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1.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068110

RESUMO

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Cromonas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Inibidores Enzimáticos/farmacologia , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Potencial da Membrana Mitocondrial , Necrose , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Células Tumorais Cultivadas
2.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064275

RESUMO

Sigma-1 receptor (chaperone Sigma1R) is an intracellular protein with chaperone functions, which is expressed in various organs, including the brain. Sigma1R participates in the regulation of physiological mechanisms of anxiety (Su, T. P. et al., 2016) and reactions to emotional stress (Hayashi, T., 2015). In 2006, fabomotizole (ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was registered in Russia as an anxiolytic (Seredenin S. and Voronin M., 2009). The molecular targets of fabomotizole are Sigma1R, NRH: quinone reductase 2 (NQO2), and monoamine oxidase A (MAO-A) (Seredenin S. and Voronin M., 2009). The current study aimed to clarify the dependence of fabomotizole anxiolytic action on its interaction with Sigma1R and perform a docking analysis of fabomotizole interaction with Sigma1R. An elevated plus maze (EPM) test revealed that the anxiolytic-like effect of fabomotizole (2.5 mg/kg i.p.) administered to male BALB/c mice 30 min prior EPM exposition was blocked by Sigma1R antagonists BD-1047 (1.0 mg/kg i.p.) and NE-100 (1.0 mg/kg i.p.) pretreatment. Results of initial in silico study showed that fabomotizole locates in the active center of Sigma1R, reproducing the interactions with the site's amino acids common for established Sigma1R ligands, with the ΔGbind value closer to that of agonist (+)-pentazocine in the 6DK1 binding site.


Assuntos
Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Benzimidazóis/farmacologia , Chaperonas Moleculares/metabolismo , Morfolinas/farmacologia , Receptores sigma/metabolismo , Animais , Anisóis/farmacologia , Sítios de Ligação/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Etilenodiaminas/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Propilaminas/farmacologia , Federação Russa
3.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071138

RESUMO

Schwann cells play an important role in peripheral nerve function, and their dysfunction has been implicated in the pathogenesis of diabetic neuropathy and other demyelinating diseases. The physiological functions of insulin in Schwann cells remain unclear and therefore define the aim of this study. By using immortalized adult Fischer rat Schwann cells (IFRS1), we investigated the mechanism of the stimulating effects of insulin on the cell proliferation and expression of myelin proteins (myelin protein zero (MPZ) and myelin basic protein (MBP). The application of insulin to IFRS1 cells increased the proliferative activity and induced phosphorylation of Akt and ERK, but not P38-MAPK. The proliferative potential of insulin-stimulated IFRS1 was significantly suppressed by the addition of LY294002, a PI3 kinase inhibitor. The insulin-stimulated increase in MPZ expression was significantly suppressed by the addition of PD98059, a MEK inhibitor. Furthermore, insulin-increased MBP expression was significantly suppressed by the addition of LY294002. These findings suggest that both PI3-K/Akt and ERK/MEK pathways are involved in insulin-induced cell growth and upregulation of MPZ and MBP in IFRS1 Schwann cells.


Assuntos
Insulina/farmacologia , Células de Schwann/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Cromonas/farmacologia , Neuropatias Diabéticas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptor de Insulina/biossíntese , Receptor de Insulina/genética , Transdução de Sinais/efeitos dos fármacos
4.
Cell Prolif ; 54(7): e13072, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34031939

RESUMO

OBJECTIVES: Induction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel-like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown. MATERIALS AND METHODS: KLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator-activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14-overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)-treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ-6438 was utilized to treat TAA-induced rat liver fibrosis. RESULTS: KLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2-regulated histone H3 lysine 27 trimethylation. Adenovirus-mediated KLF14 overexpression ameliorated TAA-induced rat liver fibrosis in PPARγ-dependent manner. Furthermore, EPZ-6438 dramatically alleviated TAA-induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation. CONCLUSIONS: KLF14 exerts a critical anti-fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Cirrose Hepática/patologia , PPAR gama/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Compostos de Bifenilo/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Cirrose Hepática/metabolismo , Camundongos , Morfolinas/farmacologia , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Regiões Promotoras Genéticas , Piridonas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Tioacetamida/farmacologia
5.
Biomed Res Int ; 2021: 6612592, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977107

RESUMO

Glioblastoma is a highly invasive primary malignant tumor of the central nervous system. Cannabinoid analogue WIN 55,212-2 (WIN) exhibited a novel anticancer effect against human tumors. However, the anticancer potential and underlying mechanism of WIN against human glioma remain unclear. Herein, the anticancer efficiency and mechanism of WIN in U251 human glioma cells were investigated. The results showed that WIN dose-dependently inhibited U251 cell proliferation, migration, and invasion in vitro. WIN treatment also effectively suppressed U251 tumor spheroids growth ex vivo. Further studies found that WIN induced significant apoptosis as convinced by the caspase-3 activation and release of cytochrome C. Mechanism investigation revealed that WIN triggered ROS-mediated DNA damage and caused dysfunction of VEGF-AKT/FAK signal axis. However, ROS inhibition effectively attenuated WIN-induced DNA damage and dysfunction of VEGF-AKT/FAK signal axis and eventually improved U251 cell proliferation, migration, and invasion. Taken together, our findings validated that WIN had the potential to inhibit U251 cell proliferation, migration, and invasion and induce apoptosis by triggering ROS-dependent DNA damage and dysfunction of VEGF-AKT/FAK signal axis.


Assuntos
Benzoxazinas/farmacologia , Glioma/metabolismo , Morfolinas/farmacologia , Naftalenos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos
6.
Nat Commun ; 12(1): 1998, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790302

RESUMO

The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance.


Assuntos
Benzamidas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Xenoenxertos/efeitos dos fármacos , Morfolinas/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Xenoenxertos/metabolismo , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Resultado do Tratamento
7.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922336

RESUMO

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes' recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.


Assuntos
Benzamidas/farmacologia , Compostos de Bifenilo/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Mesotelioma/patologia , Monócitos/patologia , Morfolinas/farmacologia , Piridonas/farmacologia , Esferoides Celulares/patologia , Macrófagos Associados a Tumor/patologia , Proliferação de Células , Humanos , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Monócitos/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Células Tumorais Cultivadas , Microambiente Tumoral , Macrófagos Associados a Tumor/efeitos dos fármacos
8.
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
9.
Mar Drugs ; 19(3)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804171

RESUMO

The proteolytic processing of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase releases amyloid-ß peptide (Aß), which deposits in amyloid plaques and contributes to the initial causative events of Alzheimer's disease (AD). In the present study, the regulatory mechanism of APP processing of three phlorotannins was elucidated in Swedish mutant APP overexpressed N2a (SweAPP N2a) cells. Among the tested compounds, dieckol exhibited the highest inhibitory effect on both intra- and extracellular Aß accumulation. In addition, dieckol regulated the APP processing enzymes, such as α-secretase (ADAM10), ß-secretase, and γ-secretase, presenilin-1 (PS1), and their proteolytic products, sAPPα and sAPPß, implying that the compound acts on both the amyloidogenic and non-amyloidogenic pathways. In addition, dieckol increased the phosphorylation of protein kinase B (Akt) at Ser473 and GSK-3ß at Ser9, suggesting dieckol induced the activation of Akt, which phosphorylated GSK-3ß. The specific phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 triggered GSK-3ß activation and Aß expression. In addition, co-treatment with LY294002 noticeably blocked the effect of dieckol on Aß production, demonstrating that dieckol promoted the PI3K/Akt signaling pathway, which in turn inactivated GSK-3ß, resulting in the reduction in Aß levels.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Benzofuranos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Animais , Linhagem Celular , Cromonas/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taninos/farmacologia
10.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924053

RESUMO

Previous studies have investigated the inhibitory effect of BMI-1026 on cyclin-dependent kinase 1 in vitro. However, the molecular mechanisms by which BMI-1026 treatment leads to cancer cell death remain unclear. This study was conducted to investigate the anticancer mechanisms of BMI-1026 on human renal carcinoma Caki cells. BMI-1026 induced apoptosis in association with the cleavage of poly(ADP-ribose) polymerase and pro-caspase-3 and the release of apoptosis-inducing factor and cytochrome c from mitochondria in Caki cells. BMI-1026-induced apoptosis was inhibited by the pan-caspase inhibitor z-VAD-fmk. Furthermore, BMI-1026 downregulated Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) at the transcriptional level and Mcl-1 (L) and cellular FADD-like IL-1ß-converting enzyme inhibitory protein (c-FLIP (L)) at the post-transcriptional level. Interestingly, Mcl-1 (L) and c-FLIP (L), but not Bcl-2 or XIAP, played important roles in BMI-1026-induced Caki cell apoptosis. Although the constitutively active form of Akt did not attenuate BMI-1026-induced apoptosis, blockade of the PI3K/Akt pathway using a subcytotoxic concentration of the PI3K/Akt inhibitor LY294002 enhanced Caki cell apoptosis induced by BMI-1026. Electrophysiological safety was confirmed by determining the cardiotoxicity of BMI-1026 via left ventricular pressure analysis. These results suggest that BMI-1026 is a potent multitarget anticancer agent with electrophysiological safety and should be further investigated.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma de Células Renais/metabolismo , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Cromonas/farmacologia , Regulação para Baixo , Citometria de Fluxo , Células HCT116 , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo
11.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799789

RESUMO

It is challenging to overcome the low response rate of everolimus in the treatment of patients with hepatocellular carcinoma (HCC). To overcome this challenge, we combined everolimus with Ku0063794, the inhibitor of mTORC1 and mTORC2, to achieve higher anticancer effects. However, the precise mechanism for the synergistic effects is not clearly understood yet. To achieve this aim, the miRNAs were selected that showed the most significant variation in expression according to the mono- and combination therapy of everolimus and Ku0063794. Subsequently, the roles of specific miRNAs were determined in the processes of the treatment modalities. Compared to individual monotherapies, the combination therapy significantly reduced viability, increased apoptosis, and reduced autophagy in HepG2 cells. The combination therapy led to significantly lower expression of miR-4790-3p and higher expression of zinc finger protein225 (ZNF225)-the predicted target of miR-4790-3p. The functional study of miR-4790-3p and ZNF225 revealed that regarding autophagy, miR-4790-3p promoted it, while ZNF225 inhibited it. In addition, regarding apoptosis, miR-4790-3p inhibited it, while ZNF225 promoted it. It was also found that HCC tissues were characterized by higher expression of miR-4790-3p and lower expression of ZNF225; HCC tissues were also characterized by higher autophagic flux. We, thus, conclude that the potentiated anticancer effect of the everolimus and Ku0063794 combination therapy is strongly associated with reduced autophagy resulting from diminished expression of miR-4790-3p, as well as higher expression of ZNF225.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Everolimo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Morfolinas/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/farmacologia , Apoptose/genética , Autofagia/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sinergismo Farmacológico , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo
12.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804063

RESUMO

Besides its insulinotropic actions on pancreatic ß cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron-IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.


Assuntos
Neuropatias Diabéticas/tratamento farmacológico , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Animais , Movimento Celular/genética , Sobrevivência Celular/genética , Cromonas/farmacologia , Técnicas de Cocultura , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/patologia , Exenatida/genética , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Morfolinas/farmacologia , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/lesões
13.
Biomed Res Int ; 2021: 6615502, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33778070

RESUMO

Cardiac hypertrophy (CH) is a common cause of sudden cardiac death and heart failure, resulting in a significant medical burden. The present study is aimed at exploring potential CH-related pathways and the key downstream effectors. The gene expression profile of GSE129090 was obtained from the Gene Expression Omnibus database (GEO), and 1325 differentially expressed genes (DEGs) were identified, including 785 upregulated genes and 540 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis of DEGs were then performed. Although there were no pathways enriched by downregulated genes, many CH-related pathways were identified by upregulated genes, including PI3K-Akt signaling pathway, extracellular matrix- (ECM-) receptor interaction, regulation of actin cytoskeleton, and hypertrophic cardiomyopathy (HCM). In the deeper analysis of PI3K-Akt signaling pathway, we found all the signaling transduction pointed to B cell lymphoma-2- (Bcl-2-) mediated cell survival. We then demonstrated that PI3K-Akt signaling pathway was indeed activated in cardiac hypertrophy. Furthermore, no matter LY294002, an inhibitor of the PI3K/AKT signaling pathway, or Venetoclax, a selective Bcl-2 inhibitor, protected against cardiac hypertrophy. In conclusion, these data indicate that Bcl-2 is involved in cardiac hypertrophy as a key downstream effector of PI3K-Akt signaling pathway, suggesting a potential therapeutic target for the clinical management of cardiac hypertrophy.


Assuntos
Cardiomegalia/metabolismo , Bases de Dados de Ácidos Nucleicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/prevenção & controle , Cromonas/farmacologia , Masculino , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia
14.
FASEB J ; 35(4): e21264, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33715230

RESUMO

Enhanced glucose uptake is coupled with elevated aerobic glycolysis (the Warburg effect) in cancer cells and is closely correlated with increased tumor aggressiveness and poor prognosis. We previously discovered that ATM, a protein kinase deficient in Ataxia-telangiectasia (A-T) disease, is an insulin-responsive protein that participates in insulin-mediated glucose uptake in muscle cells by stimulating glucose transporter 4 (GLUT4) translocation. However, the role of ATM in glucose uptake and tumorigenesis of cancer cells is unclear. In the present study, we found that aggressive breast and prostate cancer cell lines with overactivated Akt activity exhibit enhanced glucose uptake and GLUT1 translocation upon insulin treatment, and KU-55933, a specific inhibitor of ATM, inhibits insulin-mediated glucose uptake by blocking translocation of GLUT1 to the cell surface. KU-55933 also inhibits aerobic glycolysis and ATP production in these cells. Moreover, KU-55933 induces apoptosis and inhibits motility of cancer cells by inhibiting glucose uptake. Our results showed that while high concentration of glucose and insulin promote the expression of a mesenchymal biomarker (vimentin) in these cancer cells, KU-55933 strongly inhibits its expression as well as epithelial to mesenchymal transition. The roles of ATM in stimulating glucose uptake, glycolysis, motility, and proliferation of cancer cells were demonstrated by knocking-down ATM in these cells. KU-55933 treatment also inhibits tumor growth and metastasis in vivo in mouse mammary tumors through inhibition of GLUT1 translocation and vimentin expression. These results suggest that ATM acts as a promoter of tumorigenesis in cancer cells with overactivated Akt, and KU-55933 induces apoptosis and inhibits motility by blocking GLUT1-mediated glucose uptake and glycolysis in these cancer cells, which may lead to the use of KU-55933 and its analogs as new preventive or therapeutic agents against cancer.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pironas/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/genética
15.
Nat Commun ; 12(1): 1876, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: covidwho-1152854

RESUMO

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.


Assuntos
COVID-19/patologia , Ciclo do Ácido Cítrico/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular , Chlorocebus aethiops , Glucose/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Pulmão/virologia , Morfolinas/farmacologia , Naftiridinas/farmacologia , Pirimidinas/farmacologia , Piruvato Carboxilase/biossíntese , SARS-CoV-2/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
16.
Psychopharmacology (Berl) ; 238(6): 1657-1669, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33715044

RESUMO

RATIONALE: Nucleus cuneiformis (NC), a reticular nucleus of the midbrain, is a part of the descending pain modulatory system and therefore has an important role in pain perception. OBJECTIVES: Considering the abundance of GABAA and cannabinoid receptors in the NC and also the bidirectional roles for GABA in controlling nociception, the present study examined the effects of bilateral intra-NC microinjection of different doses of the GABAA receptor agonist, muscimol, and the GABAA receptor antagonist, bicuculline, on pain modulation using formalin test. We also assessed interaction between canabinergic and GABAergic systems in the NC during this test. METHODS: Rats were exposed to intra-NC microinjection of bicuculline (50,100, and 200 ng/side) or muscimol (60, 120, and 240 ng/side) and then subjected to the formalin test. In another set of experiments, the effects of muscimol (60 ng/side) or bicuculline (50 ng/side) administration 5 min before a cannabinoid receptor agonist WIN 55,212-2 (5, 10, and 20 µg/side) microinjection into NC on the formalin test were evaluated. RESULTS: Microinjection of bicuculline and muscimol into the NC decreased and increased pain responses, respectively, in a dose-dependent manner during both phases of the test. Microinjection of WIN 55,212-2 into the NC significantly reduced pain responses in a dose-dependent manner. Microinjection of bicuculline or muscimol in combination with WIN 55,212-2 into the NC respectively potentiated and attenuated WIN 55,212-2-induced antinociception in the formalin test. CONCLUSIONS: This study shows that GABA in the NC is involved in pain modulation and suggests the existence of a GABAA-mediated inhibitory system in the NC on pain control. Furthermore, it seems that the antinociceptive effect of WIN 55,212-2 in the formalin test is mediated partly by the activity of local GABAA receptors in the NC.


Assuntos
Benzoxazinas/farmacologia , Bicuculina/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Morfolinas/farmacologia , Muscimol/farmacologia , Naftalenos/farmacologia , Animais , Bicuculina/administração & dosagem , Canabinoides/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Nociceptividade/efeitos dos fármacos , Dor/tratamento farmacológico , Medição da Dor , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo
17.
Nat Neurosci ; 24(5): 658-666, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33737752

RESUMO

Cannabinoids reduce tremor associated with motor disorders induced by injuries and neurodegenerative disease. Here we show that this effect is mediated by cannabinoid receptors on astrocytes in the ventral horn of the spinal cord, where alternating limb movements are initiated. We first demonstrate that tremor is reduced in a mouse model of essential tremor after intrathecal injection of the cannabinoid analog WIN55,212-2. We investigate the underlying mechanism using electrophysiological recordings in spinal cord slices and show that endocannabinoids released from depolarized interneurons activate astrocytic cannabinoid receptors, causing an increase in intracellular Ca2+, subsequent release of purines and inhibition of excitatory neurotransmission. Finally, we show that the anti-tremor action of WIN55,212-2 in the spinal cords of mice is suppressed after knocking out CB1 receptors in astrocytes. Our data suggest that cannabinoids reduce tremor via their action on spinal astrocytes.


Assuntos
Astrócitos/metabolismo , Tremor Essencial/metabolismo , Interneurônios/metabolismo , Receptores de Canabinoides/metabolismo , Medula Espinal/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Benzoxazinas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Modelos Animais de Doenças , Interneurônios/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Naftalenos/farmacologia , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
18.
Life Sci ; 274: 119363, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33737083

RESUMO

AIMS: Post-fracture calcium and phosphorus excretion is greater than influx, which might be caused by stress. Glucocorticoid is known to enhance calcium and phosphorous excretion, and hydrogen sulfide (H2S) has been shown to exert inhibitory effects on glucocorticoid. Therefore, this study explored whether H2S could inhibit calcium and phosphorus loss after fracture by regulating glucocorticoid and/or its receptor. MAIN METHODS: The following properties were analyzed in rats with femur fractures: serum and urinary calcium and phosphorus (by colorimetry); bone turnover markers alkaline phosphatase, serum type 1 collagen amino terminal peptide, type 1 procollagen carboxy terminal peptide, and anti-tartaric acid phosphatase (by ELISA); factors related to calcium-phosphorus metabolism including glucocorticoid, parathyroid hormone, calcitonin, fibroblast growth factor 23, and 1,25(OH)2D3 (by ELISA); and sulfhydration of glucocorticoid receptor α in the kidney (by immunoprecipitation linked biotin-switch assay), after supplementing with mifepristone, the H2S donor GYY4137 or H2S generating enzyme inhibitors aminooxyacetic acid and propargylglycine. KEY FINDINGS: Serum H2S decreased and glucocorticoid secretion increased in rats post-fracture. The glucocorticoid receptor inhibitor mifepristone partly blunted calcium and phosphorus loss. Furthermore, supplementation with GYY4137 reduced glucocorticoid secretion; inhibited glucocorticoid receptor α activity by sulfhydration; downregulated vitamin D 1α-hydroxylase expression; and upregulated 24-hydroxylase, calbindin-D28k, and sodium phosphate cotransporter 2a expression in the kidney; thereby inhibiting calcium and phosphorus loss induced by fracture. Moreover, inhibiting endogenous H2S generation showed opposite effects. SIGNIFICANCE: Our findings suggest that H2S antagonized calcium and phosphorus loss after fracture by reducing glucocorticoid secretion and inhibiting glucocorticoid receptor α activity by sulfhydration.


Assuntos
Cálcio/metabolismo , Fraturas do Fêmur/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Fósforo/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/patologia , Gasotransmissores/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley
19.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669790

RESUMO

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7-23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14-16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7-23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal "dual action" for ß-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Imidazóis/farmacologia , Morfolinas/farmacologia , Sítio Alostérico , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Imidazóis/síntese química , Imidazóis/química , Ligantes , Testes de Sensibilidade Microbiana , Conformação Molecular , Simulação de Acoplamento Molecular , Morfolinas/síntese química , Morfolinas/química , Solubilidade , Relação Estrutura-Atividade , Água
20.
Nat Commun ; 12(1): 1876, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767183

RESUMO

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.


Assuntos
COVID-19/patologia , Ciclo do Ácido Cítrico/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular , Chlorocebus aethiops , Glucose/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Pulmão/virologia , Morfolinas/farmacologia , Naftiridinas/farmacologia , Pirimidinas/farmacologia , Piruvato Carboxilase/biossíntese , SARS-CoV-2/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
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