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1.
Anticancer Res ; 39(11): 6241-6247, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704853

RESUMO

BACKGROUND/AIM: We performed multimodality therapy comprising preoperative chemotherapy, extrapleural pneumonectomy (EPP), and radiation therapy for patients with malignant pleural mesothelioma (MPM). Although multimodality therapy resulted in good prognosis, further improvement is required. Therefore, herein, we analysed the prognostic factors using surgical specimens and searched for suitable molecular targets to improve the prognosis after multidisciplinary treatment. PATIENTS AND METHODS: Forty-six patients with MPM underwent multimodality therapy. Paraffin-embedded surgical samples were used for immunohistochemistry to evaluate the expression of phosphorylated (p-) AKT, extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and S6 ribosomal protein (S6RP). RESULTS: On univariate and multivariate analyses, significant differences were observed according to the histological type, pathological stage, and p-mTOR expression rate. CONCLUSION: The prognosis of MPM is affected by p-mTOR expression, suggesting that molecular-targeted treatment might be used during multimodal therapy for MPM.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Pulmonares/enzimologia , Mesotelioma/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/análise , Neoplasias Pleurais/enzimologia , Serina-Treonina Quinases TOR/análise , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Cisplatino/administração & dosagem , Terapia Combinada/métodos , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Masculino , Mesotelioma/mortalidade , Mesotelioma/terapia , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Terapia de Alvo Molecular , Pemetrexede/administração & dosagem , Neoplasias Pleurais/mortalidade , Neoplasias Pleurais/terapia , Pneumonectomia , Prognóstico , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo
2.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3429-3434, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602905

RESUMO

The aim of this paper was to observe the concentration,time and mechanism of autophagy induced by triptolide( TP) in ovarian granulosa cells( OGCs). CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat OGCs and IC50 was calculated. The effects of TP at different concentrations and time points on the expression of OGCs autophagy factor protein and the cascade of PI3 K/AKT/m TOR pathway were detected by Western blot. The effects of TP,autophagy inducer( brefeldin A) and PI3 K/m TOR inhibitor( NVP-BEZ235) on the expression of PI3 K/AKT/m TOR cascade and autophagy related factor protein were detected by Western blot. The results show that the IC50 of different concentrations of TP on OGCs of rat ovary was14. 65 µmol·L-1,and the minimum inhibitory concentration of TP was 0. 1 µmol·L-1( 100 nmol·L-1). Compared with the control group,the expression levels of beclin1 and LC3Ⅱ in each group were significantly higher than those in the control group( P<0. 05 or P<0. 01). After 12 hours of treatment with TP,brefeldin A and NVP-BEZ235,respectively,compared with the control group,TP could significantly promote the expression level of downstream autophagy effect or molecule beclin1,LC3Ⅱ and inhibit the expression level of LC3Ⅰ,p62 protein( P<0. 05 or P< 0. 01). Moreover,the expression of beclin1 and LC3Ⅱ/LC3Ⅰ in TP group was higher than that in brefeldin A group( P<0. 05 or P<0. 01),and the expression of p62 in TP group was lower than that in brefeldin A group( P<0. 05 or P<0. 01). At the same time,TP could significantly inhibit the expression of p-PI3 K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group. This study suggests that 100 nmol·L-1 TP could induce OGCs autophagy successfully in cultured rat ovary for 12 h; TP may induce OGCs autophagy by inhibiting PI3 k/Akt/m TOR signaling pathway.


Assuntos
Autofagia , Diterpenos/farmacologia , Células da Granulosa/efeitos dos fármacos , Fenantrenos/farmacologia , Transdução de Sinais , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Compostos de Epóxi/farmacologia , Feminino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo
3.
Chem Biol Interact ; 313: 108818, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494106

RESUMO

Diabetic nephropathy (DN) is a common complication of diabetes that remains the major cause of end-stage renal disease (ESRD). Forkhead box P1 (FOXP1) is a member of FOX family involved in the progression of diabetes. However, the pathogenic role of FOXP1 in DN remains unclear. This study was aimed to explore the effects of FOXP1 on glomerular mesangial cells (MCs) in response to high glucose (HG) stimulation. We found that HG stimulation markedly inhibited the FOXP1 expression in MCs in dose-and time-dependent manner. CCK-8 assay proved that FOXP1 overexpression attenuated HG-induced cell proliferation in MCs. FOXP1 exhibited anti-oxidative activity in HG-induced MCs, as proved by the decreased production of ROS and expressions of ROS producing enzymes, NADPH oxidase (NOX) 2 and NOX4. Besides, FOXP1 suppressed the expression and secretion of extracellular matrix (ECM) proteins including collagen IV (Col IV) and fibronectin (FN). Furthermore, FOXP1 overexpression significantly prevented HG-induced activation of Akt/mTOR signaling in MCs, and Akt activator blocked FOXP1-mediated cell proliferation, ROS production and ECM accumulation in MCs. Collectively, FOXP1 prevented HG-induced proliferation, oxidative stress, and ECM accumulation in MCs via inhibiting the activation of Akt/mTOR signaling pathway. The findings suggested that FOXP1 might be a therapeutic target for the treatment of DN.


Assuntos
Matriz Extracelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Células Mesangiais/metabolismo , Proteínas Repressoras/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/metabolismo , Fatores de Transcrição Forkhead/genética , Glucose/administração & dosagem , Glucose/farmacologia , Humanos , Células Mesangiais/efeitos dos fármacos , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo
4.
Int J Nanomedicine ; 14: 6249-6268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496684

RESUMO

Purpose: To develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel. Methods: Rap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement. Results: R-CL and R-FL were successfully prepared, at approximately <160 nm, 42% entrapment efficiency, and 57 µg/mg drug loading. FL cellular uptake was enhanced over 2-fold than that of CL; folate receptor-mediated endocytosis was confirmed using a competitive assay with folic acid pretreatment. In vitro cytotoxic effects increased dose-dependently. Rap-loaded liposomes inhibited mTOR signaling and induced autophagy in urothelial carcinoma cells. With gelation time of <30 seconds and gel duration of >12 hrs, both R-CL/P407 and R-FL/P407 preparations transformed into gel immediately after instillation into the mouse bladder. Drug release from the liposomal gel was erosion controlled. In orthotopic bladder cancer mouse model, statistically significant differences in ROI values were found between R-CL/P407 and R-FL/P407 groups at day 11 (P=0.0273) and day 14 (P=0.0088), indicating the highest tumor growth inhibition by R-FL/P407. Conclusion: Intravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.


Assuntos
Ácido Fólico/química , Hidrogéis/química , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Temperatura Ambiente , Administração Intravesical , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Coloides , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Feminino , Receptores de Folato com Âncoras de GPI/metabolismo , Humanos , Lipossomos , Camundongos , Tamanho da Partícula , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico
5.
J Agric Food Chem ; 67(41): 11364-11372, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31542934

RESUMO

The extensive use of pesticide caused an amount of pressure on the environment and increased the potential human health risk. Glyphosate-based herbicide (GBH) is one of the most widely used pesticides based on a 5-enolpyruvylshikimate-3-phosphate synthase target, which does not exist in vertebrates. Here, we study autophagic effects of the most famous commercial GBH Roundup (RDP) on human A549 cells in vitro. Intracellular biochemical assay indicated opening of mitochondrial permeability transition pore, LC3-II conversion, up-regulation of beclin-1, down-regulation of p62, and the changes in the phosphorylation of AMPK and mTOR induced by RDP in A549 cells. Further experimental results indicated that all the effects induced by RDP were related to its adjuvant polyethoxylated tallow amine, not its herbicidal active ingredient glyphosate isopropylamine salt. All these results showed that RDP has the ability to induce AMPK/mTOR-mediated cell autophagy in human A549 cells. This study would provide a theoretical basis for understanding RDP's autophagic effects on human A549 cells and attract attention on the potential human health risks induced by the adjuvant.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Glicina/análogos & derivados , Herbicidas/toxicidade , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Proteínas Quinases Ativadas por AMP/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Linhagem Celular , Glicina/toxicidade , Humanos , Serina-Treonina Quinases TOR/genética
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 619-624, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537247

RESUMO

Objective To investigate the effects of Astragalus polysaccharide (APS) on autophagy and expression of microtubule-associated protein 1 light chain 3B (LC3B), mammalian target of rapamycin (mTOR) and beclin1 in xanthine oxidase (XOD)-induced autophagic model of non-small cell lung cancer A549 cells. Methods A549 cells were divided into five groups: control group, model group, 100, 200 and 400 µg/mL APS-treated group. Except for control group, all groups were administered XOD for 24 hours to establish autophagic models. Morphology of autophagosome was detected by transmission electron microscopy (TEM) and the number was counted by monodansylcadaverine (MDC) staining. The expression levels of LC3B, beclin1 and mTOR were detected by Western blot analysis. Results Compared with the control group, the number of autophagosome in the model group increased; the expression of LC3B and beclin1 significantly increased; while the expression of mTOR significantly decreased. Compared with the model group, the number of autophagosome decreased remarkably; the expression of LC3B and beclin1 severely decreased, and the expression of mTOR obviously increased in 200 or 400 µg/mL APS-treated group. Conclusion APS reduces the level of autophagy, down-regulates the expression of LC3B and beclin1, and increases mTOR expression in the autophagic model of A549 cells induced by XOD.


Assuntos
Astrágalo (Planta)/química , Autofagia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Polissacarídeos/farmacologia , Células A549 , Proteínas Relacionadas à Autofagia , Proteína Beclina-1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Xantina Oxidase
7.
J Agric Food Chem ; 67(37): 10513-10520, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31475823

RESUMO

Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected ß-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of SREBP-1c gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.


Assuntos
Aminoácidos/metabolismo , Bovinos/metabolismo , Células Epiteliais/metabolismo , Gorduras/metabolismo , Glândulas Mamárias Animais/citologia , Leite/metabolismo , Fatores de Transcrição/metabolismo , Animais , Bovinos/genética , Feminino , Glândulas Mamárias Animais/metabolismo , Camundongos , Fosforilação , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética
8.
Life Sci ; 235: 116824, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476305

RESUMO

AIMS: Leflunomide is a disease modifying anti-rheumatic drug (DMARD) beneficial in refractory cases of rheumatoid arthritis. Since leflunomide approval, hepatotoxicity and instructions of liver function monitoring have been recommended. The current work aimed to explore the possible role of inflammation in leflunomide-induced hepatotoxicity with a focus on the TLR4-mediated stimulation of PI3K/mTOR/NFκB pathway. MAIN METHODS: Forty-eight male albino mice were allocated into four different groups (n; 12 mice/group). Group (i): normal mice, Group (ii-iv) mice received escalating dosed/s of leflunomide (2.5, 5 or 10 mg/kg, p.o.) every 48 h for eight weeks. At the end of the study, mice were sacrificed, and blood samples were collected for determination of liver enzymes. Liver samples were collected; (1) formalin-fixed for histologic examination, (2) frozen for PI3K and mTOR genes PCR assays. KEY FINDINGS: Results indicated a significant elevation of liver enzymes in leflunomide-treated mice (10 mg/kg); AST and ALT activities were 218.17 ±â€¯6.83 U/L and 99.83 ±â€¯9.82 U/L versus 130.5 ±â€¯12.79 U/L and 44.72 ±â€¯3.58 U/L in the vehicle group. Additionally, histopathological examination revealed higher necro-inflammatory scores in leflunomide-treated mice. Immunohistochemistry indicated dose-dependent increased staining of TLR4 and caspase 3. Furthermore, leflunomide-treated mice (5 or 10 mg/kg) showed greater staining for NFκB compared to vehicle control. RT-PCR results revealed upregulations in genes expressing PI3K and mTOR by leflunomide. SIGNIFICANCE: The current study highlights the possible role of TLR4-PI3K/mTOR/NFκB in the pathogenesis of leflunomide-induced hepatic injury. A better understanding of mechanisms of leflunomide-induced hepatotoxicity may be of translational implication for the predictive, preventive and therapeutic purposes.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Leflunomida/efeitos adversos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Caspase 3/metabolismo , Relação Dose-Resposta a Droga , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
9.
Ecotoxicol Environ Saf ; 183: 109480, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31382115

RESUMO

Spinetoram is one of the most extensively used bio-pesticide in the world. The effects of pesticide in human health are mainly caused by its residue in food. The liver is the direct target of pesticides exposure, however the study of cytotoxicity on human liver cells caused by spinetoram remains unclear. The aim of the present study was to evaluate the cytotoxic effects of the spinetoram in human liver cells in vitro. We demonstrated that spinetoram could inhibit the proliferation of human liver HepG2 cells and induce the oxidative DNA damage. Intracellular biochemical assay indicated that decrease of mitochondrial membrane potential, LC3-II conversion, accumulation of Beclin-1, degradation of p62 and the changes in the phosphorylation of AMPK, mTOR are contributed to the toxic effects of Spinetoram on HepG2 cells. These results showed that the cytotoxicity of spinetoram may be associated with the activity of AMPK/mTOR-mediated autophagy pathway. Meanwhile, the generation of 8-oxodG caused by the spinetoram suggested it has a potential genotoxic effect on human liver cells. We conclude that spinetoram has a significant cytotoxic effect by inducing AMPK/mTOR-mediated autophagy and oxidative DNA damage. This study would provide a theoretical basis for understanding its mechanisms of toxicity and supply an indication for recognizing the safety of spinetoram to human beings.


Assuntos
Autofagia/efeitos dos fármacos , Dano ao DNA , Inseticidas/toxicidade , Macrolídeos/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Proliferação de Células/efeitos dos fármacos , Citotoxinas/toxicidade , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo , Fosforilação/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
10.
Biol Res ; 52(1): 44, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426858

RESUMO

BACKGROUND: Free fatty acid receptor 1 (FFAR1) is G-protein coupled receptor predominantly expressed in pancreatic ß-cells that is activated by a variety of free fatty acids (FFAs). Once activated, it promotes glucose-stimulated insulin secretion (GSIS). However, increased levels of FFAs lead to lipotoxicity, inducing loss of ß-cell function. FFAR1 plays a key role in the development of type 2 diabetes (T2D), and previous studies have indicated the importance of developing anti-diabetic therapies against FFAR1, although its role in the regulation of ß-cell function remains unclear. The present study investigated the role of FFAR1 under lipotoxic conditions using palmitic acid (PA). The rat insulinoma 1 clone 832/13 (INS-1 832/13) cell line was used as a model as it physiologically resembles native pancreatic ß-cells. Key players of the insulin signaling pathway, such as mTOR, Akt, IRS-1, and the insulin receptor (INSR1ß), were selected as candidates to be analyzed under lipotoxic conditions. RESULTS: We revealed that PA-induced lipotoxicity affected GSIS in INS-1 cells and negatively modulated the activity of both IRS-1 and Akt. Reduced phosphorylation of both IRS-1 S636/639 and Akt S473 was observed, in addition to decreased expression of both INSR1ß and FFAR1. Moreover, transient knockdown of FFAR1 led to a reduction in IRS-1 mRNA expression and an increase in INSR1ß mRNA. Finally, PA affected localization of FFAR1 from the cytoplasm to the perinucleus. CONCLUSIONS: In conclusion, our study suggests a novel regulatory involvement of FFAR1 in crosstalk with mTOR-Akt and IRS-1 signaling in ß-cells under lipotoxic conditions.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Linhagem Celular , Células Secretoras de Insulina/metabolismo , Ratos , Transdução de Sinais
11.
Adv Exp Med Biol ; 1152: 283-292, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456190

RESUMO

Based on the insights gleaned from decades of research, it seems clear that mechanistic target of rapamycin (mTOR) is an essential signaling node that integrates environmental clues for regulation of cell survival, metabolism and proliferation of the cells. However, overwhelmingly increasing scientific evidence has added a new layer of intricacy to already complicated and versatile signaling pathway of mTOR. Deregulation of spatio-temporally controlled mTOR-driven pathway played contributory role in breast cancer development and progression. Pharmacologists and molecular biologists have specifically emphasized on the identification and development of mTOR-pathway inhibitors. In this chapter we have attempted to provide an overview of the most recent findings related to therapeutic targeting of mTOR-associated mTORC1 and mTORC2 in breast cancer. We have also comprehensively summarized regulation of mTOR and its partners by microRNAs in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Feminino , Humanos , MicroRNAs/genética , Terapia de Alvo Molecular , Metástase Neoplásica
12.
Chem Biol Interact ; 311: 108793, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421117

RESUMO

Polyphyllin I (PPI), a bioactive component extracted from Paris polyphylla, was reported to have potent anticancer activities in previous studies. However, there were few reports on the effects and underlying mechanism of PPI in human acute myeloid leukemia cells. The present study demonstrated that PPI had an inhibitory effect through inducing apoptosis and autophagy in THP-1 and NB4 cells. PPI induced apoptosis via activating JNK pathway, as evidenced by the decreased Bcl-2 levels and increased Bax, cleaved-caspase-3 and phosphorylated-JNK expressions. In addition, PPI promoted autophagy as evidenced with increased expressions of LC3-II and Beclin-1 in western blot and autophagic vacuoles in MDC staining, which was associated with the inhibition of AKT-mTOR pathway. Furthermore, JNK inhibitor SP600125 and autophagy inhibitor 3-MA were employed to evaluate the role of apoptosis and autophagy in PPI-induced cell death. We found that autophagy and apoptosis were both causes of cell death induced by PPI. These data suggested that PPI could be a potent therapeutic agent for the treatment of human acute myeloid leukemia.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diosgenina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Diosgenina/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Life Sci ; 233: 116748, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31412263

RESUMO

AIMS: Resveratrol is a polyphenolic compound that has received much attention for its use in ameliorating various systemic pathological conditions. The present study was performed to investigate whether the resveratrol alleviated cardiac hypertrophy and functional remodelling by regulating autophagy. MATERIALS AND METHODS: Male rats were exposed to CIH 8 h/day for five weeks and/or intragastric administration of resveratrol daily. The morphological and echocardiography were used to evaluate the cardiac protective effects. The apoptosis was detected by TUNEL staining. The biochemical assessments were used to evaluate oxidative stress. Further, the effect of resveratrol on autophagy and PI3K/AKT/mTOR pathway was investigated. KEY FINDINGS: The CIH group exhibited increased heart weight/body weight and left ventricle weight/body weight ratios, which was accompanied by left ventricular remodelling. Echocardiography analysis showed that CIH-treated rats had significantly higher left ventricular posterior wall thickness, ejection fraction and fractional shortening than those of controls. In addition, the apoptosis index and oxidative markers were significantly elevated in the CIH group versus the control. The autophagy marker Beclin-1 was elevated, while p62 was decreased by CIH treatment. Resveratrol treatment significantly improved cardiac function and alleviated cardiac hypertrophy, oxidative stress, and apoptosis in CIH rats. Further results indicated that PI3K/AKT pathway-mediated inhibition of the mammalian target of rapamycin (mTOR) pathway played a role in the activation of autophagy by resveratrol after CIH stimulation. SIGNIFICANCE: In conclusion, resveratrol supplementation during CIH upregulates autophagy by targeting the PI3K/AKT/mTOR pathway, which appears to be beneficial for resisting cardiac hypertrophy.


Assuntos
Cardiomegalia/prevenção & controle , Regulação da Expressão Gênica/efeitos dos fármacos , Hipóxia/complicações , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Resveratrol/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Apoptose , Autofagia , Cardiomegalia/etiologia , Cardiomegalia/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
14.
Adv Exp Med Biol ; 1155: 923-934, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468457

RESUMO

Diabetic neuropathy (DN) is the most common chronic complication of DM and its major pathological changes show axonal dysfunction, atrophy and loss. However, there are few reports that taurine promotes neurite growth of dorsal root ganglion (DRG) cells. In current study, DRG neurons were exposed to high glucose (HG) with or without taurine. The neurite outgrowth of DRG neurons was observed by fluorescent immunohistochemistry method. Expression of Gap-43, Akt, phosphorylated Akt, mTOR and phosphorylated mTOR was determined by Western blot assay. Our results showed that HG significantly decreased the neurite outgrowth and expression of Gap-43 in DRG neurons. Moreover, phosphorylated levels of Akt and mTOR were downregulated in DRG neurons exposed to HG. On the contrary, taurine supplementation significantly reversed the decreased neurite outgrowth and Gap-43 expression, and the downregulated phosphorylated levels of Akt and mTOR. However, the protective effects of taurine were blocked in the presence of PI3K antagonists LY294002 or Akt antagonists Perifosine. These results indicate that taurine promotes neurite outgrowth of DRG neurons exposed to HG via activating Akt/mTOR signal pathway.


Assuntos
Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Taurina/farmacologia , Células Cultivadas , Proteína GAP-43/metabolismo , Glucose , Humanos , Neuritos/efeitos dos fármacos , Neurônios/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
15.
Nat Cell Biol ; 21(9): 1093-1101, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451768

RESUMO

Mechanistic target of rapamycin (mTOR) kinase functions in two multiprotein complexes: lysosomal mTOR complex 1 (mTORC1) and mTORC2 at the plasma membrane. mTORC1 modulates the cell response to growth factors and nutrients by increasing protein synthesis and cell growth, and repressing the autophagy-lysosomal pathway1-4; however, dysfunction in mTORC1 is implicated in various diseases3,5,6. mTORC1 activity is regulated by phosphoinositide lipids7-10. Class I phosphatidylinositol-3-kinase (PI3K)-mediated production of phosphatidylinositol-3,4,5-trisphosphate6,11 at the plasma membrane stimulates mTORC1 signalling, while local synthesis of phosphatidylinositol-3,4-bisphosphate by starvation-induced recruitment of class II PI3K-ß (PI3KC2-ß) to lysosomes represses mTORC1 activity12. How the localization and activity of PI3KC2-ß are regulated by mitogens is unknown. We demonstrate that protein kinase N (PKN) facilitates mTORC1 signalling by repressing PI3KC2-ß-mediated phosphatidylinositol-3,4-bisphosphate synthesis downstream of mTORC2. Active PKN2 phosphorylates PI3KC2-ß to trigger PI3KC2-ß complex formation with inhibitory 14-3-3 proteins. Conversely, loss of PKN2 or inactivation of its target phosphorylation site in PI3KC2-ß represses nutrient signalling via mTORC1. These results uncover a mechanism that couples mTORC2-dependent activation of PKN2 to the regulation of mTORC1-mediated nutrient signalling by local lipid signals.


Assuntos
Lipídeos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/metabolismo , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
16.
Cell Physiol Biochem ; 53(2): 301-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31343125

RESUMO

BACKGROUND/AIMS: Propolis is one of the most promising natural products, exhibiting not only therapeutic but also prophylactic actions. Propolis has several biological and pharmacological properties, including hepatoprotective activities. The present study aimed to investigate the underlying molecular mechanisms of propolis against CCl4-mediated liver fibrosis. METHODS: Three groups of male BALB/c mice (n=15/ group) were used: group 1 comprised control mice; groups 2 and 3 were injected with CCl4 for the induction of liver fibrosis. Group 3 was then orally supplemented with propolis (100 mg/kg body weight) for four weeks. Different techniques were used to monitor the antifibrotic effects of propolis, including histopathological investigations using H&E, Masson's trichrome and Sirius red staining; Western blotting; flow cytometry; and ELISA. RESULTS: We found that the induction of liver fibrosis by CCl4 was associated with a significant increase in hepatic collagen and α-smooth muscle actin (α-SMA) expression. Moreover, CCl4-treated mice also exhibited histopathological alterations in the liver architecture. Additionally, the liver of CCl4-treated mice exhibited a marked increase in proinflammatory signals, such as increased expression of HSP70 and increased levels of proinflammatory cytokines and ROS. Mechanistically, the liver of CCl4-treated mice exhibited a significant increase in the phosphorylation of AKT and mTOR; upregulation of the expression of BAX and cytochrome C; downregulation of the expression of Bcl2; a significant elevation in the levels of TGF-ß followed by increased phosphorylation of SMAD2; and a marked increase in the expression of P53 and iNOS. Interestingly, oral supplementation of CCl4-treated mice with propolis significantly abolished hepatic collagen deposition, abrogated inflammatory signals and oxidative stress, restored CCl4-mediated alterations in the signaling cascades, and hence repaired the hepatic architecture nearly to the normal architecture observed in the control mice. CONCLUSION: Our findings revealed the therapeutic potential and the underlying mechanisms of propolis against liver fibrosis.


Assuntos
Apoptose/efeitos dos fármacos , Cirrose Hepática Experimental/patologia , Própole/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Tetracloreto de Carbono/toxicidade , Citocinas/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Smad2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
17.
Scand J Immunol ; 90(5): e12810, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31325389

RESUMO

MicroRNAs (miRNAs) play a vital role in the occurrence and development of many human diseases, including systemic lupus erythematosus (SLE). SLE is an autoimmune disease characterized by the production of autoantibodies against nuclear antigens and multiorgan involvement. Study of miRNAs involved in SLE provides new insights into the pathogenesis of SLE and might lead to the identification of new therapeutic interventions. The aim of this study was to investigate the effect of miR-183 injection on the progression of SLE by using MRL/lpr mouse model. The expression levels of miR-183 and mTOR mRNA were detected by quantitative real-time PCR assay. The effect of miR-183 on the course of spontaneous disease progression in the MRL/lpr mice was examined by intraperitoneal injection of miR-183 into mice and followed by monitoring lifespan, anti-dsDNA antibody levels, urinary albumin levels, blood urea nitrogen (BUN) levels, and Tregs and Th17 cell population. We found that miR-183 injection resulted in reduction of anti-DNA antibody and immune complex component levels, restoration of Tregs and Th17 cell population and prolongation of survival. Our findings suggest that miR-183 injection may serve as an effective therapeutic treatment for delaying or easing pathologic features of SLE.


Assuntos
Nefrite Lúpica/terapia , MicroRNAs/genética , MicroRNAs/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Adulto , Albuminúria/urina , Animais , Anticorpos Antinucleares/sangue , Nitrogênio da Ureia Sanguínea , Modelos Animais de Doenças , Feminino , Humanos , Nefrite Lúpica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/genética , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/metabolismo , Células Th17/imunologia
18.
Nat Commun ; 10(1): 2943, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270333

RESUMO

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Adenosina/metabolismo , Adenilato Quinase/metabolismo , Células-Tronco Adultas/ultraestrutura , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Autofagia , Transporte Biológico , Diferenciação Celular , Autorrenovação Celular , Metabolismo Energético , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fenótipo , Ribonucleotídeos/farmacologia , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo
19.
Chem Biol Interact ; 310: 108726, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31255635

RESUMO

Tetrandrine (TET) and cepharanthine (CEP) are two bisbenzylisoquinoline alkaloids isolated from the traditional herbs. Recent molecular investigations firmly supported that TET or CEP would be a potential candidate for cancer chemotherapy. Prognosis of patients with glucocorticoid resistant T cell acute lymphoblastic leukemia (T-ALL) remains poor; here we examined the anti-T-ALL effects of TET and CEP and the underlying mechanism by using the glucocorticoid resistant human leukemia Jurkat T cell line in vitro. TET and CEP significantly inhibited cell viabilities and induced apoptosis in dose- and time-dependent manner. Further investigations showed that TET or CEP not only upregulated the expression of initiator caspases such as caspase-8 and 9, but also increased the expression of effector caspases such as caspase-3 and 6. As the important markers of apoptosis, p53 and Bax were both upregulated by the treatment of TET and CEP. However, TET and CEP paradoxically increased the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, and activated the survival protein NF-κB, leading to high expression of p-NF-κB. Cell cycle arrest at S phase accompanied by increase in the amounts of cyclin A2 and cyclin B1, and decrease in cylcin D1 amount in cells treated with TET or CEP will be another possible mechanism. During the process of apoptosis in Jurkat T cells, treatment with TET or CEP also increased the phosphorylation of JNK and p38. The PI3K/Akt/mTOR signaling pathway modification appears to play significant role in the Jurkat T cell apoptosis induced by TET or CEP. Moreover, TET and CEP seemed to downregulate the expressions of p-PI3K and mTOR in an independent way from Akt, since these two drugs strongly stimulated the p-Akt expression. These results provide fundamental insights into the clinical application of TET or CEP for the treatment of patients with relapsed T-ALL.


Assuntos
Apoptose/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Benzilisoquinolinas/uso terapêutico , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Células Jurkat , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
20.
Nat Cell Biol ; 21(7): 889-899, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263264

RESUMO

The c-Myc oncogene drives malignant progression and induces robust anabolic and proliferative programmes leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. Here we reveal an essential role for activating transcription factor 4 (ATF4) in survival following MYC activation. MYC upregulates ATF4 by activating general control nonderepressible 2 (GCN2) kinase through uncharged transfer RNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC-target genes, primarily those regulating amino acid and protein synthesis, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation. 4E-BP1 relieves MYC-induced proteotoxic stress and is essential to balance protein synthesis. 4E-BP1 activity is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation and inhibition of mTORC1 signalling rescues ATF4-deficient cells from MYC-induced endoplasmic reticulum stress. Acute deletion of ATF4 significantly delays MYC-driven tumour progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC activity, which ensures that enhanced translation rates are compatible with survival and tumour progression.


Assuntos
Fator 4 Ativador da Transcrição/genética , Genes myc/genética , Ativação Transcricional/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Estresse do Retículo Endoplasmático/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Transgênicos , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas/fisiologia , Serina-Treonina Quinases TOR/metabolismo
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