coupled with mass spectrometry, and plasma NFL and Aß concentrations, measured using single molecule array (Simoa) assays, were investigated in elderly individuals aged 65-90 years, with normal global cognition (Mini-Mental State Examination Score ≥ 26) from the Kerr Anglican Retirement Village Initiative in Ageing Health cohort. RESULTS: A positive correlation between NFL and the kynurenine to tryptophan ratio (K/T) reflecting indoleamine 2,3-dioxygenase activity was observed (r = .451, p < .0001). Positive correlations were also observed between NFL and kynurenine (r = .364, p < .0005), kynurenic acid (r = .384, p < .0001), 3-hydroxykynurenine (r = .246, p = .014), anthranilic acid (r = .311, p = .002), and quinolinic acid (r = .296, p = .003). Further, significant associations were observed between plasma Aß40 and the K/T (r = .375, p < .0005), kynurenine (r = .374, p < .0005), kynurenic acid (r = .352, p < .0005), anthranilic acid (r = .381, p < .0005), and quinolinic acid (r = .352, p < .0005). Significant associations were also observed between plasma Aß42 and the K/T ratio (r = .215, p = .034), kynurenic acid (r = .214, p = .035), anthranilic acid (r = .278, p = .006), and quinolinic acid (r = .224, p = .027) in the cohort. On stratifying participants based on their neocortical Aß load (NAL) status, NFL correlated with KP metabolites irrespective of NAL status; however, associations between plasma Aß and KP metabolites were only pronounced in individuals with high NAL while associations in individuals with low NAL were nearly absent. CONCLUSIONS: The current study shows that KP metabolite changes are associated with biomarker evidence of neurodegeneration. Additionally, the association between KP metabolites and plasma Aß seems to be NAL status dependent. Finally, the current study suggests that an association between neurodegeneration and neuroinflammation manifests in the periphery, suggesting that preventing cytoskeleton cytotoxicity by KP metabolites may have therapeutic potential.
Assuntos
Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/sangue , Biomarcadores/sangue , Cinurenina/metabolismo , Proteínas de Neurofilamentos/sangue , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , MasculinoRESUMO
Poly(amidoamine) dendrimer (PAMAM) is well-known for its high efficiency as a drug delivery vehicle. However, the intrinsic cytotoxicity and lack of a detectable signal to facilitate tracking have impeded its practical applications. Herein, we have developed a novel label-free fluorescent and biocompatible PAMAM derivative by simple surface modification of PAMAM using acetaldehyde. The modified PAMAM possessed a strong green fluorescence, which was generated by the C=N bonds of the resulting Schiff Bases via n-π* transition, while the intrinsic cytotoxicity of PAMAM was simultaneously ameliorated. Through further PEGylation, the fluorescent PAMAM demonstrated excellent intracellular tracking in human melanoma SKMEL28 cells. In addition, our PEGylated fluorescent PAMAM derivative achieved enhanced loading and delivery efficiency of the anticancer drug doxorubicin (DOX) compared to the original PAMAM. Importantly, the accelerated kinetics of DOX-encapsulated fluorescent PAMAM nanocomposites in an acidic environment facilitated intracellular drug release, which demonstrated comparable cytotoxicity to that of the free-form doxorubicin hydrochloride (DOX·HCl) against melanoma cells. Overall, our label free fluorescent PAMAM derivative offers a new opportunity of traceable and controlled delivery for DOX and other drugs of potential clinical importance.
Assuntos
Antineoplásicos/administração & dosagem , Dendrímeros/química , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Nanocompostos/química , Poliaminas/química , Acetaldeído/química , Antineoplásicos/química , Linhagem Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Corantes Fluorescentes/química , Células HEK293 , Humanos , Nanocompostos/toxicidade , Polietilenoglicóis/química , Bases de Schiff/químicaRESUMO
Deferasirox is an orally effective iron (Fe) chelator currently used for the treatment of iron-overload disease and has been implemented as an alternative to the gold standard chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO exhibits anticancer activity due to its ability to deplete cancer cells of iron. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines compared with DFO. Furthermore, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular (59)Fe and inhibiting iron uptake from human transferrin depending on the cell type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the antitumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control, and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein N-myc downstream-regulated gene 1 and upregulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1. Collectively, we demonstrate that deferasirox is an orally effective antitumor agent against solid tumors.
Assuntos
Antineoplásicos/farmacologia , Benzoatos/farmacologia , Quelantes de Ferro/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Triazóis/farmacologia , Administração Oral , Animais , Antígenos CD/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzoatos/uso terapêutico , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Cobre/metabolismo , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Deferasirox , Feminino , Humanos , Ferro/metabolismo , Quelantes de Ferro/uso terapêutico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Tumores Neuroectodérmicos Primitivos Periféricos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores da Transferrina/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Transplante Heterólogo , Triazóis/uso terapêutico , Zinco/metabolismoRESUMO
Dysregulation of the kynurenine pathway (KP) is believed to play a significant role in neurodegenerative and cognitive disorders. While some evidence links the KP to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), further studies are needed to clarify the overall picture of how inflammation-driven KP disturbances may contribute to symptomology in ME/CFS. Here, we report that plasma levels of most bioactive KP metabolites differed significantly between ME/CFS patients and healthy controls in a manner consistent with their known contribution to symptomology in other neurological disorders. Importantly, we found that enhanced production of the first KP metabolite, kynurenine (KYN), correlated with symptom severity, highlighting the relationship between inflammation, KP dysregulation, and ME/CFS symptomology. Other significant changes in the KP included lower levels of the downstream KP metabolites 3-HK, 3-HAA, QUIN, and PIC that could negatively impact cellular energetics. We also rationalized KP dysregulation to changes in the expression of inflammatory cytokines and, for the first time, assessed levels of the iron (Fe)-regulating hormone hepcidin that is also inflammation-responsive. Levels of hepcidin in ME/CFS decreased nearly by half, which might reflect systemic low Fe levels or possibly ongoing hypoxia. We next performed a proteomics screen to survey for other significant differences in protein expression in ME/CFS. Interestingly, out of the seven most significantly modulated proteins in ME/CFS patient plasma, 5 proteins have roles in maintaining gut health, which considering the new appreciation of how gut microbiome and health modulates systemic KP could highlight a new explanation of symptomology in ME/CFS patients and potential new prognostic biomarker/s and/or treatment avenues.
RESUMO
Thiosemicarbazones are a group of compounds that have received comprehensive investigation as anticancer agents. The antitumor activity of the thiosemicarbazone, 3-amino-2-pyridinecarboxaldehyde thiosemicarbazone (3-AP; triapine), has been extensively assessed in more than 20 phase I and II clinical trials. These studies have demonstrated that 3-AP induces methemoglobin (metHb) formation and hypoxia in patients, limiting its usefulness. Considering this problem, we assessed the mechanism of metHb formation by 3-AP compared with that of more recently developed thiosemicarbazones, including di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). This was investigated using intact red blood cells (RBCs), RBC lysates, purified oxyhemoglobin, and a mouse model. The chelation of cellular labile iron with the formation of a redox-active thiosemicarbazone-iron complex was found to be crucial for oxyhemoglobin oxidation. This observation was substantiated using a thiosemicarbazone that cannot ligate iron and also by using the chelator, desferrioxamine, that forms a redox-inactive iron complex. Of significance, cellular copper chelation was not important for metHb generation in contrast to its role in preventing tumor cell proliferation. Administration of Dp44mT to mice catalyzed metHb and cardiac metmyoglobin formation. However, ascorbic acid administered together with the drug in vivo significantly decreased metHb levels, providing a potential therapeutic intervention. Moreover, we demonstrated that the structure of the thiosemicarbazone is of importance in terms of metHb generation, because the DpT analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), does not induce metHb generation in vivo. Hence, DpC represents a next-generation thiosemicarbazone that possesses markedly superior properties. This investigation is important for developing more effective thiosemicarbazone treatment regimens.
Assuntos
Antineoplásicos/farmacologia , Metemoglobina/metabolismo , Piridinas/farmacologia , Tiossemicarbazonas/farmacologia , Animais , Ácido Ascórbico , Proliferação de Células/efeitos dos fármacos , Desferroxamina/farmacologia , Interações Medicamentosas , Eritrócitos/efeitos dos fármacos , Humanos , Hipóxia/induzido quimicamente , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Cinética , Camundongos , Oxirredução/efeitos dos fármacos , Oxiemoglobinas/metabolismoRESUMO
Glioblastoma (GBM) is a central nervous system tumor with poor prognosis due to the rapid development of resistance to mono chemotherapy and poor brain targeted delivery. Chemoimmunotherapy (CIT) combines chemotherapy drugs with activators of innate immunity that hold great promise for GBM synergistic therapy. Herein, we chose temozolomide, TMZ, and the epigenetic bromodomain inhibitor, OTX015, and further co-encapsulated them within our well-established erythrocyte membrane camouflaged nanoparticle to yield ApoE peptide decorated biomimetic nanomedicine (ABNM@TMZ/OTX). Our nanoplatform successfully addressed the limitations in brain-targeted drug co-delivery, and simultaneously achieved multidimensional enhanced GBM synergistic CIT. In mice bearing orthotopic GL261 GBM, treatment with ABNM@TMZ/OTX resulted in marked tumor inhibition and greatly extended survival time with little side effects. The pronounced GBM treatment efficacy can be ascribed to three key factors: (i) improved nanoparticle-mediated GBM targeting delivery of therapeutic agents by greatly enhanced blood circulation time and blood-brain barrier penetration; (ii) inhibited cellular DNA repair and enhanced TMZ sensitivity to tumor cells; (iii) enhanced anti-tumor immune responses by inducing immunogenic cell death and inhibiting PD-1/PD-L1 conjugation leading to enhanced expression of CD4+ and CD8+ T cells. The study validated a biomimetic nanomedicine to yield a potential new treatment for GBM.
RESUMO
Glioblastoma (GBM) is an intractable malignancy with high recurrence and mortality. Combinatorial therapy based on temozolomide (TMZ) and cisplatin (CDDP) shows promising potential for GBM therapy in clinical trials. However, significant challenges include limited blood-brain-barrier (BBB) penetration, poor targeting of GBM tissue/cells, and systemic side effects, which hinder its efficacy in GBM therapy. To surmount these challenges, new GBM-cell membrane camouflaged and pH-sensitive biomimetic nanoparticles (MNPs) inspired by the fact that cancer cells readily pass the BBB and localize with homologous cells, are developed. This study's results show that MNPs can efficiently co-load TMZ and CDDP, transport these across the BBB to specifically target GBM. Incorporation of pH-sensitive polymer then allows for controlled release of drug cargos at GBM sites for combination drug therapy. Mice bearing orthotopic U87MG or drug-resistant U251R GBM tumor and treated with MNPs@TMZ+CDDP show a potent anti-GBM effect, greatly extending the survival time relative to mice receiving single-drug loaded nanoparticles. No obvious side effects are apparent in histological analyses or blood routine studies. Considering these results, the study's new nanoparticle formulation overcomes multiple challenges currently limiting the efficacy of combined TMZ and CDDP GBM drug therapy and appears to be a promising strategy for future GBM combinatorial chemotherapy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Camundongos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Selective targeting of elevated copper (Cu) in cancer cells by chelators to induce tumor-toxic reactive oxygen species (ROS) may be a promising approach in the treatment of glioblastoma multiforme (GBM). Previously, the Cu chelator di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) attracted much interest due to its potent anti-tumor activity mediated by the formation of a highly redox-active Cu-Dp44mT complex. However, its translational potential was limited by the development of toxicity in murine models of cancer reflecting poor selectivity. Here, we overcame the limitations of Dp44mT by incorporating it in new biomimetic nanoparticles (NPs) optimized for GBM therapy. Biomimetic design elements enhancing selectivity included angiopeptide-2 functionalized red blood cell membrane (Ang-M) camouflaging of the NPs carrier. Co-loading Dp44mT with regadenoson (Reg), that transiently opens the blood-brain-barrier (BBB), yielded biomimetic Ang-MNPs@(Dp44mT/Reg) NPs that actively targeted and traversed the BBB delivering Dp44mT specifically to GBM cells. To further improve selectivity, we innovatively pre-loaded GBM tumors with Cu. Oral dosing of U87MG-Luc tumor bearing mice with diacetyl-bis(4-methylthiosemicarbazonato)-copperII (Cu(II)-ATSM), significantly enhanced Cu-level in GBM tumor. Subsequent treatment of mice bearing Cu-enriched orthotopic U87MG-Luc GBM with Ang-MNPs@(Dp44mT/Reg) substantially prevented orthotopic GBM growth and led to maximal increases in median survival time. These results highlighted the importance of both angiopeptide-2 functionalization and tumor Cu-loading required for greater selective cytotoxicity. Targeting Ang-MNPs@(Dp44mT/Reg) NPs also down-regulated antiapoptotic Bcl-2, but up-regulated pro-apoptotic Bax and cleaved-caspase-3, demonstrating the involvement of the apoptotic pathway in GBM suppression. Notably, Ang-MNPs@(Dp44mT/Reg) showed negligible systemic drug toxicity in mice, further indicating therapeutic potential that could be adapted for other central nervous system disorders.
Assuntos
Antineoplásicos , Glioblastoma , Nanopartículas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Biomimética , Caspase 3 , Linhagem Celular Tumoral , Quelantes/farmacologia , Cobre/metabolismo , Diacetil , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Tiossemicarbazonas , Proteína X Associada a bcl-2RESUMO
Pancreatic cancer is an aggressive neoplasm, with a mortality rate close to 100%. The most successful agent for pancreatic cancer treatment is gemcitabine, although the overall effect in terms of patient survival remains very poor. This study was initiated to evaluate a novel class of anticancer agents against pancreatic cancer. This group of compounds belongs to the dipyridyl thiosemicarbazone class that have been shown to have potent and selective activity against a range of different neoplasms in vitro and in vivo. We demonstrate for the first time in pancreatic cancer that these agents increase the expression of the growth and metastasis suppressor N-myc downstream-regulated gene 1 and its phosphorylation at Ser330 and Thr346 that is important for its activity against this tumor. In addition, these agents increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), whereas decreasing cyclin D1 in pancreatic cancer cells. Together, these molecular alterations account, in part, for the pronounced antitumor activity observed. Indeed, these agents had significantly higher antiproliferative activity in vitro than the established treatments for pancreatic cancer, namely gemcitabine and 5-fluorouracil. Studies in vivo demonstrated that a novel thiosemicarbazone, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone hydrochloride, completely inhibited the growth of pancreatic cancer xenografts with no evidence of marked alterations in normal tissue histology. Together, our studies have identified molecular effectors of a novel and potent antitumor agent that could be useful for pancreatic cancer treatment.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Quelantes de Ferro/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Tiossemicarbazonas/farmacologia , Regulação para Cima/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quelantes de Ferro/química , Quelantes de Ferro/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Tiossemicarbazonas/química , Tiossemicarbazonas/uso terapêutico , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 transcription. Here we have analyzed a group of novel di-2-pyridylketone thiosemicarbazone- and 2-benzoylpyridine thiosemicarbazone-based iron chelators that exhibit marked anticancer activity in vitro and in vivo (Proc Natl Acad Sci USA 103:7670-7675, 2006; J Med Chem 50:3716-3729, 2007). Several of these iron chelators, in particular 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), inhibited HIV-1 transcription and replication at much lower concentrations than did 311 and ICL670. Neither Bp4aT nor Bp4eT were toxic after a 24-h incubation. However, longer incubations for 48 h or 72 h resulted in cytotoxicity. Analysis of the molecular mechanism of HIV-1 inhibition showed that the novel iron chelators inhibited basal HIV-1 transcription, but not the nuclear factor-κB-dependent transcription or transcription from an HIV-1 promoter with inactivated SP1 sites. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1, suggesting that inhibition of CDK9 may contribute to the inhibition of HIV-1 transcription. Our study suggests the potential usefulness of Bp4aT or Bp4eT in antiretroviral regimens, particularly where resistance to standard treatment occurs.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Quelantes de Ferro/farmacologia , Tiossemicarbazonas/farmacologia , Células Cultivadas , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Células HEK293 , HIV-1/metabolismo , Humanos , Ferro/antagonistas & inibidores , Ferro/metabolismo , Quelantes de Ferro/química , Tiossemicarbazonas/química , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
The kynurenine pathway (KP) is a major route for L-tryptophan (L-TRP) metabolism, yielding a variety of bioactive compounds including kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and picolinic acid (PIC). These tryptophan catabolites are involved in the pathogenesis of many neuropsychiatric disorders, particularly when the KP becomes dysregulated. Accordingly, the enzymes that regulate the KP such as indoleamine 2,3-dioxygenase (IDO)/tryptophan 2,3-dioxygenase, kynurenine aminotransferases (KATs), and kynurenine 3-monooxygenase (KMO) represent potential drug targets as enzymatic inhibition can favorably rebalance KP metabolite concentrations. In addition, the galantamine-memantine combination, through its modulatory effects at the alpha7 nicotinic acetylcholine receptors and N-methyl-D-aspartate receptors, may counteract the effects of KYNA. The aim of this review is to highlight the effectiveness of IDO-1, KAT II, and KMO inhibitors, as well as the galantamine-memantine combination in the modulation of different KP metabolites. KAT II inhibitors are capable of decreasing the KYNA levels in the rat brain by a maximum of 80%. KMO inhibitors effectively reduce the central nervous system (CNS) levels of 3-HK, while markedly boosting the brain concentration of KYNA. Emerging data suggest that the galantamine-memantine combination also lowers L-TRP, kynurenine, KYNA, and PIC levels in humans. Presently, there are only 2 pathophysiological mechanisms (cholinergic and glutamatergic) that are FDA approved for the treatment of cognitive dysfunction for which purpose the galantamine-memantine combination has been designed for clinical use against Alzheimer's disease. The alpha7 nicotinic-NMDA hypothesis targeted by the galantamine-memantine combination has been implicated in the pathophysiology of various CNS diseases. Similarly, KYNA is well capable of modulating the neuropathophysiology of these disorders. This is known as the KYNA-centric hypothesis, which may be implicated in the management of certain neuropsychiatric conditions. In line with this hypothesis, KYNA may be considered as the "conductor of the orchestra" for the major pathophysiological mechanisms underlying CNS disorders. Therefore, there is great opportunity to further explore and compare the biological effects of these therapeutic modalities in animal models with a special focus on their effects on KP metabolites in the CNS and with the ultimate goal of progressing to clinical trials for many neuropsychiatric diseases.
RESUMO
Iron is a critical nutrient for DNA synthesis and cellular proliferation. Targeting iron in cancer cells using specific chelators is a potential new strategy for the development of novel anticancer agents. One such chelator, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), possesses potent and selective anticancer activity (J Med Chem 50:3716-3729, 2007). To elucidate the mechanisms of its potent antitumor activity, Bp4eT was labeled with (14)C. Its efficacy was then compared with the (14)C-labeled iron chelator pyridoxal isonicotinoyl hydrazone (PIH), which exhibits low anticancer activity. The ability of these ligands to permeate the cell membrane and their cellular retention was examined under various conditions using SK-N-MC neuroepithelioma cells. The rate of [(14)C]PIH uptake into cells was significantly (p < 0.001) lower than that of [(14)C]Bp4eT at 37°C, indicating that the increased hydrophilicity of [(14)C]PIH reduced membrane permeability. In contrast, the efflux of [(14)C]PIH was significantly (p < 0.05) higher than that of [(14)C]Bp4eT, leading to increased cellular retention of [(14)C]Bp4eT. In addition, the uptake and release of the (14)C-labeled chelators was not reduced by metabolic inhibitors, indicating that these processes were energy-independent. No significant differences were evident in the uptake of [(14)C]Bp4eT at 37 or 4°C, demonstrating a temperature-independent mechanism. Furthermore, adjusting the pH of the culture medium to model the tumor microenvironment did not affect [(14)C]Bp4eT membrane transport. It can be concluded that [(14)C]Bp4eT more effectively permeated the cell membrane and evaded rapid efflux in contrast to [(14)C]PIH. This property, in part, accounts for the more potent anticancer activity of Bp4eT relative to PIH.
Assuntos
Antineoplásicos/metabolismo , Líquido Intracelular/metabolismo , Quelantes de Ferro/metabolismo , Tiossemicarbazonas/metabolismo , Antineoplásicos/química , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Humanos , Quelantes de Ferro/química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tiossemicarbazonas/químicaRESUMO
The overexpression of phosphodiesterase 4 (PDE4) enzymes is reported in several neurodegenerative diseases. PDE4 depletes cyclic 3'-5' adenosine monophosphate (cAMP) and, in turn, cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF), the key players in cognitive function. The present study was undertaken to investigate the mechanism behind the protective effects of roflumilast (ROF), a cAMP-specific PDE4 inhibitor, against quinolinic acid (QUIN)-induced neurotoxicity using human primary cortical neurons. Cytotoxicity was analyzed using an MTS assay. Reactive oxygen species (ROS) and mitochondrial membrane potential were measured by DCF-DA and JC-10 staining, respectively. Caspase 3/7 activity was measured using an ApoTox-Glo Triplex assay kit. cAMP was measured using an ELISA kit. The protein expression of CREB, BDNF, SAP-97, synaptophysin, synapsin-I, and PSD-95 was analyzed by the Western blotting technique. QUIN exposure down-regulated CREB, BDNF, and synaptic protein expression in neurons. Pretreatment with ROF increased the intracellular cAMP, mitochondrial membrane potential, and nicotinamide adenine dinucleotide (NAD+) content and decreased the ROS and caspase 3/7 levels in QUIN-exposed neurons. ROF up-regulated the expression of synapse proteins SAP-97, synaptophysin, synapsin-I, PSD-95, and CREB and BDNF, which indicates its potential role in memory. This study suggests for the first time that QUIN causes pre- and postsynaptic protein damage. We further demonstrate the restorative effects of ROF on the mitochondrial membrane potential and antiapoptotic properties in human neurons. These data encourage further investigations to reposition ROF in neurodegenerative diseases and their associated cognitive deficits.
Assuntos
Inibidores da Fosfodiesterase 4 , Aminopiridinas , Benzamidas , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ciclopropanos , Humanos , Neurônios/metabolismo , Neurotoxinas , Estresse Oxidativo , Inibidores da Fosfodiesterase 4/farmacologia , Ácido Quinolínico , Sinapses/metabolismoRESUMO
BACKGROUND/OBJECTIVE: Hepcidin, an iron-regulating hormone, suppresses the release of iron by binding to the iron exporter protein, ferroportin, resulting in intracellular iron accumulation. Given that iron dyshomeostasis has been observed in Alzheimer's disease (AD) together with elevated serum hepcidin levels, the current study examined whether elevated serum hepcidin levels are an early event in AD pathogenesis by measuring the hormone in cognitively normal older adults at risk of AD, based on high neocortical amyloid-ß load (NAL). METHODS: Serum hepcidin levels in cognitively normal participants (nâ=â100) aged between 65-90 years were measured using ELISA. To evaluate NAL, all participants underwent 18F-florbetaben positron emission tomography. A standard uptake value ratio (SUVR)<1.35 was classified as low NAL (nâ=â65) and ≥1.35 (nâ=â35) was classified as high NAL. RESULTS: Serum hepcidin was significantly higher in participants with high NAL compared to those with low NAL before and after adjusting for covariates: age, gender, and APOEÉ4 carriage (pâ<â0.05). A receiver operating characteristic curve based on a logistic regression of the same covariates, the base model, distinguished high from low NAL (area under the curve, AUCâ=â0.766), but was outperformed when serum hepcidin was added to the base model (AUCâ=â0.794) and further improved with plasma Aß42/40 ratio (AUCâ=â0.829). CONCLUSION: The present findings indicate that serum hepcidin is increased in individuals at risk for AD and contribute to the body of evidence supporting iron dyshomeostasis as an early event of AD. Further, hepcidin may add value to a panel of markers that contribute toward identifying individuals at risk of AD; however, further validation studies are required.
Assuntos
Peptídeos beta-Amiloides/sangue , Cognição/fisiologia , Hepcidinas/sangue , Neocórtex/metabolismo , Fragmentos de Peptídeos/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos Transversais , Feminino , Humanos , Masculino , Tomografia por Emissão de Pósitrons/métodosRESUMO
The aim of this study was to develop and validate HPLC methods for the determination in plasma of two novel thiosemicarbazone anti-tumour drugs developed in our laboratories (Dp44mT and N4mT). The appropriate separations were achieved using a HS F5 HPLC column with the mobile phase composed of a mixture of either acetate buffer/EDTA or EDTA and acetonitrile (62:38 and 50:50, v/v, respectively). The plasma samples were pretreated with SPE (phenyl and C18, respectively). Furthermore, these methods were successfully applied to in vitro plasma stability experiments. The investigation has clearly shown that both thiosemicarbazones are markedly more stable in plasma than their aroylhydrazone forerunners.
Assuntos
Antineoplásicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Naftalenos/sangue , Tiossemicarbazonas/sangue , Análise de Variância , Animais , Interpretação Estatística de Dados , Estabilidade de Medicamentos , Humanos , Isoniazida/análogos & derivados , Isoniazida/análise , Isoniazida/metabolismo , Piridoxal/análogos & derivados , Piridoxal/análise , Piridoxal/metabolismo , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SuínosRESUMO
Upregulation of the kynurenine pathway (KP) of tryptophan metabolism is commonly observed in neurodegenerative disease. When activated, L-kynurenine (KYN) increases in the periphery and central nervous system where it is further metabolised to other neuroactive metabolites including 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA) and quinolinic acid (QUIN). Particularly biologically relevant metabolites are 3-HK and QUIN, formed downstream of the enzyme kynurenine 3-monooxygenase (KMO) which plays a pivotal role in maintaining KP homeostasis. Indeed, excessive production of 3-HK and QUIN has been described in neurodegenerative disease including Alzheimer's disease and Huntington's disease. In this study, we characterise KMO activity in human primary neurons and identified new mechanisms by which KMO activation mediates neurotoxicity. We show that while transient activation of the KP promotes synthesis of the essential co-enzyme nicotinamide adenine dinucleotide (NAD+), allowing cells to meet short-term increased energy demands, chronic KMO activation induces production of reactive oxygen species (ROS), mitochondrial damage and decreases spare-respiratory capacity (SRC). We further found that these events generate a vicious-cycle, as mitochondrial dysfunction further shunts the KP towards the KMO branch of the KP to presumably enhance QUIN production. These mechanisms may be especially relevant in neurodegenerative disease as neurons are highly sensitive to oxidative stress and mitochondrial impairment.
Assuntos
Sobrevivência Celular/fisiologia , Quinurenina 3-Mono-Oxigenase/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Trifosfato de Adenosina/metabolismo , Encéfalo/metabolismo , Células HEK293 , Humanos , Ácido Cinurênico/metabolismo , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Doenças Mitocondriais/metabolismo , NAD/metabolismo , Cultura Primária de Células , Ácido Quinolínico/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Chronic kynurenine pathway (KP) activation is implicated in Alzheimer's disease (AD) pathophysiology and results in quinolinic acid-induced excitotoxic stimulation of the N-methyl-D-aspartate receptor. However, most studies focus on plasma and it is unclear if peripheral concentrations reflect brain concentrations and how these may correlate to the AD biomarkers amyloid-ß, total-tau (t-tau), or phosphorylated-tau (p-tau). We characterized the KP in matched plasma and cerebrospinal fluid (CSF) samples from 20 AD patients and 18 age-matched control subjects. Plasma concentrations of kynurenine (KYN), 3-hydroxykynurenine, anthranilic acid, picolinic acid, and neopterin significantly correlated with their respective CSF levels. In patients with AD, plasma KYN (r = -0.48, p = 0.033) and picolinic acid (r = -0.57, p = 0.009) inversely correlated with CSF p-tau and t-tau, respectively. Furthermore, in AD CSF, increased 3-hydroxykynurenine/KYN ratio correlated with t-tau (r = 0.58, p = 0.009) and p-tau (r = 0.52, p = 0.020). These data support KP involvement in AD pathogenesis and add to the case for the therapeutic modulation of the KP in AD.