RESUMO
INTRODUCTION: Preeclampsia, a specific complication of pregnancy, is a leading cause of perinatal and maternal mortality worldwide. N6-methyladenosine (m6A) is a prevalent and reversible modification of mammalian mRNAs, and is known to play an important role in various physiological and pathological processes. However, little is known about its possible effects on trophoblasts in preeclampsia. METHODS: Colorimetric RNA m6A methylation quantification assay and dot blotting were used to assess the levels of global RNA m6A modification in placental tissues collected from females with normal pregnancy and preeclampsia, while the mRNA levels of major m6A methyltransferases/demethylases were investigated by quantitative real-time polymerase chain reaction. The effects of methyltransferase-like 14 (METTL14) on trophoblasts were evaluated using cell counting kit-8, transwell invasion assay, autophagic flux assay, and Annexin V/propidium iodide apoptosis assay. The molecular mechanism underlying the regulation of forkhead box O3a (FOXO3a) expression by METTL14 was determined using methylated RNA immunoprecipitation and transcription inhibition assays. RESULTS: Global RNA m6A methylation and METTL14 expression were significantly increased in placental tissues obtained from patients with preeclampsia. In vitro studies showed that overexpression of METTL14 in HTR-8/SVneo cells inhibited trophoblast proliferation and invasion, but induced trophoblast autophagy and apoptosis. We further demonstrated that METTL14 epigenetically elevated FOXO3a expression via an m6A-dependent mechanism. FOXO3a inhibition effectively prevented the impairment of trophoblast proliferation and invasion, and diminished the induction of trophoblast autophagy and apoptosis in METTL14-overexpressing HTR-8/SVneo cells. DISCUSSION: Increased METTL14-mediated m6A modification results in an adverse impact on trophoblast function by elevating FOXO3a expression.
Assuntos
Metiltransferases , Pré-Eclâmpsia , Trofoblastos , Linhagem Celular , Movimento Celular , Feminino , Proteína Forkhead Box O3/biossíntese , Proteína Forkhead Box O3/genética , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Placenta/metabolismo , Placenta/patologia , Pré-Eclâmpsia/enzimologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologia , Regulação para CimaRESUMO
BACKGROUND AND AIMS: Endothelial cell injury causes vascular barrier dysfunction and leukocyte recruitment to the underlying tissue. Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor that exerts pro-inflammatory effects on the endothelium. Here, we investigated the effects of BMP-4 on endothelial cell (EC) migration following balloon injury in SD rats. METHODS: An intimal hyperplasia model was established using balloon injury. Hematoxylin-eosin staining (HE) and silver staining were used to detect the alteration of endothelial cells recovery after balloon injury. Serum BMP-4 levels were assessed by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured. MTT assay was used to measure cell viability. Protein expression was detected by Western blot. Intracellular reactive oxygen species (ROS) was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA). HUVECs migration was measured via transwell assay and scratch wound assay. RESULTS: The results indicated that BMP-4 inhibition significantly decreased total plasma activity of BMP-4 and reduced neointimal hyperplasia by stimulating endothelial cell migration, but did not affect the medial area following balloon injury. BMP-4 suppressed the formation of ROS via forkhead box O3 (FoXO-3)/superoxide dismutase 1 (SOD-1). In vitro, a high level of ROS induced by BMP-4 impeded HUVECs migration. CONCLUSIONS: The results suggest that BMP-4 inhibition is a potential means of preventing intimal hyperplasia formation after balloon injury.
Assuntos
Proteína Morfogenética Óssea 4 , Células Endoteliais da Veia Umbilical Humana , Animais , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 4/sangue , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Movimento Celular , Células Cultivadas , Proteína Forkhead Box O3/biossíntese , Proteína Forkhead Box O3/sangue , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Hiperplasia , Neointima/sangue , Neointima/metabolismo , Neointima/patologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/sangue , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase-1/sangueRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) remains a prevalent chronic airway inflammatory disease. Circular RNAs (circRNAs) are associated with inflammation regulation; therefore, we examined distinct effects of circRNA FOXO3 (circFOXO3) against pneumonic inflammatory processes in COPD. METHODS: We first quantified and localized circFOXO3 in mouse lung epithelial cell line MLE12 by quantitative reverse-transcription PCR and in situ hybridization. Next, circFOXO3 was suppressed by therapeutic administration of circFOXO3 knockdown lentivirus in mice exposed to air or cigarette smoke (CS) for 12 weeks, and several hallmarks of COPD were evaluated. RESULTS: We noticed that circFOXO3 is upregulated in CS-exposed lungs and cigarette smoke extract (CSE)-treated murine alveolar epithelial cells. Knockdown of circFOXO3 attenuated the release of CXCL1 and IL-6 as well as inflammatory processes in the lungs of CS-exposed mice. In addition, we identified miR-214-3p as a circFOXO3-targeted microRNA. MiR-214-3p overexpression exerted protective effects against pneumonic inflammation after CS exposure. Silencing of circFOXO3 downregulated IKK-ß mRNA (miR-214-3p's target), resulting in the dysfunction of the NF-κB signaling pathway and attenuation of CSE-induced inflammatory-cytokine expression. CONCLUSIONS: Collectively, these findings reveal a crucial function of circFOXO3 in the pathological remodeling related to CS-induced inflammatory processes. Hence, circFOXO3 might be a good target for the treatment of inflammatory disorders similar to CS-induced lung inflammation.
Assuntos
Fumar Cigarros/efeitos adversos , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica , Lesão Pulmonar/genética , RNA Circular/genética , Animais , Modelos Animais de Doenças , Proteína Forkhead Box O3/biossíntese , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Sarcopenia is an aging associated disorder involving skeletal muscle atrophy and a reduction in muscle strength, and there are no pharmaceutical interventions available thus far. Moreover, conditions such as hyperglycaemia are known to further intensify muscle degradation. Therefore, novel strategies to attenuate skeletal muscle loss are essential to enhance muscle function and thereby improve the quality of life in diabetic individuals. In this study, we have investigated the efficiency of a potato peptide hydrolysate PPH902 for its cytoprotective effects in skeletal muscle cells. PPH902 treatment in C2C12 cells showed the dose-dependent activation of the Akt/mTOR signalling pathway that is involved in skeletal myogenesis. According to Western blotting analysis, PPH902 induced the phosphorylation of Akt, mTOR proteins and induced the myogenic differentiation of C2C12 myoblasts in a differentiation medium. The phosphorylation myogenic transcription factor Foxo3A was also found to be increased in the cells treated with PPH902. In addition, treatment with PPH902 ameliorated the high glucose induced reduction in cell viability in a dose-dependent manner. Moreover, the number of myotubes in a differentiation medium reduced upon high glucose challenge, but treatment with PPH902 increased the number of differentiated myotubes. Further, the phosphorylations of AMPK and mitochondrial-related transcription factors such as PGC-1α were suppressed upon high glucose challenge but PPH902 treatment restored the protein levels. We demonstrate, for the first time, that a specific potato peptide has a therapeutic effect against sarcopenia. In addition, PPH902 improved the myogenic differentiation and their mitochondrial biogenesis and further improved myogenic protein and inhibited muscle protein degradation in C2C12 cells challenged under a high glucose condition.
Assuntos
Proteína Forkhead Box O3/biossíntese , Glucose/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Proteína Forkhead Box O3/química , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Hidrolisados de ProteínaRESUMO
Gefitinib is tyrosine kinase inhibitor of epidermal growth factor receptor, which exhibits notable clinical efficacy in non-small-cell lung cancer (NSCLC) treatment. However, gefitinib resistance is a critical obstacle for NSCLC targeted therapy. Here, we investigated the biological functions and mechanisms of lncRNA CASC9 in NSCLC gefitinib resistance. Screening analysis and RT-qPCR demonstrated that CASC9 was up-regulated in the gefitinib-resistant NSCLC cells (PC9/GR). Moreover, high-expression of CASC9 acted as an unfavorable factor for NSCLC patients. Functionally, CASC9 promoted the proliferation and gefitinib resistance of PC9/GR cells in vitro, and knockdown of CASC9 repressed the tumor growth in vivo. Mechanistically, CASC9 epigenetically promoted the FOXO3 expression via inhibiting miR-195-5p. In turn, transcription factor FOXO3 bound with the promoter region of CASC9 to enhance CASC9 transcriptional level, thereby forming CASC9/miR-195-5p/FOXO3 positive feedback loop. In conclusion, our research identified the regulation of CASC9/miR-195-5p/FOXO3 feedback loop on NSCLC gefitinib resistance, which might help researchers develop potential therapeutic targets for NSCLC.
Assuntos
Carcinogênese/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteína Forkhead Box O3/biossíntese , Gefitinibe/farmacologia , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/biossíntese , Células A549 , Idoso , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/fisiologia , Feminino , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-IdadeRESUMO
Liver fibrosis is a critical health issue in the world due to its rapidly increasing prevalence. It is of great demand to develop effective drugs for the treatment of liver fibrosis. 5-methoxytryptophan (5-MTP) has been reported to play an important role in anti-inï¬ammatory, anti-cancer, myocardial-protective eï¬ects. However, the anti-fibrotic eï¬ect of 5-MTP is never covered in liver. Here, we investigated anti-fibrotic effects of 5-MTP on liver fibrosis and its underlying mechanism. In vitro, 5-MTP treatment could inhibit TGF-ß1-induced elevated levels of collagen I, collagen III, fibronectin and α-smooth muscle actin (SMA) by stimulating autophagy process. Mechanically, the expression of FOXO3a was enhanced by 5-MTP and then repressed the level of miR-21, eventually leading to a restoration of autophagy-related gene ATG5. Furthermore, rescue experiments showed 5-MTP could activate autophagy process and suppress the activation of LX-2 cells by regulating FOXO3a/miR-21/ATG5 pathway. Consistently, 5-MTP significantly attenuated CCl4-induced hepatic fibrosis in rat model. In conclusion, our research discovered that 5-MTP effectively alleviated liver fibrosis in vitro and in vivo, which provided new insights into the application of 5-MTP for liver fibrosis.
Assuntos
Proteína 5 Relacionada à Autofagia/biossíntese , Autofagia/efeitos dos fármacos , Proteína Forkhead Box O3/biossíntese , Cirrose Hepática/metabolismo , MicroRNAs/biossíntese , Triptofano/análogos & derivados , Animais , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/genética , Tetracloreto de Carbono/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Proteína Forkhead Box O3/genética , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Triptofano/farmacologia , Triptofano/uso terapêuticoRESUMO
This study attempted to analyze the alterations in the mRNA expression levels of autophagy- and apoptosis-related genes in the forkhead box transcription factor O (FOXO) pathway in schizophrenia patients before and after olanzapine treatment. For a total of 32 acute schizophrenic inpatients, clinical data with PANSS were obtained before and after four weeks of olanzapine treatment (mean dose 14.24 ± 4.35 mg/d) along with data from 32 healthy volunteers. The mRNA expression levels of the FOXO pathway genes were measured by real-time qPCR after fasting venous blood was collected and analyzed. The mRNA expression levels of FOXO1, FOXO3A, FASLG, and BCL2L11 were observed to be significantly decreased in acute schizophrenia patients. After four weeks of olanzapine treatment, the expression levels of the first three genes were further reduced, but BCL2L11 expression levels were not significantly changed. The pairwise correlations between the mRNA expression level of FASLG and those of the other three genes were not observed in acute schizophrenia patients, while these relationships were observed in healthy controls. After olanzapine treatment, the FASLG mRNA expression level was restored and exhibited a pairwise correlation with the FOXO3A and BCL2L11 mRNA expression levels but not with the FOXO1 mRNA expression level, and FASLG mRNA expression was also correlated with the duration of the disease. The statuses and correlations of the mRNA expression levels of FOXO pathway-related genes were altered in schizophrenia patients and were affected by olanzapine treatment and the duration of the disease.
Assuntos
Antipsicóticos/uso terapêutico , Apoptose/fisiologia , Autofagia/fisiologia , Fatores de Transcrição Forkhead/biossíntese , Olanzapina/uso terapêutico , Esquizofrenia/tratamento farmacológico , Adolescente , Adulto , Antipsicóticos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/genética , Feminino , Proteína Forkhead Box O1/biossíntese , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O3/biossíntese , Proteína Forkhead Box O3/genética , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Olanzapina/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Esquizofrenia/sangue , Esquizofrenia/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Autophagy is an integral component of cellular homeostasis and metabolism. The exact mechanism of impaired autophagy in diabetes mellitus is unknown. Forkhead Box O3 (FOXO3α) is a key regulator of oxidative stress-related responses. We hypothesize FOXO3α is a direct upstream regulator of the autophagy pathway, and its upregulation is compromised in diabetic patients during stress of cardiopulmonary bypass (CPB). METHODS: The study enrolled 32 diabetic and 33 nondiabetic patients undergoing a cardiac surgical procedure on CPB. Right atrial tissue and serum samples were collected before and after CPB per protocol. A set of key components were quantitatively assessed and compared by microarray, immunoblotting, and immunohistochemistry studies. Data were analyzed using paired or unpaired student test. A P of <.05 or less was considered significant. RESULTS: Serum microarray showed FOXO3α was upregulated in the diabetic vs nondiabetic group after CPB (P = .033), autophagy-related 4B gene and Beclin 1 gene were greatly upregulated in the nondiabetic group (P = .028 and P = .002, respectively). On immunoblotting, there was upregulation of FOXO3α in the nondiabetic patients after CPB (P = .003). There were increased levels of Beclin-1, Bcl-2, and light chain 3B after CPB in the nondiabetic group only (P = .016, P = .005, P = .002, respectively). Sirtuin 1, Unc-51-like autophagy activating kinase 1 (ULK1), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), and mammalian target of rapamycin (mTOR) were not significantly changed in the nondiabetic group after CPB. CONCLUSIONS: Compared with nondiabetic patients, there was no significant upregulation of FOXO3α in diabetic patients, which could possibly explain the lack of upregulation of the autophagy process after CPB. FOXO3α could potentially serve as a therapeutic target to improve cellular homeostasis.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Diabetes Mellitus/genética , Proteína Forkhead Box O3/genética , Miocárdio/metabolismo , Estresse Oxidativo/genética , RNA/genética , Regulação para Cima , Idoso , Apoptose , Autofagia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Feminino , Proteína Forkhead Box O3/biossíntese , Humanos , Immunoblotting , Masculino , Miocárdio/patologiaRESUMO
Prostate cancer (PCa) incidence is surging in United States and other parts of the world. Synthetic and natural compounds have been explored as potential modulators of PI3K/Akt signaling that is known to drive PCa growth. Here, we evaluated the efficacy of a series of triphenyltin (IV) carboxylate derivatives against PCa. From this library, triphenylstannyl 2-(benzylcarbamoyl)benzoate (Ch-319) resulted in reduced viability and induction of cell cycle arrest in PTEN-/- PC3M and PTEN+/- DU145 cells. In parallel, downregulation of PI3K p85/p110 subunits, dephosphorylation of Akt-1 and increase in FOXO3a expression were observed. In silico studies indicated binding interactions of Ch-319 within the ATP binding site of Akt-1 at Met281, Phe442 and Glu234 residues. Elevated po-pulation of apoptotic cells, activation of Bax and reduced Bcl2 expression indicated apoptosis by Ch-319. Pre-sensitization of PCa cells with Ch-319 augmented the effect of cabazitaxel, a commonly used taxane in patients with castration-resistant PCa. Next, in a prostate-specific PTENp-/- mice, Ch-319 showed reduced weights of genitourinary apparatus as compared to DMSO treated controls. Histological studies indicated absence of neoplastic foci in Ch-319 treated prostates. Consistently, dephosphorylation of Akt-1, reduced expression of PRAS40 and androgen receptor and increase in FOXO3a were observed in treated group. Notably, no overt organ toxicity was noted in Ch-319 treated animals. Our studies identify Akt/FOXO3a signaling as a target of triphenyltin (IV) carboxylate Ch-319 and provide a molecular basis of its growth inhibitory effect in PCa cells. We propose that Ch-319 has the potential to be developed as an anticancer agent against PCa.
Assuntos
Progressão da Doença , Proteína Forkhead Box O3/biossíntese , Compostos Orgânicos de Estanho/química , Compostos Orgânicos de Estanho/farmacologia , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Compostos Orgânicos de Estanho/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Major depressive disorder (MDD) is a life-threatening illness characterized by mood changes and high rates of suicide. Although the role of neuroinflammation in MMD has been studied, the mechanistic interplay between antidepressants, neuroinflammation, and autophagy is yet to be investigated. The present study investigated the effect of melatonin on LPS-induced neuroinflammation, depression, and autophagy impairment. Our results showed that in mice, lipopolysaccharide (LPS) treatment induced depressive-like behaviors and caused autophagy impairment by dysregulating ATG genes. Moreover, LPS treatment significantly increased the levels of cytokines (TNFα, IL-1ß, IL-6), enhanced NF-á´B phosphorylation, caused glial (astrocytes and microglia) cell activation, dysregulated FOXO3a expression, increased the levels of redox signaling molecules such as ROS/TBARs, and altered expression of Nrf2, SOD2, and HO-1. Melatonin treatment significantly abolished the effects of LPS, as demonstrated by improved depressive-like behaviors, normalized autophagy-related gene expression, and reduced levels of cytokines. Further, we investigated the role of autophagy in LPS-induced depressive-like behavior and neuroinflammation using autophagy inhibitors 3-MA and Ly294002. Interestingly, inhibitor treatment significantly abolished and reversed the anti-depressive, pro-autophagy, and anti-inflammatory effects of melatonin. The present study concludes that the anti-depressive effects of melatonin in LPS-induced depression might be mediated via autophagy modulation through FOXO3a signaling.
Assuntos
Astrócitos/metabolismo , Transtorno Depressivo Maior , Proteína Forkhead Box O3/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Melatonina/farmacologia , Microglia/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Animais , Astrócitos/patologia , Autofagia/efeitos dos fármacos , Transtorno Depressivo Maior/induzido quimicamente , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/metabolismo , Transtorno Depressivo Maior/patologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Masculino , CamundongosRESUMO
The pathological effects of obesity are often severe in aging condition. Although exercise training is found to be advantageous, the intensity of exercise performed is limited in aging condition. Therefore in this study we assessed the effect of a combined treatment regimen with a short-peptide IF isolated from alcalase potato-protein hydrolysates and a moderate exercise training for 15 weeks in a 6 month old HFD induced obese senescence accelerated mouse-prone 8 (SAMP8) mice model. Animals were divided into 6 groups (n=6) (C:Control+BSA); (HF:HFD+BSA); (EX:Control+ BSA+Exercise); (HF+IF:HFD+ IF); (HF+EX:HFD+Exercise); (HF+EX+IF:HFD+Exercise+IF). A moderate incremental swimming exercise training was provided for 6 weeks and after 3 weeks of exercise, IF was orally administered (1 mg/kg body Weight). The results show that combined administration of IF and exercise provides a better protection to aging animals by reducing body weight and regulated tissue damage. IF intake and exercise training provided protection against cardiac hypertrophy and maintains the tissue homeostasis in the heart and liver sections. Interestingly, IF and exercise training showed an effective upregulation in pAMPK/ SIRT1/ PGC-1α/ pFOXO3 mechanism of cellular longevity. Therefore, exercise training with IF intake is a possible strategy for anti-obesity benefits and superior cardiac and hepatic protection in aging condition.
Assuntos
Envelhecimento , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica , Obesidade/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , RNA/genética , Sirtuína 1/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Proteína Forkhead Box O3/biossíntese , Masculino , Camundongos , Obesidade/metabolismo , Obesidade/terapia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Condicionamento Físico Animal , Sirtuína 1/biossínteseRESUMO
OBJECTIVE: The aim of this study was to investigate the expression level of circ-DONSON in glioma and to explore its effect on glioma metastasis and the underlying mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine circ-DONSON expression in 40 paired glioma tumor tissues and adjacent tissues. Meanwhile, the relation between circ-DONSON level and clinical parameters of glioma and the prognosis of patients was analyzed. The expression of circ-DONSON in glioma cell lines was analyzed by qRT-PCR as well. In addition, circs-DONSON silencing model was constructed in glioma cell lines. Cell counting kit-8 (CCK-8), cell scratch, and transwell migration assays were performed to investigate the effect of circ-DONSON on biological functions of glioma cells. Finally, the interplay between FOXO3 and circ-DONSON was explored. RESULTS: QRT-PCR results revealed that the expression level of circ-DONSON in glioma tumor tissues was remarkably higher than that of adjacent tissues, and the difference was statistically significant (p<0.05). Compared with patients with low expression of circ-DONSON, significantly higher prevalence of lymph node or distant metastasis and worse prognosis were observed in patients with high expression of circ-DONSON (p<0.05). The proliferation and migration abilities of glioma cells in circ-DONSON silenced group were remarkably suppressed when compared with NC group (p<0.05). Additionally, FOXO3 expression was remarkably down-regulated in glioma cell lines and tissues. FOXO3 expression was negatively correlated with circ-DONSON expression. In addition, cell reverse experiment demonstrated that circ-DONSON and FOXO3 can regulate each other, thereby together affecting the malignant progression of glioma. CONCLUSIONS: Circ-DONSON was remarkably associated with lymph node or distant metastasis, as well as poor prognosis of patients with glioma. Furthermore, it promoted the metastasis of glioma cells via regulating FOXO3.
Assuntos
Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteína Forkhead Box O3/biossíntese , Glioma/metabolismo , Metástase Linfática , Proteínas Nucleares/biossíntese , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Proteína Forkhead Box O3/antagonistas & inibidores , Proteína Forkhead Box O3/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Glioma/genética , Glioma/patologia , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , PrognósticoRESUMO
OBJECTIVE: The objective of this paper is to determine whether SIRT3 could retard intervertebral disc degeneration and study the mechanism. MATERIALS AND METHODS: We chose the 3-month mice to establish intervertebral disc degeneration model and study the effect of SIRT3 on the intervertebral disc by Western blotting, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), immunohistochemistry. Mouse nucleus pulposus cells were cultured to study the exact mechanism. RESULTS: The expression of SIRT3 was decreased in degenerated human nucleus pulposus. Intervertebral discs of mice treated with theacrine expressed more collagen II and less collagen X. In addition, nucleus pulposus cells stimulated with interleukin-1ß (IL-1ß) expressed less SIRT3 than that in the control group and nucleus pulposus cells with SIRT3 overexpress vectors expressed more collagen II FOXO3a and superoxide dismutase 2 (SOD2), indicating that SIRT3 could improve the intervertebral disc degeneration by anti-oxidative stress. CONCLUSIONS: SIRT3 is a protective factor for intervertebral discs and can reduce oxidative stress in the intervertebral disc.
Assuntos
Proteína Forkhead Box O3/biossíntese , Degeneração do Disco Intervertebral/fisiopatologia , Sirtuína 3/fisiologia , Superóxido Dismutase/biossíntese , Animais , Colágeno/biossíntese , Colágeno Tipo II/biossíntese , Humanos , Interleucina-1beta/farmacologia , Disco Intervertebral , Degeneração do Disco Intervertebral/metabolismo , Camundongos , Núcleo Pulposo , Estresse Oxidativo/fisiologia , Fatores de Proteção , Transdução de Sinais/fisiologia , Sirtuína 3/biossínteseRESUMO
Gene amplified in esophageal cancer 1 (GAEC1) expression and copy number changes are frequently associated with the pathogenesis of colorectal carcinomas. The current study aimed to identify the pathway and its transcriptional factors with which GAEC1 interacts within colorectal cancer, to gain a better understanding of the mechanics by which this gene exercises its effect on colorectal cancer. Two colonic adenocarcinoma cell lines (SW48 and SW480) and a nonneoplastic colon epithelial cell line (FHC) were transfected with GAEC1 to assess the oncogenic potential of GAEC1 overexpression. Multiple in vitro assays, including cell proliferation, wound healing, clonogenic, apoptosis, cell cycle, and extracellular flux, were performed. Western blot analysis was performed to identify potential gene-interaction partners of GAEC1 in vitro. Results showed that the overexpression of GAEC1 significantly increased cell proliferation, migration, and clonogenic potential ( P < 0.05) of colonic adenocarcinoma. Furthermore, GAEC1 portrayed its ability to influence mitochondrial respiration changes. The observations were in tandem with a significant increase in the expression of phosphorylated protein kinase B, forkhead box O3, and matrix metallopeptidase 9. Thus, GAEC1 has a role in regulating gene pathways, potentially in the Akt pathway. This could help in developing targeted therapies in the future.
Assuntos
Adenocarcinoma/genética , Carcinogênese/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Nucleares/genética , Adenocarcinoma/patologia , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/patologia , Variações do Número de Cópias de DNA/genética , Células Epiteliais/patologia , Proteína Forkhead Box O3/biossíntese , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , TransfecçãoRESUMO
OBJECTIVE: This study aims at investigating whether TUG1 (Taurine Upregulated Gene 1) can regulate FOXO3 expression through competitive binding to microRNA-9, thus leading to increased neuronal death and promoting the occurrence and development of acute cerebral infarction. MATERIALS AND METHODS: TUG1 and FOXO3 expressions in cerebral cortical neurons of MCAO mice, control mice and primary neurons were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The effects of TUG1 and FOXO3 on neuronal apoptosis were determined by TUNEL after cerebral infarction area was stained with TTC. The binding condition of microRNA-9, TUG1 and FOXO3 was verified by the Luciferase reporter gene assay. Western blot was performed to detect the protein expressions of B-cell lymphoma-2 (BCL-2) and BCL2-Associated X (BAX) after altering the TUG1 or FOXO3 expression in primary neurons. RESULTS: TUG1 and FOXO3 were overexpressed in cerebral cortical neurons of MCAO mice and primary neurons. The inhibition of TUG1 or FOXO3 resulted in less neuronal apoptosis. Luciferase reporter gene assay demonstrated that TUG1 regulates FOXO3 via TUG1/microRNA-9/FOXO3 regulatory network. Besides, TUG1 inhibited BCL-2 but promoted BAX expression in primary neurons. CONCLUSIONS: The overexpression of TUG1 can promote neuronal death after cerebral infarction in mice by competitive binding to microRNA-9 and promotion of FOXO3 expression.
Assuntos
Morte Celular/genética , Infarto Cerebral/genética , Infarto Cerebral/patologia , MicroRNAs/genética , Neurônios/patologia , RNA Longo não Codificante/biossíntese , Animais , Animais Recém-Nascidos , Apoptose/genética , Proteína Forkhead Box O3/biossíntese , Proteína Forkhead Box O3/genética , Genes bcl-2/genética , Camundongos , MicroRNAs/biossíntese , Cultura Primária de Células , RNA Longo não Codificante/genética , Regulação para Cima/genética , Proteína X Associada a bcl-2/genéticaRESUMO
BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Lapatinib/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteína Forkhead Box O3/biossíntese , Expressão Gênica/efeitos dos fármacos , Genes erbB-2 , Humanos , Lapatinib/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêuticoRESUMO
The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body ß-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.
Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilidade/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Oócitos/metabolismo , Ácido 3-Hidroxibutírico/biossíntese , Ácido 3-Hidroxibutírico/genética , Acetilação , Animais , Blastocisto/citologia , Bovinos , Linhagem Celular , Células do Cúmulo/metabolismo , Feminino , Proteína Forkhead Box O3/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Técnicas de Transferência Nuclear , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/biossíntese , GravidezRESUMO
PURPOSE: Osteosarcoma is a common primary malignant bone tumour, and its cure rate has stagnated over the past 25-30 years. Brazilin, a purified natural product from sappan wood (Caesalpinia sappan L.), has been proved to possess potent anti-cancer effects. In this study, we investigated the anti-cancer effect of brazilin on human osteosarcoma and elucidated the underlying mechanisms. METHODS: We exposed MG-63 cells to different concentrations of brazilin (5, 10 and 20 µM) for 24 h. Western blotting, immunocytofluorescence, luciferase reporter assays, and RT-PCR were used to evaluate whether brazilin activates FOXO family-dependent autophagy. RESULTS: Brazilin increased autophagic flux in the human osteosarcoma cell line MG-63, as evidenced by the upregulation of LC3-II and the downregulation of P62/SQSTM1. Moreover, the pharmacological or genetic blockade of autophagy decreased brazilin-induced cell death, indicating that brazilin triggered autophagic cell death in MG-63 cells. Specifically, brazilin induced FOXO3A(Ser7) phosphorylation, activated FOXO3A nuclear translocation and increased FOXO3A reporter activity, which contributed to the expression of autophagy-related genes and subsequently initiated autophagic cell death in MG-63 cells. Importantly, the increased expression and nuclear translocation of FOXO3A were tightly related to the disturbance of calcium homeostasis, which could be prevented by chelating intracellular calcium. CONCLUSIONS: Taken together, these data demonstrate that brazilin induces osteosarcoma cell death by inducing excessive autophagy, which is mediated through the Ca2+-FOXO3A pathway. Our study provides a new anti-tumour mechanism for brazilin treatment in osteosarcoma patients.
Assuntos
Benzopiranos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Sinalização do Cálcio/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Osteossarcoma/tratamento farmacológico , Autofagia/efeitos dos fármacos , Benzopiranos/antagonistas & inibidores , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Proteína Forkhead Box O3/biossíntese , Proteína Forkhead Box O3/genética , Inativação Gênica , Humanos , Imuno-Histoquímica , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosforilação/efeitos dos fármacosRESUMO
OBJECTIVE: Colorectal cancer is a common malignant tumor of the digestive tract. It frequently occurs at the junction of the rectum and sigmoid colon. It is characterized by high mortality and poor prognosis. Bcl-2 interacting mediator of cell death (BIM) plays a role in the regulation of cell proliferation and apoptosis, and involves in the pathogenesis of colorectal cancer. The transcription factor forkhead, transcription factor O subfamily 3a (FoxO3a) plays a role in the regulation of BIM expression and is associated to the pathogenesis of colorectal cancer. Bioinformatics analysis suggests that there is a targeted relationship between FoxO3a and microRNA-223 (miR-223). This study aims to investigate effects of miR-223 on the regulation of FoxO3a/BIM signaling pathway and colorectal cancer cell proliferation and apoptosis. MATERIALS AND METHODS: Colorectal cancer cell line SW620 and normal colorectal epithelial cell line NCM460 were cultured in vitro. Dual luciferase reporter assay was used to validate the relationship between miR-223 and FoxO3a. Flow cytometry was adopted to detect apoptosis. EdU staining was applied to test cell proliferation. Western blot was selected to determine FoxO3a and BIM protein expressions. RESULTS: There was targeted regulatory relationship between miR-223 and FoxO3a. MiRa-223 up-regulated, FoxO3a and BIM expressions reduced, and cell proliferation was enhanced in SW620 cells compared with NCM460 cells. MiR-223 inhibitor or pIRES2-FoxO3a transfection significantly increased FoxO3a and BIM expressions, attenuated cell proliferation, and enhanced cell apoptosis. CONCLUSIONS: MiR-223 targeted inhibited expression of FoxO3. Down-regulating the expression of miR-223, it increased the expressions of FoxO3a and BIM, weakened SW620 cells proliferation and induced apoptosis.
Assuntos
Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2/biossíntese , Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , Proteína Forkhead Box O3/biossíntese , MicroRNAs/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Proteína Forkhead Box O3/genética , Células HEK293 , Humanos , MicroRNAs/genéticaRESUMO
The HSF and FOXO families of transcription factors play evolutionarily conserved roles in stress resistance and lifespan. In humans, the rs2802292 G-allele at FOXO3 locus has been associated with longevity in all human populations tested; moreover, its copy number correlated with reduced frequency of age-related diseases in centenarians. At the molecular level, the intronic rs2802292 G-allele correlated with increased expression of FOXO3, suggesting that FOXO3 intron 2 may represent a regulatory region. Here we show that the 90-bp sequence around the intronic single nucleotide polymorphism rs2802292 has enhancer functions, and that the rs2802292 G-allele creates a novel HSE binding site for HSF1, which induces FOXO3 expression in response to diverse stress stimuli. At the molecular level, HSF1 mediates the occurrence of a promoter-enhancer interaction at FOXO3 locus involving the 5'UTR and the rs2802292 region. These data were confirmed in various cellular models including human HAP1 isogenic cell lines (G/T). Our functional studies highlighted the importance of the HSF1-FOXO3-SOD2/CAT/GADD45A cascade in cellular stress response and survival by promoting ROS detoxification, redox balance and DNA repair. Our findings suggest the existence of an HSF1-FOXO3 axis in human cells that could be involved in stress response pathways functionally regulating lifespan and disease susceptibility.