Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 180
Filtrar
1.
Phytomedicine ; 80: 153355, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33039730

RESUMO

BACKGROUND: Lung cancer has the highest incidence and cancer-related mortality of all cancers worldwide. Its treatment is focused on molecular targeted therapy. c-MET plays an important role in the development and metastasis of various human cancers and has been identified as an attractive potential anti-cancer target. Podophyllotoxin (PPT), an aryltetralin lignan isolated from the rhizomes of Podophyllum species, has several pharmacological activities that include anti-viral and anti-cancer effects. However, the mechanism of the anti-cancer effects of PPT on gefitinib-sensitive (HCC827) or -resistant (MET-amplified HCC827GR) non-small cell lung cancer (NSCLC) cells remains unexplored. PURPOSE: In the present study, we investigated the underlying mechanisms of PPT-induced apoptosis in NSCLC cells and found that the inhibition of c-MET kinase activity contributed to PPT-induced cell death. METHODS: The regulation of c-MET by PPT was examined by pull-down assay, ATP-competitive binding assay, kinase activity assay, molecular docking simulation, and Western blot analysis. The cell growth inhibitory effects of PPT on NSCLC cells were assessed using the MTT assay, soft agar assay, and flow cytometry analysis. RESULTS: PPT could directly interact with c-MET and inhibit kinase activity, which further induced the apoptosis of HCC827GR cells. In contrast, PPT did not significantly affect EGFR kinase activity. PPT significantly inhibited the cell viability of HCC827GR cells, whereas the PPT-treated HCC827 cells showed a cell viability of more than 80%. PPT dose-dependently induced G2/M cell cycle arrest, as shown by the downregulation of cyclin B1 and cdc2, and upregulation of p27 expression in HCC827GR cells. Furthermore, PPT treatment induced Bad expression and downregulation of Mcl-1, survivin, and Bcl-xl expression, subsequently activating multi-caspases. PPT thereby induced caspase-dependent apoptosis in HCC827GR cells. CONCLUSION: These results suggest the potential of PPT as a c-MET inhibitor to overcome tyrosine kinase inhibitor resistance in lung cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Podofilotoxina/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Gefitinibe/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Podofilotoxina/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo
2.
PLoS One ; 15(10): e0241299, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112928

RESUMO

Epidermal growth factor receptor (EGFR) plays a vital role in cell division and survival signaling pathways. EGFR is activated in nearly every cancer type, and its high expression in tumors is correlated with poor patient outcome. Altogether, EGFR is a prime candidate as a therapeutic target. While targeted EGFR therapy is initially effective in 75% of patients, a majority of patients relapse within the first year due to poorly understood mechanisms of resistance. p120-catenin (p120ctn) has recently been implicated as a biomarker for EGFR therapy. In previous studies, we demonstrated that p120ctn is a tumor suppressor and its loss is capable of inducing cancer. Furthermore, p120ctn down-regulation synergizes with EGFR overexpression to cause a highly invasive cell phenotype. The purpose of this present study was to investigate whether p120ctn down-regulation induced EGFR therapeutic resistance. Using human esophageal keratinocytes, we have found that EGFR-targeting compounds are toxic to cells overexpressing EGFR. Interestingly, these therapies do not cause toxicity in cells with EGFR overexpression and decreased p120ctn expression. These data suggest that decreased p120ctn causes resistance to EGFR therapy. We believe these findings are of utmost importance, as there is an unmet need to discover mechanisms of EGFR resistance.


Assuntos
Cateninas/deficiência , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Terapia de Alvo Molecular , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Cateninas/metabolismo , Linhagem Celular Tumoral , Cetuximab/farmacologia , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/farmacologia , Esôfago/patologia , Gefitinibe/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Resultado do Tratamento
3.
Oncogene ; 39(44): 6856-6870, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32978523

RESUMO

Recent findings suggested a benefit of anti-EGFR therapy for basal-like muscle-invasive bladder cancer (MIBC). However, the impact on bladder cancer with substantial squamous differentiation (Sq-BLCA) and especially pure squamous cell carcinoma (SCC) remains unknown. Therefore, we comprehensively characterized pure and mixed Sq-BLCA (n = 125) on genetic and protein expression level, and performed functional pathway and drug-response analyses with cell line models and isolated primary SCC (p-SCC) cells of the human urinary bladder. We identified abundant EGFR expression in 95% of Sq-BLCA without evidence for activating EGFR mutations. Both SCaBER and p-SCC cells were sensitive to EGFR tyrosine kinase inhibitors (TKIs: erlotinib and gefitinib). Combined treatment with anti-EGFR TKIs and varying chemotherapeutics led to a concentration-dependent synergism in SCC cells according to the Chou-Talalay method. In addition, the siRNA knockdown of EGFR impaired SCaBER viability suggesting a putative "Achilles heel" of Sq-BLCA. The observed effects seem Sq-BLCA-specific since non-basal urothelial cancer cells were characterized by poor TKI sensitivity associated with a short-term feedback response potentially attenuating anti-tumor activity. Hence, our findings give further insights into a crucial, Sq-BLCA-specific role of the ERBB signaling pathway proposing improved effectiveness of anti-EGFR based regimens in combination with chemotherapeutics in squamous bladder cancers with wild-type EGFR-overexpression.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células de Transição/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Feminino , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Receptor ErbB-4/antagonistas & inibidores , Receptor ErbB-4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
4.
Anticancer Res ; 40(10): 5621-5630, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988886

RESUMO

BACKGROUND: Targeted therapies in the treatment of head and neck squamous cell carcinoma (HNSCC) are subject to extensive research. Different mutations of genes belonging to the fibroblast growth factor (FGF) family have been detected in HNSCC. In this study, we examined the expression of FGF1 and FGF2 after treatment with small-molecule tyrosine kinase inhibitors (TKIs) and an inhibitor of mechanistic target of rapamycin (mTOR) in vitro using human papillomavirus (HPV)-positive and -negative SCC lines. MATERIALS AND METHODS: Cells of two human HPV-negative cell lines (UMSCC-11A/-14C) and one HPV-positive cell line (CERV196) were incubated with 20 µmol/l of erlotinib, gefitinib, nilotinib, dasatinib, or everolimus for 24-96 h. Cell proliferation was assessed by proliferation assay and the protein concentrations of FGF1 and FGF2 by sandwich enzyme-linked immunosorbent assay. For statistical analysis, the results were compared with those for untreated HPV-negative SCC cells. RESULTS: FGF1 and FGF2 were detected in all three tested cell lines. The tested TKIs significantly (p<0.05 reduced) FGF1 expression in the UMSCC-11A cell line within the first 24 h. At later time points, the tested TKIs and everolimus significantly (p<0.05) increased FGF1 and FGF2 expression in HPV-negative and -positive cancer cell lines. The effect was stronger in the HPV-positive cell line. CONCLUSION: Alterations in FGF signalling are considered to be relevant drivers of tumourigenesis in some HNSCCs. Our results show that the expression of FGF1 and -2 can be influenced effectively by small-molecule TKIs and everolimus. Based on our data, future research should include combinations of specific FGF inhibitors, mTOR inhibitors and other TKIs in the treatment of HNSCC and research on FGF-mediated drug escape mechanisms.


Assuntos
Everolimo/farmacologia , Fatores de Crescimento de Fibroblastos/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Serina-Treonina Quinases TOR/genética , Linhagem Celular Tumoral , Dasatinibe/farmacologia , Cloridrato de Erlotinib/farmacologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Gefitinibe/farmacologia , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/patogenicidade , Humanos , Papillomaviridae/efeitos dos fármacos , Papillomaviridae/patogenicidade , Inibidores de Proteínas Quinases/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
5.
Oncogene ; 39(39): 6190-6202, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32820249

RESUMO

Glioma reported to be refractory to EGFR tyrosine kinase inhibitor is the most common malignant tumor in central nervous system. Our research showed the low expression of miR-450a-5p and high expression of EGFR in glioma tissues. MiR-450a-5p was also observed to synergize with gefitinib to inhibit the proliferation, migration and invasion and induce the apoptosis and autophagy of glioma cells. Furthermore, miR-450a-5p was demonstrated to target 3'UTR of EGFR, and regulated EGFR-induced PI3K/AKT/mTOR signaling pathway. Moreover, the above effects induced by miR-450a-5p in glioma cells were reversed by WIPI1 silencing. The inhibition role of miR-450a-5p on glioma growth was also confirmed in vivo by subcutaneous and intracranial tumor xenografts. Therefore, we conclude that miR-450a-5p synergizes with gefitinib to inhibit the glioma tumorigenesis through inducing autophagy by regulating the EGFR-induced PI3K/AKT/mTOR signaling pathway, thereby enhancing the drug sensitivity of gefitinib.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Gefitinibe/farmacologia , Glioma/tratamento farmacológico , MicroRNAs/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
PLoS One ; 15(8): e0238155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841278

RESUMO

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.


Assuntos
Antineoplásicos/farmacologia , Sinalização do Cálcio , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Gefitinibe/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Estrenos/farmacologia , Humanos , Fatores de Transcrição NFATC/biossíntese , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
7.
Anticancer Res ; 40(7): 3847-3855, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620624

RESUMO

BACKGROUND/AIM: The effects of tyrosine kinase inhibitors (TKI) in head and neck squamous cell cancer (HNSCC) are not fully understood. We investigated the effects of selective TKIs erlotinib, gefitinib, nilotinib, and dasatinib and the mTOR-inhibitor everolimus on the expression of insulin-like growth factor 1 receptor (IGF1R) in HPV-positive and HPV-negative squamous cancer cell lines. MATERIALS AND METHODS: HPV-negative UMSCC-11A and UMSCC-14C cells and HPV-positive CERV196 cells were treated with TKIs or everolimus. Protein concentration of IGF1R was measured using ELISA. RESULTS: IGF1R expression was significantly reduced by all tested TKIs and everolimus in both HPV-negative cancer cell lines. In HPV-positive squamous cancer cells we observed significant protein inhibition. CONCLUSION: The crosstalk between epidermal growth factor receptors and IGF1R could be of central interest for the development of novel medical approaches for individualized therapy.


Assuntos
Everolimo/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Papillomavirus Humano 16/isolamento & purificação , Infecções por Papillomavirus/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dasatinibe/farmacologia , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Infecções por Papillomavirus/virologia , Pirimidinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia
8.
J Pharmacol Exp Ther ; 374(2): 295-307, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32393528

RESUMO

Gefitinib and erlotinib are epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) with activity against metastatic non-small cell lung cancer. Aldehyde oxidase-1 (AOX1) is a cytosolic drug-metabolizing enzyme. We conducted an experimental and molecular docking study on the effect of gefitinib, erlotinib, and select metabolites on the in vitro catalytic activity of AOX1, as assessed by carbazeran 4-oxidation, and determined the impact of AOX1 inhibition on hepatic metabolism of zaleplon and methotrexate. Gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib inhibited human hepatic cytosolic carbazeran 4-oxidation by a competitive mode, with inhibition constants in submicromolar or low micromolar concentrations. Desmethylgefitinib did not affect AOX1 catalytic activity. A similar pattern was obtained when investigated with human kidney cytosol or recombinant AOX1. The differential effect of gefitinib on human, rat, and mouse hepatic AOX1 catalytic activity suggests species-dependent chemical inhibition of AOX1. Erlotinib was considerably more potent than gefitinib in decreasing hepatic cytosolic zaleplon 5-oxidation and methotrexate 7-oxidation. Molecular docking analyses provided structural insights into the interaction between EGFR-TKIs and AOX1, with key residues and bonds identified, which provided favorable comparison and ranking of potential inhibitors. Based on the US Food and Drug Administration guidance to assess the risk of drug-drug interactions, the calculated R1 values indicate that further investigations are warranted to determine whether gefitinib and erlotinib impact AOX1-mediated drug metabolism in vivo. Overall, erlotinib desmethylerlotinib, didesmethylerlotinib, gefitinib, and desmorpholinopropylgefitinib are potent inhibitors of human AOX1 catalytic function and hepatic metabolism of zaleplon and methotrexate, potentially affecting drug efficacy or toxicity. SIGNIFICANCE STATEMENT: As epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib are first-line pharmacotherapy for metastatic non-small cell lung cancer. Our experimental findings indicate that clinically relevant concentrations of gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib, but not desmethylgefitinib, inhibit human aldehyde oxidase (AOX1) catalytic activity and hepatic cytosolic metabolism of zaleplon and methotrexate. Molecular docking analysis provide structural insights into the key AOX1 interactions with these EGFR-TKIs. Our findings may trigger improved strategies for new EGFR-TKI design and development.


Assuntos
Acetamidas/metabolismo , Aldeído Oxidase/antagonistas & inibidores , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Fígado/efeitos dos fármacos , Metotrexato/metabolismo , Simulação de Acoplamento Molecular , Pirimidinas/metabolismo , Aldeído Oxidase/química , Aldeído Oxidase/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Cloridrato de Erlotinib/metabolismo , Gefitinibe/metabolismo , Humanos , Fígado/metabolismo , Conformação Proteica
9.
Oncogene ; 39(25): 4844-4853, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32439863

RESUMO

In response to nutrient deficiency, autophagy degrades cytoplasmic materials and organelles in lysosomes, which is nutrient recycling, whereas activation of EGFR mediates autophagy suppression in response to growth factors. It is unclear whether PPARδ could be the regulator of autophagy in response to active EGFR. Here we found that EGFR induced PPARδ phosphorylation at tyrosine-108 leading to increased binding of LC3 to PPARδ by its LIR (LC3 interacting region) motif, consequently, inhibited autophagic flux. Conversely, EGFR inhibitor treatment reversed this event. Furthermore, EGFR-mediated PPARδ phosphorylation at tyrosine-108 led to autophagy inhibition and tumor growth. These findings suggest that PPARδ serves as a regulator of autophagy by its phosphorylation.


Assuntos
Autofagia/fisiologia , PPAR delta/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Células HCT116 , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , PPAR delta/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Tirosina/genética , Tirosina/metabolismo
10.
Endocrinology ; 161(8)2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32473019

RESUMO

Tyrosine kinase inhibitors (TKIs) used in cancer are also being investigated in diabetes. TKIs can improve blood glucose control in diabetic cancer patients, but the specific kinases that alter blood glucose or insulin are not clear. We sought to define the role of Receptor Interacting Serine/Threonine Kinase 2 (RIPK2) in mouse models of insulin resistance. We tested the TKI gefitinib, which inhibits RIPK2 activity, in wild-type (WT), Nod1-/-, Nod2-/-, and Ripk2-/- mice fed an obesogenic high-fat diet. Gefitinib lowered blood glucose during a glucose tolerance test (GTT) in a nucleotide-binding oligomerization domain (NOD)-RIPK2-independent manner in all obese mice. However, gefitinib lowered glucose-stimulated insulin secretion only in obese Ripk2-/- mice. Gefitinib had no effect on insulin secretion in obese WT, Nod1-/-, or Nod2-/- mice. Hence, genetic deletion of Ripk2 promoted the insulin-sensitizing potential of gefitinib, since this TKI lowered both blood glucose and insulin only in Ripk2-/- mice. Gefitinib did not alter the inflammatory profile of pancreas, adipose, liver, or muscle tissues in obese Ripk2-/- mice compared with obese WT mice. We also tested imatinib, a TKI that does not inhibit RIPK2 activity, in obese WT mice. Imatinib lowered blood glucose during a GTT, consistent with TKIs lowering blood glucose independently of RIPK2. However, imatinib increased glucose-stimulated insulin secretion during the glucose challenge. These data show that multiple TKIs lower blood glucose, where actions of TKIs on RIPK2 dictate divergent insulin responses, independent of tissue inflammation. Our data show that RIPK2 limits the insulin sensitizing effect of gefitinib, whereas imatinib increased insulin secretion.


Assuntos
Secreção de Insulina/efeitos dos fármacos , Secreção de Insulina/genética , Obesidade/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/fisiologia , Adiposidade/efeitos dos fármacos , Adiposidade/genética , Animais , Glicemia/efeitos dos fármacos , Glicemia/genética , Glicemia/metabolismo , Dieta Hiperlipídica , Gefitinibe/farmacologia , Insulina/metabolismo , Resistência à Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Proteína Adaptadora de Sinalização NOD1/fisiologia , Proteína Adaptadora de Sinalização NOD2/fisiologia , Obesidade/etiologia , Obesidade/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
11.
Anticancer Res ; 40(4): 1855-1866, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234873

RESUMO

BACKGROUND/AIM: The resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib or erlotinib, is considered a major challenge in the treatment of patients with non-small cell lung cancer (NSCLC). Herein, we identified the critical roles of anterior gradient 2 (AGR2) in gefitinib (Gef) resistance of mutant NSCLC cells. MATERIALS AND METHODS: Using datasets from a pair of NSCLC-sensitive and NSCLC-resistant cells, immunoblotting, immunofluorescence and immunohistochemistry, and cell viability assays were applied to identify the effects of AGR2. RESULTS: AGR2 was found to be significantly over-expressed in Gef-resistant cells and was highly associated with drug resistance, proliferation, migration, and invasion of cancer cells. Moreover, AGR2 and ADAMTS6 formed a negative feedback loop in drug-resistant cells. CONCLUSION: Modulation of overexpression of AGR2 in mutant NSCLC cells may be an attractive therapeutic strategy for the treatment of EGFR-TKI-resistant NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Mucoproteínas/genética , Proteínas Oncogênicas/genética , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Quinazolinas/farmacologia
12.
DNA Cell Biol ; 39(7): 1111-1118, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32343915

RESUMO

Advanced nonsmall-cell lung cancer (NSCLC) patients with mutated epidermal growth factor receptor (EGFR) can remarkably benefit from target therapy of EGFR-tyrosine kinase inhibitors (TKIs). However, increasing drug sensitivity and improving outcomes of NSCLC patients to EGFR-TKI therapy remains a challenge. Several studies have shown a link between microRNAs and drug resistance in cancer. In this study, we hypothesized that the rs12740674 single nucleotide polymorphism in the enhancer of miR-1262 may affect its expression, which may impact the outcome of NSCLC patients treated with EGFR-TKIs. The rs12740674 polymorphism was genotyped in two independent cohorts, including 319 EGFR-TKI treated stage IIIB/IV NSCLC patients. The allele-specific regulation on miR-1262 transcription by rs12740674 and impacts of miR-1262 on gefitinib sensitivity were evaluated in vitro and in vivo. Cox regression analyses indicated that the rs12740674 T allele was significantly associated with short survival time in both cohorts (p < 0.05). Luciferase assays demonstrated that the rs12740674 T allelic enhancer showed weaker capability to promote miR-1262 transcription compared with the C allelic enhancer, which may be due to reduced transcription factor binding according to electrophoretic mobility shift assays. Furthermore, significantly decreased miR-1262 expression in NSCLC and nontumor lung tissues of T allele carriers was observed compared with levels in C allele carriers. Moreover, miR-1262 expression enhanced the anticancer effects of gefitinib on NSCLC cells. Our data indicate that miR-1262 may be a potential therapeutic target for NSCLC.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Receptores ErbB/antagonistas & inibidores , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Inibidores de Proteínas Quinases/farmacologia , Transcrição Genética/efeitos dos fármacos , Alelos , Linhagem Celular Tumoral , Estudos de Coortes , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Estadiamento de Neoplasias
13.
J Cancer Res Clin Oncol ; 146(7): 1737-1749, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32342201

RESUMO

PURPOSE: The usual first-line strategy of wild-type EGFR (wtEGFR) non-small cell lung cancer (NSCLC) remains cisplatin-based chemotherapy. However, cisplatin often loses effectiveness because most tumors acquire drug resistance over time. As EGFR is the most important pro-survival/proliferation signal receptor in NSCLC cells, we aimed at investigating whether cisplatin resistance is related to EGFR activation and further evaluating the combined effects of cisplatin/gefitinib (EGFR-tyrosine kinase inhibitor, EGFR-TKI) on cisplatin-resistant wtEGFR NSCLC cells. MATERIALS AND METHODS: EGFR activation was analysed in parental and cisplatin-resistant wtEGFR NSCLC cell lines (H358 and H358R, A549 and A549R). Cellular proliferation and apoptosis of H358R/A549R cells treated with cisplatin or gefitinib, alone or in combination were investigated, and the related effector protein was detected by western blot analysis. Anti-tumor effect of two drugs combined was evaluated in animal models of H358R xenografts in vivo. RESULTS: EGFR was significantly phosphorylated in cisplatin-resistant wtEGFR NSCLC cells H358R and A549R than their parental cells. In H358R and A549R cells, anti-proliferative ability of gefitinib was further improved, and gefitinib combined with cisplatin enhanced inhibition of cellular survive/proliferation, and promotion of apoptosis in vitro. The combined effects were also associated with the inhibition of EGFR downstream effector proteins. Similarly, in vivo, gefitinib and cisplatin in combination significantly inhibited tumor growth of H358R xenografts. CONCLUSION: Abnormal activation of EGFR may induce wtEGFR NSCLC cell resistance to cisplatin. The combined effects of cisplatin/gefitinib suggest that gefitinib, as a combination therapy for patients with cisplatin-resistant wtEGFR NSCLC should be considered.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Biochem Biophys Res Commun ; 526(3): 568-573, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32247613

RESUMO

Gefitinib is an ATP-competitive inhibitor of receptor tyrosine kinases known to repress the progression of various types of cancers. Emerging evidence has shown that this molecule modulates endothelial cells to inhibit angiogenesis. However, the biological effects of gefitinib have not been comprehensively investigated in endothelial cells. In this study, gefitinib-mediated regulation of cell proliferation, migration, cell attachment, cytoskeletal actin filament reorganization, tubular-like structure formation and angiogenesis in vivo was examined along with the corresponding mechanisms. G1-phase cell cycle arrest was detected and led to a decrease in 3H-labeled thymidine incorporation under sublethal doses of gefitinib. Endothelial cell migration was blocked in both wound-healing and transwell assays. In addition, gefitinib simultaneously inhibited collagen- and fibronectin-dependent cell attachment. Importantly, we first observed that the gefitinib-induced phenotypes might be partially due to the abnormal retrograde flow of actin filaments. The results of this study revealed the antivasculogenic effects of gefitinib at sublethal doses and indicated that the treatment of cancer patients with this drug might impair angiogenesis in the tumor microenvironment to reduce the cancer metastasis rate in addition to the direct therapeutic benefits of repressing epidermal growth factor receptor signaling in cancer cells.


Assuntos
Inibidores da Angiogênese/farmacologia , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Gefitinibe/farmacologia , Fibras de Estresse/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galinhas , Membrana Corioalantoide/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Ratos
15.
Nat Commun ; 11(1): 1923, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317663

RESUMO

Drug resistance mediated by clonal evolution is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at a cost of decreased fecundity or increased sensitivity to another drug. These evolutionary trade-offs can be exploited using 'evolutionary steering' to control the tumour population and delay resistance. However, recapitulating cancer evolutionary dynamics experimentally remains challenging. Here, we present an approach for evolutionary steering based on a combination of single-cell barcoding, large populations of 108-109 cells grown without re-plating, longitudinal non-destructive monitoring of cancer clones, and mathematical modelling of tumour evolution. We demonstrate evolutionary steering in a lung cancer model, showing that it shifts the clonal composition of the tumour in our favour, leading to collateral sensitivity and proliferative costs. Genomic profiling revealed some of the mechanisms that drive evolved sensitivity. This approach allows modelling evolutionary steering strategies that can potentially control treatment resistance.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Evolução Molecular , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Evolução Clonal , Biologia Computacional , Simulação por Computador , Gefitinibe/farmacologia , Genótipo , Humanos , Neoplasias Pulmonares/patologia , Modelos Teóricos , Medicina Molecular , Piridonas/farmacologia , Pirimidinonas/farmacologia , Processos Estocásticos
16.
BMC Cancer ; 20(1): 315, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293355

RESUMO

BACKGROUND: The enrichment of cancer stem cell-like cells (CSCs) has been considered to be responsible for tumor progression after an initial response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung adenocarcinoma (NSCLC/ADC). CSCs with ALDH1A1bright /CD44high expression contribute to the TKIs resistance in NSCLC/ADC cells. All-trans retinoic acid (ATRA) has been shown to be a potential targeted therapy against CSCs due to its ability to inhibit ALDH1A1 activity. We therefore investigated whether ATRA could circumvent the resistance to improve the response to gefitinib in NSCLC/ADC cells. METHODS: Treatment of NSCLC/ADC A549 and H1650 cells with gefitinib enriched the gefitinib surviving cells (GSCs). The expression of ALDH1A1 and CD44 and the IC50 values for gefitinib were determined by flow cytometry (FCM) and crystal violet assay in GSCs and ATRA-treated GSCs, respectively. Using DEAB as the positive control, direct inhibitory effect of ATRA on ALDH1A1 activity was determined by ALDEFLUOR assay, RESULTS: GSCs showed higher expression of ALDH1A1 and CD44 and IC50 values for gefitinib than their respective parental cells, suggesting that gefitinib can lead to propagation of CSC-enriched gefitinib-resistant cells. Treatment with ATRA was found to significantly reduce the increased expression of ALDH1A1 and CD44 and the IC50 values for gefitinib in A549GSC and H1650GSC cells, and ATRA could directly inhibit active ALDH1A1 as compared to DEAB. CONCLUSION: Our findings suggest that combination treatment with ATRA prevents gefitinib-induced enrichment of ALDH1A1bright/CD44high CSCs and enhances gefitinib-induced growth inhibition of NSCLC/ADC cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Tretinoína/farmacologia , Células A549 , /metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Concentração Inibidora 50 , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Regulação para Cima/efeitos dos fármacos
17.
Sci Rep ; 10(1): 6367, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286420

RESUMO

Triple-negative breast cancers (TNBC) are unlikely to respond to hormonal therapies and anti-HER2-targeted therapies. TNBCs overexpress EGFR and exhibit constitutive activation of the PI3K/AKT/mTOR signalling pathway. We hypothesized that simultaneously blocking EGFR and mTOR could be a potential therapeutic strategy for the treatment of TNBC. We examined the antitumour activity of the mTOR inhibitor everolimus combined with the EGFR tyrosine kinase inhibitor gefitinib in TNBC cell with or without activating mutations in the PI3K/AKT/mTOR signalling pathway. We demonstrated that everolimus and gefitinib induced synergistic growth inhibition in the PI3K and PTEN-mutant CAL-51 cell line but not in the PTEN-null HCC-1937 cell line. The antiproliferative effect was associated with synergistic inhibition of mTOR and P70S6K phosphorylation, as well as a significant reduction in 4E-BP1 activation in the CAL-51 cell line. We also showed that combination therapy significantly inhibited cell cycle progression and increased apoptosis in this cell line. Gene and protein expression analysis revealed significant downregulation of cell cycle regulators after exposure to combined treatment. Collectively, these results suggested that dual inhibition of mTOR and EGFR may be an effective treatment for TNBC with activating mutations of PI3K.


Assuntos
Everolimo/farmacologia , Gefitinibe/farmacologia , Serina-Treonina Quinases TOR/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
18.
Biochem Biophys Res Commun ; 526(1): 191-198, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32201076

RESUMO

Collagen type I (Col I) is one of the major extracellular matrix proteins in the cancer tissue. Previously, we have reported that Col I induces epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance by mTOR activation through Akt and ERK1/2 independent pathway. In this study, we aimed to elucidate the molecular mechanism of Col I induced EGFR-TKI resistance. First, we demonstrated the uptake of fluorescently labeled Col I by EGFR-mutated lung cancer cell line PC-9 cells using confocal microscopy and flow cytometry. Metabolome analysis revealed that the metabolic profiles of PC-9 cells was influenced by Col I treatment. Uptake of Col I into PC-9 cells was not inhibited by MMP inhibitor, GM6001, and endocytosis inhibitors, Pitstop2 and Dyngo4a; however, macropinocytosis inhibitor EIPA prevented its uptake. Moreover, the combination of EIPA and EGFR-TKI abrogated Col I-induced EGFR-TKI resistance in PC-9 cells. Inhibition of Rac1, which is essential for micropinocytosis, also decreased the uptake of Col I in PC-9 cells and restored their sensitivity to EGFR-TKI. Thus, EGFR mutated lung cancer cells could develop EGFR-TKI resistance by Col I uptake by macropinocytosis route.


Assuntos
Antineoplásicos/farmacologia , Colágeno Tipo I/metabolismo , Resistencia a Medicamentos Antineoplásicos , Pinocitose , Serina-Treonina Quinases TOR/metabolismo , Aminoácidos/metabolismo , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metabolômica , Pinocitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
Mol Carcinog ; 59(7): 691-700, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115801

RESUMO

Triple-negative breast cancer (TNBC) lacks a well-defined molecular target and is associated with poorer outcomes compared to other breast cancer subtypes. Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy shows a 10% to 20% response rate in TNBC patients. Our previous studies show that PD-L1 proteins are heavily glycosylated in TNBC, and the glycosylation plays an important role in the PD-L1 protein's stability and immunosuppressive function. However, a strategy for PD-L1 deglycosylation in TNBC is poorly defined. Here we found that a saccharide analog, 2-deoxy- d-glucose (2-DG), inhibits glycosylation of PD-L1 and its immunosuppressive function by combining with EGFR inhibitor, gefitinib. Interestingly, 2-DG/gefitinib-induced deglycosylation of PD-L1 decreased the expression level of PD-L1 protein as well as its binding with PD-1. However, there was no significant decrease in 4-1BB expression and its binding with 4-1BBL by 2-DG/gefitinib. Furthermore, we demonstrated that the combination treatment of 2-DG/gefitinib and 4-1BB antibody enhances antitumor immunity in TNBC syngeneic murine models. Together, our results suggest a new immunotherapeutic strategy to enhance antitumor immunity by PD-L1 deglycosylation and 4-1BB stimulation in TNBC.


Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Desoxiglucose/farmacologia , Glucose/farmacologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Animais , Anticorpos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Gefitinibe/farmacologia , Células HEK293 , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C
20.
Surg Today ; 50(9): 1099-1106, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32052182

RESUMO

PURPOSE: Exosomes and their cargo microRNAs play a significant role in various biological processes in cancer. We hypothesized that microRNAs in exosomes secreted by gefitinib-resistant lung cancer cells might induce resistant phenotypes in otherwise gefitinib-sensitive lung cancer cells. METHODS: We isolated exosomes generated by the gefitinib-resistant human lung adenocarcinoma cell line PS-9/ZD. PC-9, which is a gefitinib-sensitive cell line, was treated with the PC-9/ZD exosomes, and these PC-9 cells were analyzed for cell proliferation after treatment with gefitinib. miRNA arrays were analyzed in PC-9 and PC-9/ZD cells, and we isolated microRNAs that were expressed at elevated levels in PC-9/ZD cells. Furthermore, we transfected these microRNAs into PC-9 cells and analyzed the effects on the cells' sensitivity to gefitinib. RESULTS: Exosomes isolated from PC-9/ZD cells significantly increased the proliferation of PC-9 cells during gefitinib treatment. A microRNA array analysis showed that miR-564, miR-658, miR-3652, miR-3126-5p, miR-3682-3p and miR-6810-5p were significantly upregulated in PC-9/ZD cells. PC-9 cells transfected with miR-564 or miR-658 showed chemo-resistant phenotypes. CONCLUSION: Exosomal miR-564 and miR-658 derived from gefitinib-resistant lung cancer cells induce drug resistance in sensitive cells. Cell-to-cell interaction via exosomal microRNAs may be a novel mechanism and therapeutic target of resistance against gefitinib.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Comunicação Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/genética , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MicroRNAs , Adenocarcinoma de Pulmão/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Terapia de Alvo Molecular , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA