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1.
Cell Physiol Biochem ; 54(1): 126-141, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017483

RESUMO

BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). METHODS: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. RESULTS: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of ß-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. CONCLUSION: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Serotonina/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Células CACO-2 , Carbazóis/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , beta-Naftoflavona/administração & dosagem
2.
Chem Biol Interact ; 312: 108816, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505164

RESUMO

Indirubins E804 (indirubin-3'-(2,3 dihydroxypropyl)-oximether) and 7BIO (7-Bromoindirubin-3'-oxime) are synthetic derivatives of natural indirubin, the active compound in Danggui Longhui Wan, a traditional Chinese remedy for cancer and inflammation. Herein, we explore E804 and 7BIO for their potential to modulate key pro-inflammatory genes and cytokines in LN-18 and T98G glioblastoma cells. High grade gliomas typically secrete large amounts of inflammatory cytokines and growth factors that promote tumor growth in an autocrine fashion. Inflammation is emerging as a key concern in the success of new treatment modalities for glioblastomas. Studies indicate that select indirubin derivatives bind and activate signaling of the AHR pathway, as well as inhibit cyclin-dependent kinases and STAT3 signaling. AHR signaling is involved in hematopoiesis, immune function, cell cycling, and inflammation, and thus may be a possible target for glioma treatment. To determine the significance of the AHR pathway in LN-18 and T98G glioma inflammatory profiles, and on the effects of E804 and 7BIO on these profiles, we used 6,2',4'-trimethoxyflavone (TMF), a putative selective AHR antagonist. It was confirmed that E804 and 7BIO activates the AHR leading to cyp1b1 expression, and that TMF antagonizes expression. We then employed a commercial cancer inflammation and immunity crosstalk qRT-PCR array to screen for anti-inflammatory related properties. TMF alone inhibited expression of ifng, ptsg2, il12b, tnfa, il10, il13, the balance between pd1 and pdl1, and even expression of mhc1a/b. E804 was very potent in suppressing many pro-inflammatory genes, including il1a, il1b, il12a, ptgs2, tlr4, and others. E804 also affected expression of il6, vegfa, and stat3. Conversely, 7BIO induced cox2, but suppressed a different selection of pro-inflammatory genes including nos2, tnfa, and igf1. Secretion of IL-6 protein, an iconic inflammatory cytokine, was decreased by E804. VEGF (vascular endothelial growth factor) protein secretion was upregulated by 7BIO, yet downregulated by E804 and E804 plus TMF. Thus, E804 is both an AHR ligand and regulator of important pro-inflammatory cytokines such as IL-6 and oncogene STAT3, among others. Our results point to the use of E804 and TMF in combination as a promising new treatment for glioblastoma.


Assuntos
Indóis/farmacologia , Oximas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Citocinas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Indóis/química , Oximas/química , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
J Exp Clin Cancer Res ; 38(1): 335, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370872

RESUMO

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilcolantreno/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/química , Receptores Estrogênicos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
4.
Int J Mol Sci ; 20(16)2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434333

RESUMO

Left ventricular hypertrophy (LVH) can be adaptive, as arising from exercise, or pathological, most commonly when driven by hypertension. The pathophysiology of LVH is consistently associated with an increase in cytochrome P450 (CYP)1B1 and mitogen-activated protein kinases (MAPKs) and a decrease in sirtuins and mitochondria functioning. Treatment is usually targeted to hypertension management, although it is widely accepted that treatment outcomes could be improved with cardiomyocyte hypertrophy targeted interventions. The current article reviews the wide, but disparate, bodies of data pertaining to LVH pathoetiology and pathophysiology, proposing a significant role for variations in the N-acetylserotonin (NAS)/melatonin ratio within mitochondria in driving the biological underpinnings of LVH. Heightened levels of mitochondria CYP1B1 drive the 'backward' conversion of melatonin to NAS, resulting in a loss of the co-operative interactions of melatonin and sirtuin-3 within mitochondria. NAS activates the brain-derived neurotrophic factor receptor, TrkB, leading to raised trophic signalling via cyclic adenosine 3',5'-monophosphate (cAMP)-response element binding protein (CREB) and the MAPKs, which are significantly increased in LVH. The gut microbiome may be intimately linked to how stress and depression associate with LVH and hypertension, with gut microbiome derived butyrate, and other histone deacetylase inhibitors, significant modulators of the melatonergic pathways and LVH more generally. This provides a model of LVH that has significant treatment and research implications.


Assuntos
Citocromo P-450 CYP1B1/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Animais , Citocromo P-450 CYP1B1/genética , Microbioma Gastrointestinal/fisiologia , Humanos , Hipertrofia Ventricular Esquerda/fisiopatologia , Melatonina/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sirtuínas/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-31373329

RESUMO

E-cigarette aerosol contains lower levels of most known carcinogens than tobacco smoke, but many users of e-cigarettes are also smokers, and these individuals may be vulnerable to possible promoting and/or cocarcinogenic effects of e-cigarettes. We investigated the possibility that a condensate of e-cigarette aerosol (EAC) enhances the metabolism of the tobacco carcinogen, benzo(a)pyrene (BaP), to genotoxic products in a human oral keratinocyte cell line. Cells were pretreated with EAC from two popular e-cigs and then with BaP. Metabolism to its ultimate carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro B[a]P (BPDE), was assayed by measuring isomers of its spontaneous hydrolysis products, BaP tetrols. The pretreatment of cells with EAC enhanced the rate of BaP tetrol formation several fold. Pretreatment with the e-liquid resulted in a smaller enhancement. The treatment of cells with EAC induced CYP1A1/1B1 mRNA and protein. The enhancement of BaP tetrol formation was inhibited by the aryl hydrocarbon receptor (AhR) inhibitor, α-napthoflavone, indicating EAC likely induces CYP1A1/1B1 and enhances BaP metabolism by activating the AhR. To our knowledge, this is first report demonstrating that e-cigarettes can potentiate the genotoxic effects of a tobacco smoke carcinogen.


Assuntos
Aerossóis/efeitos adversos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Benzo(a)pireno/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Mutagênicos/efeitos adversos , Receptores de Hidrocarboneto Arílico/genética , Fumaça/efeitos adversos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Sistemas Eletrônicos de Liberação de Nicotina , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo
6.
Chem Biol Interact ; 309: 108700, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31176714

RESUMO

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as ß-naphthoflavone (BNF), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding the ligand, AhR triggers induction of the expression of phase I and phase II drug-metabolizing genes, together with numerous other genes that are not directly involved in the metabolism of xenobiotics. Several studies have shown that AhR plays a role in tumor initiation, promotion and progression, but the molecular mechanisms involved in these processes are not fully understood. A previous study from our laboratory indicated that the SERPINB2 gene is presumably regulated by AhR. To prove that such induction is really AhR-dependent, in the present study we knocked down the expression of AhR by stable transfection of a laryngeal squamous cell carcinoma cell line (UT-SCC-34) with shRNA, resulting in 92% reduction of BNF-induced expression of SERPINB2. However, in silico analysis did not reveal AhR-dependent responsive elements in the promoter of the SERPINB2 gene. Therefore, to address this problem, we have used cycloheximide, an inhibitor of translation, and our results clearly indicate that an additional, newly synthesized protein is involved in AhR-dependent induction of SERPINB2 expression by BNF. So, to exclude that AhR binds to the putative xenobiotic-responsive elements (XREs) localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays. As expected, we found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene, further demonstrating the indirect SERPINB2 induction by AhR. However, the further analysis demonstrated that the expression of the enhancer RNA encoded by the region of DNA 20 kbp upstream from the SERPINB2 gene was AhR-dependent. Although AhR-mediated SERPINB2 induction clearly requires the synthesis of an additional protein, the kinetics of SERPINB2 induction is as fast as the kinetics of CYP1A1 and CYP1B1 induction (both genes directly regulated by AhR). Therefore, given previous studies regarding the induction of SERPINB2 expression by bacterial lipopolysaccharides (LPS), we think that, similarly, the interaction with pause-release proteins may be responsible for AhR-dependent regulation of SERPINB2 expression.


Assuntos
Receptores de Hidrocarboneto Arílico/metabolismo , Serpinas/metabolismo , Benzo(a)pireno/farmacologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Serpinas/genética , beta-Naftoflavona/farmacologia
7.
Mol Biol Rep ; 46(3): 3299-3306, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989560

RESUMO

Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. The disease also has genetic liability and is influenced by risk factors such as hormones and obesity. This study investigates the haplotypes of the Cytochrome P450 1B1 gene (CYP1B1) related to hormones and coiled-coil domain containing 57 gene (CCDC57) related to obesity in Afro-Caribbean females. Each haplotype was constructed from unphased sequence data using PHASE v.2.1 software and Haploview v.4.2 was used for linkage disequilibrium (LD) studies. There were contrasting LD observed among the single nucleotide polymorphisms of CYP1B1 and CCDC5. Accordingly, the GTA haplotype of CYP1B1 was significantly associated with UL risk (P = 0.02) while there was no association between CCDC57 haplotypes and UL (P = 0.2) for the ATG haplotype. As such, our findings suggest that the Asp449Asp polymorphism and GTA haplotype of CYP1B1 may contribute to UL susceptibility in women of Afro-Caribbean ancestry in this population.


Assuntos
Citocromo P-450 CYP1B1/genética , Leiomioma/genética , Adulto , Grupo com Ancestrais do Continente Africano/genética , Alelos , Região do Caribe , Estudos de Casos e Controles , Citocromo P-450 CYP1B1/metabolismo , Grupos Étnicos , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença , Haplótipos/genética , Humanos , Desequilíbrio de Ligação/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Neoplasias Uterinas/genética
8.
Int J Biochem Cell Biol ; 110: 111-121, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30858141

RESUMO

The extrahepatic CYP enzymes, CYP1B1 and CYP2U1, have been predominantly found in both astrocytes and brain microvessels. We investigated the alteration in the production of hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) mainly via CYP1B1 and CYP2U1 by glutamate. CYP1B1 and CYP2U1 mRNA levels were dose-dependently induced by glutamate in human U251 glioma cells and hCMEC/D3 blood-brain barrier cells. The increases in the CYP1B1 and CYP2U1 mRNA levels and the binding of CREB to CYP1B1 and CYP2U1 promoters following glutamate treatment were attenuated by mGlu5 receptor antagonist. The mRNA levels of CYP1B1 and CYP2U1 were increased in the cortex, hippocampus, and cerebellum from adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7 days of age; meanwhile, the protein levels of CYP1B1 and CYP2U1 in the astrocytes were induced by glutamate. Glutamate treatment significantly increased the production of 5-HETE, 8-HETE, 11-HETE, and 20-HETE in the cortex and cerebellum. These data suggested that the neuron-astrocyte reciprocal signaling can change the CYP-mediated AA metabolism (e.g. EETs and HETEs) in astrocytes via its specific receptor.


Assuntos
Ácido Araquidônico/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Família 2 do Citocromo P450/metabolismo , Ácido Glutâmico/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Hidroxilação/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
9.
Artigo em Inglês | MEDLINE | ID: mdl-30744811

RESUMO

We investigated the mechanism responsible for the oxidative stress and pro-inflammatory response triggered by PM2.5 collected from Nanjing of China. Two human bronchial epithelia cell lines (HBE and BEAS-2B) were used. Human gene expression profile microarray was performed to investigate the alteration of gene expression in PM2.5-treated HBE cells. The results of ROS assay and ELISA indicated that PM2.5 (150 µg/ml) increased the level of cellular reactive oxygen species (ROS) and promoted the release of interleukin-6 (IL-6) in HBE cells. CYP1A1 and CYP1B1 were the top two up-regulated genes by PM2.5 (150 µg/ml, 48 h of exposure) in HBE cells. Co-knockdown of CYP1A1/1B1 by siRNA substantially inhibited PM2.5-induced ROS generation, IL-6/IL-8 secretion and STAT3/P-STAT3 expression. Similarly, the knockdown of STAT3 also effectively inhibited PM2.5-induced rise in ROS level and IL-6/IL-8 secretion. In summary, PM2.5 mediated oxidative stress and pro-inflammatory response via up-regulating the expression of CYP1A1/1B1 in two human bronchial epithelial cell lines.


Assuntos
Brônquios/patologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Células Epiteliais/patologia , Mediadores da Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/farmacologia , Poluentes Atmosféricos/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/metabolismo , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Espécies Reativas de Oxigênio
10.
Eur J Pharm Sci ; 131: 177-194, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776468

RESUMO

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in 'suspension' for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.


Assuntos
Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1B1 , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Quinazolinonas , Antineoplásicos/farmacologia , Benzo(a)pireno/toxicidade , Bioensaio , Linhagem Celular , Cisplatino/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inibidores , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Quinazolinonas/química , Quinazolinonas/farmacologia
11.
Nanomedicine (Lond) ; 14(4): 447-464, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30694105

RESUMO

AIM: Enzyme CYP1B1 (CYP1B1) is usually overexpressed in multidrug resistance (MDR) breast cancer cells, which could metabolically inactivate docetaxel (DTX). MATERIALS & METHODS: The cationic core-shell nanoparticles (hyaluronic acid/polyethyleneimine nanoparticles [HA/PEI NPs]) modified with hyaluronic acid (HA) were developed and coloaded with DTX and α-napthtoflavone (ANF, a CYP1B1 inhibitor) to overcome MDR in breast cancer induced by CYP1B1. Physicochemical characterization, MDR reversing effect in vitro and pharmacokinetics in vivo of HA/PEI NPs were evaluated. RESULTS: The HA/PEI NPs exhibited spherical morphology with size of (193.6 ± 3.1) nm. The HA/PEI NPs could reverse MDR effectively by downregulating the expression of CYP1B1. The HA/PEI NPs improved the bioavailability of DTX. CONCLUSION: The HA/PEI NPs might be a promising strategy to overcome CYP1B1-mediated breast cancer MDR.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/uso terapêutico , Nanopartículas/química , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Microscopia Confocal , Ratos Sprague-Dawley
12.
PLoS One ; 14(1): e0211029, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30657791

RESUMO

Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.


Assuntos
Fatores de Transcrição Forkhead , Glaucoma , Proteínas de Homeodomínio , Herança Multifatorial , Mutação de Sentido Incorreto , Fatores de Transcrição , Substituição de Aminoácidos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Glaucoma/congênito , Glaucoma/metabolismo , Células HEK293 , Heterozigoto , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Mol Pharm ; 16(1): 409-421, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30481041

RESUMO

To reduce the pervasive toxicity of natural shikonin, alkannin, and their synthetic analogues and to enhance the selectivity of these chemotherapeutics toward cancer cells, a novel 5,8-dimethyl alkannin oxime derivative (DMAKO-20) was designed, synthesized, and evaluated for its strong antitumor activity both in vitro and in vivo. It showed potent growth inhibitory effects against HCT-15, HCT-116, and K562 cells (IC50 < 1 µM), moderate antiproliferative activity toward MDA-MB-231, HepG2, PANC, Bel7402, and MGC803 cancer cells (IC50 < 10 µM), and was nontoxic to the human normal VEC and HSF cells. In vivo efficacy studies demonstrated that DMAKO-20 (10 mg/kg, i.v. on every the other day, 8 times in 14 days) resulted in 59.3% reduction in HCT-15 xenograft volume. It was as effective as the toxic antimetabolite 5-FU but revealed neither toxicity nor death in mice. The mechanistic investigations indicated that DMAKO-20 underwent the tumor-specific CYP1B1-catalyzed bioactivation to afford nitric oxide and active naphthoquinone mono-oximes, which exhibited combined anticancer effects. It was defined as a representative of the "Multi-target Anticancer Prodrugs Activated by Specific Enzymes in cancer cells". The produced active metabolites exerted anticancer effects by the direct nucleophilic alkylation and the induction of the apoptosis of cancer cells through activation of the mitochondrial pathway. The discovery of DMAKO-20 and the illustration of its molecular mechanisms may provide a new strategy to overcome the nonselective toxicity of the current chemotherapeutics.


Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP1B1/metabolismo , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Células Hep G2 , Humanos , Células K562 , Camundongos , Naftoquinonas/química , Pró-Fármacos/química , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Biochim Biophys Acta Gen Subj ; 1863(2): 291-303, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30278240

RESUMO

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most difficult to biodegradate and the most toxic dioxin congener. Previously, we demonstrated in silico the ability of pig CYP1A1 to hydroxylate 2,7-dichlorodibenzo-p-dioxin (DiCDD), but not TCDD. To increase our knowledge concerning the low effectiveness of TCDD biodegradability, we analyzed in silico the binding selectivity and affinity between pig CYP1B1 and the two dioxins by means of molecular modeling. We also compared the effects of TCDD and DiCDD on CYP1B1 gene expression (qRT-PCR) and catalytic (EROD) activity in porcine granulosa cells. It was found that DiCDD and TCDD were stabilized within the pig CYP1B1 active site by hydrophobic interactions. The analysis of substrate channel availability revealed that both dioxins opened the exit channel S, allowing metabolites to leave the enzyme active site. Moreover, DiCDD and TCDD increased the CYP1B1 gene expression and catalytic activity in porcine granulosa cells. On the other hand, TCDD demonstrated higher than DiCDD calculated affinity to pig CYP1B1, hindering TCDD exit from the active site. The great distance between CYP1B1's heme and TCDD also might contribute to the lower hydroxylation effectiveness of TCDD compared to that of DiCDD. Moreover, the narrow active site of pig CYP1B1 may immobilize TCDD molecule, inhibiting its hydroxylation. The results of the access channel analysis and the distance from pig CYP1B1's heme to TCDD suggest that the metabolizing potential of pig CYP1B1 is higher than that of pig CYP1A1. However, this potential is probably not sufficiently high to considerably improve the slow TCDD biodegradation.


Assuntos
Citocromo P-450 CYP1B1/metabolismo , Dioxinas/metabolismo , Suínos/metabolismo , Animais , Biocatálise , Citocromo P-450 CYP1B1/química , Dioxinas/química , Modelos Moleculares , Estrutura Molecular
15.
Neurotox Res ; 35(1): 183-195, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30120713

RESUMO

Di-(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer in various plastic compounds, such as polyvinyl chloride (PVC), and products including baby toys, packaging films and sheets, medical tubing, and blood storage bags. Epidemiological data suggest that phthalates increase the risk of the nervous system disorders; however, the impact of DEHP on the brain cells and the mechanisms of its action have not been clarified. The aim of the present study was to investigate the effects of DEHP on production of reactive oxygen species (ROS) and aryl hydrocarbon receptor (AhR), as well as Cyp1a1 and Cyp1b1 mRNA and protein expression in primary mouse cortical neurons and glial cells in the in vitro mono-cultures. Our experiments showed that DEHP stimulated ROS production in both types of mouse neocortical cells. Moreover, the results strongly support involvement of the AhR/Cyp1A1 signaling pathway in the action of DEHP in neurons and glial cells. However, the effects of DEHP acting on the AhR signaling pathways in these two types of neocortical cells were different. In neurons, AhR mRNA expression did not change, but AhR protein expression decreased in response to DEHP. A similar trend was observed for Cyp1a1 and Cyp1b1 mRNA and protein expression. Failure to induce Cyp1a1 in neurons was confirmed by EROD assay. In primary glial cells, a decrease in AhR protein level was accompanied by a decrease in AhR mRNA expression. In glial cells, mRNA and protein expression of Cyp1a1 as well as Cyp1a1-related EROD activity were significantly increased. As for Cyp1b1, both in neurons and glial cells Cyp1b1 mRNA expression did not significantly change, whereas Cyp1b1 protein level were decreased. We postulate that developmental exposure to DEHP which dysregulates AhR/Cyp1a1 may disrupt defense processes in brain neocortical cells that could increase their susceptibility to environmental toxins.


Assuntos
Dietilexilftalato/toxicidade , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Camundongos , Neocórtex/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Med Princ Pract ; 28(1): 56-62, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30321868

RESUMO

OBJECTIVE: Intracranial tumors are one of the most frightening and difficult-to-treat tumor types. In addition to surgery, protocols such as chemotherapy and radiotherapy also take place in the treatment. Glutathione S-transferase (GST) and cytochrome P450 (CYP) enzymes are prominent drug-metabolizing enzymes in the human body. The aim of this study is to show the expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 in different types of brain tumors and compare our results with those in the literature. SUBJECTS AND METHODS: The expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 was analyzed using immunostaining in 55 patients with intracranial tumors in 2016-2017. For GST and CYP expression in normal brain tissue, samples of a portion of surrounding normal brain tissue as well as a matched far neighbor of tumor tissue were used. The demographic features of the patients were documented and the expression results compared. RESULTS: The mean age of the patients was 46.72 years; 29 patients were female and 26 were male. Fifty-seven specimens were obtained from 55 patients. Among them, meningioma was diagnosed in 12, metastases in 12, glioblastoma in 9, and pituitary adenoma in 5. The highest GSTP1, GSTM1, and CYP-1A1 expressions were observed in pituitary adenomas. The lowest GSTP1 expression was detected in glioblastomas and the lowest CYP1B1 expression in pituitary adenomas. CONCLUSION: GSTP1 and CYP expression is increased in intracranial tumors. These results should be confirmed with a larger series and different enzyme subtypes.


Assuntos
Neoplasias Encefálicas/enzimologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Neoplasias Encefálicas/patologia , Criança , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Turquia , Adulto Jovem
17.
Drug Metab Dispos ; 47(2): 67-70, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30420405

RESUMO

The overexpression of cytochrome P450 1B1 (CYP1B1) is a common characteristic of several diseases and conditions, such as inflammation, cancer, and cardiac hypertrophy. CYP1B1 is believed to contribute to pathogenesis of these diseases by mediating the formation of toxic compounds, either from exogenous or endogenous origin. We recently reported that an arachidonic acid metabolite, 19(S/R-)hydroxyeicosatetraenoic (HETE) acid, protects from cardiac hypertrophy by inhibiting the formation of toxic compounds, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)-HETE can directly inhibit CYP1B1. In the current study, we report that 19(S/R)-HETE enantioselectively inhibits human recombinant CYP1B1 activity measured by 7-ethoxyresorufin O-deethylation assay. 19(S)-HETE is more potent than the R enantiomer (K i = 37.3 and 89.1 nM, respectively). Noncompetitive inhibition was identified as the mechanism of CYP1B1 inhibition, which underlines the potentially important physiologic role of 19(S/R)-HETE as an endogenous CYP1B1 inhibitor; to our knowledge, 19(S/R)-HETE is the first inhibitor of its kind to be reported.


Assuntos
Citocromo P-450 CYP1B1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Citocromo P-450 CYP1B1/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/uso terapêutico , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade
18.
Toxicol Appl Pharmacol ; 364: 97-105, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30582946

RESUMO

Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole 14C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [14C]-BaP micro-dosing of humans. A 46 ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2 weeks between dosing and plasma collected over 72 h. [14C]-BaPeq PK analysis gave plasma Tmax and Cmax values of 1.25 h and 29-82 fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420 days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/1011 nucleotides) in two individuals 2-3 h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48-72 h. In volunteers the allelic variants CYP1B1*1/*⁎1, *1/*3 or *3/*3 and GSTM1*0/0 or *1 had no impact on [14C]-BaPeq PK or DNA adduction with this very limited sample. Plasma metabolites over 72 h from two individuals (one CYP1B1*1/*1 and one CYP1B1*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [14C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.


Assuntos
Benzo(a)pireno/farmacocinética , Carcinógenos/farmacocinética , Cromatografia Líquida/métodos , Espectrometria de Massas , Administração Oral , Adulto , Idoso , Benzo(a)pireno/administração & dosagem , Benzo(a)pireno/efeitos adversos , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Adutos de DNA/metabolismo , Feminino , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Variantes Farmacogenômicos , Medição de Risco , Adulto Jovem
19.
J Med Chem ; 61(23): 10901-10909, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30422652

RESUMO

Cytochrome P450 1B1 (CYP1B1) was found to be universally expressed in various tumors. Herein, we reported near-infrared fluorescent imaging probes for tumor detection via visualizing CYP1B1. After introducing the linker to a CYP1B1 selective inhibitor we found previously, we got the resulting compound 5b which kept strong inhibition ability against CYP1B1 (IC50 = 8.7 ± 1.2 nM) and high selectivity. Then, in vitro microscope studies and cell binding assay of probes indicated that the corresponding probe 6b could specifically be accumulated in CYP1B1 overexpressed colorectal cancer cell HCT-15 and showed satisfying binding affinity to target. During the in vivo noninvasive optical imaging, 6b was proved to rapidly lighten tumor in vivo as early as 6 h after injection. This work is the first attempt to visualize CYP1B1 for noninvasive imaging of tumor which could provide new approach for tumor diagnosis.


Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Citocromo P-450 CYP1B1/metabolismo , Desenho de Drogas , Microscopia de Fluorescência , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Química Sintética , Neoplasias Colorretais/patologia , Citocromo P-450 CYP1B1/química , Humanos , Camundongos , Modelos Moleculares , Sondas Moleculares/farmacocinética , Conformação Proteica , Distribuição Tecidual
20.
PLoS One ; 13(10): e0206756, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30372497

RESUMO

Cytochrome P450 1B1 (CYP1B1) is a member of the cytochrome p450 family of enzymes that catalyze mono-oxygenase reactions. Although constitutive Cyp1b1 expression is limited in hepatocytes, its expression and function in liver sinusoidal endothelial cells (LSEC) remains unknown. Here we determined the impact of Cyp1b1 expression on LSEC properties prepared from Cyp1b1+/+ and Cyp1b1-/- mice. LSEC expressed PECAM-1, VE-cadherin, and B4 lectin similar to EC from other mouse tissues. Cyp1b1 +/+ LSEC constitutively expressed significant levels of Cyp1b1, while Cyp1b1-/- LSEC lacked Cyp1b1 expression. LSEC also expressed VEGFR3, PROX-1, and LYVE-1, VEGFR1 and VEGFR2, as well as other cell adhesion molecules including ICAM-1, ICAM-2, VCAM-1, and thrombospondin-1 (TSP1) receptors, CD36 and CD47. However, the expression of PV-1 and stabilin (fenestration markers), and endoglin were limited in these cells. The Cyp1b1-/- LSEC showed limited fenestration, and decreased levels of VEGF and BMP6. Cyp1b1-/- LSEC also showed a decrease in the levels of VE-cadherin and ZO-1 impacting adherens and gap junction formation. Cyp1b1-/- LSEC were significantly more apoptotic, proliferated at a faster rate, and were less adherent and more migratory. These changes were attributed, in part, to decreased amounts of TSP1 and increased AKT and ERK activation. The expressions of integrins were also altered by the lack of Cyp1b1, but the ability of these cells to undergo capillary morphogenesis was minimally affected. Furthermore, Cyp1b1-/- LSEC expressed lower levels of inflammatory mediators MCP-1 and TNF-α. Thus, Cyp1b1 expression has a significant impact on LSEC angiogenic and inflammatory functions.


Assuntos
Capilares/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Endotélio Vascular/metabolismo , Fígado/metabolismo , Neovascularização Patológica/metabolismo , Animais , Apoptose , Western Blotting , Capilares/citologia , Endotélio Vascular/citologia , Imunofluorescência , Inflamação/metabolismo , Fígado/citologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/metabolismo
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