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1.
Int J Mol Sci ; 20(20)2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635251

RESUMO

Damage-specific DNA-binding protein 2 (DDB2) was originally identified as a DNA damage recognition factor that facilitates global genomic nucleotide excision repair (GG-NER) in human cells. DDB2 also contributes to other essential biological processes such as chromatin remodeling, gene transcription, cell cycle regulation, and protein decay. Recently, the potential of DDB2 in the development and progression of various cancers has been described. DDB2 activity occurs at several stages of carcinogenesis including cancer cell proliferation, survival, epithelial to mesenchymal transition, migration and invasion, angiogenesis, and cancer stem cell formation. In this review, we focus on the current state of scientific knowledge regarding DDB2 biological effects in tumor development and the underlying molecular mechanisms. We also provide insights into the clinical consequences of DDB2 activity in cancers.

2.
Nanoscale ; 8(9): 5268-79, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26879405

RESUMO

DDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer.


Assuntos
Neoplasias da Mama , Proteínas de Ligação a DNA/biossíntese , Módulo de Elasticidade , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/ultraestrutura , Adesão Celular , Feminino , Humanos , Células MCF-7 , Microscopia de Força Atômica , Metástase Neoplásica
3.
Front Chem ; 3: 67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26734600

RESUMO

We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects.

4.
Free Radic Biol Med ; 77: 139-51, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25224035

RESUMO

Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently reliable predictive biological markers, and this is a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation, which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O2(•-)) and hydrogen peroxide (H2O2). Particularly, they report how these reactive oxygen species may activate some signaling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests a way to define precisely the clinical interest of MnSOD as a prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well-known biomarkers, in addition to the typical clinical parameters.


Assuntos
Neoplasias da Mama/enzimologia , Superóxido Dismutase/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Indução Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Estresse Oxidativo , Polimorfismo Genético , Superóxidos/metabolismo
5.
Cancer Res ; 73(16): 5040-52, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23774208

RESUMO

The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-κB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-κB activity by upregulating expression of IκBα by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IκBα gene expression restored NF-κB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , NF-kappa B/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Células MCF-7 , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Invasividade Neoplásica , Prognóstico , Regiões Promotoras Genéticas , Transcrição Genética , Regulação para Cima/genética
6.
Free Radic Biol Med ; 50(12): 1771-9, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21419216

RESUMO

A high basal expression of manganese superoxide dismutase (MnSOD) has been reported in aggressive breast cancer cells, according to an unknown mechanism, and contributes to their invasive abilities. Here, we report the involvement of Sp1 and nuclear factor-κB (NF-κB) transcription factors in this high basal expression of MnSOD in aggressive breast cancer cells. Suppression or inactivation of Sp1 showed that it plays an essential role in the high MnSOD expression in aggressive breast cancer cells through a unique binding site identified by chromatin immunoprecipitation (ChIP) assay and functional analysis of the MnSOD proximal promoter. Treatment of cells with a specific NF-κB inhibitor peptide decreased significantly high basal MnSOD expression. A ChIP assay showed binding of a constitutive p50/p65 NF-κB complex to the MnSOD intronic enhancer element, associated with hyperacetylation of the H3 histone. Finally, high basal expression of MnSOD resulted in the lack of expression of Damaged DNA binding 2 (DDB2) protein in aggressive breast cancer cells. DDB2 overexpression prevented the binding of Sp1 as well as of NF-κB to their respective elements on the MnSOD gene. These results contribute to a better understanding of MnSOD up-regulation, which may be clinically important in the prediction of breast tumor progression.


Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Transcrição Sp1/metabolismo , Superóxido Dismutase/genética , Acetilação , Sequência de Bases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Feminino , Radicais Livres , Histonas/genética , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estudos Retrospectivos , Fator de Transcrição Sp1/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
7.
Biochem Pharmacol ; 80(2): 226-35, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20380812

RESUMO

The general strategy developed aims to favor the vascular effect of photodynamic therapy by targeting tumor vasculature. Since angiogenic endothelial cells represent an interesting target to potentiate this vascular effect, we previously described the conjugation of a photosensitizer to a peptide targeting neuropilins (NRPs) over-expressed specially in tumor angiogenic vessels and we recently characterized the mechanism of photosensitization-induced thrombogenic events. Nevertheless, in glioma-bearing nude mice, we demonstrated that the peptide moiety was degraded to various rates according to time after intravenous administration. In this study, new peptidases-resistant pseudopeptides were tested, demonstrating a molecular affinity for NRP-1 and NRP-2 recombinant chimeric proteins and devoid of affinity for VEGF receptor type 1 (Flt-1). To argue the involvement of NRP-1, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor. We evidenced a statistically significant decrease of the different peptides-conjugated photosensitizers uptake after RNA interference-mediated silencing of NRP-1. Peptides-conjugated photosensitizers allowed a selective accumulation into cells. In mice, no degradation was observed in plasma in vivo 4h after intravenous injection by MALDI-TOF mass spectrometry. This study draws attention to this potential problem with peptides, especially in the case of targeting strategies, and provides useful information for the future design of more stable molecules.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neuropilina-1/metabolismo , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Feminino , Inativação Gênica , Humanos , Neuropilina-1/genética , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Porfirinas/química , Porfirinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
J Photochem Photobiol B ; 96(2): 101-8, 2009 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-19464192

RESUMO

The strategy developed aims to favor the vascular effect of photodynamic therapy (PDT) by targeting tumor vasculature. This approach is considered by coupling a photosensitizer (PS) to an heptapeptide targeting neuropilin-1 (NRP-1). We previously demonstrated that this new conjugated PS, which binds to recombinant NRP-1 protein, was a much more potent PS compared to the non-conjugated PS in human umbilical vein endothelial cells (HUVEC) expressing NRP-1, due to the coupling of the peptide moiety. To argue the involvement of NRP-1 in the conjugated PS cellular uptake, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor, and we evidenced a significant decrease of the conjugated PS uptake after RNA interference-mediated silencing of NRP-1. In mice xenografted ectopically with U87 human malignant glioma cells, we demonstrated that only the conjugated PS allowed a selective accumulation in endothelial cells lining tumor vessels. Vascular endothelial growth factor (VEGF) plasma and tumor levels could not prevent the recognition of the conjugate by NRP-1. The vascular effect induced by the conjugated PS, was characterized by a reduction in tumor blood flow around 50% during PDT. In vivo, the photodynamic efficiency with the conjugated PS induced a statistically significant tumor growth delay compared to the non-coupled PS. The peptide-conjugated chlorin-type PS uptake involves NRP-1 and this targeting strategy favors the vascular effect of PDT in vivo.


Assuntos
Neuropilina-1/metabolismo , Peptídeos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Camundongos , Neuropilina-1/genética , Peptídeos/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Porfirinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
J Biol Chem ; 284(21): 14165-76, 2009 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-19339246

RESUMO

Manganese superoxide dismutase plays a role in breast tumor cell growth, which depends on its constitutive expression. However, the mechanisms responsible for the regulation of constitutive SOD2 gene expression at different malignant phenotype in breast cancers remain to be determined. The present study reports the identification and characterization of a DNA sequence located in the proximal promoter of the SOD2 gene, which forms a complex with a nuclear protein from breast tumor MCF-7 cells. Purification of this complex showed that it contained DDB2 (damaged DNA binding 2), a well known protein involved in nucleotide excision DNA repair and cell cycle regulation. Functional analysis of the proximal promoter of the SOD2 gene or modulation of DDB2 expression allowed us to demonstrate that DDB2 regulates negatively the constitutive expression of the SOD2 gene in breast cancer cells. We demonstrate that the binding of DDB2 was associated with the loss of acetylated H3 histones and the decrease in the binding of Sp1 but not AP-2alpha transcription factors to the SOD2 proximal promoter. In addition, we show that DDB2 exerts, at least in part, a control of breast cancer cell growth through its negative regulation of constitutive expression of the SOD2 gene. For the first time, these data give supporting evidence that DDB2 is a new transcriptional regulator, and they provide insight into the molecular function of breast cancer cell growth, which will have an important clinical interest.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Superóxido Dismutase/genética , Transcrição Genética , Sequência de Bases , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Feminino , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Superóxido Dismutase/metabolismo , Fatores de Transcrição/metabolismo
10.
Oncol Rep ; 21(3): 731-5, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19212633

RESUMO

Overexpression of epidermal growth factor receptor (EGFR) and mutation of pten tumor suppressor gene in human cancer cells leads to activated EGFR downstream signaling including PI3-kinase/AKT (PI3K/AKT) and/or mitogen-activated protein kinases (RAS/RAF/MAPK) and have been linked to resistance to anti-EGFR targeted therapies. Cetuximab is a chimeric IgG1 monoclonal antibody that binds the EGFR with high specificity and have been developed as promising therapeutic anticancer treatments in several solid tumors, including colorectal and head and neck squamous cell carcinomas. Cetuximab activity is related to PI3K/AKT and RAS/RAF/MAPK signaling pathways functionality and its activity has been shown to be higher in wild-type KRAS tumors. To study the influence of PTEN expression on cell response to cetuximab, we used wild-type KRAS, PTEN-null, EGFR overexpressing PC3 prostate cancer cells. Reintroduction of PTEN significantly reduced the constitutive overexpression of phosphorylated-AKT (p-AKT) and downstream kinases (p-GSK3beta and p-P70S6 kinase) as well as phosphorylated-ERK1/2 (p-ERK1/2) and consequently significantly restored cetuximab-induced cell growth inhibition and apoptosis induction. Taken together, the results achieved in the present study show that PTEN controls the cellular response to cetuximab in KRAS wild-type prostate carcinoma PC3 cells through the regulation of AKT phosphorylation and restoration of the functionality of EGFR downstream signaling. Extrapolation of these findings to clinical situation, suggests that the assessment of EGFR downstream signaling functionality could be proposed as a diagnostic response predictive marker for anti-EGFR targeted therapies.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Cetuximab , Citometria de Fluxo , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais/efeitos dos fármacos , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética
11.
PLoS One ; 3(4): e2002, 2008 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-18431487

RESUMO

The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.


Assuntos
Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Fase G1 , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores Estrogênicos/metabolismo , Fase S
12.
Breast Cancer Res Treat ; 108(2): 203-15, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17473980

RESUMO

Manganese superoxide dismutase (MnSOD) is known to play a role in cancer. MnSOD exerts a tumor suppressive effect in estrogen-dependent human breast cancer cells. In the present study we investigated the in vitro role of MnSOD in the growth of some aggressive and highly metastatic estrogen-independent breast cancer cells, i.e., MDA-MB231 and SKBR3 cells. We show that estrogen-independent cells expressed a significantly higher basal MnSOD level compared to estrogen-dependent human breast cancer cell lines (MCF-7 and T47D). For MDA-MB231 cells, the high-MnSOD level was accompanied by an overproduction of intracellular hydrogen peroxide (H2O2) and by a low expression of the major H2O2-detoxifying enzymes, catalase, and peroxiredoxin 3, compared to MCF-7 cells. Suppression of MnSOD expression by antisense RNA was associated with a decrease of H2O2 content and caused a stimulation of growth with a reduced cell doubling time but induced a decrease of colony formation. Furthermore, treatment of MDA-MB231 cells with H2O2 scavengers markedly reduced tumor cell growth and colony formation. In addition, MnSOD suppression or treatment with H2O2 scavengers reduced the invasive properties of MDA-MB231 cells up to 43%, with a concomitant decrease of metalloproteinase-9 activity. We conclude that MnSOD plays a role in regulating tumor cell growth and invasive properties of estrogen-independent metastatic breast cancer cells. These action are mediated by MnSOD-dependent H2O2 production. In addition, these results suggest that MnSOD up-regulation may be one mechanism that contributes to the development of metastatic breast cancers.


Assuntos
Neoplasias da Mama/enzimologia , Movimento Celular , Proliferação de Células , Superóxido Dismutase/metabolismo , Antioxidantes/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catalase/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Estrogênios/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Peroxirredoxina III , Peroxirredoxinas/metabolismo , RNA Antissenso/metabolismo , Superóxido Dismutase/genética , Fatores de Tempo , Transfecção
13.
Med Sci (Paris) ; 23(5): 515-8, 2007 May.
Artigo em Francês | MEDLINE | ID: mdl-17502068

RESUMO

The peroxisome proliferator-activated receptors (PPARs) are transcription factors and belong to the superfamily of nuclear receptors. They are encoded by three genes located on different chromosomes: PPARalpha, PPARbeta/delta and PPARgamma. PPARalpha plays a key role in the control of lipid metabolism and homeostasis. PPARbeta/delta is expressed ubiquitously and participates in skeletal muscle physiology. PPARbeta/delta and PPARgamma are important factors for placental development and function as well as for embryo implantation. In addition, PPARgamma is mainly involved in adipogenesis. PPARs also participate more or less to cell proliferation, differentiation and apoptosis. Surprisingly, the involvement of these transcription factors in cell-cell and/or cell-matrix interactions has not yet been reviewed except for their role as therapeutic agents in inflammation. Nevertheless, several pioneer reports have recently provided some new insights in this research field, by suggesting that PPARs are involved, directly or indirectly, in these interactions and that their activation by specific ligands may lead to potential therapeutic approaches.


Assuntos
Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Matriz Extracelular/fisiologia , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Apoptose , Diferenciação Celular , Divisão Celular , Humanos , Integrinas/genética , Fígado/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
14.
Biochimie ; 89(3): 329-36, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17070643

RESUMO

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with retinoid X receptor (RXR) and bind to specific PPAR-response elements. To identify novel PPAR target genes, we developed an affinity method to isolate human genomic fragments containing binding sites for PPARs. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced. One of them, ISF1029, was mapped by BLAT and BLAST searches on different human chromosomes, downstream of several POTE genes. ISF1029 contained three hexamers strongly related to the AGGTCA motif organized according to a DR0/3 motif. The latter was found to bind to PPARalpha in gel mobility shift and supershift assays and to exhibit a downregulation potentiality in transfection experiments under clofibrate treatment. POTE genes were shown to be highly expressed in human Caco-2 colorectal adenocarcinoma cells and downregulated by fenofibrate and 9-cis-retinoic acid, as attested by RT-PCR assays. Microarray analysis confirmed and extended to the human T98-G glioblastoma cells, the downregulation of several POTE genes expression by Wy-14,643, a potent PPARalpha activator. Our data provide new insights about the pleiotropic action of PPARs.


Assuntos
DNA/metabolismo , Genoma Humano , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Elementos de Resposta/genética , Sequência de Bases , Sítios de Ligação/genética , Southern Blotting , Células CACO-2 , Linhagem Celular Tumoral , DNA/imunologia , DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Células HCT116 , Humanos , Imunoprecipitação , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/imunologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Int J Oncol ; 28(4): 977-84, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16525649

RESUMO

This study tests the hypothesis that the activators of peroxisome proliferator-activated receptors (PPARs) and 9-cis-retinoic acid receptor (RXR) regulate human semaphorin 6B (Sema6B) gene expression. The human MCF-7 breast adenocarcinoma cell line was chosen because it expresses Sema6B at a high level. The Sema6B mRNA level was analyzed by RT-PCR and the semaphorin 6B protein content was determined using a polyclonal antibody that we have produced and characterized. Treatments with fenofibrate (a PPARalpha activator) and troglitazone (a PPARgamma ligand) strongly decreased the Sema6B mRNA. The drop in Sema6B mRNA level and in protein content was more important when the treatment combined the action of fenofibrate or troglitazone and 9-cis-retinoic acid. On the other hand, no significant change was observed in the Sema6B mRNA and protein levels when the cells were exposed to the combined action of GW610742 (a PPARbeta activator) and 9-cis-retinoic acid. These data suggest that PPARalpha/RXR and PPARgamma/RXR heterodimers are involved in the regulation of Sema6B gene expression and open new perspectives concerning the participation of these nuclear receptors in cell recognition and migration.


Assuntos
Cromanos/farmacologia , Fenofibrato/farmacologia , Semaforinas/genética , Tiazolidinedionas/farmacologia , Alitretinoína , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Dimerização , Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Células K562 , PPAR alfa/agonistas , PPAR alfa/química , PPAR delta/agonistas , PPAR gama/agonistas , PPAR gama/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores X Retinoide/agonistas , Receptores X Retinoide/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Semaforinas/metabolismo , Tiazóis/farmacologia , Tretinoína/farmacologia , Troglitazona
16.
Int J Mol Med ; 16(3): 483-92, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16077959

RESUMO

Hypolipidemic drugs (HP drugs) are xenobiotics belonging to the peroxisome proliferator family which are used as pharmaceuticals in the treatment of human hyperlipidemia and hypercholesterolemia. They cause hepatocarcinogenesis in rodents by increasing cell proliferation. One hypothesis is that this hepatocarcinogenic effect is caused by induced oxidative stress resulting from the overproduction of reactive oxygen species (ROS) and from a decreasing antioxidant defense. In addition, ROS play a role in hepatocellular proliferation by activation of NF-kappaB and AP-1, leading to an increase in mitogenic cytokines such as tumor necrosis factor-alpha. No liver cancer incidence has been noted in individuals treated with HP drugs for brief periods of time. However, the observation that old rats and mice are more susceptible than young individuals to the hepatocarcinogenic effect caused by long term exposure to HP drugs raises the question of a potential health risk for the human population. In vitro, HP drugs cause an apoptogenic effect in human hepatocytes. This effect is related to a moderate antioxidant response, dysfunction of mitochondria caused by an overproduction of ROS and release of apoptogenic factors. Finally, the apoptogenic effect of HP drugs is observed in human hepatomas, suggesting a clinical interest of these agents in antitumoral activity.


Assuntos
Hipolipemiantes/efeitos adversos , Fígado/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores Etários , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Hiperlipidemias/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Fígado/metabolismo , Fígado/patologia , Camundongos , NF-kappa B/metabolismo , Ratos , Superóxido Dismutase/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
Cancer Res ; 65(10): 4282-91, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15899820

RESUMO

Mitochondrial dysfunctions are frequently reported in cancer cells, but their direct involvement in tumorigenesis remains unclear. To understand this relation, we stimulated mitochondrial activity by overexpression of the mitochondrial triiodothyronine receptor (p43) in human dermal fibroblasts. In all clones, this stimulation induced morphologic changes and cell fusion in myotube-like structures associated with the expression of several muscle-specific genes (Myf5, desmin, connectin, myosin, AchRalpha). In addition, these clones displayed all the in vivo and in vitro features of cell transformation. This phenotype was related to an increase in c-Jun and c-Fos expression and extinction of tumor suppressor gene expression (p53, p21WAF1, Rb3). Lastly, reactive oxygen species (ROS) production was increased in positive correlation to the stimulation of mitochondrial activity. The direct involvement of mitochondrial activity in this cell behavior was studied by adding chloramphenicol, an inhibitor of mitochondrial protein synthesis, to the culture medium. This inhibition resulted in partial restoration of the normal phenotype, with the loss of the ability to fuse, a strong decrease in muscle-specific gene expression, and potent inhibition of the transformed phenotype. However, expression of tumor suppressor genes was not restored. Similar results were obtained by using N-acetylcysteine, an inhibitor of ROS production. These data indicate that stimulation of mitochondrial activity in human dermal fibroblasts induces cell transformation through events involving ROS production.


Assuntos
Antígenos de Neoplasias/biossíntese , Transformação Celular Neoplásica/patologia , Mitocôndrias/fisiologia , Fator Tu de Elongação de Peptídeos/biossíntese , Pele/patologia , Animais , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Cloranfenicol/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Inibidores da Síntese de Proteínas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
18.
Neuropharmacology ; 48(5): 673-84, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15814102

RESUMO

In order to approach the astroglial implication of addictive and neurotoxic processes associated with psychostimulant drug abuse, the effects of amphetamine or cocaine (1-100 microM) on redox status, AP-1 transcription factor and pro-enkephalin, an AP-1 target gene, were investigated in the human astrocyte-like U373 MG cells. We demonstrated an early increase in the generation of radical oxygen species and in the formation of 4-hydroxynonenal-adducts reflecting the pro-oxidant action of both substances. After 1 h or 96 h of treatment, Fos and Jun protein levels were altered and the DNA-binding activity of AP-1 was increased in response to both substances. Using supershift experiments, we observed that the composition of AP-1 dimer differed according to the substance and the duration of treatment. FRA-2 protein represented the main component of the chronic amphetamine- or cocaine-activated complexes, which suggests its relevance in the long-term effects of psychostimulant drugs. Concomitantly, the pro-enkephalin gene was differently regulated by either 6 h or 96 h of treatment. Because astrocytes interact extensively with the neurons in the brain, our data led us to conclude that oxidation-regulated AP-1 target genes may represent one of the molecular mechanisms underlying neuronal adaptation associated with psychostimulant dependence.


Assuntos
Anfetamina/farmacologia , Astrócitos/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Encefalinas/metabolismo , Precursores de Proteínas/metabolismo , Fator de Transcrição AP-1/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Análise de Variância , Western Blotting/métodos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Encefalinas/genética , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Fluoresceínas , Antígeno 2 Relacionado a Fos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Oxirredução/efeitos dos fármacos , Precursores de Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Fatores de Transcrição/metabolismo
19.
Biochimie ; 86(9-10): 633-42, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15556273

RESUMO

We showed that the metabolism of arachidonic acid (AA) in HepG2 cells generates reactive oxygen species (ROS), which activate the p38 mitogen-activated protein kinase (MAPK) pathway and the redox-sensitive transcription factors AP-1 and NF-kappaB, leading to the induction of the antioxidant manganese superoxide dismutase gene. The present study reports that AA decreases the HepG2 cell growth by 40% and 55% after a treatment for 24 and 48 h, respectively. This effect was blocked by an inhibitor of lipoxygenase/cytochrome P450 monooxygenase pathways and by the antioxidants. In addition, AA induced an oxidative stress, as an accumulation of malondialdehyde (MDA)-modified proteins, resulting to a generation of MDA and H(2)O(2) was observed after 24 h. This AA-induced oxidative stress was associated with the lack of an increase in the H(2)O(2)-degrading enzyme level. In contrast, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of AA, had not effect. The peroxisome proliferator-activated receptor gamma (PPARgamma) with AA metabolites as ligands was upregulated by the fatty acid but was not involved in the AA effect because its transcriptional activity estimated by reporter gene assays was negatively controlled by p38 MAPK pathway. These findings suggest that the effect of AA on human hepatoma cell growth by inducing an oxidative stress may present a clinical interest in the treatment of the liver cancer.


Assuntos
Ácido Araquidônico/farmacologia , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Araquidônico/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
20.
Free Radic Biol Med ; 35(6): 636-47, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12957656

RESUMO

Exogenous arachidonic acid (AA) has been shown to induce the antioxidant manganese superoxide dismutase gene by reactive oxygen species (ROS) derived from AA metabolism and the participation of the p38 mitogen-activated protein kinase (MAPK) pathway in human HepG2 hepatoma cells. The goal of this study was to investigate the effect of AA on the activation of the two redox-sensitive transcription factors AP-1 and NF-kappaB in HepG2 cells. Using electrophoretic mobility shift assays, DNA-binding activities of AP-1 and NF-kappaB were markedly increased in AA-treated HepG2 cells. The c-Jun and c-Fos proteins were identified as components of the AA-induced AP-1 complex and their levels were increased. AA-activated NF-kappaB complex was constituted as a p50 homodimer resulting in a nuclear translocation for this protein only. Moreover, no degradation of IkappaBalpha was observed. These results were contrasted to the interleukin-1beta-activated p50/p65 complex used as a positive control. Using 5,8,11,14-eicosatetraynoic acid and inhibitors of AA metabolism, AP-1 and NF-kappaB activation required the lipoxygenase/cytochrome P450 monooxygenase pathways. In addition, antioxidants inhibited the AA-induced AP-1 and NF-kappaB activation, suggesting a role of ROS released from the AA metabolism. In reporter gene assays, AA induced the transcriptional activity of AP-1 but not that of NF-kappaB. Further investigations showed that the AA-induced transcriptional activity of AP-1 was regulated by protein kinase C and p38 MAPK pathways. These results suggest that the functional AP-1 activated by AA and coupled to that of p38 MAPK pathway may play an important role in response to ROS induced by AA metabolism in HepG2 cells without the involvement of the NF-kappaB pathway.


Assuntos
Ácido Araquidônico/farmacologia , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
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