Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49
Filtrar
Mais filtros

Bases de dados
Tipo de documento
Intervalo de ano de publicação
1.
Int J Mol Sci ; 20(16)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426531

RESUMO

Pneumonitis is the leading cause of death associated with the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) against non-small cell lung cancer (NSCLC). However, the risk factors and the mechanism underlying this toxicity have not been elucidated. Tumor necrosis factor (TNF) has been reported to transactivate EGFR in pulmonary epithelial cells. Hence, we aimed to test the hypothesis that EGFR tyrosine kinase activity regulates TNF-mediated bronchial epithelial cell survival, and that inhibition of EGFR activity increases TNF-induced lung epithelial cell apoptosis. We used surfactant protein C (SPC)-TNF transgenic (tg) mice which overexpress TNF in the lungs. In this model, gefitinib, an EGFR-TKI, enhanced lung epithelial cell apoptosis and lymphocytic inflammation, indicating that EGFR tyrosine kinase prevents TNF-induced lung injury. Furthermore, IL-17A was significantly upregulated by gefitinib in SPC-TNF tg mice and p38MAPK activation was observed, indicative of a pathway involved in lung epithelial cell apoptosis. Moreover, in lung epithelial cells, BEAS-2B, TNF stimulated EGFR transactivation via the TNF-α-converting enzyme in a manner that requires heparin binding (HB)-EGF and transforming growth factor (TGF)-α. These novel findings have significant implications in understanding the role of EGFR in maintaining human bronchial epithelial cell homeostasis and in NSCLC treatment.


Assuntos
Apoptose , Células Epiteliais/metabolismo , Gefitinibe/efeitos adversos , Lesão Pulmonar/metabolismo , Pneumonia/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Receptores ErbB/metabolismo , Gefitinibe/uso terapêutico , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/fisiopatologia , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Transgênicos , Modelos Animais , Pneumonia/induzido quimicamente , Pneumonia/fisiopatologia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Fator de Crescimento Transformador alfa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Int J Mol Sci ; 19(11)2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30404198

RESUMO

In the past two decades, several molecular targeted inhibitors have been developed and evaluated clinically to improve the survival of patients with cancer. Molecular targeted inhibitors inhibit the activities of pathogenic tyrosine kinases. Particularly, aberrant receptor tyrosine kinase (RTK) activation is a potential therapeutic target. An increased understanding of genetics, cellular biology and structural biology has led to the development of numerous important therapeutics. Pathogenic RTK mutations, deletions, translocations and amplification/over-expressions have been identified and are currently being examined for their roles in cancers. Therapies targeting RTKs are categorized as small-molecule inhibitors and monoclonal antibodies. Studies are underway to explore abnormalities in 20 types of RTK subfamilies in patients with cancer or other diseases. In this review, we describe representative RTKs important for developing cancer therapeutics and predicting or evaluated resistance mechanisms.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/etiologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
3.
Int J Mol Sci ; 18(11)2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29140271

RESUMO

Cancer therapies targeting epidermal growth factor receptor (EGFR), such as small-molecule kinase inhibitors and monoclonal antibodies, have been developed as standard therapies for several cancers, such as non-small cell lung cancer, colorectal cancer, pancreatic cancer, breast cancer, and squamous cell carcinoma of the head and neck. Although these therapies can significantly prolong progression-free survival, curative effects are not often achieved because of intrinsic and/or acquired resistance. The resistance mechanisms to EGFR-targeted therapies can be categorized as resistant gene mutations, activation of alternative pathways, phenotypic transformation, and resistance to apoptotic cell death. Analysis of the processes that modulate EGFR signal transduction by EGFR-targeted inhibitors, such as tyrosine kinase inhibitors and monoclonal antibodies, has revealed new therapeutic opportunities and has elucidated novel mechanisms contributing to the discovery of more effective anticancer treatments. In this review, we discuss the roles of EGFR in cancer development, therapeutic strategies for targeting EGFR, and resistance mechanisms to EGFR-targeted therapies, with a focus on cancer therapies for individual patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Terapia de Alvo Molecular , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Amplificação de Genes , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
5.
J Biol Chem ; 289(6): 3148-63, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24338688

RESUMO

Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCε-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCε and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO(-/-) mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction.


Assuntos
Ácidos Eicosanoicos/farmacocinética , Células Endoteliais/metabolismo , Proteína Quinase C-épsilon/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Ácidos Eicosanoicos/metabolismo , Células Endoteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoxigenase/genética , Lipoxigenase/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Quinase C-épsilon/genética , Treonina/genética , Treonina/metabolismo , Junções Íntimas/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-2/genética , Proteína da Zônula de Oclusão-2/metabolismo
7.
Antibodies (Basel) ; 10(1)2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33535554

RESUMO

Antibody-drug conjugates (ADCs), which are currently being developed, may become promising cancer therapeutics. Folate receptor α (FOLR1), a glycosylphosphatidylinositol-anchored membrane protein, is an attractive target of ADCs, as it is largely absent from normal tissues but is overexpressed in malignant tumors of epithelial origin, including ovarian, lung, and breast cancer. In this study, we tested the effects of novel anti-FOLR1 antibody-eribulin conjugate MORAb-202 in breast cancer and non-small cell lung cancer (NSCLC) cell lines. FOLR1 expression, cell proliferation, bystander killing effects, and apoptosis were evaluated in seven breast cancer and nine NSCLC cell lines treated with MORAb-202. Tumor growth and FOLR1 expression were assessed in T47D and MCF7 orthotopic xenograft mouse models after a single intravenous administration of MORAb-202 (5 mg/kg). MORAb-202 was associated with inhibited cell proliferation, with specific selectivity toward FOLR1-expressing breast cancer cell lines. Eribulin, the payload of MORAb-202, was unleashed in HCC1954 cells, diffused into intercellular spaces, and then killed the non-FOLR1-expressing MCF7 cells in co-culture systems. In orthotopic xenograft mouse models, FOLR1-expressing T47D tumors and non-FOLR1-expressing MCF7 tumors were suppressed upon MORAb-202 administration. The novel anti-FOLR1 antibody-eribulin conjugate MORAb-202 has potential antitumor effects in breast cancer.

8.
J Biol Chem ; 284(49): 33833-40, 2009 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-19815546

RESUMO

Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cdelta inhibitor) and by the adenoviral expression of dominant negative protein kinase Cdelta. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.


Assuntos
Adipócitos/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Lisofosfatidilcolinas/metabolismo , Células 3T3-L1 , Animais , Diabetes Mellitus Experimental/metabolismo , Inibidores Enzimáticos/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Masculino , Espectrometria de Massas/métodos , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteína Quinase C-delta/metabolismo
9.
Biochem Biophys Res Commun ; 394(1): 106-11, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20184865

RESUMO

Barrier function of the epidermis is maintained by precise expression of keratinocyte-specific structural proteins to form the cornified cell envelope (CE). Loricrin, a major component of the CE, is expressed at the late stage of keratinocyte differentiation. In this study, we reveal the isoform-specific function of protein kinase C (PKC) in the regulation of loricrin expression. Both PKCdelta and PKCeta have been recognized as differentiation-promoting isoforms. However, loricrin expression was inversely controlled by PKCdelta and PKCeta in cultured keratinocytes and 3D skin culture; i.e. loricrin expression was decreased by PKCdelta and increased by PKCeta. To clarify the mechanisms that PKCdelta and PKCeta oppositely regulate the loricrin expression, we examined the expression of activator protein-1 (AP-1) family proteins, which modulate the transcription of loricrin and are downstream molecules of PKC. PKCdelta decreased c-Jun expression, whereas PKCeta increased JunD, which are positive regulators of loricrin transcription. These findings suggest that inverse effects of PKCdelta and PKCeta on loricrin expression attributes to the expression of c-Jun and JunD.


Assuntos
Regulação da Expressão Gênica , Queratinócitos/metabolismo , Proteínas de Membrana/genética , Proteína Quinase C-delta/metabolismo , Proteína Quinase C/metabolismo , Fator de Transcrição AP-1/metabolismo , Células Cultivadas , Humanos , Proteína Quinase C/genética , Proteína Quinase C-delta/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , Fator de Transcrição AP-1/genética
10.
J Cell Biol ; 164(7): 979-84, 2004 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-15051732

RESUMO

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825-835). This paper addresses a question whether transforming growth factor beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C alpha (PKCalpha) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.


Assuntos
Divisão Celular/efeitos dos fármacos , Queratinócitos/citologia , Proteína Quinase C/metabolismo , Proteínas S100/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C-alfa , Transporte Proteico , Proteínas de Ligação a RNA/metabolismo , Proteínas S100/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Nucleolina
11.
J Vis Exp ; (148)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31305510

RESUMO

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Afatinib/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Afatinib/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Éxons/genética , Dosagem de Genes , Humanos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
12.
Mol Cancer Ther ; 18(1): 112-126, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30322949

RESUMO

The critical T790M mutation in EGFR, which mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKI; gefitinib, erlotinib, and afatinib), has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib-resistant mechanisms, including emergence of T790M-EGFR, and responses to third-generation EGFR TKIs. Here, we used afatinib-resistant lung adenocarcinoma cells [AfaR (formerly AFR3) cells], carrying exon 19 deletion/T790M in EGFR To identify the novel resistance mechanisms in post-afatinib treatment, RocR1/RocR2 and OsiR1/OsiR2 cells were established using increasing concentrations of rociletinib and osimertinib, respectively. Attenuation of exon 19 deletion and T790M was confirmed in both rociletinib-resistant cells; in addition, EGFR and KRAS amplification was observed in RocR1 and RocR2, respectively. Significant KRAS amplification was observed in the osimertinib-resistant cell lines, indicating a linear and reversible increase with increased osimertinib concentrations in OsiR1 and OsiR2 cells. OsiR1 cells maintained osimertinib resistance with KRAS amplification after osimertinib withdrawal for 2 months. OsiR2 cells exhibited KRAS attenuation, and osimertinib sensitivity was entirely recovered. Phospho-EGFR (Y1068) and growth factor receptor-bound protein 2 (GRB2)/son of sevenless homolog 1 (SOS1) complex was found to mediate osimertinib resistance in OsiR1 cells with sustained KRAS activation. After 2 months of osimertinib withdrawal, this complex was dissociated, and the EGFR signal, but not the GRB2/SOS1 signal, was activated. Concomitant inhibition of MAPK kinase and EGFR could overcome osimertinib resistance. Thus, we identified a heterogeneous acquired resistance mechanism for third-generation EGFR TKIs, providing insights into the development of novel treatment strategies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Acrilamidas/farmacologia , Afatinib/farmacologia , Compostos de Anilina/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Amplificação de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Pirimidinas/farmacologia , Deleção de Sequência
13.
Mol Cell Biol ; 25(8): 3194-208, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15798205

RESUMO

It has been established that protein kinase Czeta (PKCzeta) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKCzeta remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKCzeta activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKCzeta-kinase domain also induces the activation loop phosphorylation of PKCzeta and downstream signal stimulation, as measured by p70 S6 kinase phosphorylation. Furthermore, only the PLD2-PX domain directly stimulates PKCzeta activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKCzeta. The mutant that substitutes the triple lysine residues (Lys101, Lys102, and Lys103) within the PLD2-PX domain with alanine abolishes interaction with the PKCzeta-kinase domain and activation of PKCzeta. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKCzeta activation is induced by its PX domain performing both direct activation of PKCzeta and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells.


Assuntos
Neoplasias da Mama/enzimologia , Fosfolipase D/química , Fosfolipase D/fisiologia , Proteína Quinase C/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Motivos de Aminoácidos , Animais , Neoplasias da Mama/genética , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Ativação Enzimática , Inativação Gênica , Humanos , Imunoprecipitação , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Fosfolipase D/genética , Fosforilação , Mapeamento de Interação de Proteínas , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
14.
J Clin Invest ; 109(6): 827-36, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11901191

RESUMO

We studied the role of protein kinase C isoform PKCdelta in ceramide (Cer) formation, as well as in the mitochondrial apoptosis pathway induced by anticancer drugs in prostate cancer (PC) cells. Etoposide and paclitaxel induced Cer formation and apoptosis in PKCdelta-positive LNCaP and DU145 cells but not in PKCdelta-negative LN-TPA or PC-3 cells. In contrast, these drugs induced mitotic cell cycle arrest in all PC cell lines. Treatment with Rottlerin, a specific PKCdelta inhibitor, significantly inhibited drug-induced Cer formation and apoptosis in LNCaP cells, as did overexpression of dominant negative-type PKCdelta. Overexpression of wild-type PKCdelta had an opposite effect in PC-3 cells. Notably, etoposide induced biphasic Cer formation in LNCaP cells. The early and transient Cer increase resulted from de novo Cer synthesis, while the late and sustained Cer accumulation was derived from sphingomyelin hydrolysis by neutral sphingomyelinase (nSMase). Cer, in turn, induced mitochondrial translocation of PKCdelta and stimulated the activity of this kinase, promoting cytochrome c release and caspase-9 activation. Furthermore, the specific caspase-9 inhibitor LEHD-fmk significantly inhibited etoposide-induced nSMase activation, Cer accumulation, and PKCdelta mitochondrial translocation. These results indicate that PKCdelta plays a crucial role in activating anticancer drug-induced apoptosis signaling by amplifying the Cer-mediated mitochondrial amplification loop.


Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/metabolismo , Isoenzimas/metabolismo , Mitocôndrias/metabolismo , Neoplasias da Próstata/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Acetofenonas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Benzopiranos/farmacologia , Inibidores de Caspase , Caspases/metabolismo , Fracionamento Celular , Ceramidas/química , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Isoenzimas/genética , Masculino , Paclitaxel/farmacologia , Proteína Quinase C/genética , Proteína Quinase C-delta , Esfingomielinas/metabolismo , Células Tumorais Cultivadas
15.
Mol Cell Endocrinol ; 270(1-2): 79-86, 2007 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-17416458

RESUMO

We have previously demonstrated that luteinizing hormone (LH) induces a rapid and transient expression of NGFI-B in the ovary. In this report, we investigated the signaling pathway for LH- and forskolin-induced NGFI-B expression in cultured rat granulosa cells of preovulatory follicles. LH- or forskolin-induced NGFI-B expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 microM), but not by low doses RO 31-8220 (0.1-1.0 microM) or adenylate cyclase inhibitor MDL-12,300A, implicating the involvement of atypical PKCs. Kinase assay revealed that LH treatment of granulosa cells resulted in a rapid stimulation of atypical PKCzeta activity. Interestingly, like LH, forskolin was also able to activate PKCzeta. Treatment with the cell-permeable PKCzeta-specific inhibitor pseudosubstrate peptide inhibited LH-or forskolin-induced NGFI-B expression, indicating the essential role of PKCzeta. Consistent with this promise, in granulosa cells depleted of diacylglycerol sensitive PKCs by prolonged treatment with tetradecanoylphobol-13-acetate, LH or forskolin could still induce NGFI-B expression, and RO 31-8220 or the PKCzeta pseudosubstrate peptide inhibited LH- or forskolin-induced NGFI-B expression. Furthermore, overexpression of dominant-negative PKCzeta in primary granulosa cells using a replication-defective adenovirus vector resulted in the suppression of LH- or forskolin-induced NGFI-B expression. Our findings demonstrate that PKCzeta, which is activated by LH or forskolin, contributes to the induction of NGFI-B in granulosa cells of preovulatory follicles.


Assuntos
Colforsina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/enzimologia , Hormônio Luteinizante/farmacologia , Ovulação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Adenoviridae , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Genes Dominantes , Vetores Genéticos , Indóis/farmacologia , Isoenzimas/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Ésteres de Forbol/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos
16.
Mol Cell Biol ; 23(5): 1581-9, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12588978

RESUMO

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.


Assuntos
Metaloendopeptidases/metabolismo , Músculo Liso Vascular/citologia , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/metabolismo , Acetofenonas/farmacologia , Adenoviridae/genética , Animais , Benzopiranos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Genes Dominantes , Humanos , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Janus Quinase 2 , Modelos Biológicos , Oxirredução , Testes de Precipitina , Proteína Quinase C-delta , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de Tempo , Transfecção
17.
Mol Biol Cell ; 13(11): 3976-88, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12429840

RESUMO

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.


Assuntos
Caveolinas/metabolismo , Membrana Celular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fosfolipase D/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Caveolina 1 , Fracionamento Celular , Membrana Celular/química , Ativação Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fosfolipase D/genética , Fosforilação , Mutação Puntual , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Transporte Proteico/fisiologia , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/metabolismo
18.
J Vis Exp ; (126)2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28829429

RESUMO

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.


Assuntos
Adenocarcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Gefitinibe , Humanos , Indóis/farmacologia , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Quinazolinas/farmacologia , Sulfonas/farmacologia
19.
Mol Cancer Res ; 15(7): 915-928, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28289161

RESUMO

EGFR tyrosine kinase inhibitors (TKI) are associated with significant responses in non-small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations. However, acquired resistance to reversible EGFR-TKIs remains a major obstacle. In particular, although the second-generation irreversible EGFR-TKI afatinib is currently used for treating NSCLC patients, the mechanisms underlying acquired afatinib resistance remain poorly understood. Here, heterogeneous mechanisms of acquired resistance were identified following long-term exposure to increasing doses of afatinib in EGFR-mutant lung adenocarcinoma PC-9 cells. Notably, three resistant cell lines, PC-9AFR1, PC-9AFR2, and PC-9AFR3 (AFR1, AFR2, and AFR3, respectively) employed distinct mechanisms for avoiding EGFR inhibition, with increased EGFR expression being detected in all resistant cell lines. Moreover, an activating EGFR mutation was partially lost in AFR1 and AFR2 cells. AFR1 cells exhibited afatinib resistance as a result of wild-type KRAS amplification and overexpression; however, these cells showed a progressive decrease and eventual loss of the acquired KRAS dependence, as well as resensitization to afatinib, following a drug holiday. Meanwhile, AFR2 cells exhibited increased expression of insulin-like growth factor-binding protein 3 (IGFBP3), which promoted insulin-like growth factor 1 receptor (IGF1R) activity and subsequent AKT phosphorylation, thereby indicating a potential bypass signaling pathway associated with IGFR1. Finally, AFR3 cells harbored the secondary EGFR mutation T790M. Our findings constitute the first report showing acquired wild-type KRAS overexpression and attenuation of afatinib resistance following a drug holiday.Implications: The heterogeneous mechanisms of afatinib resistance should facilitate the development of more effective therapeutic strategies for NSCLC patients. Mol Cancer Res; 15(7); 915-28. ©2017 AACR.


Assuntos
Adenocarcinoma/tratamento farmacológico , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinazolinas/administração & dosagem , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Afatinib , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Quinazolinas/efeitos adversos , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Biochem Biophys Rep ; 5: 1-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28955801

RESUMO

Syndecan-4 (SDC4) is a cell-surface proteoglycan associated with cell adhesion, motility, and intracellular signaling. Here, we present that SDC4 functions as a positive regulator of the transforming growth factor (TGF)-ß1-induced epithelial to mesenchymal transition (EMT) via Snail in lung adenocarcinoma, A549 cells. TGF-ß1 up-regulated the expression of SDC4, accompanied by the induction of EMT. Wound-healing and transwell chemotaxis assay revealed that SDC4 promoted cell migration and invasion. SDC4 knockdown recovered the E-cadherin and decreased vimentin and Snail expression in EMT-induced A549 cells. However, depletion of SDC4 resulted in little change of the Slug protein expression and mesenchymal cell morphology induced by TGF-ß1. The double knockdown of SDC-4 and Slug was required for reversal of epithelial morphology; it did not occur from the SDC4 single knockdown. These findings suggest that Snail is a transcriptional factor downstream of SDC4, and SDC4 regulates TGF-ß1-induced EMT by cooperating with Slug. Our data provide a novel insight into cellular mechanisms, whereby the cell-surface proteoglycan modulated TGF-ß1-induced EMT in lung adenocarcinoma, A549 cells.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA