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1.
Int J Mol Sci ; 23(9)2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35562862

RESUMO

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.


Assuntos
Astrócitos , Glioblastoma , Astrócitos/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transição Epitelial-Mesenquimal , Glioblastoma/patologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismo
2.
Clin Epigenetics ; 14(1): 68, 2022 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-35606887

RESUMO

The epigenetic changes associated with melanoma progression to advanced and metastatic stages are still poorly understood. To shed light on the CpG methylation dynamics during melanoma development, we analyzed the methylome profiles of a four-stage cell line model of melanoma progression: non-tumorigenic melanocytes (melan-a), premalignant melanocytes (4C), non-metastatic melanoma cells (4C11-), and metastatic melanoma cells (4C11+). We identified 540 hypo- and 37 hypermethylated gene promoters that together characterized a malignancy signature, and 646 hypo- and 520 hypermethylated promoters that distinguished a metastasis signature. Differentially methylated genes from these signatures were correlated with overall survival using TCGA-SKCM methylation data. Moreover, multivariate Cox analyses with LASSO regularization identified panels of 33 and 31 CpGs, respectively, from the malignancy and metastasis signatures that predicted poor survival. We found a concordant relationship between DNA methylation and transcriptional levels for genes from the malignancy (Pyroxd2 and Ptgfrn) and metastasis (Arnt2, Igfbp4 and Ptprf) signatures, which were both also correlated with melanoma prognosis. Altogether, this study reveals novel CpGs methylation markers associated with malignancy and metastasis that collectively could improve the survival prediction of melanoma patients.


Assuntos
Metilação de DNA , Melanoma , Ilhas de CpG , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/metabolismo , Prognóstico , Regiões Promotoras Genéticas
3.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374840

RESUMO

BACKGROUND: Although it has been previously demonstrated that acute inflammation can promote the tumor growth of a sub-tumorigenic dose of melanoma cells through of 5-lipoxygenase inflammatory pathway and its product leukotriene B4, and also that the peritumoral treatment with eicosapentaenoic acid and its product, leukotriene B5, reduces the tumor development, the effect of the treatment by gavage with omega-3 and omega-6 in the tumor microenvironment favorable to melanoma growth associated with acute inflammation has never been studied. METHODS: C57BL/6 mice were coinjected with 1 × 106 apoptotic cells plus 1 × 103 viable melanoma cells into the subcutaneous tissue and treated by gavage with omega-3-rich fish oil or omega-6-rich soybean oil or a mixture of these oils (1:1 ratio) during five consecutive days. RESULTS: The treatment by gavage with a mixture of fish and soybean oils (1:1 ratio) both reduced the melanoma growth and the levels of leukotriene B4 (LTB4), prostaglandin E2 (PGE2), PGE2/prostaglandin E3 (PGE3) ratio, and CXC ligand 1 (CXCL1) and increased the levels of interleukin 10 (IL-10) to IL-10/CXCL1 ratio in the melanoma microenvironment. CONCLUSION: The oral administration of a 1:1 mixture of fish oil and soybean oil was able to alter the release of inflammatory mediators that are essential for a microenvironment favorable to the melanoma growth in mice, whereas fish oil or soybean oil alone was ineffective.


Assuntos
Ácidos Graxos Ômega-3/uso terapêutico , Ácidos Graxos Ômega-6/uso terapêutico , Inflamação/tratamento farmacológico , Melanoma/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Anti-Inflamatórios/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Óleos de Peixe/uso terapêutico , Inflamação/imunologia , Inflamação/patologia , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Óleo de Soja/uso terapêutico
4.
Front Immunol ; 9: 197, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29483913

RESUMO

Epigenetics refers to the study of mechanisms controlling the chromatin structure, which has fundamental role in the regulation of gene expression and genome stability. Epigenetic marks, such as DNA methylation and histone modifications, are established during embryonic development and epigenetic profiles are stably inherited during mitosis, ensuring cell differentiation and fate. Under the effect of intrinsic and extrinsic factors, such as metabolic profile, hormones, nutrition, drugs, smoke, and stress, epigenetic marks are actively modulated. In this sense, the lifestyle may affect significantly the epigenome, and as a result, the gene expression profile and cell function. Epigenetic alterations are a hallmark of aging and diseases, such as cancer. Among biological systems compromised with aging is the decline of immune response. Different regulators of immune response have their promoters and enhancers susceptible to the modulation by epigenetic marks, which is fundamental to the differentiation and function of immune cells. Consistent evidence has showed the regulation of innate immune cells, and T and B lymphocytes by epigenetic mechanisms. Therefore, age-dependent alterations in epigenetic marks may result in the decline of immune function and this might contribute to the increased incidence of diseases in old people. In order to maintain health, we need to better understand how to avoid epigenetic alterations related to immune aging. In this review, the contribution of epigenetic mechanisms to the loss of immune function during aging will be discussed, and the promise of new means of disease prevention and management will be pointed.


Assuntos
Envelhecimento/imunologia , Metilação de DNA , Epigênese Genética , Envelhecimento/genética , Animais , Linfócitos B/imunologia , Diferenciação Celular , Regulação da Expressão Gênica , Histonas/genética , Humanos , Imunidade Inata , Camundongos , Neoplasias/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologia
5.
Cancers (Basel) ; 9(4)2017 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-28430130

RESUMO

High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and ß1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generated under phosphatidylinositol-3-kinase (PI3K) activation, enables the recruitment and activation of protein kinase B (PKB/AKT) and phosphoinositide-dependent kinase 1 (PDK1) at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC) inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased anoikis resistance, clonogenicity, dacarbazine resistance, and in vivo tumor growth and lung colonization. In metastatic cells, pAKTThr308 is highly expressed, contributing to anoikis resistance. We showed that PDK1Ser241 and PKCßIISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.

6.
Oncotarget ; 8(70): 114540-114553, 2017 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-29383100

RESUMO

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.

7.
Biochem J ; 473(6): 703-15, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26699902

RESUMO

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.


Assuntos
Glutamina/metabolismo , Melanócitos/fisiologia , Melanoma/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Glucose/metabolismo , Glutaminase/metabolismo , Glutamina/genética , Lactatos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Potenciais da Membrana/fisiologia , Metabolismo , Camundongos , Oxirredução , Fenótipo
9.
PLoS One ; 10(4): e0119234, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875202

RESUMO

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.


Assuntos
Epigênese Genética , Infecções por HIV/genética , HIV-1/fisiologia , Leucócitos Mononucleares/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Clonais , Infecções por HIV/imunologia , Histonas/análise , Histonas/genética , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Processamento de Proteína Pós-Traducional
10.
PLoS One ; 8(5): e64453, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691222

RESUMO

Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.


Assuntos
Melanoma/prevenção & controle , Metástase Neoplásica/prevenção & controle , Receptor B1 da Bradicinina/metabolismo , Análise de Variância , Western Blotting , Linhagem Celular Tumoral , Corantes Fluorescentes , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Kisspeptinas/metabolismo , Melanoma/fisiopatologia , Receptor B1 da Bradicinina/agonistas , Sais de Tetrazólio , Tiazóis
11.
Mol Cancer ; 12: 22, 2013 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-23522389

RESUMO

BACKGROUND: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet. METHODS: The ß1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and ß1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR. RESULTS: Differential association among Timp1, CD63 and ß1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity. CONCLUSIONS: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and ß1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.


Assuntos
Anoikis , Integrina beta1/metabolismo , Melanoma/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tetraspanina 30/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Anoikis/efeitos dos fármacos , Anoikis/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Progressão da Doença , Expressão Gênica , Humanos , Antígeno MART-1/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/genética , Melanoma/patologia , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Metástase Neoplásica , Fenótipo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Ligação Proteica , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/farmacologia
12.
Pigment Cell Melanoma Res ; 25(3): 354-69, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22372875

RESUMO

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.


Assuntos
Catecóis/farmacologia , Melanoma/patologia , Inibidores de Proteassoma , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Sequestradores de Radicais Livres/farmacologia , Humanos , Modelos Biológicos , Inibidores de Proteases/farmacologia , Células Tumorais Cultivadas
13.
Free Radic Biol Med ; 50(10): 1263-73, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21362470

RESUMO

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH4, and increased by the inhibitor of BH4 synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. The eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. The inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. The maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation.


Assuntos
Melanócitos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Animais , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/metabolismo , Células Tumorais Cultivadas
14.
Hypertension ; 57(5): 965-72, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21422380

RESUMO

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.


Assuntos
Angiotensina II/metabolismo , Sinalização do Cálcio/fisiologia , Peptidil Dipeptidase A/metabolismo , Análise de Variância , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Citometria de Fluxo , Lisinopril/farmacologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Epigenetics ; 6(4): 450-64, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21343701

RESUMO

Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.


Assuntos
Transformação Celular Neoplásica/genética , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Melanócitos/fisiologia , Animais , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Regulação Neoplásica da Expressão Gênica , Camundongos
16.
Transl Oncol ; 2(4): 329-40, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19956395

RESUMO

Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2'-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation.

17.
Melanoma Res ; 18(3): 172-83, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18477891

RESUMO

Trypanosoma cruzi infection is known to confer resistance to tumor development in mice, and in-vitro studies have shown the toxic effects of parasite extracts on cancer cell cultures. Investigations in which T. cruzi molecules exhibit antitumor activity have just begun. Here, we used a tumorigenic cell line Tm5, derived from mouse melanocytes melan-a, to test the effect of J18, a recombinant protein based on T. cruzi surface molecule gp82 fused to glutathione-S-transferase (GST). J18 induced actin cytoskeleton disruption in Tm5 but not in melan-a cells. Several changes indicative of apoptosis were detected in Tm5 melanoma cells but not in melan-a cells treated with J18, such as the flipping of phosphatidylserine from the inner to the external side of the plasma membrane, altered nuclear morphology, DNA fragmentation, increase in mitochondria depolarization, and in caspase-3 activity. Retention of NF-kappaB in the cytoplasm was another alteration observed specifically in J18-treated Tm5 cells. No such alterations were found in Tm5 cells treated with GST. In-vivo experiments showed that C57BL/6 mice inoculated with Tm5 cells, treated at the site of tumor cell inoculation with J18, developed tumors of smaller size than mice treated with phosphate-buffered saline or GST and survived longer.


Assuntos
Apoptose/efeitos dos fármacos , Melanoma/patologia , Proteínas de Protozoários/farmacologia , Trypanosoma cruzi , Glicoproteínas Variantes de Superfície de Trypanosoma/farmacologia , Animais , Antígenos de Superfície/farmacologia , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas
18.
Clin Exp Metastasis ; 25(1): 65-73, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-17932775

RESUMO

The incidence of melanoma is increasing worldwide. It is one of the leading cancers in pregnancy and the most common malignancy to metastasize to placenta and fetus. There are no publications about experimental models of melanoma and pregnancy. We propose a new experimental murine model to study the effects of melanoma on pregnancy and its metastatic process. We tested several doses of melanoma cells until we arrived at the optimal dose, which produced tumor growth and allowed animal survival to the end of pregnancy. Two control groups were used: control (C) and stress control (SC) and three different routes of inoculation: intravenous (IV), intraperitoneal (IP) and subcutaneous (SC). All the fetuses and placentas were examined macroscopically and microscopically. The results suggest that melanoma is a risk factor for intrauterine growth restriction but does not affect placental weight. When inoculated by the SC route, the tumor grew only in the site of implantation. The IP route produced peritoneal tumoral growth and also ovarian and uterine metastases in 60% of the cases. The IV route produced pulmonary tumors. No placental or fetal metastases were obtained, regardless of the inoculation route. The injection of melanoma cells by any route did not increase the rate of fetal resorptions. Surprisingly, animals in the IV groups had no resorptions and a significantly higher number of fetuses. This finding may indicate that tumoral factors released in the host organism to favor tumor survival may also have a pro-gestational action and consequently improve the reproductive performance of these animals.


Assuntos
Modelos Animais de Doenças , Melanoma Experimental/patologia , Doenças Placentárias/patologia , Complicações na Gravidez/patologia , Animais , Transplante de Células , Embrião de Mamíferos , Feminino , Feto , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Gravidez , Resultado da Gravidez
19.
Neoplasia ; 9(12): 1111-21, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18084618

RESUMO

Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenicmelanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hcy), an element in the methionine (universal methyl donor) cycle. This alteration was accompanied by increase in glutathione (GSH) levels and methylated DNA content. Furthermore, a significant increase in dnmt1 and 3b expression was identified along melan-a anchorage blockade. L(G)-Nitro-L-arginine methyl esther (L-NAME), known as a nitric oxide synthase (NOS) inhibitor, and N-acetyl-L-cysteine (NAC) prevented the increase in global DNA methylation, as well as the increase in dnmt1 and 3b expression, observed during melan-a detachment. Interestingly, both L-NAME and NAC did not inhibit nitric oxide (NO) production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism.


Assuntos
Transformação Celular Neoplásica/genética , Metilação de DNA , Melanócitos/metabolismo , Estresse Oxidativo , Acetilcisteína/farmacologia , Animais , Anoikis/genética , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Cisteína/metabolismo , Metilação de DNA/efeitos dos fármacos , Regulação da Expressão Gênica , Glutationa/metabolismo , Homocisteína/metabolismo , Peroxidação de Lipídeos , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Sefarose/farmacologia , Superóxidos/metabolismo , Tripsina/farmacologia
20.
Cell Biochem Funct ; 25(1): 109-14, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16850525

RESUMO

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 murine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice.


Assuntos
Guanina/análogos & derivados , Melanoma/metabolismo , Melanoma/patologia , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Feminino , Guanina/química , Guanina/farmacologia , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Fator de Iniciação de Tradução Eucariótico 5A
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