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1.
Arch Biochem Biophys ; 679: 108156, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629711

RESUMO

Recently, growing attention has been given to new classes of bioactive lipid mediators derived from ω-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), especially in the context of their role as endogenous signal modulators. One such molecule is 17-oxo-DHA, generated from DHA by the action of COX2 and a dehydrogenase. The redox-sensitive transcription factor, Nrf2 plays a key role in cellular stress responses. In the present study, the effects of 17-oxo-DHA on Nrf2-mediated expression of cytoprotective enzymes were examined in mouse skin in vivo and cultured murine epidermal JB6 cells. Topical application of 17-oxo-DHA markedly elevated the nuclear localization of Nrf2 and expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 in hairless mouse skin. In contrast to 17-oxo-DHA, the non-electrophilic metabolic precursor 17-hydroxy-DHA was a much weaker inducer of Nrf2 activation and its target protein expression. Likewise, 17-oxo-DHA significantly enhanced nuclear translocation and transcriptional activity of Nrf2 with concomitant upregulation of HO-1 expression in cultured JB6 cells. 17-Oxo-DHA was a much stronger inducer of Nrf2-mediated antioxidant response than its parent molecule, DHA. HO-1 expression was abolished in Nrf2 knockdown JB6 cells or embryo fibroblasts from Nrf2 knock out mice. 17-Oxo-DHA also markedly reduced the level of Keap1 protein by inducing ubiquitination. Mutation of Cys151 and Cys273 in Keap1 abrogated 17-oxo-DHA-induced ubiquitination and proteasome-mediated degradation of Keap1 as well as HO-1 expression, suggesting that these cysteine residues are putative sites for 17-oxo-DHA binding. Further, Keap1 degradation stimulated by 17-oxo-DHA coincided with accumulation of the autophagy substrate, p62/SQSTM1.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Epiderme/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Ácidos Docosa-Hexaenoicos/metabolismo , Epiderme/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica
2.
Bioorg Med Chem Lett ; 28(5): 915-921, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29422388

RESUMO

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [18F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [18F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.


Assuntos
Acetazolamida/química , Anidrase Carbônica IX/análise , Inibidores da Anidrase Carbônica/química , Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Tomografia por Emissão de Pósitrons , Acetazolamida/síntese química , Acetazolamida/farmacocinética , Animais , Anidrase Carbônica IX/biossíntese , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/farmacocinética , Carcinoma de Células Renais/diagnóstico , Radioisótopos de Flúor , Humanos , Neoplasias Renais/diagnóstico , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/enzimologia , Distribuição Tecidual
3.
Mol Pharmacol ; 90(5): 530-539, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27573669

RESUMO

α-Pinene is a major monoterpene of the pine tree essential oils. It has been reported that α-pinene shows anxiolytic and hypnotic effects upon inhaled administration. However, hypnotic effect by oral supplementation and the molecular mechanism of α-pinene have not been determined yet. By combining in vivo sleep behavior, ex vivo electrophysiological recording from brain slices, and in silico molecular modeling, we demonstrate that (-)-α-pinene shows sleep enhancing property through a direct binding to GABAA-benzodiazepine (BZD) receptors by acting as a partial modulator at the BZD binding site. The effect of (-)-α-pinene on sleep-wake profiles was evaluated by recording electroencephalogram and electromyogram. The molecular mechanism of (-)-α-pinene was investigated by electrophysiology and molecular docking study. (-)-α-pinene significantly increased the duration of non-rapid eye movement sleep (NREMS) and reduced the sleep latency by oral administration without affecting duration of rapid eye movement sleep and delta activity. (-)-α-pinene potentiated the GABAA receptor-mediated synaptic response by increasing the decay time constant of sIPSCs in hippocampal CA1 pyramidal neurons. These effects of (-)-α-pinene on sleep and inhibitory synaptic response were mimicked by zolpidem, acting as a modulator for GABAA-BZD receptors, and fully antagonized by flumazenil, an antagonist for GABAA-BZD receptor. (-)-α-pinene was found to bind to aromatic residues of α1- and -γ2 subunits of GABAA-BZD receptors in the molecular model. We conclude that (-)-α-pinene enhances the quantity of NREMS without affecting the intensity of NREMS by prolonging GABAergic synaptic transmission, acting as a partial modulator of GABAA-BZD receptors and directly binding to the BZD binding site of GABAA receptor.


Assuntos
Benzodiazepinas/metabolismo , Movimentos Oculares/efeitos dos fármacos , Monoterpenos/farmacologia , Pinus/química , Óleos de Plantas/farmacologia , Receptores de GABA-A/metabolismo , Sono/efeitos dos fármacos , Animais , Monoterpenos Bicíclicos , Sítios de Ligação , Flumazenil/química , Flumazenil/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Modelos Moleculares , Monoterpenos/química , Pentobarbital , Piridinas/química , Piridinas/farmacologia , Sono REM/efeitos dos fármacos , Fatores de Tempo , Vigília/efeitos dos fármacos , Zolpidem
4.
Bioorg Med Chem Lett ; 26(14): 3307-3312, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27241691

RESUMO

A series of new 2-anilinoquinolines possessing 3-(morpholino or 4-methylpiperazin-1-yl)propoxy moiety at C5 of quinoline has been designed and synthesized as potential anticancer agents. Their antiproliferative activities were evaluated against a panel of 60 cancer cell lines at NCI and compared with gefitinib as a reference compound. Most of the tested compounds displayed potent and broad spectrum antiproliferative activities. Compounds 7d, 7f and 7g showed strong inhibitory and lethal effects at 10µM concentration. Moreover, they manifested superior potencies and efficacies than gefitinib across the most tested cell lines. Compound 7d, with 4-chloro-3-trifluoromethylphenyl group, proved to be the most potent and efficacious derivative in this series, with mean GI50 and TGI values of 1.62µM and 3.47µM, respectively. Kinase screening of 7d against a panel of 47 oncogenic kinases revealed its selective inhibitory effect (96% inhibition) towards TrkA kinase. Furthermore, the most potent compounds showed low cytotoxic effects against HFF-1 normal cell line.


Assuntos
Antineoplásicos/farmacologia , Morfolinos/farmacologia , Naftalenos/farmacologia , Fosfotransferases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Morfolinos/química , Naftalenos/química , Fosfotransferases/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinolinas/química , Relação Estrutura-Atividade
5.
J Comput Aided Mol Des ; 30(8): 625-37, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27600555

RESUMO

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe243(5.39) is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na(+) allosteric site in contrast to PAR2 agonist that showed Na(+) relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.


Assuntos
Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptor PAR-2/química , Alinhamento de Sequência
6.
J Enzyme Inhib Med Chem ; 31(1): 158-66, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25807298

RESUMO

Herein, we report compound KST9046, a new agent possessing quinazoline-urea scaffold. Preliminary biological evaluation done by the National Cancer Institute (NCI), USA, showed a great inhibitory effect of KST9046 over the 60 cell-line tumor panel. Accordingly, it was selected for a dose-response assay; a broad spectrum antiproliferative activity with GI(50) ranging from 1.3 to 3.9 µM was exerted. To explore a potential kinase inhibitory effect, KST9046 was applied at a single dose of 10 µM against a kinase panel of 347 different enzymes representing >50% of the predicted human protein kinome. Interestingly, selective inhibition of 76% was observed on DDR1 kinase. Further, KST9046 showed an IC(50) value of 4.38 µM for DDR1. A molecular docking model presented KST9046 as a potential type III inhibitor for DDR1 kinase with an allosteric mode of interaction, which may offer an explanation for its selectivity. As further investigation, CYP450 assay was carried out for KST9046, it showed a promising toxicity profile against four different isoforms. Based on these findings, KST9046 can be further evaluated as a promising safe new hit for the development of broad spectrum anticancer agents with a selectivity for DDR1 kinase.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Ureia/análogos & derivados , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptor com Domínio Discoidina 1 , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinazolinas/síntese química , Quinazolinas/química , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Ureia/síntese química , Ureia/química , Ureia/farmacologia
7.
Bioorg Med Chem Lett ; 25(10): 2162-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25881828

RESUMO

A series of novel picolinamide based benzothiazoles (17 final compounds), targeting both wild-type and the most resistant T315I mutant of Bcr-Abl kinase, has been designed and synthesized. Moreover, a selected array (8 compounds) was evaluated for its antiproliferative activity over a panel of 60 cancer cell lines. Compound 5l was the most potent derivative against both native and T315I mutant ABL with IC50 values of 18.2 and 39.9nM, respectively, and showed highly selective inhibitory activity (89.8%) towards the Bcr-Abl dependent leukemia cell (K-562) at 10µM concentration. Significance of C6-oxypicolinamide moiety and SAR study for the C2 aliphatic side chain of benzothiazole are discussed in detail.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Oncogênicas v-abl/antagonistas & inibidores , Ácidos Picolínicos/química , Amidas/química , Benzotiazóis/química , Linhagem Celular Tumoral , Humanos
8.
Bioorg Med Chem Lett ; 25(7): 1546-51, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25765911

RESUMO

A series of azacyclic compounds substituted with isoxazole and 5-substituted isoxazolines were synthesized as acyclic modifications of the oxime class M1 mACh receptor agonist. Among them, 3-(tetrahydropyrin-3-yl)-5-(2-pyrrolodin-1-yl)isoxazoline compound 4f displayed potent and selective M1 mACh receptor agonist activity in the functional calcium mobilization assay (EC50=31 nM). Introduction of 2-pyrrolidinone and 3-tetrahydropyridine groups are pivotal to the high potency. Moreover, 4f was found to facilitate non-amyloidogenic amyloid precursor protein (APP) processing by significantly increasing ERK1/2 phosphorylation and sAPPα secretion, known disease-modifying effects related to M1 mAChR agonists in Alzheimer's disease (AD).


Assuntos
Isoxazóis/farmacologia , Receptor Muscarínico M1/agonistas , Relação Dose-Resposta a Droga , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Estrutura Molecular , Relação Estrutura-Atividade
9.
Bioorg Med Chem ; 23(24): 7717-27, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26631441

RESUMO

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptor and its activation initiates diverse inflammatory responses. Recent studies suggest that antagonists of PAR2 may provide a novel therapeutic strategy for inflammatory diseases. In this study, we have developed a series of 2-aryloxy-4-amino-quinazoline derivatives as PAR2 antagonists and examined their effects against LPS-induced inflammatory responses in RAW 264.7 macrophages. Among these derivatives, compound 2f displayed the greatest antagonistic activity with the IC50 value of 2.8µM. Binding modes of the newly identified PAR2 antagonists were analyzed by molecular docking using IFD/MM-GBSA methods in the putative binding site of PAR2 homology model. Moreover, 2f demonstrated significant inhibitory effects on the LPS-activated pro-inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) through the regulation of various intracellular signaling pathways involving nuclear factor-κB (NF-κB), activator protein 1 (AP-1) and the mitogen-activated protein kinases (MAPK). Furthermore, administration of 2f significantly reduced the mortality of LPS-induced sepsis in mice. These results provide useful insights into the development of novel PAR2 antagonists with anti-inflammatory activity in vitro and in vivo.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Quinazolinas/química , Quinazolinas/uso terapêutico , Receptor PAR-2/antagonistas & inibidores , Sepse/tratamento farmacológico , Aminação , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Dinoprostona/imunologia , Interleucina-1beta/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , NF-kappa B/imunologia , Quinazolinas/farmacologia , Receptor PAR-2/imunologia , Sepse/imunologia , Fator de Necrose Tumoral alfa
10.
Bioorg Med Chem ; 23(19): 6486-96, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26337020

RESUMO

We have synthesized three categories of α,ß-unsaturated carbonyl derivatives and evaluated their MAO-A and MAO-B inhibitory activities. Among them, compound 10b including α,ß-unsaturated ketone group showed the most potent and selective MAO-B inhibitory activity (IC50 human MAO-B 16 nM, >6000-fold selective vs MAO-A) and compound 10b exhibited good reversibility compared with selegiline, a well-known irreversible MAO-B inhibitor. However, both α,ß-unsaturated amide and ester derivatives exhibited weaker MAO-B inhibition potencies. The docking studies provided insights into the possible binding modes and the key interaction sites of the synthesized MAO-B inhibitors.


Assuntos
Cetonas/química , Inibidores da Monoaminoxidase/síntese química , Monoaminoxidase/química , Sítios de Ligação , Sequestradores de Radicais Livres/síntese química , Sequestradores de Radicais Livres/química , Humanos , Peróxido de Hidrogênio/química , Cetonas/síntese química , Cetonas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
12.
Biosens Bioelectron ; 227: 115169, 2023 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-36827795

RESUMO

The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium ('SpACE-CCM'), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified 'interferon-α+alanine-aspartate' as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.


Assuntos
Técnicas Biossensoriais , COVID-19 , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Pandemias , Ligação Proteica , Anticorpos Neutralizantes/metabolismo , Técnicas de Cultura de Células , Glicoproteína da Espícula de Coronavírus
13.
Bioorg Med Chem ; 20(11): 3493-501, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22543233

RESUMO

Licorice (Glycyrrhiza glabra, GG) is one of the most frequently used herbal medicines worldwide, and its various biological activities have been widely studied. GG is reported to have neurological properties such as antidepressant, anxiolytic, and anticonvulsant effects. However, its hypnotic effects and the mechanism of GG and its active compounds have not yet been demonstrated. In this study, GG ethanol extract (GGE) dose-dependently potentiated pentobarbital-induced sleep and increased the amount of non-rapid eye movement sleep in mice without decreasing delta activity. The hypnotic effect of GGE was completely inhibited by flumazenil, which is a well-known γ-aminobutyric acid type A-benzodiazepine (GABA(A)-BZD) receptor antagonist, similar to other GABA(A)-BZD receptor agonists (e.g., diazepam and zolpidem). The major flavonoid glabrol was isolated from the flavonoid-rich fraction of GGE; it inhibited [(3)H] flumazenil binding to the GABA(A)-BZD receptors in rat cerebral cortex membrane with a binding affinity (K(i)) of 1.63 µM. The molecular structure and pharmacophore model of glabrol and liquiritigenin indicate that the isoprenyl groups of glabrol may play a key role in binding to GABA(A)-BZD receptors. Glabrol increased sleep duration and decreased sleep latency in a dose-dependent manner (5, 10, 25, and 50mg/kg); its hypnotic effect was also blocked by flumazenil. The results imply that GGE and its flavonoid glabrol induce sleep via a positive allosteric modulation of GABA(A)-BZD receptors.


Assuntos
Flavonoides/farmacologia , Glycyrrhiza/química , Hipnóticos e Sedativos/farmacologia , Extratos Vegetais/farmacologia , Receptores de GABA-A/metabolismo , Animais , Relação Dose-Resposta a Droga , Etanol/química , Movimentos Oculares/efeitos dos fármacos , Flavanonas/química , Flavanonas/farmacologia , Flavonoides/química , Flavonoides/isolamento & purificação , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Hipnóticos e Sedativos/química , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Pentobarbital/farmacologia , Extratos Vegetais/química , Preparações de Plantas , Sono/efeitos dos fármacos
14.
J Cancer Prev ; 27(1): 68-76, 2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35419302

RESUMO

Cancer-associated fibroblasts (CAFs) represent a major component of the tumor microenvironment and interplay with cancer cells by secreting cytokines, growth factors and extracellular matrix proteins. When estrogen receptor-negative breast cancer MDA-MB-231 cells were treated with the CAF-conditioned medium (CAF-CM), Akt and STAT3 involved in cell proliferation and survival were activated through phosphorylation. CAFs secrete fibroblast growth factor 2 (FGF2), thereby stimulating breast cancer cell progression. Akt activation induced by CAF-CM in MDA-MB-231 cells was abolished when FGF2-neutralizing antibody was added. Treatment of MDA-MB-231 cells directly with FGF2 enhanced the phosphorylation of Akt and the FGF receptor (FGFR) substrate, FRS2α. These events were abrogated by siRNA-mediated silencing of FGFR1. In a xenograft mouse model, co-injection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced activation of Akt. Stable knockdown of FGFR1 blunted Akt phosphorylation in xenograft tumors. MDA-MB-231 cells co-cultured with CAFs or directly stimulated with FGF2 exhibited enhanced nuclear localization of FGFR1. Notably, FGF2 stimulation produced reactive oxygen species (ROS) accumulation in MDA-MB-231 cells, and FGF2-induced nuclear accumulation of FGFR1 was abrogated by the ROS scavenging agent, N-acetylcysteine.

15.
J Cancer Prev ; 26(3): 207-217, 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34703823

RESUMO

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

16.
Cancer Lett ; 500: 147-160, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33278500

RESUMO

Persistent activation of STAT3 and Nrf2 is considered to stimulate the aggressive behavior of basal-like breast cancer (BLBC). However, the precise mechanism underlying sustained overactivation of these transcription factors and their roles in breast cancer progression remain elusive. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and Nrf2 mRNA were correlated with elevated expression of P-STAT3Y705 and Nrf2 target proteins in breast cancer patients. Our present study demonstrates a unique interaction between Nrf2 and STAT3 in the maintenance and progression of BLBC. RNA sequencing analysis identified the gene encoding IL-23A upregulated by concurrent binding of STAT3 and Nrf2 to its promoter. IL-23A depletion also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, the STAT3-Nrf2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer.


Assuntos
Neoplasias da Mama/genética , Interleucina-23/genética , Fator 2 Relacionado a NF-E2/genética , Fator de Transcrição STAT3/genética , Animais , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Ativação Transcricional/genética
17.
FEBS Lett ; 595(5): 604-622, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33452674

RESUMO

Signal transducer and activator of transcription 3 (STAT3) has been considered as a potential target for development of anticancer therapeutics. Here, we report a novel mechanism by which the cyclopentenone prostaglandin, 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ) functions as an allosteric inhibitor of STAT3. 15d-PGJ2 inhibits phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3 in H-Ras-transformed human mammary epithelial cells (MCF10A-Ras) through the Michael addition reaction at cysteine 259 of STAT3. Comparative studies with 15d-PGJ2 analogues reveal that both C12-C13 and C9-C10 double bonds conjugated to the carbonyl group in the cyclopentenone ring of 15d-PGJ2 are essential for STAT3 binding. Antiproliferative and pro-apoptotic activities of 15d-PGJ2 in MCF10A-Ras cells are attributable to covalent modification of STAT3 on Cys259, and mimic the effects induced by mutation of this amino acid.


Assuntos
Antineoplásicos/farmacologia , Cisteína/química , Células Epiteliais/efeitos dos fármacos , Prostaglandina D2/análogos & derivados , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Sequência de Aminoácidos , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cisteína/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Fosforilação/efeitos dos fármacos , Prostaglandina D2/química , Prostaglandina D2/farmacologia , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica
18.
Theranostics ; 11(18): 8755-8770, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34522210

RESUMO

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes. Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models. Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the ß-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype. Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.


Assuntos
Neoplasias Colorretais/metabolismo , Antígenos Comuns de Leucócito/genética , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/fisiopatologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/fisiologia , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Estudos Retrospectivos , Células-Tronco/metabolismo , Transcriptoma/genética
19.
Biomedicines ; 8(10)2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053804

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an α,ß-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.

20.
Biochem Pharmacol ; 173: 113820, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31972171

RESUMO

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,ß-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,ß-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.


Assuntos
Curcumina/farmacologia , Cisteína/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Curcumina/química , Curcumina/metabolismo , Embrião de Mamíferos/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos Knockout , Simulação de Acoplamento Molecular , Estrutura Molecular , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , Pele/efeitos dos fármacos , Pele/metabolismo
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