RESUMO
Leucine is a branched-chain amino acid that is present in protein, and it is an essential factor in activating the mechanistic target of the rapamycin complex 1 signaling pathway and increasing muscle protein synthesis. However, the loss of digestive function after total gastrectomy leads to impaired protein absorption, potentially failing to stimulate muscle protein synthesis. Therefore, this study aimed to investigate whether muscle protein synthesis is enhanced by oral skim milk administration after total gastrectomy. Male Sprague Dawley rats were divided into total gastrectomy (TG) and sham surgery (S) groups. After five weeks postoperatively, we orally administered skim milk to achieve 3.1 g protein/kg body weight and collected blood and gastrocnemius muscle. The gastrocnemius muscle weight was significantly lower in the TG group than in the S group (p < 0.05). The increase in plasma leucine concentration was significantly lower in the TG group than in the S group (p < 0.05). The skeletal muscle protein synthesis and the phosphorylation of p70S6K and 4E-BP1 showed a similar increase in both groups. Even after TG, muscle protein synthesis was stimulated by consuming skim milk, accompanied by a sufficient rise in plasma leucine concentration.
Assuntos
Gastrectomia , Leucina , Leite , Proteínas Musculares , Músculo Esquelético , Ratos Sprague-Dawley , Animais , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Proteínas Musculares/biossíntese , Proteínas Musculares/metabolismo , Leucina/administração & dosagem , Leucina/farmacologia , Leite/química , Fosforilação , Ratos , Administração Oral , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Intrinsic resistance to targeted therapeutics in PTEN-deficient glioblastoma (GBM) is mediated by redundant signaling networks that sustain critical metabolic functions. Here, we demonstrate that coordinated inhibition of the ribosomal protein S6 kinase 1 (S6K1) and the receptor tyrosine kinase AXL using LY-2584702 and BMS-777607 can overcome network redundancy to reduce GBM tumor growth. This combination of S6K1 and AXL inhibition suppressed glucose flux to pyrimidine biosynthesis. Genetic inactivation studies to map the signaling network indicated that both S6K1 and S6K2 transmit growth signals in PTEN-deficient GBM. Kinome-wide ATP binding analysis in inhibitor-treated cells revealed that LY-2584702 directly inhibited S6K1, and substrate phosphorylation studies showed that BMS-777607 inactivation of upstream AXL collaborated to reduce S6K2-mediated signal transduction. Thus, combination targeting of S6K1 and AXL provides a kinase-directed therapeutic approach that circumvents signal transduction redundancy to interrupt metabolic function and reduce growth of PTEN-deficient GBM. SIGNIFICANCE: Therapy for glioblastoma would be advanced by incorporating molecularly targeted kinase-directed agents, similar to standard of care strategies in other tumor types. Here, we identify a kinase targeting approach to inhibit the metabolism and growth of glioblastoma.
Assuntos
Receptor Tirosina Quinase Axl , Glioblastoma , PTEN Fosfo-Hidrolase , Proteínas Proto-Oncogênicas , Pirimidinas , Receptores Proteína Tirosina Quinases , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Humanos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pirimidinas/farmacologia , Linhagem Celular Tumoral , Animais , Transdução de Sinais/efeitos dos fármacos , Camundongos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacos , Aminopiridinas , PiridonasRESUMO
BACKGROUND: Gain of function disturbances in nutrient sensing are likely the largest component in human age-related disease. Mammalian target of rapamycin complex 1 (mTORC1) activity affects health span and longevity. The drugs ketamine and rapamycin are effective against chronic pain and depression, and both affect mTORC1 activity. Our objective was to measure phosphorylated p70S6K, a marker for mTORC1 activity, in individuals with psychiatric disease to determine whether phosphorylated p70S6K could predict medication response. METHODS: Twenty-seven females provided blood samples in which p70S6K and phosphorylated p70S6K were analyzed. Chart review gathered biometric measurements, clinical phenotypes, and medication response. Questionnaires assessed anxiety, depression, autism traits, and mitochondrial dysfunction, to determine neuropsychiatric disease profiles. Univariate and multivariate statistical analyses were used to identify predictors of medication response. RESULTS: mTORC1 activity correlated highly with both classical biometrics (height, macrocephaly, pupil distance) and specific neuropsychiatric disease profiles (anxiety and autism). Across all cases, phosphorylated p70S6K was the best predictor for ketamine response, and also the best predictor for rapamycin response in a single instance. CONCLUSIONS: The data illustrate the importance of mTORC1 activity in both observable body structure and medication response. This report suggests that a simple assay may allow cost-effective prediction of medication response.
Assuntos
Ketamina , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Quinases S6 Ribossômicas 70-kDa , Humanos , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Ketamina/farmacologia , Adulto , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fosforilação , Transtornos Mentais/metabolismo , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Ansiedade/metabolismo , Adulto Jovem , IdosoRESUMO
The mutations in Caenorhabditis elegans (C. elegans) that extend lifespan slow down aging by interfering with several signaling pathways, including the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR). The tumor suppressor pRb (retinoblastoma protein) is believed to be involved in almost all human cancers. Lin-35, the C. elegans orthologue of the tumor suppressor pRb, was included in the study to explore the effects of insulin and IGF-1 because it has been linked to cancer-related pRb function in mammals and exhibits a tumor suppressor effect by inhibiting mTOR or IIS signaling. According to our results, IGF-1 or insulin increased the lifespan of lin-35 worms compared to N2 worms by increasing fertilization efficiency, also causing a significant increase in body size. It was concluded that the expression of daf-2 and rsks-1 decreased after insulin or IGF-1 administration, thus extending the lifespan of C. elegans lin-35 worms through both IIS and mTOR-dependent mechanisms. This suggests that it was mediated by the combined effect of the TOR and IIS pathways. These results, especially obtained in cancer-associated mutant lin-35 worms, will be useful in elucidating the C. elegans cancer model in the future.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Fator de Crescimento Insulin-Like I , Insulina , Longevidade , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genética , Longevidade/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genética , Receptor de Insulina/metabolismo , Receptor de Insulina/genética , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.
Assuntos
NF-kappa B , Neoplasias da Próstata , Tolerância a Radiação , Proteína Sequestossoma-1 , Transdução de Sinais , Masculino , Animais , Humanos , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/tratamento farmacológico , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Transdução de Sinais/efeitos dos fármacos , Camundongos , Tolerância a Radiação/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genéticaRESUMO
Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Colangiocarcinoma , Dasatinibe , Isocitrato Desidrogenase , Mutação , Quinases da Família src , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/tratamento farmacológico , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Dasatinibe/farmacologia , Isocitrato Desidrogenase/metabolismo , Isocitrato Desidrogenase/genética , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo , Quinases da Família src/antagonistas & inibidores , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismoRESUMO
A high-salt diet (HSD) has been associated with various health issues, including hypertension and cardiovascular diseases. However, recent studies have revealed a potential link between high salt intake and cognitive impairment. This study aims to investigate the effects of high salt intake on autophagy, tau protein hyperphosphorylation, and synaptic function and their potential associations with cognitive impairment. To explore these mechanisms, 8-month-old male C57BL/6 mice were fed either a normal diet (0.4% NaCl) or an HSD (8% NaCl) for 3 months, and Neuro-2a cells were incubated with normal medium or NaCl medium (80 mM). Behavioral tests revealed learning and memory deficits in mice fed the HSD. We further discovered that the HSD decreased autophagy, as indicated by diminished levels of the autophagy-associated proteins Beclin-1 and LC3, along with an elevated p62 protein level. HSD feeding significantly decreased insulin-like growth factor-1 receptor (IGF1R) expression in the brain of C57BL/6 mice and activated mechanistic target of rapamycin (mTOR) signaling. In addition, the HSD reduced synaptophysin and postsynaptic density protein 95 (PSD95) expression in the hippocampus and caused synaptic loss in mice. We also found amyloid ß accumulation and hyperphosphorylation of tau protein at different loci both in vivo and in vitro. Overall, this study highlights the clinical significance of understanding the impact of an HSD on cognitive function. By targeting the IGF1R/mTOR/p70S6K pathway or promoting autophagy, it may be possible to mitigate the negative effects of high salt intake on cognitive function.
Assuntos
Disfunção Cognitiva , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1 , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais , Cloreto de Sódio na Dieta , Serina-Treonina Quinases TOR , Animais , Masculino , Serina-Treonina Quinases TOR/metabolismo , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/etiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Cloreto de Sódio na Dieta/efeitos adversos , Receptor IGF Tipo 1/metabolismo , Proteínas tau/metabolismo , Autofagia/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacosRESUMO
Evidence is accumulating that osteal macrophages, in addition to bone-resorbing osteoclasts and bone-forming osteoblasts, participate vitally in bone remodeling process. Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodeling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We have reported that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in MC3T3-E1 osteoblast-like cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase are involved in the OPG synthesis. The present study aims to investigate the effect of OSM on the synthesis of OPG stimulated by BMP-4 in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells. Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK was affected by OSM. On the other hand, the phosphorylation of p70 S6 kinase stimulated by BMP-4 was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblast-like cells.
Assuntos
Proteína Morfogenética Óssea 4 , Oncostatina M , Osteoblastos , Osteoprotegerina , Proteínas Quinases S6 Ribossômicas 70-kDa , Animais , Camundongos , Oncostatina M/farmacologia , Oncostatina M/metabolismo , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/citologia , Osteoprotegerina/metabolismo , Osteoprotegerina/biossíntese , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Linhagem CelularRESUMO
Although recent studies increasingly suggest the potential anti-cancer effect of quercetin, the exact underlying mechanism remains poorly demonstrated in oral squamous cell carcinoma (oSCC). Therefore, our research explored the impacts of quercetin on the ferroptosis and mTOR/S6KP70 axis in oSCC cell lines. After treating oSCC cells with quercetin or indicated compounds and transfection with SLC7A11- or S6KP70-overexpressing plasmid, cell viability was detected by CCK-8 assay. The level of ferroptosis in oSCC cells was assessed by measuring ROS and GSH levels. The activation of mTOR/S6KP70 axis was assessed by Western blotting. Quercetin promoted ferroptosis in an mTOR/S6KP70-dependent manner to inhibit tumor growth in oSCC cells. Mechanistically, we revealed that quercetin induced lipid peroxidation and reduced GSH levels by repressing SLC7A11 expression in oSCC cells. Specifically, the effects of quercetin on ferroptosis and mTOR and S6KP70 phosphorylation were partially blocked by both mTOR agonist and S6KP70 overexpression. Moreover, mTOR inhibitor promoted ferroptosis in quercetin-treated oSCC cells. Our findings showed that ferroptosis may be a new anti-tumor mechanism of quercetin. Additionally, we identified that quercetin can target mTOR/S6KP70 cascade to inhibit the growth of oSCC cells.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Ferroptose , Neoplasias Bucais , Quercetina , Serina-Treonina Quinases TOR , Animais , Humanos , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Camundongos Nus , Neoplasias Bucais/patologia , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Quercetina/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of the two groups: supplementation with either 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were downregulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in the liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in the tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.
Soybean meal is the major source of dietary protein for growing pigs in many regions of the world, including North America, South America, and Asia. However, this conventional feedstuff is recognized to be severely deficient in glycine (the most abundant amino acid in the bodies of animals, including pigs). Compared to pigs with normal birth weights (NBW), pigs with intrauterine growth restriction (IUGR) have a lower ability to synthesize glycine and reduced growth performance between weaning and market weights. Results of recent studies with cultured muscle cells have revealed that glycine stimulates the mechanistic target of rapamycin (mTOR) cell signaling pathway (the master activator of initiation of protein synthesis in tissues) to promote protein synthesis and cell growth. There is also evidence that the mTOR pathway is impaired in the skeletal muscle of young IUGR pigs. Thus, dietary glycine supplementation may play an important role in maintaining an active mTOR cell signaling pathway for the growth of tissues, particularly skeletal muscle. Results of this study indicated that market-weight IUGR pigs had lower abundances of both total and phosphorylated mTOR, as well as its downstream target proteins in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum, when compared with NBW pigs. In contrast, neither IUGR nor glycine supplementation affected the mTOR cell signaling pathway in the liver of pigs. Taken together, these novel findings indicate that IUGR pigs have insufficient cell signaling via the mTOR cell pathway in a tissue-specific manner during their growing-finishing phases of development and that this negative impact of IUGR can be mitigated by supplementing corn- and soybean meal-based diets with 1% glycine.
Assuntos
Ração Animal , Dieta , Suplementos Nutricionais , Retardo do Crescimento Fetal , Glicina , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Retardo do Crescimento Fetal/veterinária , Suínos , Ração Animal/análise , Dieta/veterinária , Glicina/administração & dosagem , Glicina/farmacologia , Feminino , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Masculino , Doenças dos SuínosRESUMO
Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.
Assuntos
Fator de Iniciação 4A em Eucariotos , Alvo Mecanístico do Complexo 1 de Rapamicina , Biossíntese de Proteínas , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais , Ubiquitinação , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genéticaRESUMO
Glioma, particularly glioblastomas (GBM), is incurable brain tumor. The most targeted receptor tyrosine kinase (RTKs) drugs did not bring benefit to GBM patients. The mechanism of glioma growth continues to be explored to find more effective treatment. Here, we reported that Ser/Thr protein kinase YANK2 (yet another kinase 2) is upregulated in glioma tissues and promotes the growth and proliferation of glioma in vitro and in vivo. Further, we confirmed that oncogene Fyn directly activated YANK2 through phosphorylation its Y110, and Fyn-mediated YANK2 phosphorylation at Y110 site promotes glioma growth by increasing its stability. Finally, YANK2 was proved to be a novel upstream kinase of p70S6K and promotes glioma growth by directly phosphorylating p70S6K at T389. Taken together, we found a new mTOR-independent p70S6K activation pathway, Fyn-YANK2-p70S6K, which promotes glioma growth, and YANK2 is a potential oncogene and serves as a novel therapeutic target for glioma.
Assuntos
Proliferação de Células , Glioma , Proteínas Proto-Oncogênicas c-fyn , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Humanos , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Serina-Treonina Quinases TOR/metabolismo , Glioma/metabolismo , Glioma/patologia , Glioma/genética , Animais , Linhagem Celular Tumoral , Fosforilação , Carcinogênese/genética , Carcinogênese/metabolismo , Camundongos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Camundongos Nus , Regulação Neoplásica da Expressão GênicaRESUMO
The manifestations of tuberous sclerosis complex (TSC) in humans include epilepsy, autism spectrum disorders (ASD) and intellectual disability. Previous studies suggested the linkage of TSC to altered cerebral blood flow and metabolic dysfunction. We previously reported a significant elevation in cerebral blood flow in an animal model of TSC and autism of young Eker rats. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin could restore normal oxygen consumption and cerebral blood flow. In this study, we investigated whether inhibiting a component of the mTOR signaling pathway, p70 ribosomal S6 kinase (S6K1), would yield comparable effects. Control Long Evans and Eker rats were divided into vehicle and PF-4708671 (S6K1 inhibitor, 75 mg/kg for 1 h) treated groups. Cerebral regional blood flow (14C-iodoantipyrine) was determined in isoflurane anesthetized rats. We found significantly increased basal cortical (+ 32%) and hippocampal (+ 15%) blood flow in the Eker rats. PF-4708671 significantly lowered regional blood flow in the cortex and hippocampus of the Eker rats. PF-4708671 did not significantly lower blood flow in these regions in the control Long Evans rats. Phosphorylation of S6-Ser240/244 and Akt-Ser473 was moderately decreased in Eker rats but only the latter reached statistical significance upon PF-4708671 treatment. Our findings suggest that moderate inhibition of S6K1 with PF-4708671 helps to restore normal cortical blood flow in Eker rats and that this information might have therapeutic potential in tuberous sclerosis complex and autism.
Assuntos
Transtorno Autístico , Esclerose Tuberosa , Animais , Humanos , Ratos , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/metabolismo , Mamíferos/metabolismo , Fosforilação , Ratos Long-Evans , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/uso terapêutico , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Esclerose Tuberosa/tratamento farmacológico , Esclerose Tuberosa/metabolismoRESUMO
Dietary salt has been associated with cognitive impairment in mice, possibly related to damaged synapses and tau hyperphosphorylation. However, the mechanism underlying how dietary salt causes cognitive dysfunction remains unclear. In our study, either a high-salt (8%) or normal diet (0.5%) was used to feed C57BL/6 mice for three months, and N2a cells were cultured in normal medium, NaCl medium (80 mM), or NaCl (80 mM) + Liraglutide (200 nM) medium for 48 h. Cognitive function in mice was assessed using the Morris water maze and shuttle box test, while anxiety was evaluated by the open field test (OPT). Western blotting (WB), immunofluorescence, and immunohistochemistry were utilized to assess the level of Glucagon-like Peptide-1 receptor (GLP-1R) and mTOR/p70S6K pathway. Electron microscope and western blotting were used to evaluate synapse function and tau phosphorylation. Our findings revealed that a high salt diet (HSD) reduced the level of synaptophysin (SYP) and postsynaptic density 95 (PSD95), resulting in significant synaptic damage. Additionally, hyperphosphorylation of tau at different sites was detected. The C57BL/6 mice showed significant impairment in learning and memory function compared to the control group, but HSD did not cause anxiety in the mice. In addition, the level of GLP-1R and autophagy flux decreased in the HSD group, while the level of mTOR/p70S6K was upregulated. Furthermore, liraglutide reversed the autophagy inhibition of N2a treated with NaCl. In summary, our study demonstrates that dietary salt inhibits the GLP-1R/mTOR/p70S6K pathway to inhibit autophagy and induces synaptic dysfunction and tau hyperphosphorylation, eventually impairing cognitive dysfunction.
Assuntos
Disfunção Cognitiva , Liraglutida , Camundongos , Animais , Liraglutida/farmacologia , Cloreto de Sódio na Dieta/efeitos adversos , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Cloreto de Sódio/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Serina-Treonina Quinases TOR/metabolismo , CogniçãoRESUMO
Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.
Assuntos
Apoptose , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Valeriana/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17ß-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.
Assuntos
Proteínas Quinases Ativadas por AMP , Nefropatias Diabéticas , Rim , Animais , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/prevenção & controle , Feminino , Masculino , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Rim/metabolismo , Camundongos Knockout , Fosforilação , Estradiol/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Diabetes Mellitus Experimental/metabolismoRESUMO
ß-Hydroxy-ß-methylbutyrate (HMB) is a breakdown product of leucine, which promotes muscle growth. Although some studies indicate that HMB activates AKT and mTOR, others show activation of the downstream effectors, P70S6K and S6, independent of mTOR. Our aim was to study the metabolic effect of HMB around the circadian clock in order to determine more accurately the signaling pathway involved. C2C12 myotubes were treated with HMB and clock, metabolic and myogenic markers were measured around the clock. HMB-treated C2C12 myotubes showed no activation of AKT and mTOR, but did show activation of P70S6K and S6. Activation of P70S6K and S6 was also found when myotubes were treated with HMB combined with metformin, an indirect mTOR inhibitor, or rapamycin, a direct mTOR inhibitor. The activation of the P70S6K and S6 independent of AKT and mTOR, was accompanied by increased activation of phospholipase D2 (PLD). In addition, HMB led to high amplitude and advanced circadian rhythms. In conclusion, HMB induces myogenesis in C2C12 by activating P70S6K and S6 via PLD2, rather than AKT and mTOR, leading to high amplitude advanced rhythms.
Assuntos
Ritmo Circadiano , Fibras Musculares Esqueléticas , Fosfolipase D , Valeratos , Valeratos/farmacologia , Animais , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Camundongos , Fosfolipase D/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Linhagem Celular , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Desenvolvimento Muscular/efeitos dos fármacosRESUMO
The influence of SGLT-1 on perivascular preadipocytes (PVPACs) and vascular remodeling is not well understood. This study aimed to elucidate the role and mechanism of SGLT-1-mediated PVPACs bioactivity. PVPACs were cultured in vitro and applied ex vivo to the carotid arteries of mice using a lentivirus-based thermosensitive in situ gel (TISG). The groups were treated with Lv-SGLT1 (lentiviral vector, overexpression), Lv-siSGLT1 (RNA interference, knockdown), or specific signaling pathway inhibitors. Assays were conducted to assess changes in cell proliferation, apoptosis, glucose uptake, adipogenic differentiation, and vascular remodeling in the PVPACs. Protein expression was analyzed by Western blotting, immunocytochemistry, and/or immunohistochemistry. The methyl thiazolyl tetrazolium (MTT) assay and Hoechst 33342 staining indicated that SGLT-1 overexpression significantly promoted PVPACs proliferation and inhibited apoptosis in vitro. Conversely, SGLT-1 knockdown exerted the opposite effect. Oil Red O staining revealed that SGLT-1 overexpression facilitated adipogenic differentiation, while its inhibition mitigated these effects. 3H-labeled glucose uptake experiments demonstrated that SGLT-1 overexpression enhanced glucose uptake by PVPACs, whereas RNA interference-mediated SGLT-1 inhibition had no significant effect on glucose uptake. Moreover, RT-qPCR, Western blotting, and immunofluorescence analyses revealed that SGLT-1 overexpression upregulated FABP4 and VEGF-A levels and activated the Akt/mTOR/p70S6K signaling pathway, whereas SGLT-1 knockdown produced the opposite effects. In vivo studies corroborated these findings and indicated that SGLT-1 overexpression facilitated carotid artery remodeling. Our study demonstrates that SGLT-1 activation of the Akt/mTOR/p70S6K signaling pathway promotes PVPACs proliferation, adipogenesis, glucose uptake, glucolipid metabolism, and vascular remodeling.NEW & NOTEWORTHY SGLT-1 is expressed in PVPACs and can affect preadipocyte glucolipid metabolism and vascular remodeling. SGLT-1 promotes the biofunctions of PVPACs mediated by Akt/mTOR/p70S6K signaling pathway. Compared with caudal vein or intraperitoneal injection, the external application of lentivirus-based thermal gel around the carotid artery is an innovative attempt at vascular remodeling model, it may effectively avoid the transfection of lentiviral vector into the whole body of mice and the adverse effect on experimental results.
Assuntos
Adipócitos , Proliferação de Células , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais , Transportador 1 de Glucose-Sódio , Serina-Treonina Quinases TOR , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Camundongos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Adipócitos/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Transportador 1 de Glucose-Sódio/genética , Masculino , Adipogenia/fisiologia , Camundongos Endogâmicos C57BL , Remodelação Vascular , Células Cultivadas , Apoptose , Diferenciação Celular , Glucose/metabolismo , Glucose/deficiênciaRESUMO
This study aimed to elucidate the anti-colon cancer mechanism of ginsenoside Rg1 in vitro and in vivo. Cell viability rate was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay. The inhibitory effect of ginsenoside Rg1 against CT26 cell proliferation gradually increased with increasing concentration. The in vivo experiments also demonstrated an antitumor effect. The monodansylcadaverine (MDC), transmission electron microscopy (TEM), and expression of autophagy marker proteins confirmed that ginsenoside Rg1 induced autophagy in vitro. Ginsenoside Rg1 induced autophagy death of CT26 cells, but this effect could be diminished by autophagy inhibitor (3-methyladenine, 3-MA). Additionally, in a xenograft model, immunohistochemical analysis of tumor tissues showed that the LC3 and Beclin-1 proteins were highly expressed in the tumors from the ginsenoside Rg1-treated nude mice, confirming that ginsenoside Rg1 also induced autophagy in vivo. Furthermoer, both in vivo and in vitro, the protein expressions of p-Akt, p-mTOR, and p-p70S6K were inhibited by ginsenoside Rg1, which was verified by Akt inhibitors. These results indicated that the mechanism of ginsenoside Rg1 against colon cancer was associated with autophagy through inhibition of the Akt/mTOR/p70S6K signaling pathway.
Assuntos
Autofagia , Neoplasias Colorretais , Ginsenosídeos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais , Serina-Treonina Quinases TOR , Ginsenosídeos/farmacologia , Autofagia/efeitos dos fármacos , Animais , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Camundongos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Proliferação de Células/efeitos dos fármacos , Humanos , Ensaios Antitumorais Modelo de Xenoenxerto , Sobrevivência Celular/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Antineoplásicos/farmacologiaRESUMO
This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. In vivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE-/- mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. In vitro, RAW 264.7 cells were exposed to 50 µg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE-/- mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.