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1.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298969

RESUMO

Cancer is a complex disease involving alterations of multiple processes, with both genetic and epigenetic features contributing as core factors to the disease. In recent years, it has become evident that non-coding RNAs (ncRNAs), an epigenetic factor, play a key role in the initiation and progression of cancer. MicroRNAs, the most studied non-coding RNAs subtype, are key controllers in a myriad of cellular processes, including proliferation, differentiation, and apoptosis. Furthermore, the expression of miRNAs is controlled, concomitantly, by other epigenetic factors, such as DNA methylation and histone modifications, resulting in aberrant patterns of expression upon the occurrence of cancer. In this sense, aberrant miRNA landscape evaluation has emerged as a promising strategy for cancer management. In this review, we have focused on the regulation (biogenesis, processing, and dysregulation) of miRNAs and their role as modulators of the epigenetic machinery. We have also highlighted their potential clinical value, such as validated diagnostic and prognostic biomarkers, and their relevant role as chromatin modifiers in cancer therapy.


Assuntos
Epigênese Genética , MicroRNAs/genética , Neoplasias/genética , RNA Neoplásico/genética , Pesquisa Médica Translacional , Biomarcadores Tumorais , Neoplasias da Mama/genética , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Código das Histonas , Humanos , Neoplasias Pulmonares/genética , MicroRNAs/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias Ovarianas/genética , Prognóstico , Processamento Pós-Transcricional do RNA , RNA Neoplásico/biossíntese , Neoplasias Gástricas/genética
2.
Clin Cancer Res ; 27(6): 1807-1820, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33376098

RESUMO

PURPOSE: Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells. EXPERIMENTAL DESIGN: Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo, efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34+-NSG-SGM3). RESULTS: Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo, a single intratumoral Delta-24-RGD injection (107 or 108 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD-treated tumors revealed increased CD8+ T-cell infiltration. CONCLUSIONS: Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.

3.
J Clin Med ; 9(12)2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33255857

RESUMO

Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detected.

4.
Br J Haematol ; 189(6): 1064-1073, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32130737

RESUMO

Anti-cluster of differentiation 20 (CD20) monoclonal antibodies (mAbs) have shown promise in follicular lymphoma (FL) as post-induction therapy, by enhancing antibody-dependent cellular cytotoxicity (ADCC). However, cytotoxic cells are reduced after this treatment. We hypothesised that ex vivo expanded lymphokine-activated killer (LAK) cells administered to FL-remission patients are safe and improve anti-CD20 efficacy. This open, prospective, phase II, single-arm study assessed safety and efficacy of ex vivo expanded LAK cells in 20 FL-remission patients following rituximab maintenance. Mononuclear cells were obtained in odd rituximab cycles and stimulated with interleukin 2 (IL-2) for 8 weeks, after which >5 × 108 LAK cells were injected. Patients were followed-up for 5 years. At the end of maintenance, peripheral blood cells phenotype had not changed markedly. Natural killer, LAK and ADCC activities of mononuclear cells increased significantly after recombinant human IL-2 (rhIL-2) stimulation in all cycles. Rituximab significantly enhanced cytotoxic activity. No patients discontinued treatment. There were no treatment-related serious adverse events. Three patients had progressed by the end of follow-up. After a median (interquartile range) follow-up of 59.4 (43.8-70.9) months, 85% of patients remained progression free. No deaths occurred. Quality-of-life improved throughout the study. Post-induction LAK cells with rituximab seem safe in the long term. Larger studies are warranted to confirm efficacy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Células Matadoras Ativadas por Linfocina/transplante , Linfoma Folicular/terapia , Quimioterapia de Manutenção , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Feminino , Seguimentos , Humanos , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Estudos Prospectivos , Rituximab/administração & dosagem , Rituximab/efeitos adversos , Vincristina/administração & dosagem , Vincristina/efeitos adversos
5.
Transpl Infect Dis ; 19(6)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28921749

RESUMO

The use of preemptive antiviral therapy to prevent cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients might result in over-treatment, inducing drug-related toxicity and viral resistance. A search for predictive markers is needed to determine requirement for antiviral therapy. Clinical follow-up, in combination with the use of streptamers (STs) and cytokine-intracellular staining, could help to identify patients at high risk for CMV reactivations. To study the immune response and reactivation control by CMV-specific CD8+ T-cell (CMV-CTL) populations, we monitored 25 patients who have undergone allo-HSCT by using ST multimer and intracellular cytokine staining. Our study has revealed that the presence of functional CMV-specific T cells, determined by early interferon γ production or by significant T-cell expansion after first CMV reactivation, correlated with short CMV viremia duration and low number of CMV reactivations. By contrast, the absence of functional CMV-CTLs does correlate with CMV recurrence. These results support that behavior of CMV-specific subpopulations after reactivation influences reactivations and can guide preemptive therapy.


Assuntos
Antibioticoprofilaxia/efeitos adversos , Linfócitos T CD8-Positivos/virologia , Infecções por Citomegalovirus/sangue , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Ativação Viral , Adulto , Antibioticoprofilaxia/métodos , Antivirais/efeitos adversos , Biomarcadores/sangue , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/isolamento & purificação , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Seguimentos , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Transplantados , Transplante Homólogo/efeitos adversos , Viremia/prevenção & controle
6.
Oncotarget ; 8(16): 26755-26770, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28460460

RESUMO

CDC42 is an oncogenic Rho GTPase overexpressed in colorectal cancer (CRC). Although CDC42 has been shown to regulate gene transcription, the specific molecular mechanisms regulating the oncogenic ability of CDC42 remain unknown. Here, we have characterized the transcriptional networks governed by CDC42 in the CRC SW620 cell line using gene expression analysis. Our results establish that several cancer-related signaling pathways, including cell migration and cell proliferation, are regulated by CDC42. This transcriptional signature was validated in two large cohorts of CRC patients and its clinical relevance was also studied. We demonstrate that three CDC42-regulated genes offered a better prognostic value when combined with CDC42 compared to CDC42 alone. In particular, the concordant overexpression of CDC42 and silencing of the putative tumor suppressor gene CACNA2D2 dramatically improved the prognostic value. The CACNA2D2/CDC42 prognostic classifier was further validated in a third CRC cohort as well as in vitro and in vivo CRC models. Altogether, we show that CDC42 has an active oncogenic role in CRC via the transcriptional regulation of multiple cancer-related pathways and that CDC42-mediated silencing of CACNA2D2 is clinically relevant. Our results further support the use of CDC42 specific inhibitors for the treatment of the most aggressive types of CRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Canais de Cálcio/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Reprodutibilidade dos Testes
7.
Cytometry B Clin Cytom ; 92(2): 153-160, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-26918565

RESUMO

BACKGROUND: Multimer technology is widely used to screen antigen-specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus-specific T-cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen-specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice. METHODS: CMVpp65495-503 -specific HLA-A*02:01 CD8+ T lymphocytes (CTLA *02:01 -CMVpp65495-503 ) were examined with pentamer and streptamer in peripheral blood cells of 77 healthy volunteers. Quantitative and qualitative analyses were performed to compare the precision and repeatability, sensitivity and accuracy and specificity of both technologies by flow cytometry. RESULTS: Standard deviation for both techniques was less than 0.05 showing that they are repetitive and precise. Both techniques significantly correlated at high frequencies (rSpearman = 0.9422; P < 0.0001) but it was lost at lower levels (<1%) of CTLA *02:01 -CMVpp65495-503 (rSpearman = 0.3351; P = 0.1376). Streptamer is more accurate for the detection of CTLA *02:01 -CMVpp65495-503 providing significantly closer values to the theoretical ones (P < 0.0001) as pentamer binds unspecifically to a notable proportion of non-CMV-specific CD8+ T-cells. CONCLUSION: Our results suggest that streptamer multimer provides precise, accurate and specific results to detect CTLA *02:01 -CMVpp65495-503 by flow cytometry. Streptamer multimer can be used not only for the monitoring of early CTLA *02:01 -CMVpp65495-503 reconstitution in immunosuppressed patients following allo-HSCT but also, in conjunction with its reversibility role, for the isolation of CTLA *02:01 -CMVpp65495-503 for its future use in adoptive immunotherapy. © 2016 International Clinical Cytometry Society.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Antígenos HLA-A/imunologia , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia
8.
Cell Mol Life Sci ; 72(21): 4049-62, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26174234

RESUMO

Approximately, up to 70 % of the human population is infected with cytomegalovirus (CMV) that persists for life in a latent state. In healthy people, CMV reactivation induces the expansion of CMV-specific T cells up to 10 % of the entire T cell repertoire. On the contrary, CMV infection is a major opportunistic viral pathogen that remains a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Due to the delayed CMV-specific immune recovery, the incidence of CMV reactivation during post-transplant period is very high. Several methods are currently available for the monitoring of CMV-specific responses that help in clinical monitoring. In this review, essential aspects in the immune recovery against CMV are discussed to improve the better understanding of the immune system relying on CMV infection and, thereby, helping the avoidance of CMV disease or reactivation following hematopoietic stem cell transplantation with severe consequences for the transplanted patients.


Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/terapia , Citomegalovirus/patogenicidade , Transplante de Células-Tronco Hematopoéticas , Antivirais/farmacologia , Linfócitos T CD8-Positivos/virologia , Citocinas/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/prevenção & controle , Humanos , Hospedeiro Imunocomprometido/imunologia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Polimorfismo Genético , Receptores CCR5/genética , Receptores CCR5/imunologia , Doadores de Tecidos , Condicionamento Pré-Transplante/métodos , Transplante Homólogo , Ativação Viral
9.
Mol Oncol ; 8(3): 689-703, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24593875

RESUMO

Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target endothelial cells and abrogation of the angiogenic program by repression of proangiogenic IL-8, ICAM and CXCL1. Moreover, in vivo infusion of fluorescent labeled ELV efficiently targeted cells of the osseous compartment. Furthermore, treatment with miR-192 enriched ELV in a model of in vivo bone metastasis pre-conditioned osseous milieu and impaired tumor-induced angiogenesis, thereby reducing the metastatic burden and tumor colonization. Changes in the miRNA-cargo content within ELV represent a novel mechanism heavily influencing bone metastatic colonization, which is most likely relevant in other target organs. Mechanistic mimicry of this phenomenon by synthetic nanoparticles could eventually emerge as a novel therapeutic approach.


Assuntos
Adenocarcinoma/patologia , Neoplasias Ósseas/secundário , Osso e Ossos/patologia , Exossomos/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Osso e Ossos/metabolismo , Linhagem Celular Tumoral , Exossomos/genética , Exossomos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , MicroRNAs/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia
10.
Cell Mol Life Sci ; 71(7): 1211-24, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24077876

RESUMO

Chemotherapy and/or radiotherapy regular regimens used for conditioning of recipients of hematopoietic stem cell transplantation (SCT) induce a period of transient profound immunosuppression. The onset of a competent immunological response, such as the appearance of viral-specific T cells, is associated with a lower incidence of viral infections after haematopoietic transplantation. The rapid development of immunodominant peptide virus screening together with advances in the design of genetic and non-genetic viral- and tumoural-specific cellular selection strategies have opened new strategies for cellular immunotherapy in oncologic recipients who are highly sensitive to viral infections. However, the rapid development of cellular immunotherapy in SCT has disclosed the role of the T cell selection method in the modulation of functional cell activity and of in vivo secondary effects triggered following immunotherapy.


Assuntos
Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Suscetibilidade a Doenças , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunossupressão , Modelos Imunológicos , Viroses/complicações
11.
J Immunol Methods ; 396(1-2): 168-72, 2013 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-23933324

RESUMO

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia Adotiva , Leucócitos Mononucleares/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo
12.
Bone ; 52(1): 532-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23142363

RESUMO

Recent evidence suggests that miRNAs could be used as serum markers in a variety of normal and pathological conditions. In this study, we aimed to identify novel miRNAs associated with skeletal metastatic disease in a preclinical model of lung cancer bone metastasis. We assessed the validity of these miRNAs as reliable serum biochemical markers to monitor the extent of disease and response to treatment in comparison to imaging techniques and standard biochemical markers of bone turnover. Using a murine model of human lung cancer bone metastasis after zoledronic acid (ZA) treatment, PINP (procollagen I amino-terminal propeptide) was the only marker that exhibited a strong correlation with osteolytic lesions and tumor burden at early and late stages of bone colonization. In contrast, BGP (osteocalcin) and CTX (carboxyterminal telopeptide) demonstrated a strong correlation only at late stages. We performed qPCR based screening of a panel of 380 human miRNAs and quantified bone metastatic burden using micro-CT scans, X-rays and bioluminescence imaging. Interestingly, levels of miR-326 strongly associated with tumor burden and PINP in vehicle-treated animals, whereas no association was found in ZA-treated animals. Only miR-193 was associated with biochemical markers PINP, BGP and CTX in ZA-treated animals. Consistently, miR-326 and PINP demonstrated a strong correlation with tumor burden. Our findings, taken together, indicate that miR-326 could potentially serve as a novel biochemical marker for monitoring bone metastatic progression.


Assuntos
Neoplasias Ósseas/secundário , Remodelação Óssea , Neoplasias Pulmonares/patologia , Biomarcadores , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Microtomografia por Raio-X
13.
World J Gastroenterol ; 18(7): 637-45, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22363134

RESUMO

AIM: To investigate the angiogenesis-related protein expression profile characterizing metastatic colorectal cancer (mCRC) with the aim of identifying prognostic markers. METHODS: The expression of 44 angiogenesis-secreted factors was measured by a novel cytokine antibody array methodology. The study evaluated vascular endothelial growth factor (VEGF) and its soluble vascular endothelial growth factor receptor (sVEGFR)-1 protein levels by enzyme immunoassay (EIA) in a panel of 16 CRC cell lines. mRNA VEGF and VEGF-A isoforms were quantified by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) and vascular endothelial growth factor receptor (VEGFR)-2 expression was analyzed by flow cytometry. RESULTS: Metastasis-derived CRC cell lines expressed a distinctive molecular profile as compared with those isolated from a primary tumor site. Metastatic CRC cell lines were characterized by higher expression of angiogenin-2 (Ang-2), macrophage chemoattractant proteins-3/4 (MCP-3/4), matrix metalloproteinase-1 (MMP-1), and the chemokines interferon γ inducible T cell α chemoattractant protein (I-TAC), monocyte chemoattractant protein I-309, and interleukins interleukin (IL)-2 and IL-1α, as compared to primary tumor cell lines. In contrast, primary CRC cell lines expressed higher levels of interferon γ (IFN-γ), insulin-like growth factor-1 (IGF-1), IL-6, leptin, epidermal growth factor (EGF), placental growth factor (PlGF), thrombopoietin, transforming growth factor ß1 (TGF-ß1) and VEGF-D, as compared with the metastatic cell lines. VEGF expression does not significantly differ according to the CRC cellular origin in normoxia. Severe hypoxia induced VEGF expression up-regulation but contrary to expectations, metastatic CRC cell lines did not respond as much as primary cell lines to the hypoxic stimulus. In CRC primary-derived cell lines, we observed a two-fold increase in VEGF expression between normoxia and hypoxia as compared to metastatic cell lines. CRC cell lines express a similar pattern of VEGF isoforms (VEGF121, VEGF165 and VEGF189) despite variability in VEGF expression, where the major transcript was VEGF121. No relevant expression of VEGFR-2 was found in CRC cell lines, as compared to that of human umbilical vein endothelial cells and sVEGFR-1 expression did not depend on the CRC cellular origin. CONCLUSION: A distinct angiogenesis-related expression pattern characterizes metastatic CRC cell lines. Factors other than VEGF appear as prognostic markers and intervention targets in the metastatic CRC setting.


Assuntos
Anticorpos/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citocinas/metabolismo , Metástase Neoplásica/fisiopatologia , Neovascularização Patológica/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Perfilação da Expressão Gênica , Humanos , Hipóxia/metabolismo , Análise em Microsséries/métodos , Metástase Neoplásica/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
14.
Eur J Cancer ; 48(12): 1774-80, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22305465

RESUMO

BACKGROUND: The immunoglobulin G1 (IgG(1)) monoclonal antibody (MoAb) Cetuximab is active in metastatic colorectal cancer (mCRC) as first or subsequent lines of therapy. Efficacy seems restricted to KRAS wild-type tumours. IgG(1) may also induce antibody dependent cell mediated citotoxicity (ADCC) by recruitment of immune effector cells. ADCC is influenced by Fc gamma receptor (FcγR) polymorphisms. We investigated the association of FcγR polymorphisms and disease control rate (DCR) in mCRC patients treated with chemotherapy plus Cetuximab. PATIENTS AND METHODS: Tumour tissues from 106 patients were screened for KRAS codon 12 and 13 mutations using a sensitive multiplex assay (DxS, Manchester, United Kingdom). NRAS (codons: 12, 13 and 61), PI3K (exon 20) and BRAF (exon 15) were analysed by direct sequencing. Fcγ RIIa and Fcγ RIIIa polymorphisms were genotyped by TaqMan assays. RESULTS: DCR was significantly higher in KRAS wild-type tumours (61% versus 39%, p = 0.049). In epidermal growth factor receptor (EGFR) downstream-mutated mCRC patients, those harbouring an FcγRIIa H/H genotype had a higher DCR than alternative genotypes (67% versus 33%, p = 0.017). By multivariate analysis, FcγRIIa-131H/H remained significantly correlated with DCR (p = 0.008). CONCLUSION: FcγR polymorphisms may play a role in the clinical efficacy of Cetuximab in EGFR downstream mutated mCRC patients. Further research into Cetuximab immune-based mechanisms in KRAS-mutated patients seems warranted.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/genética , Receptores ErbB/genética , Polimorfismo Genético , Receptores de IgG/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cetuximab , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genética
15.
Int J Mol Sci ; 13(1): 840-65, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22312290

RESUMO

Recent advances in the field of RNA research have provided compelling evidence implicating microRNA (miRNA) and long non-coding RNA molecules in many diverse and substantial biological processes, including transcriptional and post-transcriptional regulation of gene expression, genomic imprinting, and modulation of protein activity. Thus, studies of non-coding RNA (ncRNA) may contribute to the discovery of possible biomarkers in human cancers. Considering that the response to chemotherapy can differ amongst individuals, researchers have begun to isolate and identify the genes responsible. Identification of targets of this ncRNA associated with cancer can suggest that networks of these linked to oncogenes or tumor suppressors play pivotal roles in cancer development. Moreover, these ncRNA are attractive drug targets since they may be differentially expressed in malignant versus normal cells and regulate expression of critical proteins in the cell. This review focuses on ncRNAs that are differently expressed in malignant tissue, and discusses some of challenges derived from their use as potential biomarkers of tumor properties.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/genética , MicroRNAs/metabolismo , RNA não Traduzido/metabolismo , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Polimorfismo de Nucleotídeo Único , Prognóstico
16.
PLoS One ; 6(11): e26740, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22069467

RESUMO

We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; N = 414), Sox2 gene amplification (8.5%; N = 492), and Sox 2 promoter hypomethylation (100%; N = 258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.


Assuntos
Metilação de DNA , Epigenômica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição SOXB1/genética , Azacitidina/farmacologia , Western Blotting , Movimento Celular , Proliferação de Células , Ilhas de CpG , DNA de Neoplasias/genética , Amplificação de Genes , Inativação Gênica , Glioma/metabolismo , Humanos , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/metabolismo , Células Tumorais Cultivadas
17.
Stem Cells ; 29(11): 1661-71, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21948564

RESUMO

Many antitumor therapies affect rapidly dividing cells. However, tumor proliferation may be driven by cancer stem cells (CSCs), which divide slowly and are relatively resistant to cytotoxic drugs. Thus, many tumors may progress because CSCs are not sensitive to the treatment. In this work, we searched for target genes whose expression is involved in proliferation and chemoresistance of CSCs. Both of these processes could be controlled simultaneously by cell regulators such as microRNAs (miRNAs). Therefore, colonospheres with properties of CSCs were obtained from different colon carcinoma cells, and miRNA profiling was performed. The results showed that miR-451 was downregulated in colonspheres versus parental cells. Surprisingly, expression of miR-451 caused a decrease in self-renewal, tumorigenicity, and chemoresistance to irinotecan of colonspheres. We identified cyclooxygenase-2 (COX-2) as an indirect miR-451 target gene involved in sphere growth. Our results indicate that miR-451 downregulation allows the expression of the direct target gene macrophage migration inhibitory factor, involved in the expression of COX-2. In turn, COX-2 allows Wnt activation, which is essential for CSC growth. Furthermore, miR-451 restoration decreases expression of the ATP-binding cassette drug transporter ABCB1 and results in irinotecan sensitization. These findings correlate well with the lower expression of miR-451 observed in patients who did not respond to irinotecan-based first-line therapy compared with patients who did. Our data suggest that miR-451 is a novel candidate to circumvent recurrence and drug resistance in colorectal cancer and could be used as a marker to predict response to irinotecan in patients with colon carcinoma.


Assuntos
MicroRNAs/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Irinotecano , MicroRNAs/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Surg Oncol ; 103(5): 411-5, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21400525

RESUMO

BACKGROUND: MicroRNAs are novel regulators of gene expression that are linked to the main oncogene networks, including the p53 pathway. p53 regulates the maturation process of miR-16 and miR-143. We analyzed the role as prognostic markers of miR-16 and miR-143 in 70 non-small-cell lung cancer (NSCLC) patients. METHODS: MicroRNAs were analyzed by TaqMan MicroRNA assays. Disease-free survival (DFS) and overall survival (OS) were examined using Kaplan-Meier curves with log-rank tests and the Cox proportional hazard model. RESULTS: When patients were classified in three groups according to their miR-16 expression levels, those with normal levels had the best outcome while those with high levels had the worst. DFS was 22.4 months for patients with high levels, 71.8 months for those with normal levels, and 55.8 months for those with low levels (P = 0.05). OS was 23.9 months for patients with high levels, 97.6 months for those with normal levels, and 63.5 months for those with low levels (P < 0.001). In the multivariate analyses, high miR-16 levels emerged as an independent prognostic factor for poor DFS (P = 0.001) and OS (<0.001). CONCLUSIONS: Our results provide the first hints that miR-16 levels in tumor samples may be a prognostic marker in NSCLC.


Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genética
19.
BJU Int ; 107(11): 1833-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20840328

RESUMO

UNLABELLED: What's known on the subject? and What does the study add? SXR and MDR1 are known as responsible for chemo and radiotherapy resistance in some cancers, like kidney cancer (MDR1). Invasive bladder cancer is an aggressive disease, with different behaviour upon its tumoral stage, and also within the same tumoral stage, therefore molecular markers are sought. This study shows a new molecular marker, which has shown as a predictor for bad prognosis cancers, therefore, allowing us for a better patient selection for aggressive therapies. OBJECTIVE: To investigate the prognostic value of steroid and xenobiotic receptor (SXR) and multidrug resistance 1 (MDR1) gene expression in relation to survival among patients with invasive bladder cancer. PATIENTS AND METHODS: The prospective study included 67 patients diagnosed with invasive bladder cancer and treated with radical cystectomy at one of two institutions. SXR and MDR1 gene expression was assessed by real-time quantitative polymerase chain reaction (RT-PCR) in tumoral and normal tissue from frozen surgical specimens. RESULTS: Patients were followed for a mean of 29 months; 31 patients (46%) had progression. In univariate analysis, significant predictors of overall survival (OS) were pathological stage, lymph node (LN) status, histological grade, vascular-lymphatic invasion, and SXR expression. In multivariate analysis, independent predictors of OS were LN status (odds ratio [OR], 2.96; P=0.034), vascular-lymphatic invasion (OR, 2.50; P=0.029), and SXR expression (OR, 1.05, P=0.03). Among the 51 patients with negative LNs (pN0), univariate predictors of OS were SXR expression, MDR1 expression, and pathological stage. In multivariate analysis, SXR expression (OR, 1.06; P=0.01) and MDR1 expression (OR, 3.27; P=0.03) were independently associated with survival. Within the pN0 group, patients with SXR expression had shorter progression-free survival than did those without expression (P=0.004). This association persisted in the N0 subgroup with stage pT3-pT4 disease (P=0.028). However, in the pN1 group SXR expression did not have any influence. CONCLUSIONS: For patients with invasive bladder cancer, SXR expression has value as a predictor of survival independent of the standard pathological predictors. Its maximum importance appears to be in patients with stage pT3-pT4 pN0 disease.


Assuntos
Cistectomia/métodos , Genes MDR/genética , Receptores de Esteroides/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Idoso , Análise de Variância , Estudos de Coortes , Cistectomia/mortalidade , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Razão de Chances , Receptor de Pregnano X , Prognóstico , Estudos Prospectivos , Medição de Risco , Análise de Sobrevida , Resultado do Tratamento , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia
20.
Mol Cancer Ther ; 9(8): 2265-75, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20647341

RESUMO

Thymidylate synthase (TYMS) is a target of the most widely used chemotherapeutic agents against gastrointestinal malignancies, the fluoropyrimidine-based therapy. TYMS expression levels have been identified as predictive biomarkers for 5-fluoruracil (FU) response in colorectal cancer, but their clinical utility remains controversial. The complexity of fluoropyrimidine response must require more mechanisms that currently have not been completely elucidated. In this context, microRNAs (miRNA) may play a role in modulating chemosensitivity. By carrying out an in silico analysis coupled to experimental validation, we detected that miR-192 and miR-215 target TYMS expression in colorectal cancer cell lines. However, downregulation of TYMS by these miRNAs does not sensitize colorectal cancer cell lines to FU treatment. The overexpression of miR-192/215 significantly reduces cell proliferation by targeting cell cycle progression. This effect was partially associated with p53 status, because reduction of cell proliferation and cell cycle arrest was associated with p21 and p27 induction. The decrease of S-phase cells by these miRNAs mitigates the effects of S phase-specific drugs and suggests that other mechanisms different from TYMS overexpression are essential to direct FU resistance. Finally, ectopic expression of miR-192/215 might have stronger impact to predict FU response than TYMS inhibition. Prospective studies to elucidate the role of these miRNAs as predictive biomarkers to FU are necessary.


Assuntos
Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/metabolismo , Timidilato Sintase/genética , Sítios de Ligação , Western Blotting , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Biologia Computacional , Humanos , MicroRNAs/genética , Biossíntese de Proteínas/genética , Transcrição Genética/efeitos dos fármacos , Transfecção
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