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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204950

RESUMO

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Apoptose/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia
2.
Cancers (Basel) ; 13(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800291

RESUMO

Dual specificity phosphatase 1 (DUSP1) is crucial in prostate cancer (PC), since its expression is downregulated in advanced carcinomas. Here, we investigated DUSP1 effects on the expression of mesenchymal marker Snail, cell migration and invasion, analyzing the underlying mechanisms mediated by mitogen-activated protein kinases (MAPKs) inhibition. To this purpose, we used different PC cells overexpressing or lacking DUSP1 or incubated with MAPKs inhibitors. Moreover, we addressed the correlation of DUSP1 expression with Snail and activated MAPKs levels in samples from patients diagnosed with benign hyperplasia or prostate carcinoma, studying its implication in tumor prognosis and survival. We found that DUSP1 downregulates Snail expression and impairs migration and invasion in PC cells. Similar results were obtained following the inhibition of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK). In clinical samples, we evidenced an inverse correlation between DUSP1 expression and Snail levels, which are further associated with JNK and ERK activation. Consequently, the pattern DUSP1high/activated JNKlow/activated ERKlow/Snaillow is associated with an overall extended survival of PC patients. In summary, the ratio between DUSP1 and Snail expression, with additional JNK and ERK activity measurement, may serve as a potential biomarker to predict the clinical outcome of PC patients. Furthermore, DUSP1 induction or inhibition of JNK and ERK pathways could be useful to treat PC.

3.
Food Chem Toxicol ; 124: 273-279, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30552915

RESUMO

Resveratrol is a polyphenol with chemopreventive properties against prostate cancer; however, the mechanisms underlying its actions are not completely understood. Previously, we demonstrated that DUSP1 induces apoptosis in prostate cancer cells; therefore in the present study we investigated the role of this phosphatase on resveratrol effects. Moreover, we analysed the efficiency of combined treatment of resveratrol and the chemotherapeutic drug cisplatin on cellular viability and apoptosis and its relation with DUSP1 in prostate cancer cells. We found that resveratrol up-regulates DUSP1 expression in androgen-independent prostate cancer cells, which in turn, is involved in the inhibition of the NF-κB pathway and Cox-2 expression. This phosphatase is required for the induction of apoptosis achieved by resveratrol, but does not regulate the effects of this compound on cell cycle. Furthermore, we show that resveratrol cooperates with cisplatin both in the up-regulation of DUSP1 levels and in the promotion of apoptosis, suggesting that DUSP1 is a major determinant of cisplatin sensitivity to apoptosis. These results reveal a novel molecular mechanism by which resveratrol induces apoptosis in prostate cancer cells, and highlight the importance of DUSP1 in future therapeutic approaches based in the use of this polyphenol and cisplatin.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Resveratrol/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo , Sinergismo Farmacológico , Fosfatase 1 de Especificidade Dupla/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , NF-kappa B/antagonistas & inibidores , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Regulação para Cima
4.
FASEB J ; 32(2): 920-934, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29054855

RESUMO

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras-/-) and control wild-type (H -ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iß protein expression in H -ras-/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras-/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3ß-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iß overexpression in H -ras-/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iß overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.


Assuntos
Angiotensina II/efeitos adversos , Cardiomegalia/enzimologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hipertensão/enzimologia , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Angiotensina II/farmacologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/prevenção & controle , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fibroblastos/enzimologia , Fibroblastos/patologia , Deleção de Genes , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/patologia , Camundongos , Camundongos Knockout
5.
Mol Cell Biochem ; 411(1-2): 253-60, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26472731

RESUMO

Somatostatin (SST) is one of the main regulators of thyroid function. It acts by binding to its receptors, which lead to the dissociation of G proteins into Gαi and Gßγ subunits. However, much less is known about the function of Gßγ in thyroid cells. Here, we studied the role of SST and Gßγ dimers released upon SST stimulation on the Ras-ERK1/2 pathway in FTRL-5 thyroid cells. We demonstrate that SST activates Ras through Gi proteins, since SST-induced Ras activation is inhibited by pertussis toxin. Moreover, the specific sequestration of Gßγ dimers decreases Ras-GTP and phosphorylated ERK1/2 levels, and overexpression of Gßγ increases ERK1/2 phosphorylation induced by SST, indicating that Gßγ dimers released after SST treatment mediate activation of Ras and ERK1/2. On the other hand, SST treatment does not modify the expression of the thyroid differentiation marker sodium/iodide symporter (NIS) through ERK1/2 activation. However, SST increases AKT activation and the inhibition of the Src/PI3K/AKT pathway increases NIS levels in SST-treated cells. Thus, we conclude that, in thyroid cells, signalling from SST receptors to ERK1/2 involves a Gßγ-mediated signal acting on a Ras-dependent pathway. Moreover, we demonstrate that SST might regulates NIS expression through a Src/PI3K/AKT-dependent mechanism, but not through ERK1/2 signalling, showing the main role of this hormone in thyroid function.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Sistema de Sinalização das MAP Quinases , Somatostatina/administração & dosagem , Glândula Tireoide/efeitos dos fármacos , Proteínas ras/metabolismo , Linhagem Celular , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo
6.
Mol Carcinog ; 55(11): 1639-1654, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26392228

RESUMO

The epithelial-mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which V600E BRAF, TGFß and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGFß and Snail expression, with a concomitant downregulation of E-cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that V600E BRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGFß-mediated effects on Snail, E-cadherin, migration and invasion. Moreover, V600E BRAF induces TGFß secretion through a MEK/ERK-dependent mechanism. In addition, TGFß activates the Src/FAK complex, which in turn regulates the expression of Snail and E-cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGFß-induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGFß is independent of V600E BRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGFß induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the V600E BRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGFß, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of V600E BRAF-expressing thyroid tumours. © 2015 Wiley Periodicals, Inc.


Assuntos
Quinase 1 de Adesão Focal/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases , Mutação , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sulfonamidas/farmacologia
7.
Mol Oncol ; 8(1): 27-38, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24080497

RESUMO

Dual specificity phosphatase 1 (DUSP1) and the transcription factor NF-κB are implicated in prostate cancer since their expression levels are altered along this disease, although there are no evidences up to date demonstrating a crosstalk between them. In this report, we show for the first time that DUSP1 over-expression in DU145 cells promotes apoptosis and decreases NF-κB activity by blocking p65/NF-κB nuclear translocation. Moreover, although DUSP1 impairs TNF-α-induced p38 MAPK and JNK activation, only the specific inhibition of p38 MAPK exerts the same effects than DUSP1 over-expression on both apoptosis and NF-κB activity. Consistently, DUSP1 promotes apoptosis and decreases NF-κB activity in cells in which p38 MAPK is induced by TNF-α treatment. These results demonstrate that p38 MAPK is specifically involved in DUSP1-mediated effects on both apoptosis and NF-κB activity. Interestingly, we show an inverse correlation between DUSP1 expression and activation of both p65/NF-κB and p38 MAPK in human prostate tissue specimens. Thus, most of apparently normal glands, benign prostatic hyperplasia and low-grade prostatic intraepithelial neoplasia samples show high DUSP1 expression and low levels of both nuclear p65/NF-κB and activated p38 MAPK. By contrast, DUSP1 expression levels are low or even absent in high-grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma samples, whereas nuclear p65/NF-κB and activated p38 MAPK are highly expressed in the same samples. Overall, our results provide evidence for a role of DUSP1 in the apoptosis of prostate cancer cells, through a mechanism involving the inhibition of p38 MAPK and NF-κB. Furthermore, our findings suggest that the ratio between DUSP1 and p65/NF-κB expression levels, rather than the individual expression of both molecules, is a better marker for diagnostic purposes in prostate cancer.


Assuntos
Apoptose , Fosfatase 1 de Especificidade Dupla/metabolismo , NF-kappa B/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Fosfatase 1 de Especificidade Dupla/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Fosforilação , Próstata/metabolismo , Neoplasias da Próstata/genética , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
8.
Cancer Lett ; 335(1): 232-41, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-23435375

RESUMO

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.


Assuntos
Caderinas/metabolismo , Carcinoma/metabolismo , Indóis/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/metabolismo , Antígenos CD , Butadienos/farmacologia , Caderinas/genética , Carcinoma/patologia , Carcinoma Papilar , Linhagem Celular Tumoral , Movimento Celular , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Mutação de Sentido Incorreto , Invasividade Neoplásica , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologia
9.
Cancer Lett ; 314(2): 244-55, 2012 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-22056813

RESUMO

BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic (V600E)BRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease.


Assuntos
Apoptose/efeitos dos fármacos , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Melanoma/patologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Serina-Treonina Quinases TOR/fisiologia
10.
J Cell Biochem ; 108(6): 1292-301, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19795387

RESUMO

Insulin receptor substrate-4 (IRS-4) transmits signals from the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS-4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS-4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS-4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor-alpha (TNF-alpha). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial-dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF-alpha amplifies the effect of Act D on HepG2 cell apoptosis increasing c-jun N-terminal kinase (JNK) activity, IkappaB-alpha proteolysis and glutathione depletion. IRS-4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS-4 action is independent of Akt, IkappaB kinase and JNK. IRS-4 down regulation, however, decreased gamma-glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF-alpha. These results suggest that IRS-4 protects HepG2 cells from oxidative stress induced by drug treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Carcinoma Hepatocelular/metabolismo , Dactinomicina/farmacologia , Proteínas Substratos do Receptor de Insulina/antagonistas & inibidores , Neoplasias Hepáticas/metabolismo , Interferência de RNA , Fator de Necrose Tumoral alfa/farmacologia , Humanos , Imuno-Histoquímica , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo
11.
Carcinogenesis ; 30(10): 1670-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19700418

RESUMO

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or (V600E)B-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the (V600E)B-RAF mutation, but not in HCT-116 and LoVo cells harbouring the (G13D)Ras mutation, or BE cells, which possess two mutations, (G13D)Ras and (G463V)B-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the (V12)RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy.


Assuntos
Apoptose/genética , Colesterol/deficiência , Genes ras/efeitos dos fármacos , Células HT29/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3 , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Colesterol/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Ativação Enzimática , Citometria de Fluxo , Genes ras/genética , Células HT29/efeitos dos fármacos , Células HT29/patologia , Humanos , Hidroxicolesteróis/farmacologia , Lipoproteínas/sangue , MAP Quinase Quinase 4/metabolismo , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Transfecção
12.
Mol Endocrinol ; 22(11): 2466-80, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18755855

RESUMO

Thyroid hormone (T3) plays a crucial role in processes such as cell proliferation and differentiation, whereas its implication on cellular apoptosis has not been well documented. Here we examined the effect of T3 on the apoptosis of GH4C1 pituitary cells and the mechanisms underlying this effect. We show that T3 produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in nuclear factor-kappaB (NF-kappaB)-dependent transcription, IkappaBalpha phosphorylation, translocation of p65/NF-kappaB to the nucleus, phosphorylation, and transactivation. Moreover, these effects were correlated with a T3-induced decrease in the expression of antiapoptotic gene products, such as members of the inhibitor of apoptosis protein and Bcl-2 families. On the other hand, ERK but not c-Jun N-terminal kinase or MAPK p38, was activated upon exposure to T3, and inhibition of ERK alone abrogated T3-mediated apoptosis. In addition, T3 increased the expression of the MAPK phosphatase, dual specificity phosphatase 1 (DUSP1), in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T3-induced inhibition of NF-kappaB-dependent transcription and p65/NF-kappaB translocation to the nucleus, as well as T3-mediated apoptosis. Overall, our results indicate that T3 induces apoptosis in rat pituitary tumor cells by down-regulating NF-kappaB activity through a mechanism dependent on the ERK/DUSP1 pathway.


Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , Tri-Iodotironina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Linhagem Celular , Meios de Cultura Livres de Soro , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla/genética , Genes bcl-2/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/genética , NF-kappa B/metabolismo , Hipófise/citologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Ratos
13.
Apoptosis ; 12(11): 2013-24, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17828457

RESUMO

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89-99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.


Assuntos
Apoptose/efeitos dos fármacos , Capsaicina/farmacologia , Ceramidas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Próstata/efeitos dos fármacos , Esfingomielina Fosfodiesterase/fisiologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Masculino , Próstata/citologia , Próstata/enzimologia
14.
J Hepatol ; 46(6): 1089-98, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17408801

RESUMO

BACKGROUNDS/AIMS: Insulin receptor substrate-4 (IRS-4) is a scaffold protein that mediates the actions of insulin-like growth factor-I (IGF-I). Its expression increases dramatically after partial hepatectomy (a liver regeneration model). Herein, we report IRS-4 expression in a human hepatoblastoma cell line (HepG2) and IGF-I-dependent IRS-4 tyrosine phosphorylation. METHODS: The role of IRS-4 in HepG2 proliferation was established by RNA interference (siRNA). After 72h of transfection with IRS-4 siRNA, we observed a specific reduction in IRS-4 expression. RESULTS: Depletion of IRS-4 levels decreased ERK phosphorylation, p70S6K phosphorylation and IGF-I-stimulated cell proliferation. Changes in ERK phosphorylation in IRS-4-depleted cells were independent of ras/raf/MEK1/2- and PI3K/Akt-cascades. IRS-4 down-regulation abolished IGF-I-, TPA- and IGF-I plus TPA-stimulated ERK and p70S6K activities. Our results suggest that PKC-epsilon mediates the effect of IRS-4 on ERK activity. Moreover, decreased IRS-4 levels diminished FBS- and IGF-I-stimulated HepG2 growth and cause stress fiber disruption in HepG2 cell line. CONCLUSIONS: Collectively, our data suggest that IRS-4 plays an important role in HepG2 proliferation/differentiation and exerts its actions through ERK and p70S6K activation in a ras/raf/MEK1/2- and PI3Kinase/Akt-independent manner and in a PKC-dependent way.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/metabolismo , Linhagem Celular Tumoral , Hepatectomia , Hepatócitos/citologia , Humanos , Proteínas Substratos do Receptor de Insulina , Fígado/patologia , Microscopia de Contraste de Fase , Modelos Biológicos , Fosforilação , Proteína Quinase C/metabolismo , Fatores de Tempo , Transfecção
15.
Cell Signal ; 18(12): 2292-301, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16806824

RESUMO

Cholesterol, p38 MAPK and NFkappaB have been shown to participate in inflammation and cellular differentiation. Here, we examined the effect of cholesterol on NFkappaB-dependent transcription and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in NFkappaB-dependent transcription, NFkappaB-DNA binding, IkappaBalpha degradation and p65/NFkappaB translocation to the nucleus, and the addition of exogenous cholesterol reversed these effects. Previously, we have shown that low cell cholesterol levels activate p38 MAPK. Here, we found that inhibition of p38 MAPK with the specific inhibitor SB203580 blocked the increase in NFkappaB activity, IkappaBalpha degradation and p65/NFkappaB translocation to the nucleus induced by cholesterol depletion. Moreover, the inhibition of the p38 MAPK downstream effector MSK1 with the specific inhibitor H89, or the overexpression of a kinase defective MSK1 abrogated the NFkappaB-dependent transcription induced by cholesterol depletion. On the other hand, the transactivation potential of p65/NFkappaB depends on phosphorylation of S276 by MSK1. We observed that cholesterol depletion increased the p65/NFkappaB transactivation capacity. This effect was reversed by cell cholesterol repletion or incubation with the SB203580 inhibitor. Moreover, the expression of a p65/NFkappaB S276A mutant was insensitive to cholesterol depletion. Together, our results demonstrate that cholesterol depletion induces NFkappaB transcriptional activity, not only by affecting the IkappaBalpha degradation and the translocation of p65/NFkappaB to the nucleus, but also regulating the p65/NFkappaB transactivating potential through a p38 MAPK/MSK1 mediated pathway.


Assuntos
Colesterol/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Colesterol/farmacologia , Meios de Cultura/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Hidroxicolesteróis/farmacologia , Proteínas I-kappa B/metabolismo , Imidazóis/farmacologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , NF-kappa B/genética , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Ativação Transcricional/genética , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
16.
Apoptosis ; 11(7): 1161-73, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16699960

RESUMO

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.


Assuntos
Apoptose/fisiologia , Colesterol/deficiência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Colesterol/metabolismo , Colesterol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hidroxicolesteróis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas/deficiência , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Fosfatos de Poli-Isoprenil/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sesquiterpenos , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética
18.
Prostate ; 63(1): 44-55, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15468165

RESUMO

BACKGROUND: Neuroendocrine (NE) differentiation in prostate cancer has been correlated with unfavorable clinical outcome. The mechanisms by which prostate cancer acquires NE properties are poorly understood, but several signaling pathways have been proposed. We have previously observed that vasoactive intestinal peptide (VIP) stimulates cAMP production mainly through VPAC(1) receptor, inducing NE differentiation in LNCaP cells. The aim of this study was to analyze the mechanisms involved in this process. METHODS: Reverse transcriptase (RT)-polymerase chain reaction (PCR), quantitative real-time RT-PCR, Western blotting, and immunocytochemistry were performed. RESULTS: LNCaP cells produce VIP, as demonstrated by RT-PCR and immunocytochemistry. VIP induced NE differentiation of LNCaP cells at a time as short as 1 hr of treatment, and the same occurred with the expression and secretion of neuronal-specific enolase (NSE, a NE differentiation marker). These effects were faster than those exerted by serum-deprivation. VIP induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and NE differentiation by PKA-dependent and independent pathways, since the PKA inhibitor H89 partially blocked VIP-induced NE differentiation and did not affect ERK1/2 phosphorylation. mitogen-activated protein kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) appear to be also involved since the inhibitors PD98059 and wortmannin abolished ERK1/2 phosphorylation and decreased NE differentiation induced by VIP. Moreover, VIP activated Ras suggesting the involvement of a Ras-dependent pathway. CONCLUSIONS: VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing NE differentiation through PKA, ERK1/2, and PI3K.


Assuntos
Carcinoma Neuroendócrino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Neoplasias da Próstata , Peptídeo Intestinal Vasoativo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
19.
Oncogene ; 23(37): 6292-8, 2004 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-15208680

RESUMO

B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.


Assuntos
Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-raf/metabolismo , Benzenossulfonatos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Melanoma/metabolismo , Melanoma/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Piridinas/uso terapêutico , Sorafenibe
20.
Cell Signal ; 15(1): 131-8, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12401528

RESUMO

Previous results from our laboratory showed that GH(4)C(1) cells with low-cholesterol cell content had increased adenylyl cyclase (AC) activity with a parallel increase in G protein alpha subunits associated to the plasma membrane. This effect was directly related to mevalonate availability. In the present report, we characterized the high-affinity GTPase activity present in GH(4)C(1) cell membranes and studied its regulation by cholesterol cell content. The high-affinity GTPase activity, measured as the [gamma32P]GTP hydrolysis rate, was both time-dependent and protein concentration-dependent. Cultured cells with lipoprotein-deficient serum (LPDS) showed decreased cholesterol cell content and decreased GTPase activity. The kinetic analysis, as interpreted by Lineweaver-Burk plots, indicated that low-cholesterol cell content had no effect on the apparent affinity for GTP, but resulted in a 47% decrease in the maximal velocity of the reaction. Addition of 25-hydroxycholesterol (25-HC), an inhibitor of the expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and synthetase to cells in LPDS, further decreased GTPase activity in a dose-dependent manner. This effect was reverted by exogenous cholesterol, but not by mevalonate. Studies with bacterial toxins revealed that neither cholera toxin (CTX) nor pertussis toxins (PTX) were able to revert the inhibition produced by low-cholesterol cell content. These results allowed us to postulate that cholesterol modulates GTPase activity in both Gs and Gi protein families. To analyse further the mechanism of modulation of GTPase activity by cholesterol cell content, [35S]GTPgammaS binding in membranes of GH(4)C(1) cells was studied. Changes in cholesterol cell content did not have any effect on GTP binding. Data demonstrated that high-affinity GTPase activity in plasma membrane of GH(4)C(1) cells is direct stimulated by cholesterol cell content and not by mevalonate availability. This example provides a mechanism by which cholesterol cell content can modulate signal transduction mediating by G proteins.


Assuntos
Membrana Celular/enzimologia , Colesterol/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Animais , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Meios de Cultura , Relação Dose-Resposta a Droga , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hidroxicolesteróis/farmacologia , Cinética , Ácido Mevalônico/farmacologia , Hipófise/metabolismo , Ratos , Células Tumorais Cultivadas
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