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1.
Dis Model Mech ; 16(5)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35855640

RESUMO

When pathological hypertrophy progresses to heart failure (HF), the prognosis is often very poor. Therefore, it is crucial to find new and effective intervention targets. Here, myocardium-specific Trim44 knockout rats were generated using CRISPR-Cas9 technology. Cardiac phenotypic observations revealed that Trim44 knockout affected cardiac morphology at baseline. Rats with Trim44 deficiency exhibited resistance to cardiac pathological changes in response to stimulation via isoproterenol (ISO) treatment, including improvement of cardiac remodeling and dysfunction by morphological and functional observations, reduced myocardial fibrosis and reduced expression of molecular markers of cardiac stress. Furthermore, signal transduction validation associated with growth and hypertrophy development in vivo and in vitro demonstrated that Trim44 deficiency inhibited the activation of signaling pathways involved in myocardial hypertrophy, especially response to pathological stress. In conclusion, the present study indicates that Trim44 knockout attenuates ISO-induced pathological cardiac remodeling through blocking the AKT/mTOR/GSK3ß/P70S6K signaling pathway. This is the first study to demonstrate the function and importance of Trim44 in the heart at baseline and under pathological stress. Trim44 could be a novel therapeutic target for prevention of cardiac hypertrophy and HF.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Remodelação Ventricular , Animais , Cardiomegalia/genética , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Isoproterenol/uso terapêutico , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo , Remodelação Ventricular/fisiologia
2.
Mediators Inflamm ; 2022: 7236118, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36133744

RESUMO

Infiltration and aggregation of lymphocytes in exocrine glands are the basic pathological manifestations of Sjögren's syndrome (SS), and the incidence of SS has been increasing year by year in recent years. To explore the potential signaling pathway of Runzaoling (RZL) in alleviating SS, the possible targets of RZL in SS were firstly explored through network pharmacology, and then, the regulation of PI3K/AKT/mTOR signaling in NOD mice and Th17 cells was verified. 75 8-week-old NOD mice were casually classified into 5 groups: model; hydroxychloroquine; high, medium, and low dose RZL groups, with 15 in each; and 15 BALB/c mice were employed as control group. After 10 weeks of continuous intragastric administration in mice and 24 hours of drugs intervention in Th17 cells, histopathology was observed by HE staining, and the gene transcription levels were identified by real-time quantitative PCR (RT-qPCR). The protein expressions were detected by western blotting (WB). The findings showed that high and medium dose RZL group could attenuate the submandibular gland tissue damage. The results indicated that the mRNA expressions of PI3K, AKT, mTOR, STAT3, and IL-17 in SS mice and in IL-17 stimulation of Th17 cells were dramatically increased compared with control group and decreased to varying degrees after RZL intervention. The trend of phosphorylated PI3K/AKT/mTOR and STAT3 and IL-17 protein expression in NOD mice and Th17 cells were consistent with mRNA. RZL can downregulate STAT3 and IL-17 expressions in the submandibular gland of NOD mice and in Th17 cells via regulating the PI3K/AKT/mTOR signaling pathway. Moreover, RZL could reduce the activation of CD4+ T lymphocyte differentiation to Th17 cells.


Assuntos
Síndrome de Sjogren , Animais , Modelos Animais de Doenças , Hidroxicloroquina , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro , Transdução de Sinais , Síndrome de Sjogren/metabolismo , Serina-Treonina Quinases TOR/metabolismo
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 42(8): 1256-1262, 2022 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-36073227

RESUMO

OBJECTIVE: The purpose of this study was to determine whether xenon post-conditioning affects mTOR signaling as well as endoplasmic reticulum stress (ERS)-apoptosis pathway in rats with spinal cord ischemia/reperfusion injury. METHODS: Fifty male rats were randomized equally into sham-operated group (Sham group), I/R model group (I/R group), I/R model+ xenon post-conditioning group (Xe group), I/R model+rapamycin (a mTOR signaling pathway inhibitor) treatment group (I/R+ Rapa group), and I/R model + xenon post- conditioning with rapamycin treatment group (Xe + Rapa group).. In the latter 4 groups, SCIRI was induced by clamping the abdominal aorta for 85 min followed by reperfusion for 4 h. Rapamycin (or vehicle) was administered by daily intraperitoneal injection (4 mg/kg) for 3 days before SCIRI, and xenon post-conditioning by inhalation of 1∶1 mixture of xenon and oxygen for 1 h at 1 h after initiation of reperfusion; the rats without xenon post-conditioning were given inhalation of nitrogen and oxygen (1∶ 1). After the reperfusion, motor function and histopathologic changes in the rats were examined. Western blotting and real-time PCR were used to detect the protein and mRNA expressions of GRP78, ATF6, IRE1α, PERK, mTOR, p-mTOR, Bax, Bcl-2 and caspase-3 in the spinal cord. RESULTS: The rats showed significantly lowered hind limb motor function following SCIRI (P < 0.01) with a decreased count of normal neurons, increased mRNA and protein expressions of GRP78, ATF6, IRE1α, PERK, and caspase-3, and elevated p-mTOR/mTOR ratio and Bax/Bcl-2 ratio (P < 0.01). Xenon post-conditioning significantly decreased the mRNA and protein levels of GRP78, ATF6, IRE1α, PERK and caspase-3 (P < 0.05 or 0.01) and reduced p-mTOR/mTOR and Bax/Bcl-2 ratios (P < 0.01) in rats with SCIRI; the mRNA contents and protein levels of GRP78 and ATF6 were significantly decreased in I/R+Rapa group (P < 0.01). Compared with those in Xe group, the rats in I/R+Rapa group and Xe+Rapa had significantly lowered BBB and Tarlov scores of the hind legs (P < 0.01), and caspase-3 protein level and Bax/Bcl-2 ratio were significantly lowered in Xe+Rapa group (P < 0.05 or 0.01). CONCLUSION: By inhibiting ERS and neuronal apoptosis, xenon post- conditioning may have protective effects against SCIRI in rats. The mTOR signaling pathway is partially involved in this process.


Assuntos
Traumatismo por Reperfusão/complicações , Isquemia do Cordão Espinal/complicações , Serina-Treonina Quinases TOR/metabolismo , Xenônio/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Endorribonucleases/farmacologia , Injeções Intraperitoneais , Masculino , Neurônios/metabolismo , Neurônios/patologia , Nitrogênio/administração & dosagem , Nitrogênio/metabolismo , Oxigênio/administração & dosagem , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Isquemia do Cordão Espinal/metabolismo , Isquemia do Cordão Espinal/patologia , Xenônio/administração & dosagem , Xenônio/farmacologia , Xenônio/uso terapêutico , Proteína X Associada a bcl-2/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(38): e2204083119, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095197

RESUMO

Mammalian target of rapamycin (mTOR) is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism, and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in Saccharomyces cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a rapamycin-induced developmental delay in Drosophila.


Assuntos
Sirolimo , Proteína 1A de Ligação a Tacrolimo , Animais , Humanos , Mamíferos/metabolismo , Fosforilação , Transdução de Sinais , Sirolimo/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo
5.
Nature ; 609(7928): 822-828, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36104566

RESUMO

On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates1,2. In diseases of the central nervous system, drugs with brain-restricted pharmacology are highly desirable. Here we report a strategy to achieve inhibition of mammalian target of rapamycin (mTOR) while sparing mTOR activity elsewhere through the use of the brain-permeable mTOR inhibitor RapaLink-1 and the brain-impermeable FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in glioblastoma xenografts. We further present a general method to design cell-permeable, FKBP12-dependent kinase inhibitors from known drug scaffolds. These inhibitors are sensitive to deactivation by RapaBlock, enabling the brain-restricted inhibition of their respective kinase targets.


Assuntos
Inibidores MTOR , Proteína 1A de Ligação a Tacrolimo , Encéfalo/metabolismo , Humanos , Ligantes , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/metabolismo
6.
Mol Cancer ; 21(1): 174, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056355

RESUMO

BACKGROUND: Chemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors associated with chemoresistance and a very promising direction for cancer treatment. However, the exact molecular mechanisms of cancer stemness have not been completely elucidated. METHODS: m6A-RNA immunoprecipitation and sequencing were used to screen m6A-related mRNAs and lncRNAs. qRT-PCR and FISH were utilized to analyse DDIT4-AS1 expression. Spheroid formation, colony formation, Western blot and flow cytometry assays were performed to analyse the cancer stemness and chemosensitivity of PDAC cells. Xenograft experiments were conducted to analyse the tumour formation ratio and growth in vivo. RNA sequencing, Western blot and bioinformatics analyses were used to identify the downstream pathway of DDIT4-AS1. IP, RIP and RNA pulldown assays were performed to test the interaction between DDIT4-AS1, DDIT4 and UPF1. Patient-derived xenograft (PDX) mouse models were generated to evaluate chemosensitivities to GEM. RESULTS: DDIT4-AS1 was identified as one of the downstream targets of ALKBH5, and recruitment of HuR onto m6A-modified sites is essential for DDIT4-AS1 stabilization. DDIT4-AS1 was upregulated in PDAC and positively correlated with a poor prognosis. DDIT4-AS1 silencing inhibited stemness and enhanced chemosensitivity to GEM (Gemcitabine). Mechanistically, DDIT4-AS1 promoted the phosphorylation of UPF1 by preventing the binding of SMG5 and PP2A to UPF1, which decreased the stability of the DDIT4 mRNA and activated the mTOR pathway. Furthermore, suppression of DDIT4-AS1 in a PDX-derived model enhanced the antitumour effects of GEM on PDAC. CONCLUSIONS: The ALKBH5-mediated m6A modification led to DDIT4-AS1 overexpression in PDAC, and DDIT-AS1 increased cancer stemness and suppressed chemosensitivity to GEM by destabilizing DDIT4 and activating the mTOR pathway. Approaches targeting DDIT4-AS1 and its pathway may be an effective strategy for the treatment of chemoresistance in PDAC.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Neoplasias Pancreáticas , RNA Antissenso/metabolismo , RNA Longo não Codificante , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Helicases/genética , RNA Longo não Codificante/genética , Serina-Treonina Quinases TOR/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Regulação para Cima
7.
Mol Med ; 28(1): 116, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104669

RESUMO

BACKGROUND: Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation. METHODS: A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest. RESULTS: In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy. CONCLUSIONS: We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.


Assuntos
Catarata , Diabetes Mellitus , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Catarata/etiologia , Transição Epitelial-Mesenquimal , Glucose/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mamíferos/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
8.
PLoS Pathog ; 18(9): e1010808, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36067252

RESUMO

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.


Assuntos
Receptores de Imunoglobulina Polimérica , Vírus da Síndrome da Mancha Branca 1 , Antivirais/farmacologia , Calmodulina/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas/farmacologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/metabolismo
9.
Nutrients ; 14(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36079859

RESUMO

Abnormal vasorin (Vasn) expression occurs in multiple diseases, particularly liver cancers. Vasn knockout (KO) in mice causes malnutrition, a shortened life span, and decreased physiological functions. However, the causes and underlying mechanisms remain unknown. Here, we established Vasn KO C57BL/6J mice by using the CRISPR/Cas9 system. The animals were weighed, and histology, immunohistochemistry, electronic microscopy, and liver function tests were used to examine any change in the livers. Autophagy markers were detected by Western blotting. MicroRNA (miRNA) sequencing was performed on liver samples and analyses to study the signaling pathway altered by Vasn KO. Significant reductions in mice body and liver weight, accompanied by abnormal liver function, liver injury, and reduced glycogen accumulation in hepatocytes, were observed in the Vasn KO mice. The deficiency of Vasn also significantly increased the number of autophagosomes and the expression of LC3A/B-II/I but decreased SQSTM1/p62 levels in hepatocytes, suggesting aberrant activation of autophagy. Vasn deficiency inhibited glycogen-mediated mammalian target of rapamycin (mTOR) phosphorylation and activated Unc-51-like kinase 1 (ULK1) signaling, suggesting that Vasn deletion upregulates hepatocyte autophagy through the mTOR-ULK1 signaling pathway as a possible cause of diminished life span and health. Our results indicate that Vasn is required for the homeostasis of liver glycogen metabolism upstream of hepatocyte autophagy, suggesting research values for regulating Vasn in pathways related to liver physiology and functions. Overall, this study provides new insight into the role of Vasn in liver functionality.


Assuntos
Proteínas Reguladoras de Apoptose , Glicogênio , Proteínas de Membrana , Serina-Treonina Quinases TOR , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Hepatócitos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
10.
BMC Med Genomics ; 15(1): 194, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36100894

RESUMO

BACKGROUND: This study attempted to verify the potential of KCNJ14 as a biomarker in colorectal cancer (CRC). METHODS: Data on transcriptomics and DNA methylation and the clinical information of CRC patients were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases. Biological information analysis methods were conducted to determine the role of KCNJ14 in the prognosis, diagnosis, immune cell infiltration, and regulation mechanism of CRC patients. The effect of KCNJ14 on the proliferation and migration of HCT116 and SW480 CRC cell lines was verified by in vitro experiments (MTT, colony-forming, wound healing, and transwell assays). Western blotting was performed to detect the effect of KCNJ14 on the levels of mTOR signalling pathway-related proteins. RESULTS: KCNJ14 expression was remarkably increased in CRC tissues and cell lines, which reduced the overall survival time of patients. KCNJ14 mRNA was negatively regulated by its methylation site cg17660703, which can also endanger the prognosis of patients with CRC. Functional enrichment analysis suggested that KCNJ14 is involved in the mTOR, NOD-like receptor, and VEGF signalling pathways. KCNJ14 expression was positively correlated with the number of CD4 + T cells and negatively correlated with that of CD8 + T cells in the immune microenvironment. KCNJ14 knockdown significantly reduced not only the proliferation and migration of CRC cell lines but also the levels of mTOR signalling pathway-related proteins. CONCLUSIONS: This study not only increases the molecular understanding of KCNJ14 but also provides a potentially valuable biological target for the treatment of colorectal cancer.


Assuntos
Neoplasias Colorretais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Humanos , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral
11.
Front Immunol ; 13: 854432, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36110864

RESUMO

Natural killer (NK) cells are crucial for immune responses to viral infections. CD160 is an important NK cell activating receptor, with unknown function in HIV infection. Here, we found that CD160 expression was reduced on NK cells from HIV-infected individuals and its expression was negatively correlated with HIV disease progression. Further, GLUT1 expression and glucose uptake were higher in CD160+ NK cells, and the results of RNA-seq and flow cytometry demonstrated that CD160 positively regulated glucose metabolism through the PI3K/AKT/mTOR/s6k signaling pathway, thereby enhancing NK cell function. Moreover, we determined that reduced CD160 expression on NK cells could be attributed to the higher plasma levels of TGF-ß1 in HIV-infected individuals. Overall, these results highlight the vital role of CD160 in HIV disease progression and regulation of glucose metabolism, indicating a potential target for HIV immunotherapy.


Assuntos
Infecções por HIV , Antígenos CD/metabolismo , Progressão da Doença , Proteínas Ligadas por GPI/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Células Matadoras Naturais , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Imunológicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
12.
Cancer Biol Ther ; 23(1): 336-347, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36112948

RESUMO

Melanoma is a highly aggressive cancer that can metastasize at early stage. The aim of this study is to clarify the role of Piezo1 and its potential mechanism in regulating the malignant phenotypes of melanoma. In the present study, we first showed that Piezo1 was abnormally expressed in melanoma, which accelerated the malignant progression by activating AKT/mTOR signaling. Firstly, we found that Piezo1 was upregulated in melanoma and associated with poor survival. Additionally, Piezo1 knockdown significantly weakened intracellular calcium signal and viability of melanoma cells. Furthermore, Piezo1 knockdown inhibited the transendothelial migration and invasion in vitro, as well as metastasis in vivo. Mechanistically, we found that Piezo1 activated AKT/mTOR signaling to maintain malignant phenotypes of melanoma. Therefore, Piezo1 acts as an oncogene in melanoma cells and provides a novel candidate for melanoma diagnosis and treatment.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas c-akt , Cálcio , Humanos , Canais Iônicos/genética , Melanoma/genética , Melanoma/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
13.
Pharm Biol ; 60(1): 1781-1789, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36102594

RESUMO

CONTEXT: Polyphyllin II (PPII) is a steroidal saponin isolated from Rhizoma Paridis. It exhibits significant antitumor activity such as anti-proliferation and pro-apoptosis in lung cancer. OBJECTIVE: To explore whether PPII induce autophagy and the relationship between autophagy and apoptosis in non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: The effects of PPII (0, 1, 5, and 10 µM) were elucidated by CCK8 assay, colony formation test, TUNEL staining, MDC method, and mRFP-GFP-LC3 lentivirus transfection in A549 and H1299 cells for 24 h. DMSO-treated cells were selected as control. The protein expression of autophagy (LC3-II, p62), apoptosis (Bcl-2, Bax, caspase-3) and p-mTOR was detected by Western blotting. We explored the relationship between autophagy and apoptosis by autophagy inhibitor CQ (10 µM) and 3-MA (5 mM). RESULTS: PPII (0, 1, 5, and 10 µM) inhibited the proliferation and induced apoptosis. The IC50 values of A549 and H1299 cells were 8.26 ± 0.03 and 2.86 ± 0.83 µM. We found that PPII could induce autophagy. PPII promoted the formation of autophagosome, increased the expression of LC3-II/LC3-I (p < 0.05), while decreased p62 and p-mTOR (p < 0.05). Additionally, the co-treatment with autophagy inhibitors promoted the protein expression of c-caspase-3 and rate of Bax/Bcl-2 (p < 0.05), compared with PPII-only treatment group. Therefore, our results indicated that PPII-induced autophagy may be a mechanism to promote cell survival, although it can also induce apoptosis. CONCLUSIONS: PPII-induced apoptosis exerts its anticancer activity by inhibiting autophagy, which will hopefully provide a prospective compound for NSCLC treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Saponinas , Apoptose , Autofagia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saponinas/farmacologia , Transdução de Sinais , Esteroides , Serina-Treonina Quinases TOR/metabolismo , Proteína X Associada a bcl-2
14.
Pharm Biol ; 60(1): 1751-1761, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36102631

RESUMO

CONTEXT: Ursolic acid (UA) and acteoside (ATS) are important active components that have been used to treat Alzheimer's disease (AD) because of their neuroprotective effects, but the exact mechanism is still unclear. OBJECTIVE: Network pharmacology was used to explore the mechanism of UA + ATS in treating AD, and cell experiments were used to verify the mechanism. MATERIALS AND METHODS: UA + ATS targets and AD-related genes were retrieved from TCMSP, STITCH, SwissTargetPrediction, GeneCards, DisGeNET and GEO. Key targets were obtained by constructing protein interaction network through STRING. The neuroprotective effects of UA + ATS were verified in H2O2-treated PC12 cells. The subsequent experiments were divided into Normal, Model (H2O2 pre-treatment for 4 h), Control (H2O2+ solvent pre-treatment), UA (5 µM), ATS (40 µM), UA (5 µM) + ATS (40 µM). Then apoptosis, mitochondrial membrane potential, caspase-3 activity, ATG5, Beclin-1 protein expression and Akt, mTOR phosphorylation levels were detected. RESULTS: The key targets of UA + ATS-AD network were mainly enriched in Akt/mTOR pathway. Cell experiments showed that UA (ED50: 5 µM) + ATS (ED50: 40 µM) could protect H2O2-induced (IC50: 250 µM) nerve damage by enhancing cells viability, combating apoptosis, restoring MMP, reducing the activation of caspase-3, lessening the phosphorylation of Akt and mTOR, and increasing the expression of ATG5 and Beclin-1. CONCLUSIONS: ATS and UA regulates multiple targets, bioprocesses and signal pathways against AD pathogenesis. ATS and UA synergistically protects H2O2-induced neurotrosis by regulation of AKT/mTOR signalling.


Assuntos
Fármacos Neuroprotetores , Proteínas Proto-Oncogênicas c-akt , Animais , Caspase 3/metabolismo , Glucosídeos , Peróxido de Hidrogênio/toxicidade , Farmacologia em Rede , Fármacos Neuroprotetores/farmacologia , Ácido Oleanólico/análogos & derivados , Células PC12 , Polifenóis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
15.
Oxid Med Cell Longev ; 2022: 9306097, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36120601

RESUMO

Background: It has been reported that ischemia and ischemic preconditioning (IPC) have different effects on the expression of tuberous sclerosis complex 1 (TSC1), which may contribute to the tolerance to ischemia/hypoxia with the increase of autophagy. The mechanisms of TSC1 differential expression are still unclear under ischemia/IPC conditions in hippocampal Cornu Ammon 1 (CA1) and Cornu Ammon 3 (CA3) area neuronal cells. While we have shown that 5-Aza-CdR, a DNA methyltransferase inhibitor, can upregulate TSC1 and increase hypoxic tolerance by autophagy in vivo and in vitro, in this study, we examined whether DNA methylation was involved in the differential expression of TSC1 in the CA1 and CA3 regions induced by hypoxic preconditioning (HPC). Methods: Level of rapamycin (mTOR) autophagy, a downstream molecular pathway of TSC1/TSC2 complex, was detected in HPC mouse hippocampal CA1 and CA3 areas as well as in the HPC model of mouse hippocampal HT22 cells. DNA methylation level of TSC1 promoter (-720 bp~ -360 bp) was determined in CA1 and CA3 areas by bisulfite-modified DNA sequencing (BMDS). At the same time, autophagy was detected in HT22 cells transfected with GFP-LC3 plasmid. The role of TSC1 in neuroprotection was measured by cell viability and apoptosis, and the role of TSC1 in metabolism was checked by ATP assay and ROS assay in HT22 cells that overexpressed/knocked down TSC1. Results: HPC upregulated the expression of TSC1, downregulated the level of P-mTOR (Ser2448) and P-p70S6K (Thr389), and enhanced the activity of autophagy in both in vivo and in vitro. The increased expression of TSC1 in HPC may depend on its DNA hypomethylation in the promoter region in vivo. HPC also could reduce energy consumption in HT22 cells. Overexpression and knockdown of TSC1 can affect cell viability, cell apoptosis, and metabolism in HT22 cells exposed to hypoxia. Conclusion: TSC1 expression induced by HPC may relate to the downregulation of its DNA methylation level with the increase of autophagy and the decrease of energy demand.


Assuntos
Neuroproteção , Proteínas Quinases S6 Ribossômicas 70-kDa , Trifosfato de Adenosina/metabolismo , Animais , Metilação de DNA/genética , Expressão Gênica , Hipóxia/genética , Hipóxia/metabolismo , Metiltransferases/metabolismo , Camundongos , Neuroproteção/genética , Espécies Reativas de Oxigênio , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
16.
Drug Des Devel Ther ; 16: 3071-3085, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36118165

RESUMO

Background: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury. Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting. Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR). Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.


Assuntos
Ferroptose , Traumatismo por Reperfusão , Animais , Coenzima A/metabolismo , Coenzima A/farmacologia , Coenzima A/uso terapêutico , Flavonóis , Glucose/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Ferro , Ligases/metabolismo , Ligases/farmacologia , Ligases/uso terapêutico , Lipídeos/farmacologia , Mamíferos/metabolismo , Oxigênio/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/farmacologia , Proteína de Ligação a Fosfatidiletanolamina/uso terapêutico , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Fosfotransferases (Aceptor do Grupo Álcool) , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Sincalida/metabolismo , Sincalida/farmacologia , Sincalida/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Água
17.
Medicine (Baltimore) ; 101(37): e30666, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36123851

RESUMO

RATIONALE: Ovarian clear cell carcinoma (OCCC) is an uncommon malignant form of 5 subtypes of ovarian cancer, accounting for approximately 5% to 25% of all ovarian cancers. OCCC is usually diagnosed at a young age and an early stage. More than 50% of patients are associated with endometriosis. It shows less sensitivity to platinum-based chemotherapies, high recurrence, and poor prognosis, especially late. However, platinum-based chemotherapies remain the first-line treatment. Meanwhile, new treatment modalities have been explored, including immune checkpoint inhibitors and PI3K-AKT-mTOR pathway inhibitors. PATIENT CONCERN: A 48-year-old Chinese woman, Gravida2 Para1, complained of irregular and painful vaginal bleeding for 4 months. DIAGNOSIS: The patient was diagnosed with stage IC ovarian clear cell carcinoma that presented with a mutation of the phosphatidylinositol 4,5-bisphosphate 3-kinase alpha subunit (PIK3CA) gene. INTERVENTION: We performed an early diagnosis and complete surgical resection of the tumor with platinum-based chemotherapy. OUTCOME: This patient with mutation of the PIK3CA gene was sensitive to platinum-based chemotherapy, showed a significant downwards trend in tumor markers, and was in good health within the year of follow-up. LESSONS: This study described an OCCC case that presented with a PIK3CA mutation and was successfully managed with careful and complete resection of the tumor. This patient with mutation of the PIK3CA gene was sensitive to platinum-based chemotherapy, showed a significant downwards trend in tumor markers, and did not have recurrence after a year of follow-up, indicating a reasonably good prognosis. Therefore, surgery plus platinum drug chemotherapy is still the best strategy for OCCC treatment. In addition, it is recommended for such patients to undergo genetic testing as much as possible to predict the clinical treatment effect.


Assuntos
Adenocarcinoma de Células Claras , Neoplasias Ovarianas , Adenocarcinoma de Células Claras/diagnóstico , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/genética , Biomarcadores Tumorais , Carcinoma Epitelial do Ovário/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Humanos , Inibidores de Checkpoint Imunológico , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
18.
Medicine (Baltimore) ; 101(37): e30643, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36123877

RESUMO

Although N6-methyladenosine (m6A) has been implicated in various biological functions in human cancers, its role in predicting the prognosis of glioma remains unclear. In this study, the transcriptome expression profiles and the clinical data of 961 patients were derived from the Chinese Glioma Genome Atlas (CGGA). We comprehensively evaluated the association between the expression of m6A regulators and the prognosis of glioma and established a 3-gene (YTHDF2, FTO, and ALKBH5) risk signature using least absolute shrinkage and selection operator (LASSO) analysis. Patients with a high-risk signature had significantly adverse prognoses. Gene set enrichment analysis (GSEA) analysis revealed that the G2M checkpoint, MTORC1 signaling, epithelial mesenchymal transition, and PI3K-AKT-mTOR signaling were significantly enriched in the high-risk group. Univariate and multivariate Cox regression analyses confirmed the independent prognostic value of this risk signature. We then constructed a nomogram for individualized prediction of overall survival (OS) by integrating clinicopathological features (age, World Health Organization [WHO] grade), treatment information (radiotherapy, temozolomide therapy), and m6A risk signature. The calibration curves showed excellent agreement between the predicted and actual probabilities for the 1-, 3-, and 5-year OS, with a C-index of 0.780 in the training cohort and 0.717 in the validation cohort. Altogether, our study elucidated the important role of m6A regulators in glioma prognosis, which is valuable for the selection of therapeutic methods and clinical management of patients with glioma.


Assuntos
Glioma , Nomogramas , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Temozolomida/uso terapêutico
19.
Anim Sci J ; 93(1): e13769, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36127314

RESUMO

The composition of amino acid and fatty acid has a vital function on meat quality and animal health. However, the underlying mechanism of amino acid and fatty acid metabolism in sheep during different grazing periods is still unclear. In this study, a total of 12 sheep were employed in different grazing periods. Our results showed that the composition of amino acids and fatty acids in muscle and adipose tissues was significantly altered between dry grass (DG) period and green grass (GG) period. Changes in the activities of the metabolism-related enzymes including BCKD, BCAT2, ACC, SCD, HSL, GSK3ß, p-GSK3ß, and FABP4 were observed in muscle and adipose during different grazing periods. In addition, the mRNA expression levels of ACC, FAS, SCD, HSL, LPL, and DGAT1 in muscle and adipose tissue were changed markedly in different grazing periods. Furthermore, the expression levels of mTOR and ß-catenin/PPARγ/C/EBPα pathway-related proteins were predominantly altered in muscle and adipose among DG and GG. Taken together, all investigations simplified the process of amino acid and fatty acid metabolism disorders caused by different grazing periods, and the mTOR and ß-catenin/PPARγ/C/EBPα play the essential role in this process, which provided an underlying mechanism of metabolism and meat quality.


Assuntos
Aminoácidos , Ácidos Graxos , Tecido Adiposo/metabolismo , Aminoácidos/análise , Animais , Ácidos Graxos/análise , Glicogênio Sintase Quinase 3 beta , Músculos/metabolismo , PPAR gama/metabolismo , RNA Mensageiro/metabolismo , Ovinos , Serina-Treonina Quinases TOR/metabolismo , beta Catenina/metabolismo
20.
Sci Rep ; 12(1): 15084, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064574

RESUMO

Dairy cattle experience a profound nutrient deficit postpartum that is associated with immune dysfunction characterized by heightened inflammation and reduced pathogen clearance. The activation of the central nutrient-sensing mTOR pathway is comparatively reduced in leukocytes of early postpartum dairy cows during this time of most pronounced nutrient deficit. We assessed the effect of pharmacological mTOR inhibition (Torin-1, rapamycin) on differentiation of monocyte derived classically (M1) and alternatively (M2) activated macrophages (MPh) and dendritic cells (moDC) from 12 adult dairy cows. Treatment with mTOR inhibitors generated M1 MPh with increased oxidative burst and expression of IL12 subunits but decreased phagocytosis and expression of IL1B, IL6, and IL10. In M2 MPh, treatment inhibited expression of regulatory features (CD163, ARG2, IL10) skewing the cells toward an M1-like phenotype. In moDC, mTOR inhibition increased expression of pro-inflammatory cytokines (IL12A, IL12B, IL1B, IL6) and surface CD80. In co-culture with mixed lymphocytes, mTOR-inhibited moDC exhibited a cytokine profile favoring a Th1 response with increased TNF and IFNG production and decreased IL10 concentrations. We conclude that mTOR inhibition in vitro promoted differentiation of inflammatory macrophages with reduced regulatory features and generation of Th1-favoring dendritic cells. These mechanisms could contribute to immune dysregulation in postpartum dairy cows.


Assuntos
Doenças do Sistema Imunitário , Interleucina-10 , Animais , Bovinos , Citocinas/metabolismo , Células Dendríticas , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Período Pós-Parto , Serina-Treonina Quinases TOR/metabolismo
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