RESUMO
Renal ischemia-reperfusion (I/R) causes acute kidney injury (AKI). Ischemic preconditioning (IPC) attenuates I/R-associated AKI. Whole body irradiation induces renal IPC in mice. Still, the mechanisms remain largely unknown. Furthermore, the impact of kidney-centered irradiation on renal resistance against I/R has not been studied. Renal irradiation (8.5 Gy) was done in male 8- to 12-wk-old C57bl/6 mice using a small animal radiation therapy device. Left renal I/R was performed by clamping the renal pedicles for 30 min, with simultaneous right nephrectomy, at 7, 14, and 28 days postirradiation. The renal reperfusion lasted 48 h. Following I/R, blood urea nitrogen (BUN) and serum creatinine (SCr) levels were lower in preirradiated mice compared with controls; so was the histological Jablonski score of AKI. The metabolomics signature of renal I/R was attenuated in preirradiated mice. The numbers of proliferating cell nuclear antigen (PCNA)-, cluster of differentiation molecule 11b (CD11b)-, and cell surface glycoprotein F4/80-positive cells in the renal parenchyma post-I/R were reduced in preirradiated versus control groups. Such IPC was significantly observed as early as day 14 postirradiation. RNA sequencing showed an upregulation of angiogenesis- and stress response-related signaling pathways in irradiated nonischemic kidneys on day 28. Qualitative RT-PCR confirmed the increased expression of vascular endothelial growth factor (VEGF), activin receptor-like kinase 5 (ALK5), heme oxygenase-1 (HO1), platelet endothelial cell adhesion molecule-1 (PECAM1), NADPH oxidase 2 (NOX2), and heat shock proteins 70 and 27 (HSP70 and HSP27, respectively) in irradiated kidneys compared with controls. In addition, irradiated kidneys showed an increased CD31-positive vascular area compared with controls. A 14-day gavage of irradiated mice with the antiangiogenic drug sunitinib before I/R abrogated the irradiation-induced IPC at both functional and structural levels. Our observations suggest that kidney-centered irradiation activates proangiogenic pathways and induces IPC, with preserved renal function and attenuated inflammation post-I/R.NEW & NOTEWORTHY This study based on a mouse model of renal ischemia-reperfusion (I/R) aimed to 1) test whether and how irradiation strictly centered on the kidney protects against the I/R injury and 2) determine the shortest efficient delay of kidney irradiation to achieve such nephroprotection. Kidney irradiation increased the vascular surface in the renal parenchyma and conferred resistance against renal I/R damage, which highlights novel putative strategies in the field of ischemic acute kidney injury.
Assuntos
Injúria Renal Aguda , Precondicionamento Isquêmico , Traumatismo por Reperfusão , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/prevenção & controle , Animais , Isquemia/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Myoferlin, an emerging oncoprotein, has been associated with a low survival in several cancer types including pancreas ductal adenocarcinoma where it controls mitochondria structure and respiratory functions. Owing to the high susceptibility of KRAS-mutated cancer cells to iron-dependent cell death, ferroptosis, and to the high iron content in mitochondria, we investigated the relation existing between mitochondrial integrity and iron-dependent cell death. We discovered that myoferlin targeting with WJ460 pharmacological compound triggered mitophagy and ROS accumulation culminating with lipid peroxidation and apoptosis-independent cell death. WJ460 caused a reduction of the abundance of ferroptosis core regulators xc- cystine/glutamate transporter and GPX-4. Mitophagy inhibitor Mdivi1 and iron chelators inhibited the myoferlin-related ROS production and restored cell growth. Additionally, we reported a synergic effect between ferroptosis inducers, erastin and RSL3, and WJ460.
Assuntos
Ferroptose , Neoplasias Pancreáticas , Humanos , Ferro/metabolismo , Peroxidação de Lipídeos , Mitofagia , Pâncreas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.
Assuntos
Biomarcadores Tumorais/genética , Proliferação de Células , Bases de Conhecimento , Neoplasias/patologia , Software , Esferoides Celulares/patologia , Microambiente Tumoral , Técnicas de Cultura de Células/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/classificação , Neoplasias/metabolismo , RNA-Seq , Reprodutibilidade dos Testes , Esferoides Celulares/imunologia , Esferoides Celulares/metabolismo , Células Tumorais CultivadasRESUMO
Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in aging populations. Here, we applied metabolomics to human sera of patients with nAMD during an active (exudative) phase of the pathology and found higher lactate levels and a shift in the lipoprotein profile (increased VLDL-LDL/HDL ratio). Similar metabolomics changes were detected in the sera of mice subjected to laser-induced choroidal neovascularization (CNV). In this experimental model, we provide evidence for two sites of lactate production: first, a local one in the injured eye, and second a systemic site associated with the recruitment of bone marrow-derived inflammatory cells. Mechanistically, lactate promotes the angiogenic response and M2-like macrophage accumulation in the eyes. The therapeutic potential of our findings is demonstrated by the pharmacological control of lactate levels through pyruvate dehydrogenase kinase (PDK) inhibition by dichloroacetic acid (DCA). Mice treated with DCA exhibited normalized lactate levels and lipoprotein profiles, and inhibited CNV formation. Collectively, our findings implicate the key role of the PDK/lactate axis in AMD pathogenesis and reveal that the regulation of PDK activity has potential therapeutic value in this ocular disease. The results indicate that the lipoprotein profile is a traceable pattern that is worth considering for patient follow-up. KEY MESSAGES: Lactate and lipoprotein profile are associated with the active phase of AMD and CNV development. Lactate is a relevant and functional metabolite correlated with AMD progression. Modulating lactate through pyruvate dehydrogenase kinase led to a decrease of CNV progression. Pyruvate dehydrogenase kinase is a new therapeutic target for neovascular AMD.
Assuntos
Ácido Láctico/metabolismo , Redes e Vias Metabólicas , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Transdução de Sinais , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Biomarcadores , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Gerenciamento Clínico , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma , Metabolômica/métodos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.
Assuntos
Cromakalim/análogos & derivados , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular , Cromakalim/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Taxa Respiratória/efeitos dos fármacosRESUMO
Synthetic glucocorticoids such as budesonide (BUD) are potent anti-inflammatory drugs commonly used to treat patients suffering from chronic inflammatory diseases. A previous animal study reported a higher anti-inflammatory activity with a 2-hydroxypropyl-ß-cyclodextrin (HPßCD)-based formulation of BUD (BUD:HPßCD). This study investigated, on cellular models (A549 and A-THP-1), the effect of BUD:HPßD in comparison with BUD and HPßCD on the effects induced by oxidative and inflammatory stress as well as the role of cholesterol. We demonstrated the protective effect afforded by BUD:HPßCD against cytotoxicity and ROS generation induced by oxidative and inflammatory stress. The effect observed for BUD:HPßCD was comparable to that observed with HPßCD with no major effect of cholesterol content. We also demonstrated (i) the involvement of the canonical molecular pathway including ROS generation, a decrease in PI3K/Akt activation, and decrease in phosphorylated/unphosphorylated HDAC2 in the effect induced by BUD:HPßCD, (ii) the maintenance of IL-8 decrease with BUD:HPßCD, and (iii) the absence of improvement in glucocorticoid insensitivity with BUD:HPßCD in comparison with BUD, in conditions where HDAC2 was inhibited. Resulting from HPßCD antioxidant and anticytotoxic potential and protective capacity against ROS-induced PI3K/Akt signaling and HDAC2 inhibition, BUD:HPßCD might be more beneficial than BUD alone in a context of concomitant oxidative and inflammatory stress.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Anti-Inflamatórios/química , Budesonida/química , Inibidores Enzimáticos/química , Interleucina-8/metabolismo , Oxidantes/química , Espécies Reativas de Oxigênio/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina/metabolismo , Células A549 , Anti-Inflamatórios/metabolismo , Budesonida/metabolismo , Morte Celular/efeitos dos fármacos , Colesterol/química , Portadores de Fármacos/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Quimioterapia Combinada , Inibidores Enzimáticos/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Oxidantes/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células THP-1RESUMO
Gut microbiota may influence blood pressure (BP), namely via end products of carbohydrate fermentation. After informed consent, male volunteers were prospectively categorized into 3 groups upon European Society of Hypertension criteria based on 24-hour ambulatory BP measurements: (1) hypertension, (2) borderline hypertension, and (3) normotension. Stool, urine and serum samples were collected in fasting conditions. Gut microbiota was characterized by 16S amplicon sequencing. Metabolomics, including quantification of short-chain fatty acids, was conducted using nuclear magnetic resonance. Two-way ANOVA combined with Tukey post hoc test, as well as multiple permutation test and Benjamini-Hochberg-Yekutieli false discovery rate procedure, was used. The cohort included 54 males: 38 hypertensive (including 21 under treatment), 7 borderline, and 9 normotensive. No significant difference was observed between groups concerning age, body mass index, smoking habits, and weekly alcohol consumption. The genus Clostridium sensu stricto 1 positively correlated with BP levels in nontreated patients (n=33). This correlation was significant after multiple permutation tests but was not substantiated following false discovery rate adjustment. Short-chain fatty acid levels were significantly different among groups, with higher stool levels of acetate, butyrate, and propionate in hypertensive versus normotensive individuals. No difference was observed in serum and urine metabolomes. Correlation between stool metabolome and 24-hour BP levels was evidenced, with R2 reaching 0.9. Our pilot study based on 24-hour ambulatory BP measurements, 16S amplicon sequencing, and metabolomics supports an association between gut microbiota and BP homeostasis, with changes in stool abundance of short-chain fatty acids.
Assuntos
Pressão Sanguínea/fisiologia , Ácidos Graxos Voláteis/análise , Fezes/química , Microbioma Gastrointestinal/fisiologia , Hipertensão/fisiopatologia , Adulto , Humanos , Hipertensão/microbiologia , Masculino , Metaboloma , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains a deadly malignancy with no efficient therapy available up-to-date. Glycolysis is the main provider of energetic substrates to sustain cancer dissemination of PDAC. Accordingly, altering the glycolytic pathway is foreseen as a sound approach to trigger pancreatic cancer regression. Here, we show for the first time that high transforming growth factor beta-induced (TGFBI) expression in PDAC patients is associated with a poor outcome. We demonstrate that, although usually secreted by stromal cells, PDAC cells synthesize and secrete TGFBI in quantity correlated with their migratory capacity. Mechanistically, we show that TGFBI activates focal adhesion kinase signaling pathway through its binding to integrin αVß5, leading to a significant enhancement of glycolysis and to the acquisition of an invasive phenotype. Finally, we show that TGFBI silencing significantly inhibits PDAC tumor development in a chick chorioallantoic membrane assay model. Our study highlights TGFBI as an oncogenic extracellular matrix interacting protein that bears the potential to serve as a target for new anti-PDAC therapeutic strategies.
Assuntos
Carcinoma Ductal Pancreático/patologia , Movimento Celular , Proteínas da Matriz Extracelular/metabolismo , Glicólise , Neoplasias Pancreáticas/patologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Embrião de Galinha , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Inativação Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Vitronectina/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismo , Análise de Sobrevida , Fator de Crescimento Transformador beta1/genéticaRESUMO
Our study investigates the biochemical and functional impact of selective histone deacetylase 6 (HDAC6) inhibitors, a promising class of novel therapeutics, in several cancer models. Selective HDAC6 inhibitors (Tubathian A, Tubastatin A, Tubacin and Ricolinostat) and a non-selective HDAC inhibitor (Vorinostat) were evaluated on cancer cell lines derived from multiple tumour types in both an in vitro and in vivo setting as potential cancer therapeutics. Selective HDAC6 inhibitors resulted in α-tubulin acetylation with no impact on histone acetylation but failed to show any anti-cancer properties. Only the use of high concentrations of selective HDAC6 inhibitors resulted in co-inhibition of other HDAC enzymes and consequently in reduced growth, migratory and/or invasive activity of cancer cells in vitro as well as in vivo. The specificity of HDAC6 inhibition was confirmed using a CRISPR/Cas9 knockout cell line. Our results suggest that selective HDAC6 inhibitors may fall short as potential single agent anti-cancer drugs and prove that many previous data regarding this promising class of compounds need to be interpreted with great care due to their use in high concentrations resulting in low selectivity and potential off-target effects.
Assuntos
Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Desacetilase 6 de Histona/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Neoplasias/patologia , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis.
Assuntos
Quimiocina CXCL12/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Nefropatias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Movimento Celular , Metabolismo Energético , Deleção de Genes , Perfilação da Expressão Gênica , Glicólise , Homeostase , Rim/lesões , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Tretinoína/químicaRESUMO
Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.
Assuntos
Códon/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Biossíntese de Proteínas , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Códon/efeitos dos fármacos , Feminino , Humanos , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transdução de Sinais , Fatores de Elongação da Transcrição , Uridina/química , Uridina/genética , Uridina/metabolismo , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Peixe-Zebra/genéticaRESUMO
Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer, a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAFs are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer cell growth, transcriptome, metabolome, and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest, and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF1). Subsequently, RT-activated CAFs promoted survival of colorectal cancer cells, as well as a metabolic switch favoring glutamine consumption through IGF1 receptor (IGF1R) activation. RT followed by IGF1R neutralization in orthotopic colorectal cancer models reduced the number of mice with organ metastases. Activation of the downstream IGF1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from rectal cancer patients after neoadjuvant chemoradiotherapy. Taken together, our data support the notion that paracrine IGF1/IGF1R signaling initiated by RT-activated CAF worsens colorectal cancer progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival.Significance: These findings reveal that paracrine IGF1/IGF1R signaling promotes colorectal cancer progression, establishing a preclinical rationale to target this activation loop. Cancer Res; 78(3); 659-70. ©2017 AACR.
Assuntos
Fibroblastos Associados a Câncer/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/patologia , Raios gama/efeitos adversos , Comunicação Parácrina , Receptores de Somatomedina/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/radioterapia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos da radiação , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/efeitos da radiação , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/radioterapia , Feminino , Humanos , Metaboloma , Camundongos , Camundongos Nus , Prognóstico , Transdução de Sinais , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A new series of N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of >10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.
Assuntos
Benzopiranos/química , Benzopiranos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
Metabolic reprogramming toward aerobic glycolysis unavoidably induces methylglyoxal (MG) formation in cancer cells. MG mediates the glycation of proteins to form advanced glycation end products (AGEs). We have recently demonstrated that MG-induced AGEs are a common feature of breast cancer. Little is known regarding the impact of MG-mediated carbonyl stress on tumor progression. Breast tumors with MG stress presented with high nuclear YAP, a key transcriptional co-activator regulating tumor growth and invasion. Elevated MG levels resulted in sustained YAP nuclear localization/activity that could be reverted using Carnosine, a scavenger for MG. MG treatment affected Hsp90 chaperone activity and decreased its binding to LATS1, a key kinase of the Hippo pathway. Cancer cells with high MG stress showed enhanced growth and metastatic potential in vivo. These findings reinforce the cumulative evidence pointing to hyperglycemia as a risk factor for cancer incidence and bring renewed interest in MG scavengers for cancer treatment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Produtos Finais de Glicação Avançada/metabolismo , Glicólise , Proteínas de Choque Térmico HSP90/metabolismo , Metástase Neoplásica , Fosfoproteínas/metabolismo , Aldeído Pirúvico/metabolismo , Aerobiose , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Ischemia/reperfusion (I/R) is the most common cause of acute kidney injury (AKI). Its pathophysiology remains unclear. Metabolomics is dedicated to identify metabolites involved in (patho)physiological changes of integrated living systems. Here, we performed 1H-Nuclear Magnetic Resonance metabolomics using urine, serum and kidney samples from a mouse model of renal I/R. METHODS: Renal 30-min ischemia was induced in 12-week-old C57BL/6J male mice by bilaterally clamping vascular pedicles, and was followed by 6, 24 or 48-hour reperfusion (n = 12/group). Sham-operated mice were used as controls. Statistical discriminant analyses, i.e. principal component analysis and orthogonal projections to latent structures (OPLS-DA), were performed on urine, serum and kidney lysates at each time-point. Multivariate receiver operating characteristic (ROC) curves were drawn, and sensitivity and specificity were calculated from ROC confusion matrix (with averaged class probabilities across 100 cross-validations). RESULTS: Urine OPLS-DA analysis showed a net separation between I/R and sham groups, with significant variations in levels of taurine, di- and tri-methylamine, creatine and lactate. Such changes were observed as early as 6 hours post reperfusion. Major metabolome modifications occurred at 24h post reperfusion. At this time-point, correlation coefficients between urine spectra and conventional AKI biomarkers, i.e. serum creatinine and urea levels, reached 0.94 and 0.95, respectively. The area under ROC curve at 6h, 24h and 48h post surgery were 0.73, 0.98 and 0.97, respectively. Similar discriminations were found in kidney samples, with changes in levels of lactate, fatty acids, choline and taurine. By contrast, serum OPLS-DA analysis could not discriminate sham-operated from I/R-exposed animals. CONCLUSIONS: Our study demonstrates that renal I/R in mouse causes early and sustained metabolomic changes in urine and kidney composition. The most implicated pathways at 6h and 24h post reperfusion include gluconeogenesis, taurine and hypotaurine metabolism, whereas protein biosynthesis, glycolysis, and galactose and arginine metabolism are key at 48h post reperfusion.
RESUMO
2D-DIGE analysis coupled with mass spectrometry is a global, without a priori, comparative proteomic approach particularly suited to identify and quantify enzymes isoforms and structural proteins, thus making it an efficient tool for the characterization of the changes in cell phenotypes that occur in physiological and pathological conditions. In this data article in support of the research article entitled "Metabolic reprogramming in transformed mouse cortical astrocytes: a proteomic study" [1] we illustrate the changes in protein profile that occur during the metabolic reprogramming undergone by cultured mouse astrocytes in a model of in-vitro cancerous transformation [2].
RESUMO
Metabolic reprogramming is thought to play a key role in sustaining the survival and proliferation of cancer cells. These changes facilitate for example the uptake and release of nutrients required for nucleotide, protein and lipid synthesis necessary for macromolecule assembly and tumor growth. We applied a 2D-DIGE (two-dimensional differential in-gel electrophoresis) quantitative proteomic analysis to characterize the proteomes of mouse astrocytes that underwent in vitro cancerous transformation, and of their normal counterparts. Metabolic reprogramming effects on enzymatic and structural protein expression as well as associated metabolites abundance were quantified. Using enzymatic activity measurements and zymography, we documented and confirmed several changes in abundance and activity of various isoenzymes likely to participate in metabolic reprogramming. We found that after transformation, the cells increase their expression of glycolytic enzymes, thus augmenting their ability to use aerobic glycolysis (Warburg effect). An increased capacity to dispose of reducing equivalents through lactate production was also documented. Major effects on carbohydrates, amino acids and nucleotides metabolic enzymes were also observed. Conversely, the transformed cells reduced their enzymatic capacity for reactions of tricarboxylic acid oxidation, for neurotransmitter (glutamate) metabolism, for oxidative stress defense and their expression of astroglial markers. BIOLOGICAL SIGNIFICANCE: The use of a global approach based on a 2D DIGE analysis allows obtaining a comprehensive view of the metabolic reprogramming undergone by astrocytes upon cancerous transformation. Indeed, except for a few enzymes such as pyruvate carboxylase and glutaminase that were not detected in our initial analysis, pertinent information on the abundance of most enzymes belonging to pathways relevant to metabolic reprogramming was directly obtained. In this in vitro model, transformation causes major losses of astrocyte-specific proteins and functions and the acquisition of metabolic adaptations that favor intermediate metabolites production for increased macromolecule biosynthesis. Thus our approach appears to be readily applicable for the investigation of changes in protein abundance that determine various transformed cell phenotypes. It could similarly be applied to the evaluation of the effects of treatments aimed at correcting the consequences of cell transformation.
RESUMO
The molecular mechanisms responsible for the failure of antiangiogenic therapies and how tumors adapt to these therapies are unclear. Here, we applied transcriptomic, proteomic, and metabolomic approaches to preclinical models and provide evidence for tumor adaptation to vascular endothelial growth factor blockade through a metabolic shift toward carbohydrate and lipid metabolism in tumors. During sunitinib or sorafenib treatment, tumor growth was inhibited and tumors were hypoxic and glycolytic. In sharp contrast, treatment withdrawal led to tumor regrowth, angiogenesis restoration, moderate lactate production, and enhanced lipid synthesis. This metabolic shift was associated with a drastic increase in metastatic dissemination. Interestingly, pharmacological lipogenesis inhibition with orlistat or fatty acid synthase downregulation with shRNA inhibited tumor regrowth and metastases after sunitinib treatment withdrawal. Our data shed light on metabolic alterations that result in cancer adaptation to antiangiogenic treatments and identify key molecules involved in lipid metabolism as putative therapeutic targets.