RESUMO
Kaempferol (KF), which is a natural dietary flavonoid, has potential beneficial effects as a chemopreventive agent for critical health conditions, such as cancer. However, the molecular mechanisms underlying the activity of KF remain unknown. We report on the inhibition of neoplastic cell transformation by KF through the suppression of phosphatidylinositol 3-kinase (PI3K) activity. Epidermal growth factor (EGF)-induced neoplastic transformation of mouse epidermal JB6 P+ cells was inhibited by 40 microM KF. The activation of activator protein-1 and nuclear factor-kappaB induced by EGF was also attenuated by KF. The EGF-induced phosphorylation of Akt (protein kinase B) was completely suppressed by KF, although extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and p90 ribosomal S6 kinase were unaffected by KF. Kinase assay data revealed that KF bound directly to PI3K, which is upstream of Akt, and suppressed its activity. Furthermore, KF inhibited ultraviolet B (UVB)-induced PI3K activity and attenuated UVB-induced phosphorylation of Akt. Our results suggest that KF docks at the adenosine triphosphate-binding site of PI3K, which is located between the N-lobe and C-lobe of the kinase domain. Inhibition by KF of PI3K, which is an important factor in carcinogenesis, and its downstream effects may explain the chemopreventive action of KF.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Quempferóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Epiderme/efeitos dos fármacos , Epiderme/fisiologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Modelos Moleculares , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Plasmídeos , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Cyclooxygenase-2 (COX-2), a key mediator of inflammation, and its product, prostaglandin E(2) (PGE(2)), enhance carcinogenesis, particularly in skin. Ultraviolet (UV) B is the most carcinogenic component of solar irradiation, and a crucial role of COX-2 in UVB-mediated skin carcinogenesis has been reported. Here, we investigated the effects of delphinidin, an abundant dietary anthocyanin, on UVB-induced COX-2 upregulation and the underlying molecular mechanism. We found that delphinidin suppressed UVB-induced COX-2 expression in JB6 P+ mouse epidermal cells. COX-2 promoter activity and PGE(2) production were also suppressed by delphinidin treatment within non-cytotoxic concentrations. Activator protein-1 and nuclear factor-kappaB, crucial transcription factors involved in COX-2 expression, were activated by UVB and delphinidin abolished this activation. UVB-induced phosphorylation of c-Jun N-terminal kinase, p38 kinase and Akt was inhibited by delphinidin. The activities of mitogen-activated protein kinase kinase (MAPKK) 4 and phosphatidylinositol-3 kinase (PI-3K) were inhibited markedly by delphinidin. A pull-down assay using delphinidin-Sepharose beads revealed that delphinidin binds directly with MAPKK4 or PI-3K in a manner that was competitive with adenosine triphosphate. Moreover, in vivo investigations using mouse skin revealed that the upregulation of COX-2 expression, MAPKK4 activity and PI-3K activity induced by UVB was abolished with delphinidin treatment. Collectively, our results demonstrated that delphinidin targets MAPKK4 and PI-3K directly to suppress COX-2 overexpression, suggesting a potential protective role for delphinidin against UVB-mediated skin carcinogenesis.
Assuntos
Antocianinas/farmacologia , Ciclo-Oxigenase 2/biossíntese , Epiderme/enzimologia , Epiderme/efeitos da radiação , MAP Quinase Quinase 4/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Células Cultivadas , Carboidratos da Dieta/farmacologia , Dinoprostona/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Feminino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/prevenção & controle , Fator de Transcrição AP-1/metabolismo , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiaçãoRESUMO
Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in many foods, including coffee. Recent studies suggested that caffeic acid exerts anticarcinogenic effects, but little is known about the underlying molecular mechanisms and specific target proteins. In this study, we found that Fyn, one of the members of the non-receptor protein tyrosine kinase family, was required for ultraviolet (UV) B-induced cyclooxygenase-2 (COX-2) expression, and caffeic acid suppressed UVB-induced skin carcinogenesis by directly inhibiting Fyn kinase activity. Caffeic acid more effectively suppressed UVB-induced COX-2 expression and subsequent prostaglandin E(2) production in JB6 P+ mouse skin epidermal (JB6 P+) cells compared with chlorogenic acid (5-O-caffeoylquinic acid), an ester of caffeic acid with quinic acid. Data also revealed that caffeic acid more effectively induced the downregulation of COX-2 expression at the transcriptional level mediated through the inhibition of activator protein-1 (AP-1) and nuclear factor-kappaB transcription activity compared with chlorogenic acid. Fyn kinase activity was suppressed more effectively by caffeic acid than by chlorogenic acid, and downstream mitogen-activated protein kinases (MAPKs) were subsequently blocked. Pharmacological Fyn kinase inhibitor (3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and leflunomide) data also revealed that Fyn is involved in UVB-induced COX-2 expression mediated through the phosphorylation of MAPKs in JB6 P+ cells. Pull-down assays revealed that caffeic acid directly bound with Fyn and non-competitively with adenosine triphosphate. In vivo data from mouse skin also supported the idea that caffeic acid suppressed UVB-induced COX-2 expression by blocking Fyn kinase activity. These results suggested that this compound could act as a potent chemopreventive agent against skin cancer.
Assuntos
Anticarcinógenos/farmacologia , Ácidos Cafeicos/farmacologia , Café/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/biossíntese , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Animais , Linhagem Celular Tumoral , Ácido Clorogênico/farmacologia , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Feminino , Isoxazóis/farmacologia , Leflunomida , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Pirimidinas/farmacologia , Ácido Quínico/análogos & derivados , Ácido Quínico/farmacologia , Pele/efeitos dos fármacos , Pele/enzimologia , Raios Ultravioleta/efeitos adversosRESUMO
Neurodegenerative disorders such as Alzheimer's disease (AD) are strongly associated with oxidative stress, which is induced by reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)). Recent studies suggest that moderate coffee consumption may reduce the risk of neurodegenerative diseases such as AD, but the molecular mechanisms underlying this effect remain to be clarified. In this study, we investigated the protective effects of chlorogenic acid (5-O-caffeoylquinic acid; CGA), a major phenolic phytochemical found in instant decaffeinated coffee (IDC), and IDC against oxidative PC12 neuronal cell death. IDC (1 and 5 microg/ml) or CGA (1 and 5 microM) attenuated H(2)O(2)-induced PC12 cell death. H(2)O(2)-induced nuclear condensation and DNA fragmentation were strongly inhibited by pretreatment with IDC or CGA. Pretreatment with IDC or CGA also inhibited the H(2)O(2)-induced cleavage of poly(ADP-ribose) polymerase (PARP), and downregulation of Bcl-X(L) and caspase-3. The accumulation of intracellular ROS in H(2)O(2)-treated PC12 cells was dose-dependently diminished by IDC or CGA. The activation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) by H(2)O(2) in PC12 cells was also inhibited by IDC or CGA. Collectively, these results indicate that IDC and CGA protect PC12 cells from H(2)O(2)-induced apoptosis by blocking the accumulation of intracellular ROS and the activation of MAPKs.
Assuntos
Apoptose/efeitos dos fármacos , Café/química , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fenóis/farmacologia , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Caspase 3/metabolismo , Ácido Clorogênico/isolamento & purificação , Ácido Clorogênico/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/isolamento & purificação , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Fenóis/isolamento & purificação , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteína bcl-X/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS: Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. METHODS AND RESULTS: Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). CONCLUSION: Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.
Assuntos
Cacau , MAP Quinase Quinase 1/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Preparações de Plantas/farmacologia , Proantocianidinas/farmacologia , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Biflavonoides/farmacologia , Catequina/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MAP Quinase Quinase 1/efeitos dos fármacos , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Trombina/farmacologiaRESUMO
Effective treatment for glioblastoma (GBM) is limited by the presence of the blood-brain barrier (BBB) and rapid resistance to single agent therapies. To address these issues, we developed a transferrin-functionalized nanoparticle (Tf-NP) that can deliver dual combination therapies. Using intravital imaging, we show the ability of Tf-NPs to traverse intact BBB in mice as well as achieve direct tumor binding in two intracranial orthotopic models of GBM. Treatment of tumor-bearing mice with Tf-NPs loaded with temozolomide and the bromodomain inhibitor JQ1 leads to increased DNA damage and apoptosis that correlates with a 1.5- to 2-fold decrease in tumor burden and corresponding increase in survival compared to equivalent free-drug dosing. Immunocompetent mice treated with Tf-NP-loaded drugs also show protection from the effects of systemic drug toxicity, demonstrating the preclinical potential of this nanoscale platform to deliver novel combination therapies to gliomas and other central nervous system tumors.
Assuntos
Antineoplásicos Alquilantes/química , Antineoplásicos/administração & dosagem , Azepinas/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Glioma/tratamento farmacológico , Nanopartículas/química , Temozolomida/administração & dosagem , Triazóis/administração & dosagem , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Azepinas/química , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/instrumentação , Glioma/metabolismo , Glioma/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Temozolomida/química , Triazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA damaging chemotherapy is a cornerstone of current front-line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo-resistant disease; therefore, adjuvant treatments that synergize with DNA-damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide-nucleic acid complex that facilitates tumor-specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex-mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high-grade serous OC to cytotoxic chemotherapy by blocking p38/MK2-dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.
RESUMO
Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.
Assuntos
Neoplasias da Mama/patologia , Tamanho Celular , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reconhecimento Automatizado de Padrão/métodos , Técnica de Subtração , Algoritmos , Linhagem Celular Tumoral , Simulação por Computador , Feminino , Humanos , Microscopia Intravital , Modelos Biológicos , Modelos Estatísticos , Fenótipo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, multivariate robust analysis method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to an HC RNAi screen to discover novel components of the DNA damage response in an osteosarcoma cell line. M-RAM automatically selects the most informative phenotypic readouts and time points to facilitate the more efficient design of follow-up experiments and enhance biological understanding. Our method outperforms univariate hit identification and identifies relevant genes that these approaches would have missed. We found that statistical cell-to-cell variation in phenotypic responses is an important predictor of hits in RNAi-directed image-based screens. Genes that we identified as modulators of DNA damage signaling in U2OS cells include B-Raf, a cancer driver gene in multiple tumor types, whose role in DNA damage signaling we confirm experimentally, and multiple subunits of protein kinase A.
Assuntos
Ensaios de Triagem em Larga Escala , Modelos Biológicos , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Algoritmos , Animais , Linhagem Celular , Simulação por Computador , Dano ao DNA , Técnicas de Silenciamento de Genes , Humanos , Fenótipo , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Metastasis is one of the most threatening features of the oncogenic process and the main cause of cancer-related mortality. Several studies have demonstrated that matrix metalloproteinases (MMPs) are critical for tumor invasion and metastasis. Resveratrol (3,5,4'-trihydroxystilbene), a phenolic compound of red wine, has been reported to be a natural chemopreventive agent. However, the cancer preventive effects of piceatannol (3,5,3',4'-tetrahydroxystilbene), a metabolite of resveratrol and the underlying molecular mechanisms have not yet been fully elucidated. In this study, we report that piceatannol inhi-bits H-ras-induced MMP-2 activity and the invasive phenotype of MCF10A human breast epithelial cells harboring mutated H-ras (H-ras MCF10A cells) more effectively than resveratrol. Piceatannol attenuated the H-ras-induced phosphorylation of Akt in a time- and dose-dependent manner, whereas resveratrol, at the same concentrations, did not exert an inhibitory effect. In vitro kinase assays demonstrated that piceatannol significantly inhibited phosphatidylinositol 3-kinase (PI3K) activity and suppressed phospha-tidylinositol (3,4,5)-trisphosphate (PIP3) expression in the H-ras MCF10A cells. Ex vivo pull-down assays revealed that piceatannol directly bound to PI3K, inhibiting PI3K activity. Data from molecular docking suggested that piceatannol is a more tight-binding inhibitor than resveratrol due to the additional hydrogen bond between the hydroxyl group and the backbone amide group of Val882 in the ATP-binding pocket of PI3K.
Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Genes ras , Metaloproteinase 2 da Matriz/genética , Estilbenos/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Mutação , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol , Vinho/análiseRESUMO
The unregulated migration and invasion of human aortic smooth muscle cells (HASMCs) into the intima is a crucial step in the development of atherosclerosis. Recently, the oriental persimmon extract (Diospyros kaki Thunb. cv. Fuyu) has been investigated for its anti-atherogenic properties, but the molecular mechanisms involved remain unclear. We investigated the inhibitory effects of persimmon peel and flesh extract on the platelet-derived growth factor (PDGF) BB-induced MMP-1 expression using Western blot, and abnormal migration and invasion of HASMCs using a modified Boyden chamber assay and a wound healing assay. We also evaluated the inhibitory effects of persimmon peel extract on aortic vessel thickening using a rat aortic sprouting assay. Persimmon peel (PPE), but not flesh extract (PFE), inhibited PDGF-BB-induced MMP-1 expression, cell migration and invasion in HASMCs, while suppressing the rat aortic sprouting. Western blot and in vitro kinase assay data demonstrated that PPE inhibited Src kinase activity and subsequently attenuated PDGF-BB-induced phosphorylation of MAPK and Akt signalling pathways. Taken together, our results indicate that persimmon peel might possess a potential anti-atherogenic effect through attenuation of ASMCs migration and invasion and aortic sprouting by direct inhibition of the c-Src kinase activity.
Assuntos
Aorta/citologia , Movimento Celular/efeitos dos fármacos , Diospyros/química , Miócitos de Músculo Liso/citologia , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Becaplermina , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Frutas/química , Humanos , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin.
Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Poluentes Ambientais/toxicidade , Receptores ErbB/metabolismo , Rutina/farmacologia , Animais , Linhagem Celular , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Quinases raf/metabolismoRESUMO
Abnormal regulation of Ca(2+) mediates tumorigenesis and Ca(2+) channels are reportedly deregulated in cancers, indicating that regulating Ca(2+) signaling in cancer cells is considered as a promising strategy to treat cancer. However, little is known regarding the mechanism by which Ca(2+) affects cancer cell death. Here, we show that 20-O-ß-d-glucopyranosyl-20(S)-protopanaxadiol (20-GPPD), a metabolite of ginseng saponin, causes apoptosis of colon cancer cells through the induction of cytoplasmic Ca(2+). 20-GPPD decreased cell viability, increased annexin V-positive early apoptosis and induced sub-G1 accumulation and nuclear condensation of CT-26 murine colon cancer cells. Although 20-GPPD-induced activation of AMP-activated protein kinase (AMPK) played a key role in the apoptotic death of CT-26 cells, LKB1, a well-known upstream kinase of AMPK, was not involved in this activation. To identify the upstream target of 20-GPPD for activating AMPK, we examined the effect of Ca(2+) on apoptosis of CT-26 cells. A calcium chelator recovered 20-GPPD-induced AMPK phosphorylation and CT-26 cell death. Confocal microscopy showed that 20-GPPD increased Ca(2+) entry into CT-26 cells, whereas a transient receptor potential canonical (TRPC) blocker suppressed Ca(2+) entry. When cells were treated with a TRPC blocker plus an endoplasmic reticulum (ER) calcium blocker, 20-GPPD-induced calcium influx was completely inhibited, suggesting that the ER calcium store, as well as TRPC, was involved. In vivo mouse CT-26 allografts showed that 20-GPPD significantly suppressed tumor growth, volume and weight in a dose-dependent manner. Collectively, 20-GPPD exerts potent anticarcinogenic effects on colon carcinogenesis by increasing Ca(2+) influx, mainly through TRPC channels, and by targeting AMPK.
Assuntos
Antineoplásicos/farmacologia , Cálcio/metabolismo , Neoplasias do Colo/tratamento farmacológico , Ginsenosídeos/farmacologia , Panax/química , Canais de Cátion TRPC/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Morte Celular , Sobrevivência Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Transdução de Sinais , Canais de Cátion TRPC/antagonistas & inibidoresRESUMO
Epidemiologic and animal studies revealed that capsaicin can act as a carcinogen or cocarcinogen. However, the molecular mechanisms of the cancer-promoting effects of capsaicin are not clear. Here, we report that capsaicin has a cocarcinogenic effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin carcinogenesis in vivo and is mediated through the epidermal growth factor receptor (EGFR), but not the transient receptor potential vanilloid subfamily member 1 (TRPV1). Topical application of capsaicin on the dorsal skin of 7,12-dimetylbenz(a)anthracene-initiated and TPA-promoted TRPV1 wild-type (WT) and TRPV1 knockout (KO) mice induced more and larger skin tumors in TRPV1/KO mice, suggesting a TRPV1-independent mechanism. Cyclooxygenase-2 (COX-2) was highly elevated by capsaicin treatment in tumors and murine embryonic fibroblasts from TRPV1/KO mice. Inhibitors of EGFR/MEK signaling suppressed TPA/capsaicin-induced COX-2 expression in TRPV1/KO cells, indicating that activation of EGFR and its downstream signaling is involved in COX-2 elevation. Capsaicin induced a further induction of TPA-increased COX-2 expression in EGFR/WT cells, but not in EGFR/KO cells. TPA/capsaicin cotreatment caused EGFR tyrosine phosphorylation and activated EGFR downstream signaling, including ERKs and Akt in EGFR/WT, but not EGFR/KO cells. Specific inhibition of EGFR and TRPV1 indicated that capsaicin-induced ERK activation in A431 cells was dependent on EGFR, but not TRPV1. Together, these findings suggest that capsaicin might act as a cocarcinogen in TPA-induced skin carcinogenesis through EGFR-dependent mechanisms.
Assuntos
Capsaicina/toxicidade , Cocarcinogênese , Receptores ErbB/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Canais de Cátion TRPV/metabolismo , Acetato de Tetradecanoilforbol/toxicidade , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/biossíntese , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Canais de Cátion TRPV/deficiência , Quinases da Família src/metabolismoRESUMO
Abnormal expression of cyclooxygenase-2 (COX-2) and prostaglandin (PG)E(2) is an important mediator in inflammation and tumor promotion. Arsenite is a well-known metalloid carcinogen that is strongly associated with increased risk of liver cancer, but the underlying mechanism remains to be clarified. The present study demonstrates that COX-2 expression and PGE(2) secretion are up-regulated by arsenite in rat liver epithelial (RLE) cells. The possible inhibitory effect of quercetin, a naturally occurring dietary flavonol, on arsenite-induced COX-2 expression and PGE(2) production was investigated. Pretreatment with quercetin resulted in the reduction of arsenite-induced expression of COX-2 and production of PGE(2). The arsenite-induced phosphorylation of Akt, p70S6K, and extracellular signal-regulated protein kinases (ERKs), but not p38, was inhibited by quercetin treatment. An ex vivo kinase assay revealed that quercetin suppressed arsenite-induced phosphoinositide 3-kinase (PI3K) activity upstream of Akt in RLE cell lysates. Ex vivo pull-down assays demonstrated that quercetin directly bound with PI3K to inhibit PI3K activity. Moreover, LY294002 (a PI3K inhibitor) significantly attenuated COX-2 expression and PGE(2) production in arsenite-treated RLE cells. These results suggest that quercetin suppresses arsenite-induced COX-2 expression mainly by blocking the activation of the PI3K signaling pathway, which may contribute to its chemopreventive potential.
Assuntos
Arsenitos/toxicidade , Ciclo-Oxigenase 2/metabolismo , Neoplasias Hepáticas Experimentais/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Quercetina/farmacologia , Animais , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/enzimologia , Ratos , Células Tumorais CultivadasRESUMO
AIMS: Abnormal migration and proliferation of human aortic smooth muscle cells (HASMCs) to the intima causes intimal thickening of the aorta, which is strongly related to the development of atherosclerosis. Previous studies have suggested that red wine polyphenols, particularly resveratrol, have great protective effects against cardiovascular diseases. Here, we compared the anti-atherosclerotic effect of piceatannol, a metabolite of resveratrol, and its underlying mechanisms. METHODS AND RESULTS: We demonstrated that piceatannol inhibited platelet-derived growth factor (PDGF)-BB-induced cell migration using a modified Boyden chamber assay and wound healing assay. Western blot analysis showed that PDGF-BB-induced phosphorylation of Akt, p70S6K, and p38 was inhibited by piceatannol, but not resveratrol. In vitro and ex vivo phosphoinositide 3-kinase (PI3K) assays demonstrated that piceatannol suppressed PI3K activity more effectively than resveratrol. PDGF-BB-induced migration and proliferation of HASMCs were inhibited by treatment with a commercial PI3K inhibitor, LY294002. Both in vitro and ex vivo pull-down assays revealed that piceatannol directly binds with sepharose 4B-PI3K beads in an ATP-competitive manner. CONCLUSION: The results of the present study demonstrate that piceatannol directly binds with PI3K in an ATP-competitive manner and suppresses PI3K activity with anti-atherosclerotic effects.
Assuntos
Movimento Celular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Estilbenos/farmacologia , Trifosfato de Adenosina/metabolismo , Indutores da Angiogênese/farmacologia , Aorta/citologia , Becaplermina , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis , Resveratrol , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Quinase SykRESUMO
Luteolin, a flavonoid present in various vegetables including onion and broccoli, has been reported to possess anticarcinogenic effects. However, its chemopreventive effect on UV-induced skin cancer and its mechanism are not fully understood. Herein, we examined the chemopreventive effect and associated mechanisms of luteolin in the JB6 P+ cell line and the SKH-1 hairless mouse model. Luteolin suppressed UVB-induced cyclooxygenase-2 expression and activator protein-1 and nuclear factor-kappaB activity in JB6 P+ cells. Immunoblot and kinase assay data showed that luteolin attenuated protein kinase C(epsilon) (PKC(epsilon)) and Src kinase activities and subsequently inhibited UVB-induced phosphorylation of mitogen-activated protein kinases and the Akt signaling pathway. In addition, pull-down assays revealed that luteolin binds directly to PKC(epsilon) and Src in an ATP-competitive manner. Importantly, luteolin suppressed tumor incidence, multiplicity, and overall size in SKH-1 hairless mice. Analysis of the skin by immunohistochemistry and immunoblotting showed that luteolin-treated groups had a substantial reduction in the levels of cyclooxygenase-2, tumor necrosis factor-alpha, and proliferating cell nuclear antigen compared with groups treated with only UVB. Further analysis using skin lysates showed that luteolin inhibited PKC(epsilon) and Src kinase activity. Together, these data suggest that luteolin exerts potent chemopreventive activity against UVB-induced skin cancer mainly by targeting PKC(epsilon)and Src.
Assuntos
Luteolina/farmacologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/prevenção & controle , Trifosfato de Adenosina/metabolismo , Animais , Ligação Competitiva , Proteína Tirosina Quinase CSK , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos da radiação , Linhagem Celular , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Pelados , NF-kappa B/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosforilação/efeitos da radiação , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Neoplasias Cutâneas/etiologia , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família srcRESUMO
Skin cancer is the most frequently diagnosed cancer in the United States. Ultraviolet B (UVB) rays (wavelength: 280-320nm) play a pivotal role in the development of skin cancer by inducing the expression of inflammatory proteins such as cyclooxygenase-2 (COX-2). Cyanidin, the most plentiful of the plant pigments known as anthocyanidins, is a potent chemopreventive agent. In the present study, we examined the molecular mechanisms underlying the chemopreventive activity of cyanidin and identified its molecular targets. Cyanidin inhibited UVB-induced COX-2 expression and prostaglandin E(2) secretion in the epidermal skin cell line JB6 P+ by suppressing the transactivation of nuclear factor-kappaB and activator protein-1 which are well-known transcription factors regulated by mitogen-activated protein kinase. Cyanidin markedly inhibited the phosphorylation of JNK1/2, ERK1/2, and MEK1/2 than the of MKK4 and Raf-1, two upstream kinases of JNK1/2, ERK1/2, and MEK1/2. Cyanidin significantly suppressed the activities of MKK4, MEK1, and Raf-1 through direct binding. Transient transfection of a small interfering RNA specific for MKK4 inhibited the UVB-induced expression of COX-2 in JB6 P+ cells, as did the expression of a dominant-negative ERK2 mutant. We conclude that MKK4, MEK1, and Raf-1 are targets of cyanidin for the suppression of UVB-induced COX-2 expression.
Assuntos
Antocianinas/farmacologia , Ciclo-Oxigenase 2/efeitos dos fármacos , Epiderme/efeitos dos fármacos , MAP Quinase Quinase 1/efeitos dos fármacos , MAP Quinase Quinase 4/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Animais , Western Blotting , Linhagem Celular , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Epiderme/enzimologia , Epiderme/efeitos da radiação , Imunoprecipitação , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 4/fisiologia , Camundongos , Proteínas Proto-Oncogênicas c-raf/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/fisiologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Tumor necrosis factor (TNF)-alpha-mediated cyclooxygenase (COX)-2 expression plays key roles in inflammation and tumorigenesis, particularly skin carcinogenesis, and hence targeting the TNF-alpha-mediated signaling pathway might be a promising strategy for developing chemopreventive agents against skin cancer and other skin disorders. Here we report that Fyn kinase - one of the members of the nonreceptor protein tyrosine kinase family - is involved in TNF-alpha-induced COX-2 expression, and that delphinidin - a major anthocyanidin present in red wine and berries - inhibits these effects by directly inhibiting Fyn kinase activity. Delphinidin strongly inhibited TNF-alpha-induced COX-2 expression in JB6 P+ mouse epidermal (JB6 P+) cells, whereas two other major phenolic compounds (resveratrol and gallic acid) did not exert significant inhibitory effects. Delphinidin inhibited the TNF-alpha-induced phosphorylations of JNK, p38 MAP kinase, Akt, p90RSK, MSK1, and ERK, and subsequently blocked the activation of the eukaryotic transcription factors AP-1 and NF-kappaB. Kinase and pull-down assay data revealed that delphinidin inhibited Fyn kinase activity and directly bound with Fyn kinase noncompetitively with ATP. By using PP2 (a commercial inhibitor of Fyn kinase) and siRNA-Fyn, we confirmed that Fyn kinase is involved in TNF-alpha-induced COX-2 expression mainly by down-regulating JNK in JB6 P+ cells. Together these findings suggest that the targeted inhibition of Fyn kinase activity and COX-2 expression by delphinidin contributes to the chemopreventive potential of red wine and berries.
Assuntos
Antocianinas/administração & dosagem , Ciclo-Oxigenase 2/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/biossíntese , Fator de Necrose Tumoral alfa/administração & dosagem , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Plantas , Inibidores de Proteínas Quinases/administração & dosagemRESUMO
Matrix metalloproteinases (MMPs) are involved in various skin disorders, including photoaging, dermatitis, and tumorigenesis. Tumor necrosis factor (TNF)-alpha is a key proinflammatory cytokine that acts to provoke inflammation, proliferation, and tumorigenesis. The present study investigated the possible inhibitory effects of red wine polyphenols on TNF-alpha-induced upregulation of MMP-9 and on the migratory phenotype of JB6 P+ mouse epidermal (JB6 P+) cells. Red wine extract (RWE) and quercetin, which is a major flavonoid present in red wine, inhibited significantly the TNF-alpha-induced upregulation of MMP-9 and cell migration, whereas resveratrol did not have significant inhibitory effects. The inhibitory effects of RWE and quercetin were mediated by suppression of the phosphorylation of Akt and the transactivation of activator protein-1 and nuclear factor-kappaB, as determined by Western blotting and luciferase assays, respectively. Aside from Akt, quercetin had no effect on the phosphorylation of other mitogen-activated protein kinases. Direct kinase assay data revealed that RWE and quercetin inhibited phosphatidylinositol 3-kinase (PI3K) activity. The results of direct and cell-based pull-down assays demonstrated that RWE and quercetin bound to PI3K, resulting in the inhibition of PI3K activity. Using chemical inhibitors, it was confirmed that the PI3K-dependent Akt pathway was involved in TNF-alpha-induced MMP-9 upregulation and migration in JB6 P+ cells. Collectively, these results indicate that TNF-alpha-induced MMP-9 upregulation and the migratory phenotype are associated with the PI3K/Akt pathway, and that these effects are inhibited strongly by RWE and quercetin.