RESUMO
Forearm rotation (supination/pronation) alters corticospinal excitability to the biceps brachii, but it is unclear whether corticospinal excitability is influenced by joint angle, muscle length, or both. Thus the purpose of this study was to separately examine elbow joint angle and muscle length on corticospinal excitability. Corticospinal excitability to the biceps and triceps brachii was measured using motor evoked potentials (MEPs) elicited via transcranial magnetic stimulation. Spinal excitability was measured using cervicomedullary motor evoked potentials (CMEPs) elicited via transmastoid electrical stimulation. Elbow angles were manipulated with a fixed biceps brachii muscle length (and vice versa) across five unique postures: 1) forearm neutral, elbow flexion 90°; 2) forearm supinated, elbow flexion 90°; 3) forearm pronated, elbow flexion 90°; 4) forearm supinated, elbow flexion 78°; and 5) forearm pronated, elbow flexion 113°. A musculoskeletal model determined biceps brachii muscle length for postures 1-3, and elbow joint angles (postures 4-5) were selected to maintain biceps length across forearm orientations. MEPs and CMEPs were elicited at rest and during an isometric contraction of 10% of maximal biceps muscle activity. At rest, MEP amplitudes to the biceps were largest during supination, which was independent of elbow joint angle. CMEP amplitudes were not different when the elbow was fixed at 90° but were largest in pronation when muscle length was controlled. During an isometric contraction, there were no significant differences across forearm postures for either MEP or CMEP amplitudes. These results highlight that elbow joint angle and biceps brachii muscle length can each independently influence spinal excitability. NEW & NOTEWORTHY Changes in upper limb posture can influence the responsiveness of the central nervous system to artificial stimulations. We established a novel approach integrating neurophysiology techniques with biomechanical modeling. Through this approach, the effects of elbow joint angle and biceps brachii muscle length on corticospinal and spinal excitability were assessed. We demonstrate that spinal excitability is uniquely influenced by joint angle and muscle length, and this highlights the importance of accounting for muscle length in neurophysiological studies.
Assuntos
Potencial Evocado Motor , Antebraço/fisiologia , Articulações/fisiologia , Músculo Esquelético/fisiologia , Postura , Tratos Piramidais/fisiologia , Adulto , Fenômenos Biomecânicos , Humanos , Contração Isométrica , Masculino , Músculo Esquelético/anatomia & histologiaRESUMO
A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1ß from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1ß secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-κB and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1ß in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases.
Assuntos
Monócitos , Núcleosídeo-Difosfato Quinase , Humanos , Monócitos/metabolismo , Inflamassomos/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sobrevivência Celular , Interleucina-1beta/metabolismoRESUMO
Drug repurposing is a cost-effective means of targeting new therapies for cancer. We have examined the effects of the repurposed drugs, bezafibrate, medroxyprogesterone acetate and valproic acid on human osteosarcoma cells, i.e., SAOS2 and MG63 compared with their normal cell counterparts, i.e. mesenchymal stem/stromal cells (MSCs). Cell growth, viability and migration were measured by biochemical assay and live cell imaging, whilst levels of lipid-synthesising enzymes were measured by immunoblotting cell extracts. These drug treatments inhibited the growth and survival of SAOS2 and MG63 cells most effectively when used in combination (termed V-BAP). In contrast, V-BAP treated MSCs remained viable with only moderately reduced cell proliferation. V-BAP treatment also inhibited migratory cell phenotypes. MG63 and SAOS2 cells expressed much greater levels of fatty acid synthase and stearoyl CoA desaturase 1 than MSCs, but these elevated enzyme levels significantly decreased in the V-BAP treated osteosarcoma cells prior to cell death. Hence, we have identified a repurposed drug combination that selectively inhibits the growth and survival of human osteosarcoma cells in association with altered lipid metabolism without adversely affecting their non-transformed cell counterparts.
Assuntos
Bezafibrato/administração & dosagem , Neoplasias Ósseas/patologia , Proliferação de Células/efeitos dos fármacos , Acetato de Medroxiprogesterona/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteossarcoma/patologia , Ácido Valproico/administração & dosagem , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/enzimologia , Linhagem Celular Tumoral , Regulação para Baixo , Reposicionamento de Medicamentos , Quimioterapia Combinada , Ácido Graxo Sintases/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/enzimologia , Estearoil-CoA Dessaturase/metabolismo , Regulação para CimaRESUMO
The loss of anti-proliferative responsiveness in prostate cancer cell lines toward ligands for vitamin D receptor, retinoic acid receptors/retinoid X receptors and peroxisome proliferator activated receptor (PPAR)alpha/gamma may entail underlying epigenetic events, as ligand insensitivity reflects significantly altered messenger RNA expression of corepressors and histone-modifying enzymes. Expression patterns were dependent on phases of the cell cycle and associated with repressed basal gene expression of vitamin D receptor and PPARalpha/gamma target genes, for example CDKN1A [encodes p21((waf1/cip1))]. Elevated nuclear corepressor 1 (NCOR1) and nuclear corepressor 2/silencing mediator of retinoic acid and thyroid hormone receptor protein levels were detected in prostate cancer cell lines compared with non-malignant counterparts. Knockdown of the corepressor NCOR1 significantly elevated basal expression of a cohort of target genes, including CDKN1A. Both chemical [histone deacetylases inhibitor (HDACi)] and NCOR1 knockdown targeting enhanced anti-proliferative sensitivity toward PPARalpha/gamma ligands in prostate cancer cell lines. Pursuing PPARalpha/gamma signaling, microarray approaches were undertaken to identify pathways and genes regulated uniquely by a combination of PPARalpha/gamma activation and HDAC inhibition. Again, HDACi and knockdown approaches demonstrated that elevated NCOR1 expression and activity distorted PPARalpha/gamma gene targets centered on, for example cell cycle control, including CDKN1A and TGFBRAP1. Quantitative real time polymerase chain reaction validation and chromatin immunoprecipitation assays both confirmed that elevated NCOR1 disrupted the ability of PPARalpha/gamma to regulate key target genes (CDKN1A and TGFBRAP1). Interrogation of these relationships in prostate cancer samples using principal component and partial correlation analyses established significant interdependent relationships between NCOR1-PPARalpha/gamma and representative target genes, independently of androgen receptor expression. Therefore, we conclude that elevated NCOR1 distorts the actions of PPARalpha/gamma selectively and generates a potential epigenetic lesion with diagnostic and prognostic significance.
Assuntos
Biomarcadores Tumorais/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Epigênese Genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Correpressor 1 de Receptor Nuclear/metabolismo , PPAR alfa/metabolismo , PPAR gama/metabolismo , Neoplasias da Próstata/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Western Blotting , Ciclo Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Perfilação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Correpressor 1 de Receptor Nuclear/antagonistas & inibidores , Correpressor 1 de Receptor Nuclear/genética , Análise de Sequência com Séries de Oligonucleotídeos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
BACKGROUND: The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells. RESULTS: Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and beta-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription.Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex. CONCLUSION: We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate that CSN2 loss results in a phenotype distinct from that of cells lacking CSN5 possibly as a consequence of altered CSN5 activity within a resultant CSN subcomplex. Our data present the first evidence for the sequential loss of F-box proteins upon CSN manipulation and are the first to identify a potential link between CSN function and autophagy.
Assuntos
Autofagia , Caseínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo do Signalossomo COP9 , Caseínas/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeo Hidrolases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Burkitt/tratamento farmacológico , Adolescente , Bezafibrato/administração & dosagem , Linfoma de Burkitt/epidemiologia , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos , Doenças Endêmicas , Feminino , Humanos , Malaui/epidemiologia , Masculino , Acetato de Medroxiprogesterona/administração & dosagem , RecidivaRESUMO
Blood serum is commonly used for clinical diagnostics because its protein composition bears a wealth of information about the health of an organism. More recently the analysis of the small molecule composition, the metabolome, has received increased attention because the metabolite composition is influenced by many diseases, by the administration of drugs and toxins, and by the diet and life style of an individual. When nuclear magnetic resonance spectroscopy is used as an analytical tool it is often preferable to remove catalytically active proteins, in particular for longer measurements, because metabolite concentrations are otherwise in constant flux. Here we have compared different protocols for the separation of proteins and metabolites, including precipitation methods and ultrafiltration. Whereas most extraction methods involving protein precipitation deplete some metabolites, ultrafiltration is superior in retaining metabolite concentrations and offers excellent reproducibility. We also describe a new method to recover the hydrophobic fraction for ultrafiltration with good reproducibility.
Assuntos
Análise Química do Sangue/métodos , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Actiemil , Animais , Bovinos , Precipitação Química , Hidrogênio/química , Interações Hidrofóbicas e Hidrofílicas , Reprodutibilidade dos Testes , UltrafiltraçãoRESUMO
Following our previous description of the serotonin transporter (SERT) acting as a conduit to 5-hydroxytryptamine (5-HT)-mediated apoptosis, specifically in Burkitt's lymphoma, we now detail its expression among a broad spectrum of B cell malignancy, while exploring additional SERT substrates for potential therapeutic activity. SERT was readily detected in derived B cell lines with origins as diverse as B cell precursor acute lymphoblastic leukemia, mantle cell lymphoma, diffuse large B cell lymphoma, and multiple myeloma. Concentration and timecourse kinetics for the antiproliferative and proapoptotic activities of the amphetamine derivatives fenfluramine (an appetite suppressant) and 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") revealed them as being similar to the endogenous indoleamine. A tricyclic antidepressant, clomipramine, instead mirrored the behavior of the selective serotonin reuptake inhibitor fluoxetine, both being effective in the low micromolar range. A majority of neoplastic clones were sensitive to one or more of the serotonergic compounds. Dysregulated bcl-2 expression, either by t(14;18)(q32;q21) translocation or its introduction as a constitutively active transgene, provided protection from proapoptotic but not antiproliferative outcomes. These data indicate a potential for SERT as a novel anti-tumor target for amphetamine analogs, while evidence is presented that the seemingly more promising antidepressants are likely impacting malignant B cells independently of the transporter itself.
Assuntos
Antineoplásicos/farmacologia , Linfoma de Burkitt/química , Linfoma de Células B/química , Psicotrópicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/tratamento farmacológico , Linhagem Celular Tumoral , Clomipramina/farmacologia , Fenfluramina/farmacologia , Fluoxetina/farmacologia , Humanos , Linfoma de Células B/tratamento farmacológico , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/fisiologiaRESUMO
It is becoming increasingly well established that nonsteroidal anti-inflammatory drugs (NSAID) protect against tumors of the gastrointestinal tract and that they may also protect against a variety of other tumors. These activities have been widely attributed to the inhibition of cylooxygenases (COX) and, in particular, COX-2. However, several observations have indicated that other targets may be involved. Besides targeting COX, certain NSAID also inhibit enzymes belonging to the aldo-keto reductase (AKR) family, including AKR1C3. We have demonstrated previously that overexpression of AKR1C3 acts to suppress cell differentiation and promote proliferation in myeloid cells. However, this enzyme has a broad tissue distribution and therefore represents a novel candidate for the target of the COX-independent antineoplastic actions of NSAID. Here we report on the X-ray crystal structures of AKR1C3 complexed with the NSAID indomethacin (1.8 A resolution) or flufenamic acid (1.7 A resolution). One molecule of indomethacin is bound in the active site, whereas flufenamic acid binds to both the active site and the beta-hairpin loop, at the opposite end of the central beta-barrel. Two other crystal structures (1.20 and 2.1 A resolution) show acetate bound in the active site occupying the proposed oxyanion hole. The data underline AKR1C3 as a COX-independent target for NSAID and will provide a structural basis for the future development of new cancer therapies with reduced COX-dependent side effects.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Ácido Flufenâmico/metabolismo , Hidroxiprostaglandina Desidrogenases/química , Indometacina/metabolismo , Sequência de Aminoácidos , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação , Cristalização , Inibidores de Ciclo-Oxigenase/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
We and others have demonstrated expression of the aldo-keto reductase AKR1C3 in myeloid leukemia cell lines and that inhibitors of the enzyme, including nonsteroidal anti-inflammatory drugs (NSAIDs), promote HL-60 differentiation in response to all-trans retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3). Here, we demonstrate that overexpression of AKR1C3 reciprocally desensitizes HL-60 cells to ATRA and D3, thus confirming the enzyme as a novel regulator of cell differentiation. AKR1C3 possesses marked 11-ketoreductase activity converting prostaglandin (PG) D2 to PGF2alpha. Supplementing HL-60 cultures with PGD2 mimicked treatment with AKR1C3-inhibitors by enhancing the differentiation of the cells in response to ATRA. However, PGD2 is chemically unstable, being converted first to PGJ2 and then stepwise to 15-deoxy-Delta(12,14)-prostaglandin J2(15Delta-PGJ2), a natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). Consistent with this, PGD2 was rapidly converted to PGJ2 under normal tissue culture conditions but not in the presence of recombinant AKR1C3 when PGF2alpha was predominantly formed. In addition, PGJ2 but not PGF2alpha recapitulated the potentiation of HL-60 differentiation by PGD2 and AKR1C3 inhibitors. Furthermore, the capacity of all of these treatments to potentiate HL-60 cell differentiation was significantly reduced in the presence of the PPARgamma-antagonist GW 9662. We conclude that AKRIC3 protects HL-60 cells against ATRA and D3-induced cell differentiation by limiting the production of natural PPARgamma ligands via the diversion of PGD2 toward PGF2alpha and away from PGJ2. In addition, these observations identify AKR1C3 as plausible target for the non-cyclooxygenase-dependent antineoplastic actions of NSAIDs.
Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Prostaglandina D2/análogos & derivados , 3-Hidroxiesteroide Desidrogenases/biossíntese , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica) , Androstano-3,17-diol/metabolismo , Androstano-3,17-diol/farmacologia , Anilidas/farmacologia , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Di-Hidrotestosterona/metabolismo , Di-Hidrotestosterona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Células HL-60 , Humanos , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacologia , Transgenes , Tretinoína/farmacologiaRESUMO
BACKGROUND: The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. METHODS: In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. RESULTS: These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. CONCLUSIONS: These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer.
Assuntos
3-Hidroxiesteroide Desidrogenases/genética , Epigênese Genética , Hidroxiprostaglandina Desidrogenases/genética , Neoplasias da Próstata/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Hidroxiprostaglandina Desidrogenases/metabolismo , Masculino , PPAR gama/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , VorinostatRESUMO
BACKGROUND: The role of anaplerotic nutrient entry into the Krebs cycle via pyruvate carboxylase has been the subject of increased scrutiny and in particular whether this is dysregulated in cancer. Here, we use a tracer-based NMR analysis involving high-resolution (1)H-(13)C-HSQC spectra to assess site-specific label incorporation into a range of metabolite pools, including malate, aspartate and glutamate in the acute myeloid leukaemia cell line K562. We also determine how this is affected following treatment with the redeployed drug combination of the lipid-regulating drug bezafibrate and medroxyprogesterone (BaP). RESULTS: Using the tracer-based approach, we assessed the contribution of pyruvate carboxylase (PC) vs. pyruvate dehydrogenase (PDH) activity in the derivation of Krebs cycle intermediates. Our data show that PC activity is indeed high in K562 cells. We also demonstrate a branched entry to the Krebs cycle of K562 cells with one branch running counterclockwise using PC-derived oxaloacetate and the other clockwise from the PDH activity. Finally, we show that the PC activity of K562 cells exclusively fuels the ROS-induced decarboxylation of oxaloacetate to malonate in response to BaP treatment; resulting in further Krebs cycle disruption via depletion of oxaloacetate and malonate-mediated inhibition of succinate dehydrogenase (SDH) resulting in a twofold reduction of fumarate. CONCLUSIONS: This study extends the interest in the PC activity in solid cancers to include leukaemias and further demonstrates the value of tracer-based NMR approaches in generating a more accurate picture of the flow of carbons and metabolites within the increasingly inappropriately named Krebs cycle. Moreover, our studies indicate that the PC activity in cancer cells can be exploited as an Achilles heel by using treatments, such as BaP, that elevate ROS production.
RESUMO
High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a 13C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-13C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-13C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of α-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.
RESUMO
High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a 13 C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-13 C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-13 C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of α-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.
RESUMO
Treatment of MSCV-GFP-transduced HL60 promyelocytic cells with all-trans retinoic acid (ATRA) resulted in a significant increase in GFP expression. The increased GFP expression was observed by 16 hr and was dependent on de novo protein production. This effect was specific to ATRA and unrelated to cell differentiation because it was not induced by dimethyl sulfoxide. Furthermore, a similar increase in GFP expression was observed in MSCV-GFP-transfected K562 cells, which do not differentiate when exposed to ATRA. Significantly increased GFP expression was seen at doses as low as 0.5 nM ATRA and was abrogated by AGN193109, an antagonist of retinoid signaling. We therefore conclude that this increase in gene expression is mediated by retinoic acid receptors. The long terminal repeat (LTR) region of MSCV contains candidate retinoic acid response elements and response elements for the ATRA-inducible transcription factor C/EBPalpha. We suggest that the increase in GFP expression is driven by the action of ATRA-activated host cell transcription factors. These findings offer a method to increase the expression of retroviral transgenes either in vitro or in vivo by treatment with low doses of retinoic acid that are clinically achievable and well tolerated. This use of inducible host cell transcription factors offers an alternative to engineering novel LTR regulatory sequences in order to increase transgene expression.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Vírus da Leucemia Murina/genética , Transgenes/fisiologia , Tretinoína/farmacologia , Animais , Sequência de Bases , Diferenciação Celular , Proteínas de Fluorescência Verde/genética , Células HL-60 , Humanos , Células K562 , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Naftalenos/farmacologia , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/metabolismo , Células-Tronco/virologia , Fatores de Transcrição , Transdução Genética , Proteínas do Envelope Viral/genéticaRESUMO
Studies in the 1990s identified a link between extracellular Nm23 proteins and acute myeloid leukaemia (AML). Confidence in the importance of these observations was undermined by a lack of appreciation that extracellular Nm23 proteins were relevant to either normal or pathophysiology coupled with the lack of demonstrable activity of Nm23 proteins against human AML cell lines. However, independent studies have highlighted the importance of Nm23-H1 in AML and have identified an elaborate Nm23-H1-mediated cross talk between cells within the AML clone. In other studies, roles for Nm23-H1 have now also been implicated in the maintenance of the stem cell state of embryonic stem (ES) cells and induced pluripotent stem (IPS) cells. In this review, we have generated a unifying model of the action of Nm23-H1 in AML, including previously unpublished data from our laboratory, and provide arguments as to why we consider this role to be distinct from that in ES and IPS cells.
Assuntos
Leucemia Mieloide Aguda , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Animais , Progressão da Doença , Humanos , Leucemia Mieloide Aguda/metabolismo , Mucina-1/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
B cell non-Hodgkin lymphomas (B-NHLs) are the most common adult hematological cancers and many remain incurable. Development of chemotherapy regimens is confounded by the prevalence of B-NHL in older, frailer patients and the chemo-protective tumor microenvironment. Although biological therapies such as rituximab have significantly improved outcomes and selective kinase inhibitors are showing promise, the rate of new drug discovery remains disappointing, the treatments expensive and long-term benefits uncertain. An alternative strategy is redeployment of available, inexpensive and non-toxic drugs. Here, we demonstrate the antiproliferative and mitochondrial superoxide (MSO) driven pro-apoptotic activities of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against B-NHL cells, with a bias toward MZL, in the presence and absence of the microenvironmental signal CD40L. Our study is the first to confirm the presence of CD40L within the lymph node of B-NHL and its capacity to drive B-NHL proliferation. These findings implicate BEZ + MPA as a potential therapeutic strategy in B-NHL.
Assuntos
Apoptose/efeitos dos fármacos , Bezafibrato/farmacologia , Ligante de CD40/farmacologia , Acetato de Medroxiprogesterona/farmacologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Hormonais/farmacologia , Ligante de CD40/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Células L , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an 'ideal' fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm. METHODS: We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial. FINDINGS: Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies. CONCLUSIONS: These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.
Assuntos
Acetilcarnitina/sangue , Biomarcadores Tumorais/sangue , Mieloma Múltiplo/sangue , Acetilcarnitina/urina , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/urina , Carnitina/sangue , Carnitina/urina , Ensaios Clínicos Fase III como Assunto , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Metaboloma , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Mieloma Múltiplo/urina , Análise Multivariada , Estadiamento de Neoplasias , Indução de Remissão , Resultado do TratamentoRESUMO
The concept of the haematopoietic stem cell (HSC) niche was formulated by Schofield in the 1970s, as a region within the bone marrow containing functional cell types that can maintain HSC potency throughout life. Since then, ongoing research has identified numerous cell types and a plethora of signals that not only maintain HSCs, but also dictate their behaviour with respect to homeostatic requirements and exogenous stresses. It has been proposed that there are endosteal and vascular niches within the bone marrow, which are thought to regulate different HSC populations. However, recent data depicts a more complicated picture, with functional crosstalk between cells in these two regions. In this review, recent research into the endosteal/vascular cell types and signals regulating HSC behaviour are considered, together with the possibility of a single subcompartmentalised niche.
RESUMO
Nm23-H1 plays complex roles in the development of diverse cancers including breast carcinoma, high-grade lymphomas, and acute myeloid leukemia (AML). In the case of AML and lymphomas, serum Nm23-H1 protein is elevated with the highest levels correlating with poorest prognosis. A recent study identified that this association is most likely causal in AML and that Nm23-H1 acts as an AML cell survival factor. In this study, we report heterogeneity in the ability of AML samples to bind and respond to Nm23-H1, and we offer evidence that binding is essential for improved survival. Further, we show that the subset of AMLs that bind Nm23-H1 do not do so through the putative Nm23-H1 receptor MUC1*. Although rNm23-H1 promoted the survival of the most primitive blasts within responding AMLs, it was not these cells that actually bound the protein. Instead, rNm23-H1 bound to more mature CD34(lo)/CD34(-) and CD11b(+) cells, revealing an indirect survival benefit of Nm23-H1 on primitive blasts. In support of this finding, the survival of purified blast cells was enhanced by medium conditioned by more mature cells from the clone that had been stimulated by rNm23-H1. Levels of interleukin 1ß (IL1ß) and IL6 in rNm23-H1 conditioned medium mirrored the potency of the conditioned media to promote blast cell survival. Furthermore, Nm23-H1 expression was significantly associated with IL1ß and IL6 expression in primary uncultured AML samples. These findings have implications for the role of Nm23-H1 in AML and its use as a prognostic marker. Additionally, they offer the first evidence of novel cross-talk between cell populations within the tumor clone.