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1.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202704

RESUMO

The aim of this study was to evaluate the effect of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, on red blood cell parameters in the context of iron homeostasis in patients with tuberous sclerosis complex (TSC) and evaluate its effect on cell size in vitro. Everolimus has a significant impact on red blood cell parameters in patients with TSC. The most common alteration was microcytosis. The mean MCV value decreased by 9.2%, 12%, and 11.8% after 3, 6, and 12 months of everolimus treatment. The iron level declined during the first 3 months, and human soluble transferrin receptor concentration increased during 6 months of therapy. The size of K562 cells decreased when cultured in the presence of 5 µM everolimus by approximately 8%. The addition of hemin to the cell culture with 5 µM everolimus did not prevent any decrease in cell size. The stage of erythroid maturation did not affect the response to everolimus. Our results showed that the mTOR inhibitor everolimus caused red blood cell microcytosis in vivo and in vitro. This effect is not clearly related to a deficit of iron and erythroid maturation. This observation confirms that mTOR signaling plays a complex role in the control of cell size.


Assuntos
Tamanho Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adolescente , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Criança , Pré-Escolar , Índices de Eritrócitos , Eritrócitos/metabolismo , Everolimo/administração & dosagem , Everolimo/efeitos adversos , Everolimo/farmacologia , Citometria de Fluxo , Humanos , Ferro/metabolismo , Células K562 , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos
2.
Anticancer Res ; 41(7): 3287-3292, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230123

RESUMO

BACKGROUND: Osteosarcoma is the most frequent malignant bone neoplasm. The efficacy of combination therapy of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor and a mammalian-target-of-rapamycin (mTOR) inhibitor was previously reported in several cancer types. In the present study, we evaluated the efficacy of a combination of palbociclib (CDK 4/6 inhibitor) and everolimus (mTOR inhibitor) on an osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. MATERIALS AND METHODS: osteosarcoma PDOX mouse models were randomized into five treatment groups of seven mice each: Group 1, untreated control; group 2, doxorubicin treatment; group 3, palbociclib treatment; group 4, everolimus treatment; group 5, palbociclib-everolimus combination treatment. Treatment duration was 2 weeks. RESULTS: The palbociclib-everolimus combination reduced the tumor-volume ratio in the osteosarcoma PDOX mouse model compared with the control and doxorubicin (p=0.018). Everolimus alone also inhibited osteosarcoma PDOX growth compared to the control (p=0.04), but less than the combination. Palbociclib alone and doxorubicin were ineffective. There were no significant body-weight losses in any group. Only the palbociclib-everolimus combination induced extensive tumor necrosis observed histopathologically. CONCLUSION: The present study demonstrated that the combination of CDK4/6 and mTOR inhibitors can be a translatable approach for doxorubicin-resistant osteosarcoma in the clinic.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adolescente , Animais , Neoplasias Ósseas/metabolismo , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Everolimo/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Osteossarcoma/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207825

RESUMO

Non-clear cell renal cell carcinomas are a miscellaneous group of tumors that include different histological subtypes, each one characterized by peculiarity in terms of genetic alteration, clinical behavior, prognosis, and treatment response. Because of their low incidence and poor enrollment in clinical trials, alongside their heterogeneity, additional efforts are required to better unveil the pathogenetic mechanisms and, consequently, to improve the treatment algorithm. Nowadays, tyrosine kinase inhibitors, mTOR and MET inhibitors, and even cisplatin-based chemotherapy and immunotherapy are potential weapons that are still under evaluation in this setting. Various biomarkers have been evaluated for detecting progression and monitoring renal cell carcinoma, but more studies are necessary to improve this field. In this review, we provide an overview on the molecular characteristics of this group of tumors and the recently published trials, giving an insight into what might become the future therapeutic standard in this complex world of non-clear cell kidney cancers.


Assuntos
Biomarcadores Tumorais , Carcinoma de Células Renais , Neoplasias Renais , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met , Serina-Treonina Quinases TOR , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
4.
Zool Res ; 42(4): 482-486, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34235896

RESUMO

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that begins with defective rod photoreceptor function, followed by impaired cone function, and complete blindness in its late stage. To date, however, there is no effective treatment for RP. By carrying a nonsense mutation in the Pde6b gene, rd1 mice display elevated cGMP in conjunction with higher intracellular Ca 2+ in their rod photoreceptors, resulting in fast retinal degeneration. Ca 2+ has been linked to activation of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway integrates extracellular and intracellular signals to sense the supply of nutrients and plays a central role in regulating protein and lipid synthesis as well as apoptosis and autophagy. In the present study, we showed that mTOR and phosphorylated mTOR (p-mTOR, activated form of mTOR) are up-regulated in rd1 photoreceptors at postnatal day 10 (P10), a pre-degenerative stage. Moreover, the downstream effectors of mTOR, such as pS6K and S6K, are also increased, suggesting activation of the mTOR signaling pathway. Intravitreal administration of rapamycin, a negative regulator of mTOR, inhibits the mTOR pathway in rd1 photoreceptors. Consequently, the progression of retinal degeneration is slower and retinal function is enhanced, possibly mediated by activation of autophagy in the photoreceptors. Taken together, these results highlight rapamycin as a potential therapeutic avenue for retinal degeneration.


Assuntos
Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/prevenção & controle , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/patologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Camundongos , Degeneração Retiniana/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sirolimo/uso terapêutico
5.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073521

RESUMO

In this study, we investigated the effect of mTOR inhibitor (mTORi) drug-eluting biodegradable stent (DE stent), a putative restenosis-inhibiting device for coronary artery, on thermal-injury-related ureteral stricture in rabbits. In vitro evaluation confirmed the dose-dependent effect of mTORi, i.e., rapamycin, on fibrotic markers in ureteral component cell lines. Upper ureteral fibrosis was induced by ureteral thermal injury in open surgery, which was followed by insertion of biodegradable stents, with or without rapamycin drug-eluting. Immunohistochemistry and Western blotting were performed 4 weeks after the operation to determine gross anatomy changes, collagen deposition, expression of epithelial-mesenchymal transition markers, including Smad, α-SMA, and SNAI 1. Ureteral thermal injury resulted in severe ipsilateral hydronephrosis. The levels of type III collagen, Smad, α-SMA, and SNAI 1 were increased 28 days after ureteral thermal injury. Treatment with mTORi-eluting biodegradable stents significantly attenuated thermal injury-induced urinary tract obstruction and reduced the level of fibrosis proteins, i.e., type III collagen. TGF-ß and EMT signaling pathway markers, Smad and SNAI 1, were significantly modified in DE stent-treated thermal-injury-related ureteral stricture rabbits. These results suggested that intra-ureteral administration of rapamycin by DE stent provides modification of fibrosis signaling pathway, and inhibiting mTOR may result in fibrotic process change.


Assuntos
Implantes Absorvíveis , Stents Farmacológicos , Sirolimo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Obstrução Ureteral , Animais , Fibrose , Coelhos , Sirolimo/química , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Obstrução Ureteral/terapia
6.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071043

RESUMO

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR-TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Cardiomiopatia Dilatada/genética , Mutação com Ganho de Função , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Mutação Puntual , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Substituição de Aminoácidos , Animais , Autofagia , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cardiomiopatia Dilatada/terapia , Células Cultivadas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Ventrículos do Coração/citologia , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos Wistar , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
7.
Anticancer Res ; 41(6): 2885-2894, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083279

RESUMO

BACKGROUND/AIM: We evaluated the radiosensitizing effect of the combination treatment of trametinib, a MEK inhibitor, and temsirolimus, an mTOR inhibitor, on non-small-cell lung carcinoma (NSCLC) cells. MATERIALS AND METHODS: The effects of combining trametinib and temsirolimus with radiation in NSCLC cell lines were evaluated using clonogenic survival and apoptosis assays. DNA double-strand breaks and cell cycle distribution were analyzed using flow cytometry. Tumor volume was measured to determine the radiosensitivity in lung cancer xenograft models. RESULTS: Exposure of lung cancer cells to a combination of trametinib and temsirolimus reduced clonogenic survival and promoted radiation-induced apoptosis. Combined inhibition of MEK and mTOR induced prolonged expression of γH2AX after irradiation and resulted in prolonged G2/M cell cycle arrest after irradiation in A549 cells. In vivo studies revealed that co-administration of the drugs sensitizes lung cancer xenografts to radiotherapy. CONCLUSION: The combination of trametinib and temsirolimus can enhance lung cancer radiosensitivity in vitro and in vivo.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/radioterapia , MAP Quinase Quinase Quinases/metabolismo , Piridonas/farmacologia , Pirimidinonas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Quimioterapia Combinada , Humanos , Neoplasias Pulmonares/patologia , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Células Tumorais Cultivadas
8.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066490

RESUMO

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. "Adherent" cancer cell lines frequently release "free-floating" cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.


Assuntos
Modelos Biológicos , Neoplasias/patologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metástase Neoplásica , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
9.
Anticancer Res ; 41(5): 2257-2275, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33952452

RESUMO

BACKGROUND: Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models. MATERIALS AND METHODS: Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area. RESULTS: In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro. CONCLUSION: Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Everolimo/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Humanos , Camundongos Endogâmicos , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esferoides Celulares/patologia , Serina-Treonina Quinases TOR/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Nat Med ; 27(5): 851-861, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33958797

RESUMO

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAFV600E mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH.


Assuntos
Senescência Celular/genética , Histiocitose de Células de Langerhans/genética , Histiocitose de Células de Langerhans/patologia , Células de Langerhans/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Senescência Celular/efeitos dos fármacos , Citocinas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
11.
Cancer Sci ; 112(7): 2870-2883, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33931924

RESUMO

Wnt, PI3K-Akt-mTOR, and NF-κB pathways were reported to be involved in DNA damage repair (DDR). DDR-deficient cancers become critically dependent on backup DNA repair pathways. Neuritin 1 (NRN1) is reported to be involved in PI3K-Akt-mTOR, and its role in DDR remains unclear. Methylation-specific PCR, siRNA, flow cytometry, esophageal cancer cell lines, and xenograft mouse models were used to examine the role of NRN1 in esophageal cancer. The expression of NRN1 is frequently repressed by promoter region methylation in human esophageal cancer cells. NRN1 was methylated in 50.4% (510/1012) of primary esophageal cancer samples. NRN1 methylation is associated significantly with age (P < .001), tumor size (P < .01), TNM stage (P < .001), differentiation (P < .001) and alcohol consumption (P < .05). We found that NRN1 methylation is an independent prognostic factor for poor 5-y overall survival (P < .001). NRN1 inhibits colony formation, cell proliferation, migration, and invasion, and induces apoptosis and G1/S arrest in esophageal cancer cells. NRN1 suppresses KYSE150 and KYSE30 cells xenografts growth in nude mice. PI3K signaling is reported to activate ATR signaling by targeting CHK1, the downstream component of ATR. By analyzing the synthetic efficiency of NVP-BEZ235 (PI3K inhibitor) and VE-822 (an ATR inhibitor), we found that the combination of NVP-BEZ235 and VE-822 increased cytotoxicity in NRN1 methylated esophageal cancer cells, as well as KYSE150 cell xenografts. In conclusion, NRN1 suppresses esophageal cancer growth both in vitro and in vivo by inhibiting PI3K-Akt-mTOR signaling. Methylation of NRN1 is a novel synthetic lethal marker for PI3K-Akt-mTOR and ATR inhibitors in human esophageal cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Reparo do DNA , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Neuropeptídeos/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Consumo de Bebidas Alcoólicas , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Dano ao DNA , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/mortalidade , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Xenoenxertos , Humanos , Masculino , Metilação , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Transplante de Neoplasias , Neuropeptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Prognóstico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/uso terapêutico , Pirazóis/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Carga Tumoral
12.
Nat Commun ; 12(1): 2715, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976157

RESUMO

Efficient immune responses rely on heterogeneity, which in CD8+ T cells, amongst other mechanisms, is achieved by asymmetric cell division (ACD). Here we find that ageing, known to negatively impact immune responses, impairs ACD in murine CD8+ T cells, and that this phenotype can be rescued by transient mTOR inhibition. Increased ACD rates in mitotic cells from aged mice restore the expansion and memory potential of their cellular progenies. Further characterization of the composition of CD8+ T cells reveals that virtual memory cells (TVM cells), which accumulate during ageing, have a unique proliferation and metabolic profile, and retain their ability to divide asymmetrically, which correlates with increased memory potential. The opposite is observed for naive CD8+ T cells from aged mice. Our data provide evidence on how ACD modulation contributes to long-term survival and function of T cells during ageing, offering new insights into how the immune system adapts to ageing.


Assuntos
Envelhecimento/genética , Divisão Celular Assimétrica/genética , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/genética , Serina-Treonina Quinases TOR/genética , Envelhecimento/imunologia , Animais , Divisão Celular Assimétrica/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Imunidade Inata , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação Linfocitária , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/imunologia
13.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808215

RESUMO

Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Humanos , Imidazóis/farmacologia , Isoenzimas , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas/farmacologia
14.
J Food Sci ; 86(6): 2700-2712, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33908630

RESUMO

Natural compounds have been increasingly investigated as substances enhancing the effect of drugs and reducing drug-related adverse reactions. The objective of this study was to determine how a combination of cisplatin (DDP) with cyanidin-3-O-glucoside (C3G) affected malignancy features of cervical cancer cells. The results demonstrated that the proliferation of HeLa cells treated with 5 µg/ml DDP, 400 µg/ml C3G, or a combination of both (5 µg/ml DDP and 400 µg/ml C3G) was inhibited by 17.43%, 34.98%, and 63.38%, respectively. The IC50 values for DDP and the DDP/C3G combination treatments in HeLa cells were 18.53 and 6.435 µg/ml, respectively. Flow cytometry analysis indicated that treatment with DDP, C3G, or the combination induced G1 cell cycle arrest and apoptosis in HeLa cells. Furthermore, after treatment, cyclin D1 and Bcl-2 levels decreased; Bax, cleaved caspase-3, p53, and TIMP-1 were activated; and the PI3K/AKT/mTOR signaling pathway was modulated. These anticancer effects were enhanced in cells treated with the combination of DDP and C3G compared to those treated with DDP or C3G alone. Our study indicates that C3G increases the antitumor activity of DDP, suggesting a potential strategy to reduce adverse effects associated with chemotherapy in cervical cancer. PRACTICAL APPLICATION: Natural biologically active food ingredients are suggested to have a potential to enhance the effect of chemotherapy in cancer. We believe that our study makes a significant contribution to the literature because it revealed, for the first time, that C3G could increase the antitumor activity of DDP, suggesting a potential strategy to reduce adverse effects associated with chemotherapy in cervical cancer.


Assuntos
Antocianinas/farmacologia , Proliferação de Células , Cisplatino/farmacologia , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose , Regulação para Baixo , Quimioterapia Combinada , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
15.
Nucleic Acids Res ; 49(9): 5159-5176, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33893802

RESUMO

The eIF4E are a family of initiation factors that bind the mRNA 5' cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active during stress and that it selects specific mRNA populations based on 5' TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have 'moon-lighting' functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote 'stress resistance' and adaptation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Biossíntese de Proteínas , Estresse Fisiológico/genética , Animais , Células Cultivadas , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Camundongos , Naftiridinas/farmacologia , Capuzes de RNA/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores
16.
Res Vet Sci ; 136: 622-630, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33930632

RESUMO

Ammonia is a harmful gas with a pungent odor, participates in the regulation of a variety of apoptosis and autophagy, which in turn affects the growth and differentiation of cells. To test the regulation of NH3 on the apoptosis and autophagy of mammary epithelial cells, we selected NH4Cl as NH3 donor in vitro model. MTT and CCK-8 assay kits were employed to detect cell activity. Real-time quantitative PCR and western blot methods were used to detect the abundance of inflammatory molecules, apoptosis markers, and autophagy genes. We selected TUNEL kit and the Annexin-FITC/PI method to detect apoptosis. TEM analysis was used to detect autophagic vesicles, and MDC stain evaluated the formation of autophagosome. The results indicated that NH4Cl reduced cell viability in a concentration-dependent manner and promoted cell inflammatory response, apoptosis, and autophagy. NH4Cl stimulation notable increased the autophagosomes number. Interestingly, we also detected that the addition of LY294002 and Rapamycin inhibited the PI3K/Akt pathway and the mTOR pathway, respectively, resulting in changes in both apoptosis and autophagy. Therefore, we draw a conclusion that NH3 may regulate the apoptosis and autophagic response of bovine mammary epithelial cells through the PI3K/Akt/mTOR signaling pathway. Further investigations on ammonia's function in other physiological respects, will be critical to provide theoretical help for the improvement of production performance. It will be also helpful for controlling the harmful gas ammonia concentration in the livestock house to protect the health of dairy cows.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
17.
Int J Nanomedicine ; 16: 2173-2186, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33758505

RESUMO

Background: Colon cancer is a top lethal cancer in man and women worldwide and drug resistance is the major cause of cancer-related death. Combinational therapy and drug delivery with nanoparticles have been shown to effectively overcome drug resistance in many cancers. We previously reported that nanoemulsion (NE) loaded paclitaxel (PTX) and BEZ235 could synergistically inhibit colon cancer cell growth. Purpose: To investigate whether NE loaded PTX and BEZ235 can overcome drug resistance and synergistically inhibit drug-resistant colon cancer cell growth in vitro and in vivo. Methods: The in vitro treatment effect on cell viability was assayed using CCK8 kit, cell morphological change was detected by ß-tubulin immunofluorescence staining, drug resistance-related proteins were analyzed by Western blotting, and in vivo tumor growth test was performed in nude mice xeno-transplanted with 2 drug-resistant colon cancer cell lines HCT116-LOHP and HT29-DDP. Results: Both cell lines were sensitive to PTX but relatively insensitive to BEZ235. PTX combined with BEZ235 synergistically inhibited the proliferation of both cell lines. Nanoemulsion loaded PTX (NE-PTX) reduced the IC50 of PTX to approximately 2/5 of free PTX, indicating a high inhibitory efficacy of NE-PTX. When NE-PTX combined with a low concentration of BEZ235 (50 nM), the IC50 was decreased to approximately 2/3 of free PTX. Moreover, NE-PTX+BEZ235 treatment increased apoptosis, decreased Pgp and ABCC1 expression, and reduced tumor weights compared to the single drug treatment and the control group. These results suggest that nanoemulsion loaded PTX+BEZ235 can overcome drug resistance and improve the inhibitory effect on cancer cell proliferation and tumor growth. Conclusion: Our study thus provides a possible new approach to treat colon cancer patients with drug resistance.


Assuntos
Apoptose , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Imidazóis/uso terapêutico , Nanopartículas/química , Paclitaxel/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Quinolinas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Emulsões/química , Feminino , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Paclitaxel/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Ann Transplant ; 26: e929279, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33707409

RESUMO

Coronavirus disease 19 (COVID-19) has been an ongoing pandemic since December 2019. Unfortunately, kidney transplant recipients are a high-risk group during the disease course, and scientific data are still limited in this patient group. Beyond the dosage of immunosuppressive drugs, pharmacological immunosuppression may also alter the infection response in the COVID-19 course. The effects of immunosuppressive agents on the development and process of infection should not be decided only by determining how potent they are and how much they suppress the immune system; it is also thought that the direct effect of the virus, increased oxidative stress, and cytokine storm play a role in the pathogenesis of COVID-19 disease. There are data about immunosuppressive drugs like calcineurin inhibitors (CNI) or mammalian target of rapamycin inhibitors (mTORi) therapy related to their beneficial effects during any infection course. Limited data suggest that the use of CNI or mTORi may have beneficial effects on the process. In this hypothetical review, the probable impacts of CNI and mTORi on the pathogenesis of the COVID-19 were investigated.


Assuntos
COVID-19/imunologia , Inibidores de Calcineurina/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Rim , Complicações Pós-Operatórias/imunologia , Inibidores de Proteínas Quinases/uso terapêutico , Imunidade Adaptativa/efeitos dos fármacos , COVID-19/diagnóstico , Inibidores de Calcineurina/farmacologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/prevenção & controle , Síndrome da Liberação de Citocina/virologia , Rejeição de Enxerto/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Hospedeiro Imunocomprometido , Imunossupressores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/virologia , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
19.
Life Sci ; 274: 119354, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33737087

RESUMO

AIMS: Gigantol is a bibenzyl compound isolated from orchids of the genus Dendrobium. Gigantol has been demonstrated to possess various pharmacologic (including anticancer) effects. Cisplatin (DDP) has been used and studied as the first-line agent for breast cancer (BC) treatment. Often, its efficacy is jeopardized due to intolerance and organ toxicity. We investigated if gigantol could enhance the anticancer effects of DDP in BC cells and its underlying mechanism of action. MAIN METHODS: The potential pathway of gigantol in BC cells was detected by network-pharmacology and molecular-docking studies. The proliferation and apoptosis of BC cell lines were measured by the MTT assay, colony formation, Hoechst-33342 staining, and flow cytometry. Protein expression was measured by western blotting. KEY FINDINGS: Gigantol could inhibit proliferation of BC cells and enhance DDP-induced apoptosis. According to the results of western blotting, gigantol reinforced DDP-induced anticancer effects through downregulation of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway in BC cells. The effects were consistent with those of the pathway inhibitor LY294002. SIGNIFICANCE: Our data might provide new insights into the underlying antitumor effect of gigantol in BC cells. This enhancement effect in the combination of gigantol and DDP may provide many therapeutic benefits in clinical treatment regimens against BC.


Assuntos
Bibenzilas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cisplatino/farmacologia , Sinergismo Farmacológico , Guaiacol/análogos & derivados , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Guaiacol/farmacologia , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas
20.
Eur J Med Chem ; 216: 113318, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33730624

RESUMO

Identifying a pharmacological agent that targets only one of more than 500 kinases present in humans is an important challenge. One potential solution to this problem is the development of bivalent kinase inhibitors, which consist of two connected fragments, each bind to a dissimilar binding site of the bisubstrate enzyme. The main advantage of bivalent (type V) kinase inhibitors is generating more interactions with target enzymes that can enhance the molecules' selectivity and affinity compared to single-site inhibitors. Earlier type V inhibitors were not suitable for the cellular environment and were mostly used in in vitro studies. However, recently developed bivalent compounds have high kinase affinity, high biological and chemical stability in vivo. This review summarized the hetero-bivalent kinase inhibitors described in the literature from 2014 to the present. We attempted to classify the molecules by serine/threonine and tyrosine kinase inhibitors, and then each target kinase and its hetero-bivalent inhibitor was assessed in depth. In addition, we discussed the analysis of advantages, limitations, and perspectives of bivalent kinase inhibitors compared with the monovalent kinase inhibitors.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor EphA1/antagonistas & inibidores , Receptor EphA1/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
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