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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205045

RESUMO

SGLT-2i's exert direct anti-inflammatory and anti-oxidative effects on resting endothelial cells. However, endothelial cells are constantly exposed to mechanical forces such as cyclic stretch. Enhanced stretch increases the production of reactive oxygen species (ROS) and thereby impairs endothelial barrier function. We hypothesized that the SGLT-2i's empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) exert an anti-oxidative effect and alleviate cyclic stretch-induced endothelial permeability in human coronary artery endothelial cells (HCAECs). HCAECs were pre-incubated with one of the SGLT-2i's (1 µM EMPA, 1 µM DAPA and 3 µM CANA) for 2 h, followed by 10% stretch for 24 h. HCAECs exposed to 5% stretch were considered as control. Involvement of ROS was measured using N-acetyl-l-cysteine (NAC). The sodium-hydrogen exchanger 1 (NHE1) and NADPH oxidases (NOXs) were inhibited by cariporide, or GKT136901, respectively. Cell permeability and ROS were investigated by fluorescence intensity imaging. Cell permeability and ROS production were increased by 10% stretch; EMPA, DAPA and CANA decreased this effect significantly. Cariporide and GKT136901 inhibited stretch-induced ROS production but neither of them further reduced ROS production when combined with EMPA. SGLT-2i's improve the barrier dysfunction of HCAECs under enhanced stretch and this effect might be mediated through scavenging of ROS. Anti-oxidative effect of SGLT-2i's might be partially mediated by inhibition of NHE1 and NOXs.


Assuntos
Células Endoteliais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Transporte de Sódio-Glucose/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Compostos Benzidrílicos/farmacologia , Canagliflozina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucosídeos/farmacologia , Guanidinas/farmacologia , Humanos , Inflamação/genética , Inflamação/patologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Estresse Oxidativo/genética , Pirazóis/farmacologia , Piridonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Transporte de Sódio-Glucose/genética , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Trocador 1 de Sódio-Hidrogênio/genética , Estresse Mecânico , Sulfonas/farmacologia
2.
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
3.
Life Sci ; 280: 119666, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087279

RESUMO

AIMS: The preclinical evaluation of innovative drugs plays an important role in the new drugs development. As a derivative of pirfenidone (PFD), mefunidone (MFD) has shown better anti-fibrosis and anti-inflammatory activity in both cell lines and animal models. To support the clinical investigations of MFD, the metabolic characterization of MFD was initially evaluated in preclinical models. MAIN METHODS: The potential metabolites of MFD were analyzed by LC-MS/MS methods. The induction effect of MFD on CYP1A2, CYP2B6, and CYP3A4 was performed in primary human hepatocytes, and the inhibition of CYP enzymes by MFD was also evaluated in human liver microsomes. Finally, the pharmacokinetic profiles of MFD were assessed in SD rats after the rats had received multiple doses (62.5 mg/kg) of MFD. KEY FINDINGS: MFD was metabolized in three pathways including oxidation, N-demethylation, and hydroxylation. Except for slight inhibition on the activity of CYP2D6, MFD exerted no effect on other CYP enzymes. Moreover, drug accumulation of MFD was not observed in rats after repeated dosing of MFD. SIGNIFICANCE: MFD was first discovered in preclinical investigations without inducing and inhibiting metabolic enzymes. This work provides some important information about the metabolic characterization of MFD for its further clinical investigations.


Assuntos
Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Hepatócitos/metabolismo , Piperazinas/metabolismo , Piperazinas/farmacocinética , Piridonas/metabolismo , Piridonas/farmacocinética , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Piperazinas/farmacologia , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley
4.
FASEB J ; 35(7): e21497, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34152015

RESUMO

Despite the increasing understanding of the pathophysiology of hepatic fibrosis, the therapies to combat it remain inadequate. Fluorofenidone (AKF-PD) is a novel pyridone agent able to ameliorate hepatic fibrosis in an experimental hepatic fibrosis model induced by dimethylnitrosamine. However, the underlying mechanism remains to be further elucidated. In light of the critical role of the NF-κB pathway in inflammation and hepatic fibrosis, together with the preliminary finding that AKF-PD decreases the release of proinflammatory cytokines in the endotoxemia and unilateral ureteral occlusion model, the aim of this study was to explore whether AKF-PD exerts an antifibrotic effect in hepatic fibrosis by inhibiting inflammation and suppressing the activation of the NF-κB pathway in vivo and in vitro. To test this possibility, the effect of AKF-PD on hepatic fibrosis models induced by both carbon tetrachloride (CCL4 ) and porcine serum (PS) was investigated. Our results showed that AKF-PD treatment ameliorated hepatic injury and fibrosis in both models. Furthermore, the administration of AKF-PD induced a robust anti-inflammatory reaction revealed by the downregulation of the proinflammatory cytokines as well as the suppression of the infiltration of inflammatory cells in the fibrotic liver. The analysis of the mechanism of action demonstrated that the attenuation of the production of proinflammatory cytokines and chemokines mediated by AKF-PD in vivo and in vitro were accompanied by the suppression in the activation of the NF-κB signaling pathway. In conclusion, AKF-PD might be considered as an antifibrotic agent attenuating hepatic inflammation and fibrosis potentially through the suppression of the NF-κB pathway.


Assuntos
Anti-Inflamatórios/farmacologia , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Hepatopatias/prevenção & controle , NF-kappa B/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Piridonas/farmacologia , Animais , Células Cultivadas , Fibrose/metabolismo , Fibrose/patologia , Inflamação/metabolismo , Inflamação/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Ratos , Ratos Sprague-Dawley
5.
Molecules ; 26(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063645

RESUMO

Drug combinations have been the hotspot of the pharmaceutical industry, but the promising applications are limited by the unmet solubility and low bioavailability. In this work, novel cocrystals, consisting of two antithrombotic drugs with poor solubility and low bioavailability in vivo, namely, apixaban (Apx) and quercetin (Que), were developed to discover a potential method to improve the poor solubility and internal absorption of the drug combination. Compared with Apx, the dissolution behavior of Apx-Que (1:1) and Apx-Que-2ACN (1:1:2) was enhanced significantly, while the physical mixture of the chemicals failed to exhibit the advantages. The dissolution improvements of Apx-Que-2ACN could be explained by the fact that the solid dispersion-like structure and column-shaped cage of Que accelerated the access of the solvent to the inner layer of Apx. The fracture of the hydrogen bonds of Apx, which was the joint of the adjacent Que chains, facilitated the break-up of the structures. Besides, the bioavailability of Apx-Que was increased compared with the physical mixture and Apx, and Apx-Que remained stable in high temperature and illumination conditions. Therefore, a drug-drug cocrystal of two antithrombotic agents with poor solubility was developed, which exhibited greatly improved solubility, bioavailability and superior stability, indicating a novel method to overcome the shortages of drug combination.


Assuntos
Cristalização , Combinação de Medicamentos , Pirazóis/farmacologia , Piridonas/farmacologia , Quercetina/farmacologia , Solventes , Animais , Disponibilidade Biológica , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Fibrinolíticos/farmacologia , Ligação de Hidrogênio , Masculino , Preparações Farmacêuticas , Pós , Pirazóis/química , Piridonas/química , Quercetina/química , Ratos , Ratos Sprague-Dawley , Solubilidade , Temperatura , Termogravimetria , Difração de Raios X
6.
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
8.
Lancet Respir Med ; 9(5): 476-486, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33798455

RESUMO

BACKGROUND: Pirfenidone has been shown to slow disease progression in patients with idiopathic pulmonary fibrosis (IPF). However, there are few treatment options for progressive fibrotic interstitial lung diseases (ILDs)) other than IPF. In view of the pathomechanistic and clinical similarities between IPF and other progressive fibrotic ILDs, we aimed to assess the efficacy and safety of pirfenidone in patients with four non-IPF progressive fibrotic ILDs. METHODS: We did a multicentre, double-blind, randomised, placebo-controlled, parallel phase 2b trial (RELIEF) in 17 centres with expertise in ILD in Germany. Eligible participants were patients aged 18-80 years with progressive fibrotic ILD due to four diagnoses: collagen or vascular diseases (ie, connective tissue disease-associated ILDs), fibrotic non-specific interstitial pneumonia, chronic hypersensitivity pneumonitis, or asbestos-induced lung fibrosis. Other eligibility criteria included a forced vital capacity (FVC) of 40-90% predicted, a diffusing capacity of the lung for carbon monoxide of 10-90% predicted, and an annual decline of FVC of at least 5% predicted despite conventional therapy, based on at least three measurements within 6-24 months before enrolment. Patients who had received any previous antifibrotic therapy were excluded. We randomly assigned patients (1:1) to either oral pirfenidone (267 mg three times per day in week 1, 534 mg three times per day in week 2, and 801 mg three times per day thereafter) or matched placebo, added to their ongoing medication. Randomisation was done centrally using permuted block randomisation with varying block sizes stratified by the four diagnostic groups. Patients, investigators, statisticians, monitors, and the study coordinator were masked to treatment assignment until database closure. The placebo-controlled study period was 48 weeks (including up-titration). The primary endpoint was absolute change in percentage of predicted FVC (FVC % predicted) from baseline to week 48 in the intention-to-treat population, with imputation of missing data by the smallest sum of squared differences and attribution of deceased patients to the lowest rank in a rank ANCOVA model. Additionally, we did linear mixed-model repeated measures slope analyses of FVC % predicted longitudinal data over the course of the study as a prespecified sensitivity analysis and post-hoc sensitivity analyses of the primary endpoint in the intention-to-treat population using imputation methods of last observation carried forward [LOCF] and a regression-based multiple imputation procedure. Safety was assessed in all patients who received at least one dose of study medication. This trial is registered with EudraCT 2014-000861-32; DRKS00009822 and is no longer recruiting. FINDINGS: Between April 5, 2016, and Oct 4, 2018, we randomly assigned 127 patients to treatment: 64 to pirfenidone, 63 to placebo. After 127 patients had been randomised, the study was prematurely terminated on the basis of an interim analysis for futility triggered by slow recruitment. After 48 weeks and in the overall population of 127 patients, rank ANCOVA with diagnostic group included as a factor showed a significantly lower decline in FVC % predicted in the pirfenidone group compared with placebo (p=0·043); the result was similar when the model was stratified by diagnostic group (p=0·042). A significant treatment effect was also observed when applying the LOCF and multiple imputation methods to analyses of the primary endpoint. The median difference (Hodges-Lehmann estimate) between pirfenidone and placebo groups for the primary endpoint was 1·69 FVC % predicted (95% CI -0·65 to 4·03). In the linear mixed-model repeated measures slope analysis of FVC % predicted, the estimated difference between treatment and placebo groups from baseline to week 48 was 3·53 FVC % predicted (95% CI 0·21 to 6·86) with imputation of deaths as prespecified, or 2·79 FVC % predicted (95% CI 0·03 to 5·54) without imputation. One death (non-respiratory) occurred in the pirfenidone group (2%) and five deaths (three of which were respiratory) occurred in the placebo group (8%). The most frequent serious adverse events in both groups were infections and infestations (five [8%] in the pirfenidone group, ten [16%] in the placebo group); general disorders including disease worsening (two [3%] in the pirfenidone group, seven [11%] in the placebo group); and cardiac disorders (one ([2%] in the pirfenidone group, 5 [8%] in the placebo group). Adverse events (grade 3-4) of nausea (two patients on pirfenidone, two on placebo), dyspnoea (one patient on pirfenidone, one on placebo), and diarrhoea (one patient on pirfenidone) were also observed. INTERPRETATION: In view of the premature study termination, results should be interpreted with care. Nevertheless, our data suggest that in patients with fibrotic ILDs other than IPF who deteriorate despite conventional therapy, adding pirfenidone to existing treatment might attenuate disease progression as measured by decline in FVC. FUNDING: German Center for Lung Research, Roche Pharma.


Assuntos
Doenças Pulmonares Intersticiais , Fibrose Pulmonar , Piridonas/farmacologia , Testes de Função Respiratória/métodos , Anti-Inflamatórios não Esteroides/farmacologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Monitoramento de Medicamentos/métodos , Término Precoce de Ensaios Clínicos , Feminino , Humanos , Análise de Intenção de Tratamento , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/diagnóstico , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/fisiopatologia , Avaliação de Sintomas/estatística & dados numéricos
9.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922284

RESUMO

BACKGROUND: Melanoma patients stop responding to targeted therapies mainly due to mitogen activated protein kinase (MAPK) pathway re-activation, phosphoinositide 3 kinase/the mechanistic target of rapamycin (PI3K/mTOR) pathway activation or stromal cell influence. The future of melanoma treatment lies in combinational approaches. To address this, our in vitro study evaluated if lower concentrations of Celecoxib (IC50 in nM range) could still preserve the chemopreventive effect on melanoma cells treated with trametinib. MATERIALS AND METHODS: All experiments were conducted on SK-MEL-28 human melanoma cells and BJ human fibroblasts, used as co-culture. Co-culture cells were subjected to a celecoxib and trametinib drug combination for 72 h. We focused on the evaluation of cell death mechanisms, melanogenesis, angiogenesis, inflammation and resistance pathways. RESULTS: Low-dose celecoxib significantly enhanced the melanoma response to trametinib. The therapeutic combination reduced nuclear transcription factor (NF)-kB (p < 0.0001) and caspase-8/caspase-3 activation (p < 0.0001), inhibited microphthalmia transcription factor (MITF) and tyrosinase (p < 0.05) expression and strongly down-regulated the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway more significantly than the control or trametinib group (p < 0.0001). CONCLUSION: Low concentrations of celecoxib (IC50 in nM range) sufficed to exert antineoplastic capabilities and enhanced the therapeutic response of metastatic melanoma treated with trametinib.


Assuntos
Adjuvantes Farmacêuticos/farmacologia , Celecoxib/farmacologia , Inflamação/prevenção & controle , Melanoma/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Piridonas/farmacologia , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
10.
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
11.
Int J Mol Sci ; 22(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921242

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. Present-day treatments have not shown real improvements in reducing the high mortality rate and the short survival of the disease. The average survival is less than 5% after 5 years. New innovative treatments are necessary to curtail the situation. The very dense pancreatic cancer stroma is a barrier that impedes the access of chemotherapeutic drugs and at the same time establishes a pro-proliferative symbiosis with the tumor, thus targeting the stroma has been suggested by many authors. No ideal drug or drug combination for this targeting has been found as yet. With this goal in mind, here we have explored a different complementary treatment based on abundant previous publications on repurposed drugs. The cell surface protein CD44 is the main receptor for hyaluronan binding. Many malignant tumors show over-expression/over-activity of both. This is particularly significant in pancreatic cancer. The independent inhibition of hyaluronan-producing cells, hyaluronan synthesis, and/or CD44 expression, has been found to decrease the tumor cell's proliferation, motility, invasion, and metastatic abilities. Targeting the hyaluronan-CD44 pathway seems to have been bypassed by conventional mainstream oncological practice. There are existing drugs that decrease the activity/expression of hyaluronan and CD44: 4-methylumbelliferone and bromelain respectively. Some drugs inhibit hyaluronan-producing cells such as pirfenidone. The association of these three drugs has never been tested either in the laboratory or in the clinical setting. We present a hypothesis, sustained by hard experimental evidence, suggesting that the simultaneous use of these nontoxic drugs can achieve synergistic or added effects in reducing invasion and metastatic potential, in PDAC. A non-toxic, low-cost scheme for inhibiting this pathway may offer an additional weapon for treating pancreatic cancer.


Assuntos
Adenocarcinoma/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Receptores de Hialuronatos/genética , Hialuronan Sintases/genética , Ácido Hialurônico/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Bromelaínas/uso terapêutico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/antagonistas & inibidores , Hialuronan Sintases/antagonistas & inibidores , Ácido Hialurônico/antagonistas & inibidores , Himecromona/uso terapêutico , Terapia de Alvo Molecular , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Piridonas/farmacologia , Piridonas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos
12.
J Pediatr Surg ; 56(7): 1233-1239, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33863557

RESUMO

PURPOSE: We reported the in vitro and in vivo anti-tumor effects of trametinib, an MEK inhibitor, on neuroblastoma. However, long-term trametinib administration for bulky tumors failed to prevent local relapse. In this study, we established a local minimal residual disease (L-MRD) model to develop an optimal clinical protocol. METHODS: We prepared an l-MRD model by implanting neuroblastoma cells (SK-N-AS) into the renal capsule of nude mice with total tumorectomy or sham operation 14 days later. These mice received post-operative administration of trametinib or vehicle for eight weeks. Relapse was measured once weekly. Flow cytometry was performed with SK-N-AS cells treated by trametinib. RESULTS: Tumorectomy+trametinib dramatically suppressed relapse, and all mice survived during trametinib administration, while other treatments failed to suppress relapse. The survival rates for other groups were 20% in sham+trametinib, 17% in tumorectomy+vehicle, and 0% in sham+vehicle. Relapse occurred in the tumorectomy+trametinib group after withdrawal of trametinib administration. Flow cytometry revealed G1 arrest in SK-N-AS cells treated with trametinib. CONCLUSION: These findings suggested that trametinib was able to suppress relapse from minimal residual tumor cells. Therefore, we propose that trametinib be administered as an option for maintenance therapy after surgical and chemotherapeutic treatments for neuroblastoma in future clinical protocols.


Assuntos
Recidiva Local de Neoplasia , Neuroblastoma , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasia Residual , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico
13.
Aging (Albany NY) ; 13(8): 11665-11677, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879635

RESUMO

Colorectal cancer (CRC), a common malignant tumor in the digestive tract, is a leading cause of cancer-related death. SPRY4 has been reported to act as a tumor suppressor gene in various tumors. This study aims to assess the role of SPRY4 in colorectal cancer (CRC) and uncover its underlying mechanisms. Firstly, the expression levels of SPRY4 were measured in CRC cell lines. SPRY4-overexpressing or silencing plasmids were transfected into CRC cells to regulate its expression level. CCK-8, colony formation, EdU assay, wound-healing and Transwell assays were performed to determine cell proliferation, invasion and migration abilities. Then, apoptosis was measured by flow cytometry analysis, and the expression of apoptosis-related protein was analyzed by western-blotting. Next, the in vivo tumorigenesis assay was performed in nude mice. According to the results, there was a lower expression of SPRY4 in CRC cell lines compared with normal cell line, and the overexpression of SPRY4 significantly suppressed cell proliferation, migration and invasion, and promoted apoptosis in SW480 cells. Moreover, the enhanced proliferation, invasion and migration upon SPRY4 silencing was reversed by EZH2 inhibition. In addition, we found that the overexpression of SPRY4 inhibited tumorigenesis in vivo by diminishing the size and weight of the tumors. Our study indicates that SPRY4 might be a potential tumor suppressor gene and prognostic factor for patients with CRC.


Assuntos
Neoplasias Colorretais/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas do Tecido Nervoso/genética , Piridonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Methods Mol Biol ; 2265: 155-171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704713

RESUMO

Researchers often aim to incorporate microenvironmental variables such as the dimensionality and composition of the extracellular matrix into their cell-based assays. A technical challenge created by introduction of these variables is quantification of single-cell measurements and control of environmental reproducibility. Here, we detail a methodology to quantify viability and proliferation of melanoma cells in 3D collagen-based culture platforms by automated microscopy and 3D image analysis to yield robust, high-throughput results of single-cell responses to drug treatment.


Assuntos
Antineoplásicos/farmacologia , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Processamento de Imagem Assistida por Computador/métodos , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Análise de Célula Única/métodos , Sobrevivência Celular/efeitos dos fármacos , Colágeno , Imidazóis/farmacologia , Melanoma/patologia , Oximas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Esferoides Celulares
15.
Anticancer Res ; 41(3): 1387-1399, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33788730

RESUMO

BACKGROUND/AIM: Despite clinical benefit from treatment with dabrafenib and trametinib in melanoma patients with BRAF mutations, half relapse within months and one-third are unresponsive to treatment. We evaluated the anticancer potential of metformin in combination with trametinib plus paclitaxel, against four melanoma cell lines. MATERIALS AND METHODS: Metformin with trametinib and paclitaxel was tested for effects on cell viability, signaling molecules in MAPK and mTOR pathways, factors involved in epithelial-mesenchymal transition (EMT), and cell motility. RESULTS: The combination of metformin with trametinib and paclitaxel showed differential growth inhibitory effects; synergistic effects were observed in a cell line in which metformin suppresses ERK activity, whereas the combination showed antagonistic effects in a cell line with metformin-induced ERK activation. Trametinib or paclitaxel increased the expression of EMT regulators and melanoma cell motility, which were suppressed by combining metformin with trametinib and paclitaxel. CONCLUSION: The combined treatment of metformin with trametinib and paclitaxel showed divergent effects on melanoma cell viability. Metformin might be useful as a potential adjuvant against cell proliferation and metastatic activity in melanoma patients.


Assuntos
Melanoma/patologia , Metformina/farmacologia , Paclitaxel/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Cutâneas/patologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Melanoma/genética , Melanoma/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo
16.
J Med Chem ; 64(6): 3086-3099, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33689340

RESUMO

Apelin-13 is an endogenous peptidic agonist of the apelin receptor (APJ) receptor with the potential for improving cardiac function in heart failure patients. However, the low plasma stability of apelin-13 necessitates continuous intravenous infusion for therapeutic use. There are several approaches to increase the stability of apelin-13 including attachment of pharmacokinetic enhancing groups, stabilized peptides, and Fc-fusion approaches. We sought a small-molecule APJ receptor agonist approach to target a compound with a pharmacokinetic profile amenable for chronic oral administration. This manuscript describes sequential optimization of the pyrimidinone series, leading to pyridinone 14, with in vitro potency equivalent to the endogenous ligand apelin-13 and with an excellent oral bioavailability and PK profile in multiple preclinical species. Compound 14 exhibited robust pharmacodynamic effects similar to apelin-13 in an acute rat pressure-volume loop model and was advanced as a clinical candidate.


Assuntos
Receptores de Apelina/agonistas , Piridonas/química , Piridonas/farmacologia , Animais , Receptores de Apelina/metabolismo , Cães , Descoberta de Drogas , Haplorrinos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Modelos Moleculares , Piridonas/farmacocinética , Ratos , Ratos Sprague-Dawley
17.
Ann Neurol ; 89(5): 926-941, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33675084

RESUMO

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. METHODS: Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS: The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. INTERPRETATION: Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.


Assuntos
Endotélio Vascular , Malformações Arteriovenosas Intracranianas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Cognição , Dependovirus/genética , Encefalite/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Regulação da Expressão Gênica/genética , Humanos , Malformações Arteriovenosas Intracranianas/complicações , Malformações Arteriovenosas Intracranianas/psicologia , Hemorragias Intracranianas/etiologia , Hemorragias Intracranianas/genética , Imageamento por Ressonância Magnética , Camundongos , Mutação/genética , Neovascularização Patológica/etiologia , Neovascularização Patológica/genética , Desempenho Psicomotor , Piridonas/farmacologia , Pirimidinonas/farmacologia
18.
Mol Med Rep ; 23(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33786626

RESUMO

Paraquat (PQ) is a widely used herbicide that is severely toxic to humans and animals. Pulmonary fibrosis is a disorder that can result from PQ poisoning. Fluorofenidone (AKF­PD) is a novel small molecule pyridone drug with a widespread and clear anti­organ fibrosis effect; however, its mechanism of action on PQ poisoning­induced pulmonary fibrosis is not clear. The purpose of the present study was to investigate the protective effect and underlying mechanism of AKF­PD on PQ poisoning­induced pulmonary fibrosis. Human alveolar epithelial cells (HPAEpiC) and Sprague­Dawley rats were treated with AKF­PD in the presence or absence of PQ. Hematoxylin­eosin and Masson staining were used to observe the morphological changes in lung tissue. Cell Counting Kit­8 and lactate dehydrogenase assays were used to evaluate the viability of HPAEpiC cells. ELISA was used to detect inflammatory factors and the collagen content. Finally, the effects of AKF­PD on pulmonary fibrosis, as well as the underlying mechanisms, were evaluated via western blotting, reverse transcription­quantitative PCR and immunofluorescence analysis. AKF­PD effectively alleviated PQ­induced pulmonary fibrosis and reduced the expression of oxidative stress and inflammatory factors. Moreover, AKF­PD treatment effectively inhibited the PI3K/Akt/mTOR signaling pathway and upregulated autophagy. Overall, these findings suggested that AKF­PD can alleviate PQ­induced inflammation and pulmonary fibrosis by inhibiting the PI3K/Akt/mTOR signaling pathway and by upregulating autophagy.


Assuntos
Autofagia , Fosfatidilinositol 3-Quinases/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Piridonas/uso terapêutico , Transdução de Sinais , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Células Cultivadas , Humanos , Masculino , Estresse Oxidativo , Paraquat/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismo , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/metabolismo
19.
Am J Respir Cell Mol Biol ; 64(6): 722-733, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33689587

RESUMO

B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and ß-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell-secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell-mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.


Assuntos
Linfócitos B/imunologia , Movimento Celular , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/imunologia , Fibrose Pulmonar Idiopática/patologia , Antígenos/metabolismo , Linfócitos B/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Indóis/farmacologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Pneumonia/patologia , Piridonas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/metabolismo
20.
Cancer Med ; 10(8): 2840-2854, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33734579

RESUMO

MX2 is an interferon inducible gene that is mostly known for its antiviral activity. We have previously demonstrated that MX2 is also associated with the tumorigenesis process in melanoma. However, it remains unknown which molecular mechanisms are regulated by MX2 in response to interferon signaling in this disease. Here, we report that MX2 is necessary for the establishment of an interferon-induced transcriptional profile partially through regulation of STAT1 phosphorylation and other interferon-related downstream factors, including proapoptotic tumor suppressor XAF1. MX2 and XAF1 expression tightly correlate in both cultured melanoma cell lines and in patient-derived primary and metastatic tumors, where they also are significantly related with survival. MX2 mediates IFN growth-inhibitory signals in both XAF1 dependent and independent ways and in a cell type and context-dependent manner. Higher MX2 expression renders melanoma cells more sensitive to targeted therapy drugs such as vemurafenib and trametinib; however, this effect is XAF1 independent. In summary, we uncovered a new mechanism in the complex regulation of interferon signaling in melanoma that can influence both survival and response to therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferons/farmacologia , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos/farmacologia , Apoptose , Proteínas Reguladoras de Apoptose/genética , Biomarcadores Tumorais/genética , Proliferação de Células , Sinergismo Farmacológico , Humanos , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Resistência a Myxovirus/genética , Fosforilação , Piridonas/farmacologia , Pirimidinonas/farmacologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Células Tumorais Cultivadas
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