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1.
Cell Rep ; 34(10): 108831, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33691103

RESUMO

Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8+ T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Fosfogluconato Desidrogenase/metabolismo , 6-Aminonicotinamida/química , 6-Aminonicotinamida/farmacologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Granzimas/genética , Granzimas/metabolismo , Humanos , Imunoterapia , Listeria monocytogenes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/fisiologia , Fosfogluconato Desidrogenase/antagonistas & inibidores , Fosfogluconato Desidrogenase/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transplante Heterólogo
2.
Vet Res ; 51(1): 29, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32101153

RESUMO

Echinococcosis is a zoonotic disease caused by cestode species of the genus Echinococcus, which demonstrates considerable medical and veterinary concerns. The development of novel drugs for echinococcosis treatment is urgently needed. In this study, we demonstrated that lonidamine (LND) and 6-aminonicotinamide (6-AN) exhibited considerable in vitro effects against both larval- and adult-stage of E. granulosussensu stricto (s. s.) and E. multilocularis. The combination of LND and 6-AN exhibited a significantly higher activity than the single drug treatment. These results highlight the therapeutic potential of LND, 6-AN and the combination of LND and 6-AN for the treatment of echinococcosis.


Assuntos
6-Aminonicotinamida/farmacologia , Anticestoides/farmacologia , Echinococcus granulosus/efeitos dos fármacos , Echinococcus multilocularis/efeitos dos fármacos , Indazóis/farmacologia , Animais , Equinococose/tratamento farmacológico , Echinococcus granulosus/crescimento & desenvolvimento , Echinococcus multilocularis/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento
3.
Mol Cell Endocrinol ; 499: 110595, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31563469

RESUMO

The pentose phosphate pathway (PPP) plays an important role in the biosynthesis of ribonucleotide precursor and NADPH. Cancer cells frequently increase the flux of glucose into the PPP to support the anabolic demands and regulate oxidative stress. Consistently, metabolomic analyses indicate an upregulation of the PPP in thyroid cancer. In the present study, we found that the combination of glucose-6-phosphate dehydrogenase (G6PD) and transketolase inhibitors (6-aminonicotinamide and oxythiamine) exerted an additive or synergistic effect on cell growth inhibition in thyroid cancer cells. Targeting PPP significantly increased cellular reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress and apoptosis. Suppressed cell viability could be partially rescued with treatment with the ROS scavenger or apoptosis inhibitor but not ER-stress inhibitor. Taken together, dual PPP blockade leads to pharmacologic additivity or synergism and causes ROS-mediated apoptosis in thyroid cancer cells.


Assuntos
6-Aminonicotinamida/farmacologia , Oxitiamina/farmacologia , Via de Pentose Fosfato/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucosefosfato Desidrogenase , Humanos , NADP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Neoplasias da Glândula Tireoide/tratamento farmacológico
4.
EBioMedicine ; 49: 232-246, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31680002

RESUMO

BACKGROUND: Hepatitis B surface antigen (HBsAg) is one of the important clinical indexes for hepatitis B virus (HBV) infection diagnosis and sustained seroconversion of HBsAg is an indicator for functional cure. However, the level of HBsAg could not be reduced by interferons and nucleoside analogs effectively. Therefore, identification of a new drug targeting HBsAg is urgently needed. METHODS: In this study, 6-AN was screened out from 1500 compounds due to its low cytotoxicity and high antiviral activity. The effect of 6-AN on HBV was examined in HepAD38, HepG2-NTCP and PHHs cells. In addition, the antivirus effect of 6-AN was also identified in mouse model. FINDINGS: 6-AN treatment resulted in a significant decrease of HBsAg and other viral markers both in vitro and in vivo. Furthermore, we found that 6-AN inhibited the activities of HBV SpI, SpII and core promoter by decreasing transcription factor PPARα, subsequently reduced HBV RNAs transcription and HBsAg production. INTERPRETATION: We have identified a novel small molecule to inhibit HBV core DNA, HBV RNAs, HBsAg production, as well as cccDNA to a minor degree both in vitro and in vivo. This study may shed light on the development of a novel class of anti-HBV agent.


Assuntos
6-Aminonicotinamida/farmacologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Replicação Viral/efeitos dos fármacos , 6-Aminonicotinamida/química , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos , Viremia/sangue
5.
Biomed Pharmacother ; 108: 618-624, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30243096

RESUMO

Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It is well accepted that the epithelial-mesenchymal transition (EMT) plays a key role in the process of metastasis. As a cytokine that macrophage secretes, IL-6 is involved in the progression of tumors, including the invasion and metastasis via kinds of signaling pathways. However, the mechanism of interactions between IL-6, macrophage, EMT and colon cancer is not fully understood. Increased CD68+ macrophages and IL-6 level were found in colon tumor as compared to normal colon tissue. Metastatic lymph node showed even more CD68+ macrophages and higher IL-6 level than the primary tumor. These results suggested that macrophages and IL-6 play an important role in EMT of colon cancer. In order to investigate the effect of macrophage and IL-6 on EMT of colon cancer, we cultured human colon carcinoma cell line SW48 with conditioned medium (CM) from PMA-stimulated monocyte THP-1 cells and tested for IL-6 dependent EMT pathways. Wound healing assay and Transwell assay were used to analyze cell migration and invasion. Results showed that CM-treated SW48 cells increased IL-6 production and displayed elevated capacity of migration and invasion compared to untreated cells. Increased expressions of EMT markers (N-cadherin, Vimentin and ß-catenin) and decreased expression of EMT marker(E-cadherin) were found in CM-treated SW48 cells by Western Blot. The addition of an anti-IL-6 antibody significantly inhibited the increase of EMT markers (Vimentin and ß-catenin) as well as cell migration and invasion, suggesting that IL-6 played a critical role in promoting EMT of CM-treated SW48 cells. In addition, we found that the levels of p-STAT3 and p-ERK increased in CM-treated SW48 compared to untreated cells, which can be reversed by AG490, an inhibitor of JAK. In the meantime, the suppression of JAK-associated signaling pathways caused a decrease of ß-catenin. In summary, our study suggested that macrophage-induced IL-6 promotes migration and invasion of colon cancer cell via Wnt/ß-catenin pathway in STAT3/ERK-dependent way.


Assuntos
Neoplasias do Colo/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Transcrição STAT3/metabolismo , Células THP-1/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , 6-Aminonicotinamida/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metiltioinosina/farmacologia , Células THP-1/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos
6.
Chem Biol Interact ; 294: 167-177, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30170107

RESUMO

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (µM), although pre-treatment with 100 µM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.


Assuntos
6-Aminonicotinamida/farmacologia , Leishmania/metabolismo , Metabolômica , Via de Pentose Fosfato/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Interações Hidrofóbicas e Hidrofílicas , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Fosfogluconato Desidrogenase/metabolismo
7.
Int J Oncol ; 53(4): 1703-1712, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066842

RESUMO

Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway. Multiple studies have previously revealed that elevated G6PD levels promote cancer progression in numerous tumor types; however, the underlying mechanism remains unclear. In the present study, it was demonstrated that high G6PD expression is a poor prognostic factor in bladder cancer, and the levels of G6PD expression increase with increasing tumor stage. Patients with bladder cancer with high G6PD expression had worse survival rates compared with those with lower G6PD expression in resected tumors. In vitro experiments revealed that knockdown of G6PD suppressed cell viability and growth in Cell Counting Kit-8 and colony formation assays, and increased apoptosis in bladder cancer cell lines compared with normal cells. Further experiments indicated that the weakening of the survival ability in G6PD-knockdown bladder cancer cells may be explained by intracellular reactive oxygen species accumulation and protein kinase B pathway suppression. Furthermore, it was additionally revealed that 6-aminonicotinamide (6-AN), a competitive G6PD inhibitor, may be a potential therapy for bladder cancer, particularly in cases with high G6PD expression, and that the combination of cisplatin and 6-AN may optimize the clinical dose or minimize the side effects of cisplatin.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Glucosefosfato Desidrogenase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias da Bexiga Urinária/patologia , 6-Aminonicotinamida/farmacologia , 6-Aminonicotinamida/uso terapêutico , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Feminino , Técnicas de Silenciamento de Genes , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/genética , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxa de Sobrevida , Bexiga Urinária/patologia , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/terapia
8.
Pestic Biochem Physiol ; 143: 141-146, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29183584

RESUMO

Aluminum phosphide (AlP) poisoning is a severe toxicity with 30-70% mortality rate. However, several case reports presented AlP-poisoned patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and extensive hemolysis who survived the toxicity. This brought to our mind that maybe G6PD deficiency could protect the patients from severe fatal poisoning by this pesticide. In this research, we investigated the protective effect of 6-aminonicotinamide (6-AN)- as a well-established inhibitor of the NADP+- dependent enzyme 6-phosphogluconate dehydrogenase- on isolated rat hepatocytes in AlP poisoning. Hepatocytes were isolated by collagenase perfusion method and incubated into three different flasks: control, AlP, and 6-AN+ALP. Cellar parameters such as cell viability, reactive oxygen species (ROS) formation, mitochondria membrane potential collapse (MMP), lysosomal integrity, content of reduced (GSH) and oxidized glutathione (GSSG) and lipid peroxidation were assayed at intervals. All analyzed cellular parameters significantly decreased in the third group (6-AN+AlP) compared to the second group (AlP), showing the fact that G6PD deficiency induced by 6-AN had a significant protective effect on the hepatocytes. It was concluded that G6PD deficiency significantly reduced the hepatotoxicity of AlP. Future drugs with the power to induce such deficiency may be promising in treatment of AlP poisoning.


Assuntos
6-Aminonicotinamida/farmacologia , Compostos de Alumínio/toxicidade , Glucosefosfato Desidrogenase/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Praguicidas/toxicidade , Fosfinas/toxicidade , Substâncias Protetoras/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Hepatócitos/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
9.
Free Radic Biol Med ; 109: 189-200, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28192232

RESUMO

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H2O2) levels and glutathione redox potential (EGSH), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research.


Assuntos
Células Endoteliais/metabolismo , Glutationa/análise , Peróxido de Hidrogênio/análise , Miócitos Cardíacos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , 6-Aminonicotinamida/farmacologia , Animais , Animais Geneticamente Modificados , Butionina Sulfoximina/farmacologia , Sistema Cardiovascular/citologia , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citosol/metabolismo , Citosol/ultraestrutura , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Oxirredução , Via de Pentose Fosfato/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Peixe-Zebra
10.
Bioorg Med Chem ; 24(19): 4711-4722, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27544589

RESUMO

In organ transplantation, T cell-mediated immune responses play a key role in the rejection of allografts. Janus kinase 3 (JAK3) is specifically expressed in hematopoietic cells and associated with regulation of T cell development via interleukin-2 signaling pathway. Here, we designed novel 4,6-diaminonicotinamide derivatives as immunomodulators targeting JAK3 for prevention of transplant rejection. Our optimization of C4- and C6-substituents and docking calculations to JAK3 protein confirmed that the 4,6-diaminonicotinamide scaffold resulted in potent inhibition of JAK3. We also investigated avoidance of human ether-a-go-go related gene (hERG) inhibitory activity. Selected compound 28 in combination with tacrolimus prevented allograft rejection in a rat heterotopic cardiac transplantation model.


Assuntos
6-Aminonicotinamida/análogos & derivados , 6-Aminonicotinamida/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , 6-Aminonicotinamida/síntese química , 6-Aminonicotinamida/uso terapêutico , Animais , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/uso terapêutico , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Ratos
11.
Tumour Biol ; 37(5): 6027-34, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26596840

RESUMO

Pentose phosphate pathway (PPP) is a metabolic pathway that generates NADPH and pentose. PPP genes have been reported to be primarily or secondarily upregulated in many cancers. We aimed to study the general alteration of PPP in acute myelogenous leukemia (AML). We performed data mining and analysis of the Cancer Genome Atlas (TCGA) AML dataset for genetic alteration of the PPP gene set. In vitro studies including proliferation, migration, and invasion assays, together with metabolite consumption and oxidation assays, were performed. PPP genes were upregulated in 61 % of patients with AML. The majority of altered cases were expression changes measured by RNA sequencing. Expressions of critical PPP genes such as G6PD, PFKL, PFKP, and PGLS were consistently upregulated in all altered cases. Altered PPP is not associated with survival or disease relapse. PPP inhibition using 6-aminonicotinamide (6AN) increases glucose oxidative metabolism in AML. 6AN decreased the glucose oxidation and increased fatty acid oxidation. Here, we showed that PPP inhibition increased glucose oxidative metabolism in AML. PPP inhibition suppressed growth, migration, and invasion of AML, but not colony formation. PPP plays an important role in AML. Our results could contribute to the development of novel targeted treatment.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Via de Pentose Fosfato , 6-Aminonicotinamida/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Variação Genética , Glucose/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Oxirredução/efeitos dos fármacos , Prognóstico
12.
Chem Pharm Bull (Tokyo) ; 63(10): 825-32, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26423040

RESUMO

Structural optimization of 2-aminonicotinamide derivatives as ghrelin receptor inverse agonists is reported. So as to avoid mechanism-based inactivation (MBI) of CYP3A4, 1,3-benzodioxol ring of the lead compound was modified. Improvement of the main activity and lipophilicity was achieved simultaneously, leading to compound 18a, which showed high lipophilic ligand efficiency (LLE) and low MBI activity.


Assuntos
6-Aminonicotinamida/análogos & derivados , 6-Aminonicotinamida/farmacologia , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Citocromo P-450 CYP3A/metabolismo , Agonismo Inverso de Drogas , Receptores de Grelina/agonistas , 6-Aminonicotinamida/metabolismo , Fármacos Antiobesidade/metabolismo , Descoberta de Drogas , Humanos , Microssomos Hepáticos/metabolismo , Obesidade/tratamento farmacológico , Receptores de Grelina/metabolismo
13.
Sci Rep ; 5: 9925, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25894462

RESUMO

Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM.


Assuntos
Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Via de Pentose Fosfato/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , 6-Aminonicotinamida/farmacologia , Afatinib , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe , Glucose/metabolismo , Humanos , Metaboloma/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Oxigênio/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/metabolismo
14.
Exp Cell Res ; 327(1): 48-56, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-24880125

RESUMO

AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA.


Assuntos
Autofagia/fisiologia , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 6-Aminonicotinamida/farmacologia , Sequência de Aminoácidos , Autofagia/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Cloroquina/farmacologia , Células HEK293 , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Células K562 , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/fisiologia , Dados de Sequência Molecular , Proteólise/efeitos dos fármacos
15.
Am J Physiol Heart Circ Physiol ; 306(5): H709-17, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24414069

RESUMO

In vitro studies suggested that glucose metabolism through the oxidative pentose phosphate pathway (oxPPP) can paradoxically feed superoxide-generating enzymes in failing hearts. We therefore tested the hypothesis that acute inhibition of the oxPPP reduces oxidative stress and enhances function and metabolism of the failing heart, in vivo. In 10 chronically instrumented dogs, congestive heart failure (HF) was induced by high-frequency cardiac pacing. Myocardial glucose consumption was enhanced by raising arterial glycemia to levels mimicking postprandial peaks, before and after intravenous administration of the oxPPP inhibitor 6-aminonicotinamide (80 mg/kg). Myocardial energy substrate metabolism was measured with radiolabeled glucose and oleic acid, and cardiac 8-isoprostane output was used as an index of oxidative stress. A group of five chronically instrumented, normal dogs served as control. In HF, raising glycemic levels from ∼ 80 to ∼ 170 mg/dL increased cardiac isoprostane output by approximately twofold, whereas oxPPP inhibition normalized oxidative stress and enhanced cardiac oxygen consumption, glucose oxidation, and stroke work. In normal hearts glucose infusion did not induce significant changes in cardiac oxidative stress. Myocardial tissue concentration of 6P-gluconate, an intermediate metabolite of the oxPPP, was significantly reduced by ∼ 50% in treated versus nontreated failing hearts, supporting the inhibitory effect of 6-aminonicotinamide. Our study indicates an important contribution of the oxPPP activity to cardiac oxidative stress in HF, which is particularly pronounced during common physiological changes such as postprandial glycemic peaks.


Assuntos
6-Aminonicotinamida/farmacologia , Cardiotônicos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Miocárdio/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Animais , Glicemia/metabolismo , Dinoprosta/análogos & derivados , Dinoprosta/metabolismo , Modelos Animais de Doenças , Cães , Gluconatos/metabolismo , Glicólise/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Masculino , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Recuperação de Função Fisiológica , Volume Sistólico/efeitos dos fármacos , Superóxidos/metabolismo , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacos
16.
Mol Cancer Res ; 12(1): 48-57, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24296756

RESUMO

UNLABELLED: The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-κB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. IMPLICATIONS: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors.


Assuntos
6-Aminonicotinamida/farmacologia , Autofagia/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Via de Pentose Fosfato/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Caspase 1/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Reprogramação Celular , Cloroquina/farmacologia , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Hidroxicloroquina/farmacologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos A , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/metabolismo , Complexos Multiproteicos/biossíntese , NF-kappa B/biossíntese , Consumo de Oxigênio/genética , Via de Pentose Fosfato/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/biossíntese , Teratogênicos/farmacologia , Proteína 2 do Complexo Esclerose Tuberosa
17.
Reprod Fertil Dev ; 26(7): 931-42, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23859479

RESUMO

The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos/fisiologia , Via de Pentose Fosfato/fisiologia , 6-Aminonicotinamida/farmacologia , Animais , Bovinos , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Células do Cúmulo/fisiologia , Feminino , Glucose/metabolismo , Ácido Láctico/biossíntese , Meiose/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , NADP/farmacologia , Oócitos/ultraestrutura , Oxirredução , Via de Pentose Fosfato/efeitos dos fármacos
18.
Sci Rep ; 3: 2973, 2013 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24131900

RESUMO

Pellagra is a photosensitivity syndrome characterized by three "D's": diarrhea, dermatitis, and dementia as a result of niacin deficiency. However, the molecular mechanisms of photosensitivity dermatitis, the hallmark abnormality of this syndrome, remain unclear. We prepared niacin deficient mice in order to develop a murine model of pellagra. Niacin deficiency induced photosensitivity and severe diarrhea with weight loss. In addition, niacin deficient mice exhibited elevated expressions of COX-2 and PGE syntheses (Ptges) mRNA. Consistently, photosensitivity was alleviated by a COX inhibitor, deficiency of Ptges, or blockade of EP4 receptor signaling. Moreover, enhanced PGE2 production in niacin deficiency was mediated via ROS production in keratinocytes. In line with the above murine findings, human skin lesions of pellagra patients confirmed the enhanced expression of Ptges. Niacin deficiency-induced photosensitivity was mediated through EP4 signaling in response to increased PGE2 production via induction of ROS formation.


Assuntos
Dinoprostona/metabolismo , Niacina/deficiência , Transtornos de Fotossensibilidade/metabolismo , Espécies Reativas de Oxigênio/metabolismo , 6-Aminonicotinamida/farmacologia , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dermatite/etiologia , Dermatite/metabolismo , Dermatite/patologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Camundongos , Niacina/antagonistas & inibidores , Transtornos de Fotossensibilidade/etiologia , Transtornos de Fotossensibilidade/patologia , Transdução de Sinais/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos
19.
J Neurochem ; 125(3): 420-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23410250

RESUMO

Neural stem/progenitor cells (NSPCs) are multipotent cells within the embryonic and adult brain that give rise to both neuronal and glial cell lineages. Maintenance of NSPC multipotency is promoted by low oxygen tension, although the metabolic underpinnings of this trait have not been described. In this study, we investigated the metabolic state of undifferentiated NSPCs in culture, and tested their relative reliance on oxidative versus glycolytic metabolism for survival, as well as their dependence on hypoxia inducible factor-1alpha (HIF-1α) expression for maintenance of metabolic phenotype. Unlike primary neurons, NSPCs from embryonic and adult mice survived prolonged hypoxia in culture. In addition, NSPCs displayed greater susceptibility to glycolytic inhibition compared with primary neurons, even in the presence of alternative mitochondrial TCA substrates. NSPCs were also more resistant than neurons to mitochondrial cyanide toxicity, less capable of utilizing galactose as an alternative substrate to glucose, and more susceptible to pharmacological inhibition of the pentose phosphate pathway by 6-aminonicotinamide. Inducible deletion of exon 1 of the Hif1a gene improved the ability of NSPCs to utilize pyruvate during glycolytic inhibition, but did not alter other parameters of metabolism, including their ability to withstand prolonged hypoxia. Taken together, these data indicate that NSPCs have a relatively low requirement for oxidative metabolism for their survival and that hypoxic resistance is not dependent upon HIF-1α signaling.


Assuntos
Hipóxia Celular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Neurais/metabolismo , Oxigênio/metabolismo , 6-Aminonicotinamida/farmacologia , Análise de Variância , Animais , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Filamentos Intermediários/deficiência , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Proteínas do Tecido Nervoso/deficiência , Nestina , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , Fosfopiruvato Hidratase/metabolismo , Ácido Pirúvico/metabolismo , Cianeto de Sódio/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
20.
Reprod Domest Anim ; 48(4): 545-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23189959

RESUMO

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.


Assuntos
Glicólise/fisiologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Via de Pentose Fosfato/fisiologia , Suínos , 6-Aminonicotinamida/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Células do Cúmulo , Feminino , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Ácido Láctico/biossíntese , NADP/farmacologia , Oócitos/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Fluoreto de Sódio/farmacologia
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