RESUMO
Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.
Assuntos
Dieta Hiperlipídica , Metabolismo Energético , Resistência à Insulina , Camundongos Endogâmicos C57BL , Obesidade , Proteína Desacopladora 1 , Animais , Dieta Hiperlipídica/efeitos adversos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Metabolismo Energético/efeitos dos fármacos , Masculino , Camundongos , Proteína Desacopladora 1/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Chalconas/farmacologia , Camundongos Obesos , Fármacos Antiobesidade/farmacologia , Células 3T3-L1RESUMO
PURPOSE: To investigate the ciliary body anatomy and position of the implantable collamer lens (ICL) in low-vault eyes and analyze factors related to insufficient vault. SETTING: Zhongshan Ophthalmic Center, Guangzhou, China. DESIGN: Retrospective case-control observational study. METHODS: In this study, 73 eyes of 73 patients with an insufficient vault (<250 µm) were matched with 73 eyes with an ideal vault (250 to 750 µm). Ultrasound biomicroscopy was used to determine the ciliary body morphology and ICL position. The biometric parameters acquired by Scheimpflug tomography were compared. The correlation between the vault and these factors was analyzed, and the least absolute shrinkage and selection operator method was used to screen the risk factors for low vault. RESULTS: The low-vault group had a steeper corneal curvature, thicker lens thickness (LT), higher crystalline lens rise, and shorter axial length (AL) (all P < .005). The ciliary process length (CPL) and maximum ciliary body thickness (CBTmax) were significantly smaller, and the trabecular-ciliary angle (TCA), iris-ciliary angle (ICA), and ciliary sulcus width (CSW) were significantly greater in the low-vault eyes (all P < .005). The low-vault group had more ICL haptics below the ciliary process, and TCA, ICA, CPL, CBTmax, CSW, and haptic position were related to the postoperative vault (all P < .05). CPL, AL, and LT were identified as predictors of a low vault. CONCLUSIONS: Malposition of ICL haptics behind the ciliary process is a risk factor for low vault. A shorter CPL, thicker LT, and shorter AL are significant risk factors for the postoperative low vault.
Assuntos
Cristalino , Lentes Intraoculares , Lentes Intraoculares Fácicas , Humanos , Corpo Ciliar/diagnóstico por imagem , Corpo Ciliar/cirurgia , Estudos Retrospectivos , Microscopia Acústica/métodos , Cristalino/diagnóstico por imagemRESUMO
The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.
Assuntos
Benzamidas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Piperazinas/farmacologia , Pirazóis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Apoptose , Movimento Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIMS: Risk factor studies for acute kidney injury (AKI) in China are lacking, especially those regarding non-traditional risk factors, such as laboratory indicators. METHODS: All adult patients admitted to 38 tertiary and 22 secondary hospitals in China in any one month between July and December 2014 were surveyed. AKI patients were screened according to the Kidney Disease: Improving Global Outcomes' definition of AKI. Logistic regression was used to analyze the risk factors for AKI, and Cox regression was used to analyze the risk of in-hospital mortality for AKI patients; additionally, a propensity score analysis was used to reconfirm the risk factors among laboratory indicators for mortality. RESULTS: The morbidity of AKI was 0.97%. Independent risk factors for AKI were advancing age, male gender, hypertension, and chronic kidney disease. All-cause mortality was 16.5%. The predictors of mortality in AKI patients were advancing age, tumor, higher uric acid level and increases in Acute Physiologic Assessment and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores. The hazard ratio (HR) for mortality with uric acid levels > 9.1 mg/dl compared with ≤ 5.2 mg/dl was 1.78 (95% CI: 1.23 to 2.58) for the AKI patients as a group, and was 1.73 (95% CI: 1.24 to 2.42) for a propensity score-matched set. CONCLUSION: In addition to traditional risk factors, uric acid level is an independent predictor of all-cause mortality after AKI.
Assuntos
Injúria Renal Aguda/etiologia , Medição de Risco/métodos , Injúria Renal Aguda/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Mortalidade Hospitalar , Hospitalização , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Ácido Úrico/sangue , Adulto JovemRESUMO
BACKGROUND: It has been found that L-carnitine ameliorated cisplatin-induced acute kidney injury (AKI) in rats. However, the detailed role of L-carnitine in improving the renal urinary concentration function in cisplatin-induced AKI is not fully understood. METHODS: In this study, 30 Sprague-Dawley rats were divided randomly into 5 groups: control, cisplatin (CIS), L-carnitine (CAR), L-carnitine plus cisplatin (CAR + CIS), and cisplatin plus L-carnitine (CIS + CAR) groups. Cisplatin (7 mg/kg) and L-carnitine (300 mg/kg) were injected intraperitoneally. Urine (24 h) and blood samples were collected to analyze renal urinary concentrating function. Immunoblotting, confocal laser microscopy, and enzyme-linked immunosorbent assays were used to assess the level and localization of the water channel aquaporin (AQP) 2, and levels of stimulatory G protein α subunit (GSα protein), arginine vasopressin (AVP) receptor 2, adenylyl cyclase and serum AVP. RESULTS: Renal urinary concentrating function was improved by L-carnitine in rats with cisplatin-induced AKI. AQP2 expression, which decreased after cisplatin treatment, was improved by L-carnitine in different regions of the kidney. Moreover, our data indicated that L-carnitine could increase AQP2 accumulation at the apical plasma membranes of the renal-collecting ducts. Finally, intervention with L-carnitine effectively improved the expression of AQP2 upstream signaling proteins, such as GSα protein, adenylyl cyclase, and serum AVP levels in rats with cisplatin-induced AKI. CONCLUSION: L-carnitine resolves the cisplatin-induced urinary concentration defect, which may occur by increasing AVP/cyclic adenosine monophosphate/AQP2 levels, indicating the potential use of L-carnitine to ameliorate the renal urinary concentration effect in cancer patients treated with cisplatin.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Aquaporina 2/metabolismo , Carnitina/uso terapêutico , Injúria Renal Aguda/induzido quimicamente , Adenilil Ciclases/metabolismo , Animais , Arginina Vasopressina/sangue , Cisplatino/toxicidade , Creatinina/sangue , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/sangueRESUMO
BACKGROUND: There is a need for investigating the analgesic method as part of early recovery after surgery tailored for laparoscopic colorectal cancer (LCRC) surgery. In this randomized trial, we aimed to investigate the analgesic efficacy of an inverse 'v' shaped bilateral, subfascial ropivacaine continuous infusion in LCRC surgery. METHODS: Forty two patients undergoing elective LCRC surgery were randomly allocated to one of two groups to receive either 0.5% ropivacaine continuous infusion at the subfascial plane (n = 20, R group) or fentanyl intravenous patient controlled analgesia (IV PCA) (n = 22, F group) for postoperative 72 hours. The primary endpoint was the visual analogue scores (VAS) when coughing at postoperative 24 hours. Secondary end points were the VAS at 1, 6, 48, and 72 hours, time to first flatus, time to first rescue meperidine requirement, rescue meperidine consumption, length of hospital stay, postoperative nausea and vomiting, sedation, hypotension, dizziness, headache, and wound complications. RESULTS: The VAS at rest and when coughing were similar between the groups throughout the study. The time to first gas passage and time to first rescue meperidine at ward were significantly shorter in the R group compared to the F group (P = 0.010). Rescue meperidine was administered less in the R group; however, without statistical significance. Other study parameters were not different between the groups. CONCLUSIONS: Ropivacaine continuous infusion with an inverse 'v ' shaped bilateral, subfascial catheter placement showed significantly enhanced bowel recovery and analgesic efficacy was not different from IV PCA in LCRC surgery.
RESUMO
BACKGROUND: GI stromal tumours (GISTs) are clinically heterogenous exhibiting varying degrees of disease aggressiveness in individual patients. OBJECTIVES: We sought to identify genetic alterations associated with high-risk GIST, explore their molecular consequences, and test their utility as prognostic markers. DESIGNS: Exome sequencing of 18 GISTs was performed (9 patients with high-risk/metastatic and 5 patients with low/intermediate-risk), corresponding to 11 primary and 7 metastatic tumours. Candidate alterations were validated by prevalence screening in an independent patient cohort (n=120). Functional consequences of SETD2 mutations were investigated in primary tissues and cell lines. Transcriptomic profiles for 8 GISTs (4 SETD2 mutated, 4 SETD2 wild type) and DNA methylation profiles for 22 GISTs (10 SETD2 mutated, 12 SETD2 wild type) were analysed. Statistical associations between molecular, clinicopathological factors, and relapse-free survival were determined. RESULTS: High-risk GISTs harboured increased numbers of somatic mutations compared with low-risk GISTs (25.2 mutations/high-risk cases vs 6.8 mutations/low-risk cases; two sample t test p=3.1×10-5). Somatic alterations in the SETD2 histone modifier gene occurred in 3 out of 9 high-risk/metastatic cases but no low/intermediate-risk cases. Prevalence screening identified additional SETD2 mutations in 7 out of 80 high-risk/metastatic cases but no low/intermediate-risk cases (n=29). Combined, the frequency of SETD2 mutations was 11.2% (10/89) and 0% (0/34) in high-risk and low-risk GISTs respectively. SETD2 mutant GISTs exhibited decreased H3K36me3 expression while SETD2 silencing promoted DNA damage in GIST-T1 cells. In gastric GISTs, SETD2 mutations were associated with overexpression of HOXC cluster genes and a DNA methylation signature of hypomethylated heterochromatin. Gastric GISTs with SETD2 mutations, or GISTs with hypomethylated heterochromatin, showed significantly shorter relapse-free survival on univariate analysis (log rank p=4.1×10-5). CONCLUSIONS: Our data suggest that SETD2 is a novel GIST tumour suppressor gene associated with disease progression. Assessing SETD2 genetic status and SETD2-associated epigenomic phenotypes may guide risk stratification and provide insights into mechanisms of GIST clinical aggressiveness.
Assuntos
Biomarcadores Tumorais/genética , Tumores do Estroma Gastrointestinal/genética , Histona-Lisina N-Metiltransferase/genética , Mutação de Sentido Incorreto , Estudos de Casos e Controles , Códon sem Sentido/genética , Metilação de DNA/genética , Exoma/genética , Tumores do Estroma Gastrointestinal/epidemiologia , Tumores do Estroma Gastrointestinal/patologia , Histonas/genética , Humanos , Mutação de Sentido Incorreto/genética , Invasividade Neoplásica , Fenótipo , Prevalência , Prognóstico , Índice de Gravidade de Doença , Singapura/epidemiologiaRESUMO
Hypoxia-inducible factor-1α (HIF-1α) is one of the key transcription factors that mediate adaptation to hypoxia. Despite increasing evidence implicating the PKC family as potential modulators of HIF-1α, the molecular mechanisms of PKC isoform-dependent HIF-1α activity under hypoxic conditions have not been systematically elucidated in cancer cell lines. Here, we collectively investigated how each isoform of the PKC family contributes to HIF-1α accumulation in the human cervical cancer cell line HeLa. Among the abundant PKC isoforms, blockade of either PKCα or PKCδ was found to substantially reduce HIF-1α accumulation and transcriptional activity in hypoxic cells. Knockdown of PKCδ resulted in a reduction of HIF-1α mRNA levels, whereas the HIF-1α mRNA level was unchanged regardless of PKCα knockdown. Upon searching for the downstream effectors of these kinases, we found that PKCα controls HIF-1α translation via AKT-mTOR under hypoxic conditions. On the other hand, one of the well-known transcriptional regulation pathways of HIF-1α, nuclear factor-κB (NF-κB) is identified as a downstream effector of PKCδ. Taken together, our findings provide insights into the roles of PKC isoforms as additional, discrete modulators of hypoxia-stimulated HIF-1α accumulation through different signaling pathways.
Assuntos
Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína Quinase C-alfa/fisiologia , Proteína Quinase C-delta/fisiologia , Hipóxia Celular , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isoenzimas/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVE: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications. DESIGN: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice. RESULTS: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs. CONCLUSIONS: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.
Assuntos
Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Gástricas/genética , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Fator de Transcrição GATA4/biossíntese , Fator de Transcrição GATA6/biossíntese , Perfilação da Expressão Gênica/métodos , Inativação Gênica , Predisposição Genética para Doença , Xenoenxertos , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Camundongos Nus , Transplante de Neoplasias , Oncogenes/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais CultivadasRESUMO
Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1α and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1α containing plasmid. A mutant HIF-1α incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1α-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ativação Transcricional/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cinética , Ligação Proteica , Mapas de Interação de Proteínas/genética , Transdução de SinaisRESUMO
Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Ácido Etacrínico/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vitamina K 3/farmacologia , Sítios de Ligação/genética , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína p300 Associada a E1A/genética , Ácido Etacrínico/química , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Immunoblotting , Luciferases/genética , Luciferases/metabolismo , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas/métodos , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Vitamina K 3/químicaRESUMO
In this article, we present the case of a man with uremia. Laboratory testing revealed thrombocytopenia, erythrocyte fragmentation, elevated lactate dehydrogenase, and malignant hypertension, manifestations that are similar to thrombotic microangiopathy (TMA). Thromboasthenia, manifested as a decrease in the platelet aggregation rate, was also noted. Regular hemodialysis (3 times per week) improved the patient's thrombocytopenia and thromboasthenia. This case supports the conclusion that uremic toxin, which can be removed by hemodialysis, inhibits the quantity and quality of platelets. We believe that the platelet aggregation rate can be a useful tool in distinguishing uremia from TMA.
Assuntos
Agregação Plaquetária/fisiologia , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/terapia , Uremia/diagnóstico , Uremia/terapia , Diagnóstico Diferencial , Humanos , Masculino , Púrpura Trombocitopênica Trombótica/diagnóstico , Púrpura Trombocitopênica Trombótica/patologia , Diálise Renal/métodos , Medição de Risco , Índice de Gravidade de Doença , Trombocitopenia/diagnóstico , Trombocitopenia/patologia , Microangiopatias Trombóticas/patologia , Uremia/patologia , Adulto JovemRESUMO
We previously found that clioquinol (CQ) increases functional hypoxia-inducible factor-1α (HIF-1α) with enhanced transcription of its target genes. Here we report that compounds derived from 8-hydroxyquinoline including CQ, broxyquinoline (BQ), iodoquinol (IQ) and chloroacetoxyquinoline (CAQ) promote neovascularization effectively based on chick chorioallantoic membrane assays. The CQ analogues induce stabilization of HIF-1α as well as enhance HIF-1-mediated vascular endothelial growth factor transcription. These analogues also exert inhibitory effects on the activity of prolyl and asparaginyl hydroxylations of HIF-1α in vitro. Despite metal ion-dependent restoration of the inhibited HIF-1α hydroxylase activity, the cellular HIF-1α-inducing effects of the CQ analogues are reversed to varying degrees by Zn(2+) and Fe(2+). While CQ and BQ are completely reversed by Zn(2+), co-administration of Zn(2+) and IQ has only a partial reversing effect. On the other hand, CAQ-mediated stabilization of HIF-1α is reversed by Fe(2+) but not by Zn(2+). These phenomena are found to coincide with elevation of the intracellular Zn(2+) and Fe(2+) levels by the CQ analogues, suggesting that metal ion effects on HIF-1α in cells likely reflect the differential transporting capability of the analogues.
Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Hipóxia Celular/fisiologia , Clioquinol/farmacologia , Fatores de Crescimento Endotelial/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ferro/metabolismo , Zinco/metabolismo , Animais , Transporte Biológico , Cátions Bivalentes/metabolismo , Hipóxia Celular/genética , Embrião de Galinha , Membrana Corioalantoide/fisiologia , Clioquinol/análogos & derivados , Fatores de Crescimento Endotelial/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Hidroxilação , Oxigenases de Função Mista/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
PURPOSE: DZNep (3-deazaneplanocin A) depletes EZH2, a critical component of polycomb repressive complex 2 (PRC2), which is frequently deregulated in cancer. Despite exhibiting promising anticancer activity, the specific genetic determinants underlying DZNep responsiveness in cancer cells remain largely unknown. We sought to determine molecular factors influencing DZNep response in gastric cancer. EXPERIMENTAL DESIGN: Phenotypic effects of DZNep were evaluated in a panel of gastric cancer cell lines. Sensitive lines were molecularly interrogated to identify potential predictors of DZNep responsiveness. The functional importance of candidate predictors was evaluated using short hairpin RNA (shRNA) and siRNA technologies. RESULTS: DZNep depleted PRC2 pathway components in almost all gastric cancer lines, however, only a subset of lines exhibited growth inhibition upon treatment. TP53 genomic status was significantly associated with DZNep cellular responsiveness, with TP53 wild-type (WT) lines being more sensitive (P < 0.001). In TP53-WT lines, DZNep stabilized p53 by reducing ubiquitin conjugation through USP10 upregulation, resulting in activation of canonical p53 target genes. TP53 knockdown in TP53-WT lines attenuated DZNep sensitivity and p53 target activation, showing the functional importance of an intact p53 pathway in regulating DZNep cellular sensitivity. In primary human gastric cancers, EZH2 expression was negatively correlated with p53 pathway activation, suggesting that higher levels of EZH2 may repress p53 activity. CONCLUSION: Our results highlight an important role for TP53 genomic status in influencing DZNep response in gastric cancer. Clinical trials evaluating EZH2-targeting agents such as DZNep should consider stratifying patients with gastric cancer by their TP53 genomic status.
Assuntos
Adenosina/análogos & derivados , Mutação , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Adenosina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Immunoblotting , Masculino , Metilação/efeitos dos fármacos , Pessoa de Meia-Idade , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacos , Adulto JovemRESUMO
Insight into the regulation of core transcription factors is important for a better understanding of the molecular mechanisms that control self-renewal and pluripotency of human ESCs (hESCs). However, the transcriptional regulation of NANOG itself in hESCs has largely been elusive. We established a NANOG promoter luciferase reporter assay as a fast read-out for indicating the pluripotent status of hESCs. From the functional cDNA screens and NANOG promoter characterization, we successfully identified a zinc finger transcription factor KLF4 and a homeodomain transcription factor PBX1 as two novel transcriptional regulators that maintain the pluripotent and undifferentiated state of hESCs. We showed that both KLF4 and PBX1 mRNA and protein expression were downregulated during hESC differentiation. In addition, overexpression of KLF4 and PBX1 upregulated NANOG promoter activity and also the endogenous NANOG protein expression in hESCs. Direct binding of KLF4 on NANOG proximal promoter and PBX1 on a new upstream enhancer and proximal promoter were confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assay. Knockdown of KLF4/PBX1 or mutation of KLF4/PBX1 binding motifs significantly downregulated NANOG promoter activity. We also showed that specific members of the SP/KLF and PBX family are functionally redundant at the NANOG promoter and that KLF4 and PBX1 cooperated with OCT4 and SOX2, and transactivated synergistically the NANOG promoter activity. Our results show two novel upstream transcription activators of NANOG that are functionally important for the self-renewal of hESC and provide new insights into the expanded regulatory circuitry that maintains hESC pluripotency.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Células-Tronco Embrionárias , Proteínas de Homeodomínio/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Proteína Homeobox Nanog , Fator de Transcrição 1 de Leucemia de Células Pré-B , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To explore therapeutic effect of glutathione (GSH) on unilateral ureteral obstruction (UUO) induced renal interstitial fibrosis and its mechanism in rat. METHODS: Thirty-six adult healthy Wistar rats were randomly divided into 3 groups (each n=12): sham operation group, UUO group and GSH group. Rats in UUO group and GSH group underwent left unilateral ureteral ligation as described previously. Rats in sham group had their ureters manipulated but not ligated. In GSH group, GSH was injected intraperitoneally (100 mg.kg(-1).d(-1)) in different doses based on the animal's body weight, one day before UUO and then for consecutive 10 days after UUO. Meantime, same volume of physiological saline was given in sham operation and UUO groups as GSH group. Animals were sacrificed at 10 days after surgery. The pathological changes in obstructed kidney tissue were observed with hematoxylin and eosin (HE) and Masson stains. The contents of hydroxyproline (Hyp) and total anti-oxygen capability (T-AOC) were measured by chemical colorimetry method, the activity of total superoxide dismutase (T-SOD) was assayed by a modified xanthine/xanthine oxidase method, and the content of malondialdehyde (MDA) was determined by thiobarbituric acid method. RESULTS: Ten days after UUO, swelling and atrophy in renal parenchyma of obstructed kidneys were clearly observed. Fibrous material and monocyte infiltration were increased in the interstitial space. Furthermore, thickening of interstitial space of the tubular basement membrane and widening of the interstitial space of the renal cortex were noted. Hypertrophy or atrophy of juxtaglomerular tubules were also observed. There were cellular or albumin casts in a part of tubules. Interstitial fibrosis was observed in obstructed kidneys 10 days after UUO. The data indicated that ureteral obstruction significantly increased the contents of Hyp and MDA, but decreased the content of T-AOC and T-SOD activity, as compared with sham operation group [Hyp: (0.524+/-0.132) microg/mg, T-AOC: (1.48+/-0.21) U/mg, T-SOD: (12.77+/-0.76) U/mg, MDA: (2.65+/-0.32) nmol/mg, all P<0.01]. Compared with UUO group, pathological changes were milder and the contents of Hyp [(1.598+/- 0.252) microg/mg vs. (1.027+/-0.196) microg/mg, P<0.05] and MDA [(4.58+/-0.59) nmol/mg vs. (3.26+/- 0.34) nmol/mg, P<0.05] were significantly decreased in kidney of GSH group, meanwhile the content of T-AOC was increased [(0.67+/-0.19) U/mg vs. (0.94+/-0.17) U/mg, P<0.05], but the content of T-SOD did not show any change [(9.39+/-0.87) U/mg vs. (9.41+/-0.93) U/mg, P>0.05]. CONCLUSION: Reduced glutathione treatment attenuates UUO-induced renal interstitial fibrosis via decreasing content of Hyp in UUO kidney and preventing oxidation stress injury.
Assuntos
Glutationa/farmacologia , Hidroxiprolina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Obstrução Ureteral/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Rim/metabolismo , Rim/patologia , Masculino , Malondialdeído/metabolismo , Nefroesclerose/prevenção & controle , Distribuição Aleatória , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Obstrução Ureteral/complicações , Obstrução Ureteral/patologiaRESUMO
The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.