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1.
Life Sci ; 248: 117474, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112869

RESUMO

BACKGROUND/OBJECTIVES: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipocytes. NNMT expression in adipose tissue is increased in obesity and diabetes. Knockdown of NNMT prevents mice from developing diet-induced obesity, which is closely linked to insulin resistance. An early sign of systemic insulin resistance is reduced expression of glucose transporter 4 (GLUT4) selectively in adipose tissue. Adipose tissue-specific knockout and overexpression of GLUT4 cause reciprocal changes in NNMT expression. The aim of the current study was to elucidate the mechanism that regulates NNMT expression in adipocytes. METHODS: 3T3-L1 adipocytes were cultured in media with varying glucose concentrations or activators and inhibitors of intracellular pathways. NNMT mRNA and protein levels were measured with quantitative polymerase chain reaction and Western blotting. RESULTS: Glucose deprivation of 3T3-L1 adipocytes induced a 2-fold increase in NNMT mRNA and protein expression. This effect was mimicked by inhibition of glucose transport with phloretin, and by inhibition of glycolysis with the phosphoglucose isomerase inhibitor 2-deoxyglucose. Conversely, inhibition of the pentose phosphate pathway did not affect NNMT expression. Pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) pathway caused an increase in NNMT levels that was similar to the effect of glucose deprivation. Activation of mTOR with MHY1485 prevented the effect of glucose deprivation on NNMT expression. Furthermore, upregulation of NNMT levels depended on functional autophagy and protein translation. CONCLUSION: Glucose availability regulates NNMT expression via an mTOR-dependent mechanism.


Assuntos
Adipócitos/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Glucose/farmacologia , Nicotinamida N-Metiltransferase/genética , Serina-Treonina Quinases TOR/genética , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Desoxiglucose/farmacologia , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Homeostase/genética , Camundongos , Morfolinas/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Floretina/farmacologia , Biossíntese de Proteínas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Triazinas/farmacologia
2.
Zhonghua Bing Li Xue Za Zhi ; 49(2): 156-161, 2020 Feb 08.
Artigo em Chinês | MEDLINE | ID: mdl-32074729

RESUMO

Objective: To evaluate the expression of p-AKT and p-mTOR, the key proteins in PI3K/AKT/mTOR pathway in pediatric Burkitt lymphoma (BL), and to investigate the clinical and prognostic significance. Methods: Fifty-eight cases of pediatric BL and thirty cases of reactive hyperplastic lymphadenitis (RH) were collected at Children's Hospital of Fudan University from September 2011 to July 2018. Paraffin sections of tissues were immune stained for p-AKT and p-mTOR, and the expression was assessed and correlated with the clinical features and prognosis. Results: A total of 58 cases were diagnosed and 6 cases lost the follow-up. Of the remaining 52 BL patients including 43 males and 9 females, the median age was 5 years (range: 2 to 14 years). Regarding to the correlation between the two biomarkers, Spearman test showed that p-mTOR was positively associated with the expression of p-AKT (r=0.759, P<0.001). Of all BL patients, the positive rates of p-AKT and p-mTOR were 62.1% (36/58) and 60.3%(35/58) respectively, both significantly higher than control group (P=0.011, P=0.035 respectively). The presence of p-AKT was significantly associated with higher lactate dehydrogenase (LDH≥573 IU/L) level in patients of the disease (P=0.006), while p-mTOR was increased both in the higher LDH and lower ratio of albumin to globulin (A/G) group (P=0.006, P=0.034 respectively). Expression of p-AKT and p-mTOR did not show any statistical correlation with sex, age, St.jude stage, tumor size, B-symptom present or not, number of extra-nodal sites or international prognostic index (IPI) (P>0.05). Fifty-two patients had a median follow-up of 40 months (range: 5-87 months). Univariate analysis showed that p-AKT expression was significant in predicting both inferior OS (5-year estimate, 72.7% vs. 94.7%, χ(2)=4.123, P=0.042) and PFS (5-year estimate, 66.7% vs. 94.7%, χ(2)=5.822, P=0.016). The 5-year OS rate was 71.0% (22/31) for the p-mTOR positive cohort of patients compared to 95.2% (17/21) for p-mTOR negative group (χ(2)=4.881, P=0.027); however, there was no statistical significance in 5-year PFS rate (P>0.05). Especially, the 5-year OS and PFS rate of p-AKT/p-mTOR double-positive group were significantly lower than negative control group (including absence of single p-AKT or p-mTOR expression, and absence of both) (OS: 69.0% vs. 95.7%, χ(2)=6.285, P=0.012; PFS: 65.5% vs. 91.3%, χ(2)=5.405, P=0.020). The results of multivariate COX proportional risk regression analysis indicated that p-AKT/p-mTOR double-positive, higher LDH and IPI score 3-5 were independent prognostic factors for both OS and PFS, and the bulky tumor (>10 cm) for PFS of pediatric BL. Conclusion: The expression of p-AKT and p-mTOR may be a potential reference for diagnosis and the independent prognostic indicators of pediatric BL.


Assuntos
Linfoma de Burkitt , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Fosfatidilinositol 3-Quinases , Prognóstico , Proteínas Proto-Oncogênicas c-akt , Estudos Retrospectivos , Serina-Treonina Quinases TOR
3.
Adv Exp Med Biol ; 1223: 69-80, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32030685

RESUMO

The mammalian target of rapamycin (mTOR) represents a critical hub for the regulation of different processes in both normal and tumor cells. Furthermore, it is now well established the role of mTOR in integrating and shaping different environmental paracrine and autocrine stimuli in tumor microenvironment (TME) constituents. Recently, further efforts have been employed to understand how the mTOR signal transduction mechanisms modulate the sensitivity and resistance to targeted therapies, also for its involvement of mTOR also in modulating angiogenesis and tumor immunity. Indeed, interest in mTOR targeting was increased to improve immune response against cancer and to develop new long-term efficacy strategies, as demonstrated by clinical success of mTOR and immune checkpoint inhibitor combinations. In this chapter, we will describe the role of mTOR in modulating TME elements and the implication in its targeting as a great promise in clinical trials.


Assuntos
Neoplasias/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/imunologia , Microambiente Tumoral/efeitos dos fármacos
4.
Crit Rev Oncol Hematol ; 147: 102886, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32014673

RESUMO

In several tumors the PI3K/AKT/mTOR pathway is frequently disrupted, an event that results in uncontrolled cell proliferation and tumor growth. Through the years, several compounds have been developed to inhibit the pathway at different steps: the mammalian target of rapamycin (mTOR) seemed to be the most qualified target. However, this kinase has such a key role in cell survival that mechanisms of resistance are rapidly developed. Nevertheless, clinical results obtained with mTOR inhibitors in breast cancer, renal cell carcinoma, neuroendocrine tumors and mantle cell lymphoma push oncologists to actively further develop these drugs, maybe by better selecting the population to which they are offered, through the research of predictive factors of responsiveness. In this review, we aim to describe mechanisms of resistance to mTOR inhibitors, from preclinical and clinical perspectives.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linfoma de Célula do Manto , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adulto , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Humanos , Inibidores de Proteínas Quinases
5.
Life Sci ; 245: 117368, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32001270

RESUMO

AIMS: Amino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs. MAIN METHODS: An in vivo model of I/R injury was tested in control, mTOR+/+, and mTOR+/-. Mice were randomly assigned to receive BCAAs, rapamycin, or BCAAs + rapamycin. Furthermore, isolated cardiomyocytes were subjected to simulated ischemia and cell death was quantified. Biochemical and mitochondrial swelling assays were also performed. KEY FINDINGS: Mice treated with BCAAs had a significant reduction in infarct size as a percentage of the area at risk compared to controls (34.1 ± 3.9% vs. 44.7 ± 2.6%, P = 0.001), whereas mice treated with the mTOR inhibitor rapamycin were not protected by BCAA administration (42.2 ± 6.5%, vs. control, P = 0.015). This protection was not detected in our hetero knockout mice of mTOR. Western blot analysis revealed no change in AKT signaling whereas activation of mTOR was identified. Furthermore, BCAAs prevented swelling which was reversed by the addition of rapamycin. In myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA-induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001). SIGNIFICANCE: BCAA treatment exhibits a protective effect in myocardial I/R injury and that mTOR plays an important role in this preconditioning effect.


Assuntos
Aminoácidos de Cadeia Ramificada/uso terapêutico , Cardiotônicos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Western Blotting , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Wistar , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo
6.
DNA Cell Biol ; 39(3): 368-378, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31971825

RESUMO

Nuclear protein 1 (NUPR1) is a stress-related small molecule and plays important roles in various tumors, including multiple myeloma (MM). Autophagy is essential for maintaining cellular homoeostasis in response to stress and, together with apoptosis, determines cell fate. Previous studies indicate that NUPR1 is involved in cancer progression of MM, but the underlying mechanisms have not been elucidated. In this study, we confirmed that NUPR1 and basal autophagy markers were highly expressed in the bone marrow of MM patients. The overexpression of NUPR1 was correlated with staging (both by Revised International Staging System [RISS] and Durie-Salmon [D-S] Staging System), levels of hemoglobin and calcium, and bone marrow plasma cell ratio in the MM patients. NUPR1 silencing reduced autophagy activities and induced apoptosis in U266 and RPMI 8226. We further observed a decrease in NUPR1 silencing-induced apoptosis in the presence of rapamycin, while an increase in apoptosis after chloroquine and 3-methyladenine treatment. Analysis of the mechanism indicated that PI3K/AKT/mTOR pathway was involved in autophagy-mediated apoptosis upon NUPR1 knockdown. In summary, our results demonstrate that NUPR1 silencing suppresses autophagy activities and induces autophagy-mediated apoptosis in MM cells through the PI3K/AKT/mTOR pathway, which exhibits potential as a treatment strategy for MM.


Assuntos
Apoptose , Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/genética , Transdução de Sinais , Adenina/análogos & derivados , Adenina/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Inativação Gênica , Humanos , Mieloma Múltiplo/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
7.
Life Sci ; 244: 117346, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978448

RESUMO

AIM: Emerging studies have shown that application of low concentration of bioactive phytomolecules can confer anti-proliferative effects on tumour cells by inducing senescence pathways. The alkaloid berberine is recognized for its anti-cancer attributes but its potential to induce senescence in tumour cells is least understood. MATERIALS AND METHODS: The present work assessed the mechanisms pertaining to dose-dependent anti-proliferative effects of berberine in the perspective of senescence and inflammation using human non-small cell lung cancer cell line (A549). KEY FINDINGS: Amongst the different tested bioactive phytomolecules, berberine treatment suppressed the proliferation of A549 cells regardless of the concentration applied. Application of low doses of berberine induced a weak SA-ß-gal activity and p21WAF1 expression but did not show evidence of SASP activation due to absence of NF-κB activation and expression of proinflammatory genes. However, treatment with higher dose of berberine showed no evidence of SA-ß-gal activity or p21WAF1 expression, but instead induced apoptosis and suppressed the expression of cell cyclins. The proliferative capacity of berberine treated cells was at par with control cells and no SA-ß-gal activity could be observed in first generation of berberine treated cells. mTOR pathway showed no distinct activation on account of berberine treatment thereby further emphasizing that low dose of berberine induced quiescence and not senescence in A549 cells. SIGNIFICANCE: Taken together, our observations indicate that despite its strong anti-proliferative effects, low dose berberine treatment may only induce transient changes akin to quiescence that needs to be considered before implying pro-senescence attributes of berberine in cancer therapeutics.


Assuntos
Apoptose/efeitos dos fármacos , Berberina/farmacologia , Senescência Celular/efeitos dos fármacos , Ciclinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Neoplasias Pulmonares/patologia , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Proliferação de Células/efeitos dos fármacos , Ciclinas/genética , Humanos , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética
8.
Chem Biol Interact ; 317: 108943, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31926917

RESUMO

Epidemiological studies have shown that cigarette smoking is beneficial in ulcerative colitis and that nicotine may be responsible for this effect. However, the mechanism remains unclear. In a previous study, nicotine was found to induce autophagy in intestinal cells. Here, we evaluated the effect of nicotine-induced autophagy in a dextran sodium sulfate (DSS)-induced colitis mouse model. C57BL/6 adult male mice drank DSS water solution freely for seven consecutive days, and then tap water was administered. The effect of nicotine treatment was examined in the DSS model, including colon length, disease severity, histology of the colon tissue, and inflammation levels. Moreover, autophagy levels were detected by Western blot analysis (LC3II/LC3I, p62, and beclin-1). The levels of DSS-induced colitis were significantly decreased following nicotine treatment. The disease activity score, body weight, histologic damage scores, and the level of colonic inflammatory factors of nicotine-treated mice all decreased compared to those of the control mice. Additionally, nicotine enhanced the expression of LC3II/LC3I and beclin-1 but decreased the p62 protein level. Inhibiting autophagy by 3-MA attenuated the protective effects of nicotine on colitis. Additionally, both in vitro and in vivo experiments showed changes in AMPK-mTOR-P70S6K during this process. These results suggest that nicotine improved colitis by regulating autophagy and provided a protective effect against DSS-induced colitis.


Assuntos
Adenilato Quinase/metabolismo , Autofagia/efeitos dos fármacos , Colite/prevenção & controle , Nicotina/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adenilato Quinase/genética , Animais , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética
9.
Cell Prolif ; 53(2): e12738, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31957155

RESUMO

OBJECTIVES: Podocyte injury is a prediction marker of diabetic nephropathy (DN), and AKT/mTOR pathway-mediated inhibition of autophagy is widely reported to contribute to podocyte damage. Recent study stated that sperm-associated antigen 5 (SPAG5) activated AKT/mTOR signalling in bladder urothelial carcinoma, indicating SPAG5 might regulate autophagy and play a role in podocyte damage. MATERIALS AND METHODS: Apoptosis and autophagy of human podocytes (HPCs) were detected by flow cytometry and immunofluorescence (IF). Gene level was assessed by Western blot and RT-qPCR. Molecular interactions were determined by pulldown, RNA immunoprecipitation (RIP), co-immunoprecipitation (co-IP), chromatin immunoprecipitation (ChIP) and luciferase reporter assays. RESULTS: SPAG5 mRNA and protein levels were upregulated under high glucose treatment in HPCs. Silencing SPAG5 reversed the increase of apoptosis and decrease of autophagy in high glucose-treated HPCs. Later, we found a long non-coding RNA (lncRNA) SPAG5 antisense RNA1 (SPAG5-AS1) as a neighbour gene to SPAG5. Mechanistically, YY1 transcriptionally upregulated SPAG5-AS1 and SPAG5 in high glucose-treated podocytes. SPAG5-AS1 acted as a competitive endogenous RNA (ceRNA) to regulate miR-769-5p/YY1 axis and induce SPAG5. SPAG5-AS1 interacted with ubiquitin-specific peptidase 14 (USP14) and leads to de-ubiquitination and stabilization of SPAG5 protein. CONCLUSIONS: This study revealed that SPAG5-AS1 inhibited autophagy and aggravated apoptosis of podocytes via SPAG5/AKT/mTOR pathway, indicating SPAG5-AS1/SPAG5 as a potential target for the alleviation of podocyte injury and offering new thoughts for the treatments of DN.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Células Cultivadas , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Glucose/metabolismo , Humanos , Podócitos/fisiologia , RNA Longo não Codificante/metabolismo , Transcrição Genética/fisiologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/fisiologia , Regulação para Cima/fisiologia
10.
Life Sci ; 243: 117271, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31926243

RESUMO

AIMS: 1,8-Cineole is a plant-derived monoterpene and a major constituent of Eucalyptus essential oil. Previously, we demonstrated that 1,8-cineole inhibited hepatocellular carcinoma (HCC) HepG2 cell growth. However, the underlying mechanisms remain unknown. Here, we evaluated the mechanisms of action of 1,8-cineole and the potential benefits of its combination with anticancer compounds harboring "anti-senescence" properties in HepG2 cells. MAIN METHODS: Cell viability was determined by the MTT assay. Cell cycle was assessed through flow cytometry (FC) and western blot (WB). Senescence was determined by the SA-ß-galactosidase assay, and apoptosis by caspase-3 activity, WB, and TUNEL. MAPKs (ERK, JNK, and p38), AMPK, and Akt/mTOR were analyzed by WB. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were evaluated by FC and fluorescence microscopy. KEY FINDINGS: 1,8-Cineole inhibited cell proliferation by promoting G0/G1 arrest. While 1,8-cineole was unable to trigger apoptosis, it induced cellular senescence. 1,8-Cineole promoted ROS production, ΔΨm depolarization, AMPK, ERK, and p38 activation and mTOR inhibition. Antioxidants, like N-acetyl-L-cysteine and vitamins, prevented HepG2 cell growth inhibition and senescence induced by 1,8-cineole. Pre-incubation with 1,8-cineole sensitized HepG2 cells to the anti-senescence compounds, quercetin, simvastatin, U0126, and SB202190. Combinations of 1,8-cineole and each compound synergistically inhibited cell viability, and combined treatment with 1,8-cineole and simvastatin induced apoptosis. SIGNIFICANCE: 1,8-Cineole induces G0/G1 arrest and senescence in HepG2 cells through oxidative stress and MAPK, AMPK, and Akt/mTOR pathways, and sensitizes cells to anti-senescence drugs, suggesting that 1,8-cineole has potential as an antineoplastic and adjuvant compound in combination with anti-senescence drugs in HCC therapy.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Eucaliptol/farmacologia , Fase G1/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática , Eucaliptol/administração & dosagem , Células Hep G2 , Humanos , Proteínas Quinases/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
11.
DNA Cell Biol ; 39(2): 310-320, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31971826

RESUMO

Renal vascular sclerosis caused by aging plays an important role in the occurrence and development of chronic kidney disease. Clinical studies have confirmed that endurance exercise is able to delay the aging of skeletal muscle and brain tissue. However, to date, few studies have assessed whether endurance exercise is able to improve the occurrence of renal vascular sclerosis caused by natural aging and its related mechanisms. In this study, we investigated the protective effect of aerobic endurance exercise on renal vascular sclerosis in aged mice and its effect on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. The results suggested that aerobic endurance exercise preserved kidney morphology and renal function. Glomerular basement membrane thickness was evidently increased, podocyte foot processes were effaced in aged mice, and aerobic endurance exercise significantly ameliorated the overall lesion range. The protein expression of vascular endothelial growth factor (VEGF) and JG12 was lower in the senile control group (OC group). The protein expression of VEGF and JG12 was significantly increased after aerobic endurance exercise. Furthermore, aerobic endurance exercise resulted in downregulation of Bax, Caspase 3, IL-6, and senescent cells and upregulation of Bcl-2. The upregulation of PI3K and its downstream signal molecules AKT and mTOR after aerobic endurance exercise was further observed. Our observations indicated that aerobic endurance exercise may inhibit renal vascular sclerosis in aged mice by regulating the PI3K/AKT/mTOR signaling pathway.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esclerose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Condicionamento Físico Animal/métodos , Circulação Renal/fisiologia , Transdução de Sinais/fisiologia
12.
Toxicol Lett ; 323: 1-9, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31982503

RESUMO

Zearalenone (ZEA) is a prevalent non-steroidal estrogenic mycotoxin produced mainly by Fusarium contamination. Our previous study showed that ZEA induces the autophagy of Sertoli cells (SCs). However, the underlying mechanisms are still unknown. Several studies have indicated that the increasing level of cytoplasmic Ca2+ could induce autophagy through CaMKKß and AMPK pathways. Thus in order to investigate the potential mechanism underlying ZEA-induced autophagy, the activity of calmodulin-dependent kinase kinase ß(CaMKKß)and AMP-activated protein kinase (AMPK) signaling pathway in ZEA-infected TM4 cells was studied. In the present study, ZEA activated the CaMKKß and AMPK signaling pathways. The AMPK inhibitor and activator significantly inhibited and stimulated the effect of ZEA on AMPK, the transformation from LC3I to LC3II, and the distribution of LC3 dots. In addition, cytosolic calcium (Ca2+) was increased gradually with the concentration of ZEA. After treatment of ZEA-infected cells with 1, 2-bis (2-aminophenoxy) ethane-N, N, N', N'- tetraacetic acid- tetraac etoxymethyl ester (BAPTA-AM) and 2-aminoethyl diphenylborinate (2-APB), the intracellular concentration of Ca2+ reduced significantly. Also, the activities of CaMKKß and AMPK and subsequent autophagy decreased. Moreover, the antioxidant NAC significantly decreased activities of AMPK and autophagy -related protein. Therefore, it can be speculated that ROS- mediated ER-stress induced by ZEA activates AMPK via Ca2+-CaMKKß leading to autophagy in TM4 cells.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Autofagia/efeitos dos fármacos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/fisiologia , Cálcio/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Células Cultivadas , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia
13.
Nat Commun ; 11(1): 532, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988290

RESUMO

Cancer proteogenomics promises new insights into cancer biology and treatment efficacy by integrating genomics, transcriptomics and protein profiling including modifications by mass spectrometry (MS). A critical limitation is sample input requirements that exceed many sources of clinically important material. Here we report a proteogenomics approach for core biopsies using tissue-sparing specimen processing and microscaled proteomics. As a demonstration, we analyze core needle biopsies from ERBB2 positive breast cancers before and 48-72 h after initiating neoadjuvant trastuzumab-based chemotherapy. We show greater suppression of ERBB2 protein and both ERBB2 and mTOR target phosphosite levels in cases associated with pathological complete response, and identify potential causes of treatment resistance including the absence of ERBB2 amplification, insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification, and candidate resistance mechanisms including androgen receptor signaling, mucin overexpression and an inactive immune microenvironment. The clinical utility and discovery potential of proteogenomics at biopsy-scale warrants further investigation.


Assuntos
Neoplasias da Mama/genética , Proteogenômica/métodos , Receptor ErbB-2/genética , Trastuzumab/uso terapêutico , Biópsia com Agulha de Grande Calibre , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Regulação para Baixo , Humanos , Projetos Piloto , Receptor ErbB-2/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
14.
Cancer Sci ; 111(3): 962-973, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31960523

RESUMO

Tumors consist of heterogeneous cell populations that contain cancer cell subpopulations with anticancer drug-resistant properties called "persister" cells. While this early-phase drug tolerance is known to be related to the stem cell-like characteristic of persister cells, how the stem cell-related pathways contribute to drug resistance has remained elusive. Here, we conducted a single-cell analysis based on the stem cell lineage-related and gastric cell lineage-related gene expression in patient-derived gastric cancer cell models. The analyses revealed that 5-fluorouracil (5-FU) induces a dynamic change in the cell heterogeneity. In particular, cells highly expressing stem cell-related genes were enriched in the residual cancer cells after 5-FU treatment. Subsequent functional screening identified aldehyde dehydrogenase 1A3 (ALDH1A3) as a specific marker and potential therapeutic target of persister cells. ALDH1A3 was selectively overexpressed among the ALDH isozymes after treatment with 5-FU or SN38, a DNA topoisomerase I inhibitor. Attenuation of ALDH1A3 expression by RNA interference significantly suppressed cell proliferation, reduced the number of persister cells after anticancer drug treatment and interfered with tumor growth in a mouse xenograft model. Mechanistically, ALDH1A3 depletion affected gene expression of the mammalian target of rapamycin (mTOR) cell survival pathway, which coincided with a decrease in the activating phosphorylation of S6 kinase. Temsirolimus, an mTOR inhibitor, reduced the number of 5FU-tolerant persister cells. High ALDH1A3 expression correlated with worse prognosis of gastric cancer patients. These observations indicate that the ALDH1A3-mTOR axis could be a novel therapeutic target to eradicate drug-tolerant gastric cancer cells.


Assuntos
Aldeído Oxirredutases/genética , Antineoplásicos/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Serina-Treonina Quinases TOR/genética , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
15.
Bull Cancer ; 107(3): 346-351, 2020 Mar.
Artigo em Francês | MEDLINE | ID: mdl-31955867

RESUMO

Desmoid tumors (TDs) are derived from mesenchymal stem cells and their pathogenesis is strongly linked to the Wingless/Wnt cascade where the deregulation of ß-catenin plays a major role. A mutation of the CTNNB1 encoding ß-catenin is found in the majority of sporadic TD cases and constitutional mutations of APC have been described in heritable forms in patients with familial adenomatous polyposis (FAP). Estrogens could also play a role in pathogenesis and this is the basis for the use of hormone therapy. Other signaling pathways have been involved in the development of TDs such as Notch, Hedgehog, JAK/STAT, PI3 Kinase/AKT and mTOR. Metalloproteases are expressed in TDs and play a role in invasiveness. TGF-ß, as a growth factor, stimulates the transcriptional activity of ß-catenin. Future studies will need to focus on better describing and understanding the immune environment of TDs. One of the major difficulties for the experimental study of TDs is the virtual absence of a preclinical model, either in vitro or in vivo. This is partly why the interactions between the different signaling pathways presented here and their consequences for the development of TDs are still poorly understood.


Assuntos
Fibromatose Agressiva/etiologia , Transdução de Sinais/fisiologia , Polipose Adenomatosa do Colo/genética , Carcinogênese , Elafina/metabolismo , Estrogênios/metabolismo , Fibromatose Agressiva/genética , Fibromatose Agressiva/metabolismo , Fibromatose Agressiva/patologia , Genes APC , Proteínas Hedgehog/metabolismo , Humanos , Janus Quinases/metabolismo , Linfotoxina-alfa/metabolismo , Metaloproteases/metabolismo , Mutação , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição STAT/metabolismo , Serina-Treonina Quinases TOR/metabolismo , beta Catenina/genética
16.
Chem Biol Interact ; 315: 108888, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31682805

RESUMO

Relapse and drug resistance is still major challenges in the treatment of leukemia. Promethazine, an antihistaminic phenothiazine derivative, has been used to prevent chemotherapy-induced emesis, although there is no report about its antitumor potential. Thus, we evaluated the promethazine cytotoxicity against several leukemia cells and the underlying mechanisms were investigated. Promethazine exhibited potent and selective cytotoxicity against all leukemia cell types in vitro at clinically relevant concentrations. Philadelphia positive chronic myeloid leukemia (CML) K562 cells were the most sensitive cell line. The cytotoxicity of promethazine in these cells was triggered by the activation of AMPK and inhibition of PI3K/AKT/mTOR pathway. The subsequent downstream effects were NOXA increase, MCL-1 decrease, and Beclin-1 activation, resulting in autophagy-associated apoptosis. These data highlight targeting autophagy may represent an interesting strategy in CML therapy, and also the antitumor potential of promethazine by acting in AMPK and PI3K/AKT/mTOR signaling pathways. Since this drug is currently used with relative low side effects, its repurposing may represent a new therapeutic opportunity for leukemia treatment.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Prometazina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células Jurkat , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
17.
Cancer Sci ; 111(1): 47-58, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31710162

RESUMO

Breast cancer is the most prevalent malignancy among women. Although endocrine therapy is effective, the development of endocrine resistance is a major clinical challenge. The tumor microenvironment (TME) promotes tumor malignancy, and tumor-associated macrophages (TAM) within the TME play a crucial role in endocrine resistance. Herein, we aimed to elucidate the relationship between TAM and the endocrine-resistant phenotype of breast cancer. Macrophages were cultured with conditioned medium (CM) from tamoxifen-sensitive (MCF7-S) or -resistant (MCF7-R) MCF7 breast cancer cells. M2 polarization was detected by CD163 immunofluorescence. To determine the effect on endocrine resistance, MCF7 cells were cultured in the supernatant of different TAM, and then treated with tamoxifen. CC-chemokine ligand 2 (CCL2) immunohistochemistry was carried out on pathological sections from 100 patients with invasive estrogen receptor-positive breast cancer. We found that macrophages cultured in the CM of MCF7-S and MCF7-R cells were induced into TAM, with a more obvious M2 polarization in the latter. Tamoxifen resistance was increased by culture in TAM medium. TAM secreted CCL2, which increased endocrine resistance in breast cancer cells through activation of the PI3K/Akt/mTOR signaling pathway. High expression of CCL2 was correlated with infiltration of CD163+macrophages (r = 0.548, P < .001), and patients with high CCL2 expression presented shorter progression-free survival than those with low CCL2 expression (P < .05). We conclude that CCL2 secreted by TAM activates PI3K/Akt/mTOR signaling and promotes an endocrine resistance feedback loop in the TME, suggesting that CCL2 and TAM may be novel therapeutic targets for patients with endocrine-resistant breast cancer.


Assuntos
Neoplasias da Mama/genética , Quimiocina CCL2/genética , Resistencia a Medicamentos Antineoplásicos/genética , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Intervalo Livre de Progressão , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Tamoxifeno/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética
18.
Cell Prolif ; 53(1): e12714, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31696568

RESUMO

OBJECTIVES: Osteoclasts (OC) are unique terminally differentiated cells whose primary function is bone resorption. We previously showed that osteoprotegerin (OPG) inhibits OC differentiation in vitro by enhancing autophagy via the adenosine monophosphate-activated protein kinase (AMPK)/mTOR/p70S6K signalling pathway in vitro. Here, we aimed to elucidate the mechanism of AMPK mediated autophagy to regulate OPG-mediated inhibition of OC differentiation and identify potential therapeutic targets associated with bone loss. MATERIALS AND METHODS: We used the AMPK activator AICAR to determine the relationship between AMPK activation and OC differentiation, and studied the role of AMPK-mediated autophagy in OPG-mediated inhibition of OC differentiation by using autophagy inhibitors or AMPK knockdown. RESULTS: AMP-activated protein kinase activation caused LC3II accumulation and weakened OC differentiation activity. In contrast, inactivation of autophagy by 3-methyladenine or Bafilomycin A1 could attenuate OPG-mediated inhibition of OC differentiation via the AMPK/mTOR/p70S6K signalling pathway. Furthermore, the AMPK inhibitor compound C and knockdown of AMPK impaired OPG-mediated inhibition of OC differentiation by inducing autophagy. CONCLUSIONS: These results demonstrated that the AMPK signalling pathway functions as a critical regulator in the OPG-mediated inhibition of OC differentiation, by inducing autophagy. Our results provide a basis for future bone-related studies on the AMPK signalling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Aminoimidazol Carboxamida/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/citologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
19.
Cancer Sci ; 111(1): 112-126, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31675763

RESUMO

Drug repositioning is an emerging approach to developing novel cancer treatments. Vorinostat is a histone deacetylase inhibitor approved for cancer treatment, but it could attenuate its anticancer activity by activating the mTOR pathway. The HMG-CoA reductase inhibitor fluvastatin reportedly activates the mTOR inhibitor AMP-activated protein kinase (AMPK), and we thought that it would potentiate vorinostat's anticancer activity in renal cancer cells. The combination of vorinostat and fluvastatin induced robust apoptosis and inhibited renal cancer growth effectively both in vitro and in vivo. Vorinostat activated the mTOR pathway, as evidenced by the phosphorylation of ribosomal protein S6, and fluvastatin inhibited this phosphorylation by activating AMPK. Fluvastatin also enhanced vorinostat-induced histone acetylation. Furthermore, the combination induced endoplasmic reticulum (ER) stress that was accompanied by aggresome formation. We also found that there was a positive feedback cycle among AMPK activation, histone acetylation, and ER stress induction. This is the first study to report the beneficial combined effect of vorinostat and fluvastatin in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Fluvastatina/farmacologia , Neoplasias Renais/tratamento farmacológico , Vorinostat/farmacologia , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Renais/metabolismo , Fosforilação/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
20.
Int J Radiat Oncol Biol Phys ; 106(3): 564-570, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678634

RESUMO

PURPOSE: Activation of the PI3K/mTOR signaling pathway is common in head and neck squamous cell carcinoma (HNSCC). BYL719 is an α-specific PI3K inhibitor that is synergistic and efficacious when combined with cetuximab, a Food and Drug Administration-approved radiosensitizing agent in the treatment of HNSCC. The agent independently has been shown to enhance radiosensitivity. This study evaluates the addition of BYL719 to cetuximab and radiation in the treatment of locally advanced HNSCC. METHODS AND MATERIALS: This is a single-institution, phase 1 study. Patients with American Joint Committee on Cancer seventh edition stage III to IVB HNSCC received standard cetuximab (400 mg/m2 intravenous loading dose) before intensity modulated radiation therapy (IMRT) followed by 250 mg/m2 weekly infusions during IMRT. BYL719 was given orally during IMRT in 3 dose levels: (1) 200 mg/d, (2) 250 mg/d, or (3) 300 mg/d in a standard 3 + 3 dose-escalation design. RESULTS: Eleven patients were evaluable. Dose level 2 was the maximum tolerated dose for BYL719. Two patients on dose level 3 had dose-limiting toxicities of oral mucositis that required a dose reduction of BYL719. One patient on dose level 2 had a dose-limiting toxicity of nausea that led to withdrawal of on-study treatment. Related grade 3 or higher adverse events consisted of decreased lymphocyte count, oral mucositis, dysphagia, hyperglycemia, maculopapular rash, and palmar-plantar erythrodysesthesia syndrome. All 11 patients had a complete response on posttreatment imaging, and 10 remain disease free. Of the 8 patients with mutational analysis, 1 had an activating PIK3CA mutation associated with a rapid response on serial intratreatment magnetic resonance imaging scans. CONCLUSIONS: The recommended phase 2 dose of BYL719 is 250 mg/d in combination with cetuximab and IMRT in patients with locally advanced HNSCC. Further evaluation of the addition of BYL719 to the platinum-sparing regimen of cetuximab and IMRT in the treatment of locally advanced HNSCC is warranted.


Assuntos
Cetuximab/administração & dosagem , Neoplasias de Cabeça e Pescoço/terapia , Radiossensibilizantes/administração & dosagem , Radioterapia de Intensidade Modulada , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Tiazóis/administração & dosagem , Adulto , Idoso , Quimiorradioterapia/métodos , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Serina-Treonina Quinases TOR/metabolismo , Tiazóis/efeitos adversos
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