RESUMO
The effect of immunoinflammation on bone repair during the recovery process of bone defects needs to be further explored. It is reported that Mg2+ can promote bone repair with immunoregulatory effect, but the underlying mechanism on adaptive immunity is still unclear. Here, by using chitosan and hyaluronic acid-coated Mg2+ (CSHA-Mg) in bone-deficient mice, it is shown that Mg2+ can inhibit the activation of CD4+ T cells and increase regulatory T cell formation by inducing immunosuppressive dendritic cells (imDCs). Mechanistically, Mg2+ initiates the activation of the MAPK signaling pathway through TRPM7 channels on DCs. This process subsequently induces the downstream HIF-1α expression, a transcription factor that amplifies TGF-ß production and inhibits the effective T cell function. In vivo, knock-out of HIF-1α in DCs or using a HIF-1α inhibitor PX-478 reverses inhibition of bone inflammation and repair promotion upon Mg2+-treatment. Moreover, roxadustat, which stabilizes HIF-1α protein expression, can significantly promote immunosuppression and bone repair in synergism with CSHA-Mg. Thus, the findings identify a key mechanism for DCs and its HIF-1α-TGF-ß axis in the induction of immunosuppressive bone microenvironment, providing potential targets for bone regeneration.
Assuntos
Células Dendríticas , Subunidade alfa do Fator 1 Induzível por Hipóxia , Magnésio , Fator de Crescimento Transformador beta , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Magnésio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Celular/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Regeneração Óssea/efeitos dos fármacos , Isoquinolinas/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Canais de Cátion TRPM/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quitosana/farmacologia , Quitosana/química , Compostos de Mostarda , FenilpropionatosRESUMO
Some mustard compounds (mustards) are highly toxic chemical warfare agents. Some are explored as new anticancer drugs. Therefore, the fast, selective, and sensitive detection of mustards is extremely important for public security and cancer therapy. Mustards mostly target the N7 position on the guanine bases of DNA. The guanine-rich G-quadruplex DNA (G4) has been widely studied in the sensing area, and it was found that dimeric G4 (D-G4) could dramatically light up the fluorescence intensity of thioflavin T (ThT). Based on this, we used for the first time the D-G4 DNA as a selective probe for ultrasensitive fluorescence detection of nitrogen mustard (NM). When NM occupies the N7 on guanine, it can block the formation of the D-G4 structure due to the steric hindrance, and hence, it inhibits the combination of D-G4 with ThT, leading to a sharp decrease of fluorescence intensity. The proposed reaction mechanism is proved using ultraviolet-visible (UV-Vis) spectra, circular dichroism (CD) spectra, and polyacrylamide gel electrophoresis. Herein, the concentration of D-G4/ThT used is as low as 50 nM due to its highly fluorescent performance, enabling both high sensitivity and low cost. NM can be detected with a wide linear range from 10 to 2000 nM. The detection limit of NM reaches a surprisingly low concentration of 6 nM, which is 2 or 3 orders of magnitude lower than that of previously developed fluorescence methods for mustards and simulants.
Assuntos
Quadruplex G , Dicroísmo Circular , DNA/química , Corantes Fluorescentes/química , Compostos de Mostarda , Espectrometria de Fluorescência/métodosRESUMO
Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.
Assuntos
Injúria Renal Aguda/prevenção & controle , Autofagia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Traumatismo por Reperfusão/prevenção & controle , Serina-Treonina Quinases TOR/genética , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose/genética , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/metabolismo , Rim/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos de Mostarda/farmacologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Fenilpropionatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Hypoxia-inducible factor-1α (Hif1α) is activated in hypoxia and is closely related to oxidative stress, immunity and cell metabolism. Recently, it is reported that Hif1α is involved in atherosclerosis, ischemia-reperfusion (I/R) injury, alcoholic liver disease and pancreatic tumors. In this study, we found that Hif1 signal pathway is significantly changed in pancreas of acute pancreatitis (AP) mice. Meanwhile, we verified that the high expression of Hif1α injured pancreatic tissues of cerulean-induced AP mice, which prompting that Hif1α participated in the progress of histopathology on AP. We applied a Hif1α inhibitor PX478 and observed that it could alleviate histological injury of pancreas as well as the levels of serum amylase, lipase and proinflammatory cytokine in the murine model of AP induced by caerulein. In addition, PX478 could reduce the formation of necrosome (RIP3 and p-MLKL) and the generation of reactive oxygen species (ROS) in AP mice. Correspondingly, we further confirmed the effectiveness of PX478 in vitro and found that inhibiting Hif1α could mitigated the necrosis of pancreatic acinar cells via reducing the RIP3 and p-MLKL expression and the ROS production. In conclusion, inhibiting Hif1α could protect against acinar cells necrosis in AP, which may provide a new target for the prevention and treatment of AP clinically.
Assuntos
Células Acinares/efeitos dos fármacos , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Compostos de Mostarda/farmacologia , Necrose/tratamento farmacológico , Pancreatite/tratamento farmacológico , Fenilpropionatos/farmacologia , Células Acinares/metabolismo , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Necrose/metabolismo , Pancreatite/metabolismoRESUMO
Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus-oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.
Assuntos
Bovinos , Células do Cúmulo/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Células Cultivadas , Equinomicina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Compostos de Mostarda/farmacologia , Oócitos/fisiologia , Fenilpropionatos/farmacologiaRESUMO
BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms. METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells. RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment. CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Ácido Dicloroacético/farmacologia , Compostos de Mostarda/farmacologia , Fenilpropionatos/farmacologia , Células A549 , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Células HT29 , Células HeLa , Humanos , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismoRESUMO
The development of bifunctional platinum complexes with the ability to interact with DNA via different binding modes is of interest in anticancer metallodrug research. Therefore, we report platinum(II) terpyridine complexes to target DNA by coordination and/or through a tethered alkylating moiety. The platinum complexes were evaluated for their in vitro antiproliferative properties against the human cancer cell lines HCT116 (colorectal), SW480 (colon), NCI-H460 (non-small cell lung), and SiHa (cervix) and generally exhibited potent antiproliferative activity although lower than their respective terpyridine ligands. 1H NMR spectroscopy and/or ESI-MS studies on the aqueous stability and reactivity with various small biomolecules, acting as protein and DNA model compounds, were used to establish potential modes of action for these complexes. These investigations indicated rapid binding of complex PtL3 to the biomolecules through coordination to the Pt center, while PtL4 in addition alkylated 9-ethylguanine. PtL3 was investigated for its reactivity to the model protein hen egg white lysozyme (HEWL) by protein crystallography which allowed identification of the Nδ1 atom of His15 as the binding site.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , DNA/química , Compostos de Mostarda/química , Compostos de Platina/química , Piridinas/química , Alquilação , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Humanos , Ligantes , Muramidase/metabolismo , Análise Espectral/métodosRESUMO
Incomplete recovery from episodes of acute kidney injury (AKI) can predispose patients to develop chronic kidney disease (CKD). Although hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia/ischemia, the role of HIF-1α in CKD progression following incomplete recovery from AKI is poorly understood. Here, we investigated this issue using moderate and severe ischemia/reperfusion injury (I/RI) mouse models. We found that the outcomes of AKI were highly associated with the time course of tubular HIF-1α expression. Sustained activation of HIF-1α, accompanied by the development of renal fibrotic lesions, was found in kidneys with severe AKI. The AKI to CKD progression was markedly ameliorated when PX-478 (a specific HIF-1α inhibitor, 5 mg· kg-1·d-1, i.p.) was administered starting on day 5 after severe I/RI for 10 consecutive days. Furthermore, we demonstrated that HIF-1α C-terminal transcriptional activation domain (C-TAD) transcriptionally stimulated KLF5, which promoted progression of CKD following severe AKI. The effect of HIF-1α C-TAD activation on promoting AKI to CKD progression was also confirmed in in vivo and in vitro studies. Moreover, we revealed that activation of HIF-1α C-TAD resulted in the loss of FIH-1, which was the key factor governing HIF-1α-driven AKI to CKD progression. Overexpression of FIH-1 inhibited HIF-1α C-TAD and prevented AKI to CKD progression. Thus, FIH-1-modulated HIF-1α C-TAD activation was the key mechanism of AKI to CKD progression by transcriptionally regulating KLF5 pathway. Our results provide new insights into the role of HIF-1α in AKI to CKD progression and also the potential therapeutic strategy for the prevention of renal diseases progression.
Assuntos
Injúria Renal Aguda/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Oxigenases de Função Mista/metabolismo , Insuficiência Renal Crônica/etiologia , Transdução de Sinais/efeitos dos fármacos , Injúria Renal Aguda/patologia , Animais , Progressão da Doença , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Compostos de Mostarda/uso terapêutico , Fenilpropionatos/uso terapêutico , Domínios Proteicos , Insuficiência Renal Crônica/patologia , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions. METHODS: We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay. RESULTS: All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections. CONCLUSIONS: The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Ácido Dicloroacético/farmacologia , Ácido Dicloroacético/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Imidazóis/farmacologia , Imidazóis/uso terapêutico , L-Lactato Desidrogenase/antagonistas & inibidores , Compostos de Mostarda/farmacologia , Compostos de Mostarda/uso terapêutico , Fenilpropionatos/farmacologia , Fenilpropionatos/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Complexo Piruvato Desidrogenase/metabolismo , Reprodutibilidade dos TestesRESUMO
Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.
Assuntos
Estenose da Valva Aórtica/etiologia , Valva Aórtica/patologia , Calcinose/etiologia , Células Endoteliais/metabolismo , Hemorreologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , MicroRNAs/genética , Enzimas de Conjugação de Ubiquitina/biossíntese , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/metabolismo , Calcinose/patologia , Adesão Celular , Transdiferenciação Celular , Células Cultivadas , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Inflamação , MicroRNAs/agonistas , Monócitos/fisiologia , Compostos de Mostarda/farmacologia , Oligonucleotídeos/farmacologia , Técnicas de Cultura de Órgãos , Fenilpropionatos/farmacologia , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Reologia , Estresse Mecânico , Suínos , Enzimas de Conjugação de Ubiquitina/fisiologia , UbiquitinaçãoRESUMO
Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys34 residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography-electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (µLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstract.
Assuntos
Vesícula/induzido quimicamente , Substâncias para a Guerra Química/química , Compostos de Mostarda/química , Albumina Sérica Humana/química , Alquilação , Biomarcadores/sangue , Substâncias para a Guerra Química/toxicidade , Humanos , Compostos de Mostarda/toxicidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
IMPORTANCE: Retinopathy of prematurity (ROP) is an important risk factor for blindness in children due to neovascularization (NV). Hypoxia stimulates the formation of NV, as retinal hypoxia affects the stability and function of hypoxia-inducible factor (HIF) transcription factors. The purpose of this study is to study the mechanism of ROP and provide theoretical basis for clinical treatment of ROP. OBJECTIVE: In the present study, we used a mouse model of oxygen-induced retinopathy (OIR) to demonstrate the effects of the HIF-1α inhibitor PX-478 on OIR, and to determine its mechanism of action, to provide a theoretical basis for the clinical treatment of ROP. MATERIALS AND METHODS: The OIR mouse model was induced by exposing neonatal mouse pups and their mothers to 75 ± 5% oxygen from postnatal day 7 (P7) to P12, before being returned to room air from P12 to P17. Flat mount analyses were performed at P12 and P17. Hif1a, Hif2a, Hif3a, and Vegfa mRNA were detected by reverse transcription-polymerase chain reaction in OIR mice at P12 and P17. Hif1a and Vegfa mRNA were detected in OIR mice at P12 and P17 treatment with PX-478. Western blot analyses were used to assess the levels of HIF-1α, VEGF-A, and EPO before and after treatment with PX-478 at P12 and P17. RESULTS: Hif1a mRNA was increased in OIR mice at P12 and P17, while Vegfa mRNA was increased at P12 and P17. HIF-1α, VEGF-A, and EPO protein levels were increased in OIR mice at P12 and P17, as compared to control mice at the same age (all p < 0.05). Inhibition of HIF-1α by injection of PX-478 in OIR mice (P9-P16) caused a decrease in the retinal avascular area at P12 and P17 (both p < 0.05), NV areas at P17 (p < 0.05), Vegfa mRNA decreased at P12 and P17, as compared to control mice (p < 0.05), and VEGF-A and EPO protein levels at P12 and P17, as compared to control mice. Our study found that there were PX-478 both retina and vitreous body of OIR. Inhibition of HIF-1α by injection of PX-478 in OIR mice caused a decrease in the retinal avascular area at P12 and P17, NV areas decreased at P17, VEGF-A and EPO protein levels at P12 and P17. Endothelial cell migration assays and cell tube formation indication PX-478 attenuate cell migration and significantly weakened the cell cavity formation under the condition of hypoxia. CONCLUSION: HIF-1α plays a main role in OIR and can be considered a therapeutic target in OIR by suppressing downstream angiogenic factors, PX-478 decreasing the retinal avascular area and NV.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Compostos de Mostarda/farmacocinética , Fenilpropionatos/farmacocinética , Retina/metabolismo , Retinopatia da Prematuridade/tratamento farmacológico , Animais , Animais Recém-Nascidos , Western Blotting , Modelos Animais de Doenças , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , RNA/genética , Retina/efeitos dos fármacos , Retina/patologia , Retinopatia da Prematuridade/metabolismoRESUMO
Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma with poor prognosis caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Previous studies have revealed that HIF-1α, which mediates much of the cellular response to hypoxia, plays an important role in life cycle of KSHV. KSHV infection promotes HIF-1α activity, and several KSHV genes are in turn activated by HIF-1α. In this study, we investigated the effects of knocking down HIF-1α in PELs. We observed that HIF-1α knockdown in each of two PEL lines leads to a reduction in both aerobic and anaerobic glycolysis as well as lipid biogenesis, indicating that HIF-1α is necessary for maintaining a metabolic state optimal for growth of PEL. We also found that HIF-1α suppression leads to a substantial reduction in activation of lytic KSHV genes, not only in hypoxia but also in normoxia. Moreover, HIF-1α knockdown led to a decrease in the expression of various KSHV latent genes, including LANA, vCyclin, kaposin, and miRNAs, under both normoxic and hypoxic conditions. These observations provide evidence that HIF-1α plays an important role in PEL even in normoxia. Consistent with these findings, we observed a significant inhibition of growth of PEL in normoxia upon HIF-1α suppression achieved by either HIF-1α knockdown or treatment with PX-478, a small molecule inhibitor of HIF-1α. These results offer further evidence that HIF-1α plays a critical role in the pathogenesis of PEL, and that inhibition of HIF-1α can be a potential therapeutic strategy in this disease.
Assuntos
Antígenos Virais/genética , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfoma de Efusão Primária/virologia , Sarcoma de Kaposi/virologia , Antígenos Virais/imunologia , Hipóxia Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linfoma de Efusão Primária/tratamento farmacológico , MicroRNAs/metabolismo , Compostos de Mostarda/farmacologia , Fenilpropionatos/farmacologiaAssuntos
Segurança Química/legislação & jurisprudência , Substâncias para a Guerra Química/legislação & jurisprudência , Substâncias para a Guerra Química/provisão & distribuição , Guerra Química/legislação & jurisprudência , Guerra Química/prevenção & controle , Indústria Química/ética , Indústria Química/legislação & jurisprudência , Segurança Química/normas , Substâncias para a Guerra Química/intoxicação , Substâncias para a Guerra Química/toxicidade , Cloro/intoxicação , Cloro/toxicidade , Toxicologia Forense , Humanos , Compostos de Mostarda/intoxicação , Compostos de Mostarda/toxicidade , Agentes Neurotóxicos/intoxicação , Agentes Neurotóxicos/toxicidade , Organofosfatos/toxicidade , Compostos Organotiofosforados/intoxicação , SíriaRESUMO
Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Ossificação Heterotópica/genética , Ossificação Heterotópica/prevenção & controle , Ferimentos e Lesões/complicações , Receptores de Ativinas Tipo I/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Queimaduras/complicações , Queimaduras/genética , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Modelos Animais de Doenças , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Integrases/metabolismo , Medições Luminescentes , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Knockout , Modelos Biológicos , Compostos de Mostarda/farmacologia , Ossificação Heterotópica/diagnóstico por imagem , Ossificação Heterotópica/tratamento farmacológico , Fenilpropionatos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Tendões/efeitos dos fármacos , Tendões/patologia , Tendões/cirurgia , Tenotomia , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/patologia , Microtomografia por Raio-XRESUMO
Neurotensin (NT) via its receptor 1 (NTR1) modulates the development of colitis, decreases HIF-1α/PHD2 interaction, stabilizes and increases HIF-1α transcriptional activity, and promotes intestinal angiogenesis. HIF-1α induces miR-210 expression, whereas miR-210 is strongly upregulated in response to NT in NCM460 human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1). In this study, we examined whether NT activates a NTR1-HIF-1α-miR-210 cascade using in vitro (NCM460-NTR1 cells) and in vivo (transgenic mice overexpressing [HIF-1α-OE] or lacking HIF-1α [HIF-1α-knockout (KO)] in intestinal epithelial cells and mice lacking NTR1 [NTR1-KO]) models. Pretreatment of NCM460-NTR1 cells with the HIF-1α inhibitor PX-478 or silencing of HIF-1α (small interfering HIF-1α) attenuated miR-210 expression in response to NT. Intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNBS) administration (2-d model) increased colonic miR-210 expression that was significantly reduced in NTR1-KO, HIF-1α-KO mice, and wild-type mice pretreated intracolonically with locked nucleic acid anti-miR-210. In contrast, HIF-1α-OE mice showed increased miR-210 expression at baseline that was further increased following TNBS administration. HIF-1α-OE mice had also exacerbated TNBS-induced neovascularization compared with TNBS-exposed wild-type mice. TNBS-induced neovascularization was attenuated in HIF-1α-KO mice, or mice pretreated intracolonically with anti-miR-210. Intracolonic anti-miR-210 also reduced colitis in response to TNBS (2 d). Importantly, miR-210 expression was increased in tissue samples from ulcerative colitis patients. We conclude that NT exerts its proinflammatory and proangiogenic effects during acute colitis via a NTR1-prolyl hydroxylase 2/HIF-1α-miR-210 signaling pathway. Our results also demonstrate that miR-210 plays a proinflammatory role in the development of colitis.
Assuntos
Colite Ulcerativa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , Neurotensina/metabolismo , Animais , Linhagem Celular Tumoral , Colo/irrigação sanguínea , Colo/patologia , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Compostos de Mostarda/administração & dosagem , NF-kappa B/metabolismo , Fenilpropionatos/administração & dosagem , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Transdução de Sinais , Ácido Trinitrobenzenossulfônico , Regulação para Cima/efeitos dos fármacosRESUMO
In this study, the antifungal activity of yellow mustard (YMF) and oriental mustard (OMF) meal extracts against 14 strains of fungi was tested on a solid medium. The results obtained with the YMF were next confirmed in liquid medium determining the minimum inhibitory concentration (MIC) and the minimum fungicide concentration (MFC). Finally, the use of YMF as a natural preservative to extend the useful life of bread was evaluated. Breads with different concentrations of YMF (2, 4, 6 and 8 g/kg) were prepared and contaminated with Aspergillus flavus ISPA 8111 and Penicilliumnordicum CECT 2320. For 10 days the formation of mycelium was observed, and after that the fungal growth and the mycotoxins production was determined. The results obtained with the YMF were compared with breads treated with the commercial additive sodium propionate (E-281). The results showed a significant reduction of the fungal population using 6 g/kg and 8 g/kg of YMF in bread contaminated with A. flavus and with P.nordicum and an extensions of the breads shelf life of 7 and 5 days, respectively, in comparison with the control experiment. A reduction of 78% of AFB1 was observed using 6 g/kg of YMF while no AFB1 production was detected employing 8 g/kg of YMF in bread preparation.
Assuntos
Aflatoxinas/toxicidade , Antifúngicos/farmacologia , Aspergillus flavus/efeitos dos fármacos , Pão/microbiologia , Compostos de Mostarda/farmacologia , Aflatoxinas/química , Microbiologia de Alimentos , Conservantes de Alimentos/farmacologia , Armazenamento de Alimentos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Penicillium/efeitos dos fármacos , Propionatos/farmacologiaRESUMO
Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Neoplasias Mamárias Animais/terapia , Compostos de Mostarda/uso terapêutico , Fenilpropionatos/uso terapêutico , Linfócitos T Citotóxicos/imunologia , Animais , Proliferação de Células , Células Cultivadas , Terapia Combinada , Células Dendríticas/transplante , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
During a course of colitis, production of the gaseous mediator hydrogen sulfide (H2S) is markedly up-regulated at sites of mucosal damage and contributes significantly to healing and resolution of inflammation. The signaling mechanisms through which H2S promotes resolution of colitis are unknown. We hypothesized that the beneficial effects of H2S in experimental colitis are mediated via stabilization of hypoxia-inducible factor (HIF)-1α. The hapten dinitrobenzene sulfonic acid was used to induce colitis in rats and mice. This resulted in an elevated expression of the H2S-producing enzyme, cystathionine γ-lyase (CSE), and HIF-1α at sites of mucosal ulceration, and the expression of these 2 enzymes followed a similar pattern throughout the course of colitis. This represented a functionally important relationship because the loss of CSE-derived H2S production led to decreased HIF-1α stabilization and exacerbation of colitis. Furthermore, application of an H2S-releasing molecule, diallyl disulfide (DADS), stabilized colonic HIF-1α expression, up-regulated hypoxia-responsive genes, and reduced the severity of disease during peak inflammation. Importantly, the ability of DADS to promote the resolution of colitis was abolished when coadministered with an inhibitor of HIF-1α in vivo (PX-478). DADS was also able to maintain HIF-1α expression at a later point in colitis, when HIF-1α levels would have normally returned to control levels, and to enhance resolution. Finally, we found that HIF-1α stabilization inhibited colonic H2S production and may represent a negative feedback mechanism to prevent prolonged HIF-1α stabilization. Our findings demonstrate an important link between H2S and HIF-1α in the resolution of inflammation and injury during colitis and provide mechanistic insights into the therapeutic value of H2S donors.
Assuntos
Colite/metabolismo , Sulfeto de Hidrogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Compostos Alílicos/farmacologia , Animais , Benzenossulfonatos/toxicidade , Colite/tratamento farmacológico , Colite/patologia , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Dissulfetos/farmacologia , Expressão Gênica , Células HT29 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos de Mostarda/farmacologia , Fenilpropionatos/farmacologia , Estabilidade Proteica , Ratos , Ratos Wistar , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR(+/-) heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR(+/-) heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function.