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1.
Bioinformatics ; 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35104317

RESUMO

Comparisons of protein structures are critical for developing novel protein designs, annotating protein functions, and predicting protein structure. The template modeling score (TM-score) is a widely used but computationally expensive measure of protein similarity that is applicable to a wide variety of structural biology problems. We introduce TMQuery-a continuously updated database containing over 8 billion pre-computed TM-score values for every pair of proteins in the Protein Data Bank (PDB), allowing researchers to quickly query and download TM-scores via a web interface. AVAILABILITY: Publicly available at https://tmquery.gsk.com/.

2.
Transl Oncol ; 14(12): 101229, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34592589

RESUMO

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

3.
Sci Rep ; 10(1): 11329, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647229

RESUMO

Prostate cancer accounts for approximately 13.5% of all newly diagnosed male cancer cases. Significant clinical burdens remain in terms of ineffective prognostication, with overtreatment of insignificant disease. Additionally, the pathobiology underlying disease heterogeneity remains poorly understood. As the role of cancer stem cells in the perpetuation of aggressive carcinoma is being substantiated by experimental evidence, it is crucially important to understand the molecular mechanisms, which regulate key features of cancer stem cells. We investigated two methods for in vitro cultivation of putative prostate cancer stem cells based on 'high-salt agar' and 'monoclonal cultivation'. Data demonstrated 'monoclonal cultivation' as the superior method. We demonstrated that 'holoclones' expressed canonical stem markers, retained the exclusive ability to generate poorly differentiated tumours in NOD/SCID mice and possessed a unique mRNA-miRNA gene signature. miRNA:Target interactions analysis visualised potentially critical regulatory networks, which are dysregulated in prostate cancer holoclones. The characterisation of this tumorigenic population lays the groundwork for this model to be used in the identification of proteomic or small non-coding RNA therapeutic targets for the eradication of this critical cellular population. This is significant, as it provides a potential route to limit development of aggressive disease and thus improve survival rates.


Assuntos
Técnicas de Cultura de Células , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Animais , Biomarcadores Tumorais/genética , Carcinogênese , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/genética
4.
J Thorac Oncol ; 13(3): 413-425, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29191776

RESUMO

INTRODUCTION: The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. METHODS: A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). RESULTS: NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. CONCLUSION: NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.


Assuntos
Quinase do Linfoma Anaplásico/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias Torácicas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Coortes , Europa (Continente) , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Neoplasias Torácicas/patologia
5.
Blood ; 131(8): 911-916, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29282218

RESUMO

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O-glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF-/-/Asgr1-/- mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF-/-/Asgr1-/- mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1-/- mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Fator de von Willebrand/fisiologia , Animais , Receptor de Asialoglicoproteína/genética , Assialoglicoproteínas/genética , Células Cultivadas , Humanos , Lectinas Tipo C/genética , Macrófagos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido N-Acetilneuramínico/química
7.
Hum Pathol ; 70: 98-104, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29079174

RESUMO

Ovarian cancer is the fifth most common cancer in women worldwide and has the highest mortality amongst gynecological cancers. miRNAs are a class of non-coding RNAs, approximately 22 nt long, that negatively regulate gene expression and have roles in cell growth, differentiation, metabolism, apoptosis and tumorigenesis. Dysregulated miRNA-223 expression has been implicated in a wide range of cancer subtypes. SMARCD1 is an integral protein component of the SWI/SNF complex, which remodels chromatin, and which has important roles in transcriptional control, DNA replication, recombination and repair. In this study, we examined whether the expression levels of miR-223 and SMARCD1 are altered in ovarian serous neoplasia and whether miR-223 functionally regulates the gene and protein expression of SMARCD1 in vivo, as has been predicted by in silico methods. Benign, atypical proliferative serous tumors (borderline) and malignant serous tumors (n = 144) were laser-capture microdissected, and relative expression levels of miR-223 and SMARCD1 were quantified by RT-PCR. Ovarian cancer cell line OC316 was reverse transfected with a miR-223 mimic, and relative expression levels of miR-223 and SMARCD1 were quantified by reverse-transcription polymerase chain reaction; protein expression of SMARCD1 was evaluated by Western blot. miR-223 expression was up-regulated in high-grade ovarian serous carcinoma samples (median RQ = 4.8881, P = .0045), whilst SMARCD1 was down-regulated (median RQ = 0.5107, P = .0492). In OC316 cells transfected with a miR-223 mimic, SMARCD1 gene expression was down-regulated 3-fold (P = .001), and SMARCD1 protein expression was down-regulated 2-fold (P = .002). These results suggest a regulatory role for miR-223 in ovarian serous neoplasia, linking it with SMARCD1.


Assuntos
Proliferação de Células , MicroRNAs/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/etiologia , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transdução de Sinais , Fatores de Transcrição/genética
8.
Int J Mol Med ; 38(2): 433-45, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27353001

RESUMO

Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer-related deaths each year. Our group and others have previously demonstrated dysfunctional microRNA (miRNA or miR) expression in the context of thyroid cancer. The objective of the present study was to investigate the impact of synthetic manipulation of expression of miR-25 and miR-222 in benign and malignant thyroid cells. miR-25 and miR-222 expression was upregulated in 8505C (an anaplastic thyroid cell line) and Nthy-ori (a SV40-immortalised thyroid cell line) cells, respectively. A transcriptomics-based approach was utilised to identify targets of the two miRNAs and real-time PCR and western blotting were used to validate a subset of the targets. Almost 100 mRNAs of diverse functions were found to be either directly or indirectly targeted by both miR-222 and miR-25 [fold change ≥2, false discovery rate (FDR) ≤0.05]. Gene ontology analysis showed the miR-25 gene target list to be significantly enriched for genes involved in cell adhesion. Fluidigm real-time PCR technologies were used to validate the downregulation of 23 and 22 genes in response to miR-25 and miR-222 overexpression, respectively. The reduction of the expression of two miR-25 protein targets, TNF-related apoptosis­inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), was also validated. Manipulating the expression of both miR-222 and miR-25 influenced diverse gene expression changes in thyroid cells. Increased expression of miR-25 reduced MEK4 and TRAIL protein expression, and cell adhesion and apoptosis are important aspects of miR-25 functioning in thyroid cells.


Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase 4/metabolismo , MicroRNAs/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Humanos , MAP Quinase Quinase 4/genética , MicroRNAs/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia
9.
Cancer Biol Ther ; 17(5): 526-42, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26828826

RESUMO

Oncogenic mutations in BRAF are common in melanoma and thyroid carcinoma and drive constitutive activation of the MAPK pathway. Molecularly targeted therapies of this pathway improves survival compared to chemotherapy; however, responses tend to be short-lived as resistance invariably occursCell line models of melanoma and thyroid carcinoma, +/- BRAF(V600E) activating mutation, were treated with the MEK inhibitor PD0325901. Treated and naive samples were assayed for expression of key members of the MAPK pathway. Global microRNA expression profiling of naive and resistant cells was performed via next generation sequencingand indicated pluripotency pathways in resistance. Parental cell lines were progressed to holoclones to confirm the miRNA stemness profileMembers of the MIR302/373/374/520 family of embryonic stem cell specific cell cycle regulating (ESCC) microRNAs were identified as differentially expressed between resistant BRAF(V600E) melanoma and thyroid cell lines. Upregulated expression of gene and protein stemness markers, upregulated expression of MAPK pathway genes and downregulation of the ESCC MIR302 cluster in BRAF(V600E) melanoma indicated an increased stem-like phenotype in resistant BRAF(V600E) melanoma. Conversely, downregulated expression of gene and protein stemness markers, downregulated expression of MAPK pathway genes, upregulation of the ESCC MIR520 cluster, reeexpression of cell surface receptors, and induced differentiation-associated morphology in resistant BRAF(V600E) indicate a differentiated phenotype associated with MEK inhibitor resistance in BRAF(V600E) thyroid cellsThe differential patterns of resistance observed between BRAF(V600E) melanoma and thyroid cell lines may reflect tissue type or de novo differentiation, but could have significant impact on the response of primary and metastatic cells to MEK inhibitor treatment. This study provides a basis for the investigation of the cellular differentiation/self-renewal access and its role in resistance to MEK inhibition.


Assuntos
Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Células-Tronco Pluripotentes
10.
Eur Respir J ; 47(1): 212-22, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26541527

RESUMO

Disease severity in viral bronchiolitis in infancy is difficult to predict and has been linked to host innate immunity. The study aimed to investigate the innate cytokine interleukin-15 (IL-15) as a marker of disease severity.A prospective single-centre observational study was conducted in a university-affiliated paediatric teaching hospital, comparing children (0-18 months) hospitalised for viral bronchiolitis, those admitted to the paediatric intensive care unit with severe disease and healthy age-matched controls. IL-15-related parameters were compared between groups. PCR and microRNA (miRNA) sequencing was undertaken on natural killer (NK) cells collected from study participants.Samples from 88 children with viral bronchiolitis and 43 controls enrolled between 2009 and 2012 were analysed. Peripheral blood mononuclear cell (PBMC) IL-15 mRNA expression was significantly higher in those with moderate severity bronchiolitis compared with controls and those with severe disease. Serum IL-15 levels correlated with disease severity. The relative frequency of NK cells in peripheral blood was significantly reduced in participants with bronchiolitis. The NK cell miRNA transcriptome in bronchiolitis was distinct. Targets of de-regulated miRNA were differentially expressed in bronchiolitis, including JAK3, STAT5A and NFKB1 on the IL-15 signalling pathway.IL-15 is associated with disease severity in children hospitalised with viral bronchiolitis.


Assuntos
Bronquiolite Viral/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , MicroRNAs/genética , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Bronquiolite Viral/genética , Bronquiolite Viral/metabolismo , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Pediátrica , Interleucina-15/genética , Janus Quinase 3/metabolismo , Leucócitos Mononucleares/imunologia , Masculino , Subunidade p50 de NF-kappa B/metabolismo , Estudos Prospectivos , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Fator de Transcrição STAT5/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteína bcl-X/metabolismo
11.
J Soc Policy ; 44(2): 297-318, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26635422

RESUMO

The concept of the 'social investment state' refocuses attention on the productive function of social policy eclipsed for some time by the emphasis on its social protection or compensation roles. Here we distinguish between different social investment strategies, the Nordic 'heavy' and the Liberal 'light', with particular reference to the inclusive growth approach adopted in Australia. In 2007, social democrats in Australia returned to government with a clear mandate to reject the labour market deregulation and other neoliberal policies of its predecessor, and to tackle entrenched social and economic disadvantage in Australian society. For the last five years, social investment and inclusive growth has been at the centre of the Australian social policy agenda. Against this background, the article examines and critically assesses the (re)turn to 'social investment' thinking in Australia during Labor's term in office (2007-13). Analysis focuses not just on what was actually achieved, but also on the constraining role of prevailing economic and political circumstances and on the processes that were used to drive social investment reform. In many ways, the article goes some way to exposing ongoing tensions surrounding the distinctiveness of 'social investment' strategies pursued by leftist parties within the (neo)liberal state.

12.
J Clin Pathol ; 68(9): 692-702, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26038242

RESUMO

AIMS: Targeting the stem cell properties of tumor-initiating cells is an avenue through which cancer treatment may be improved. Before this can be achieved, so-called 'cancer stem cell' (CSC) models must be developed and characterized in specific malignancies. METHODS: In this study, holoclone formation assays were used to characterise stem-like molecular signatures in prostate cancer (PCa) cells. RESULTS: LNCaP and PC3 parent cells were capable of responding to stem cell differentiation morphogen retinoic acid (RA), suggesting the presence of inherent stem-like properties. LNCaP cells, which represent early, androgen-responsive disease, formed holoclones after twenty six days. PC3 cells, which represent advanced, metastatic, castration-resistant disease, formed holoclones after only six days. Holoclones displayed decreased expression of RA-genes, suggesting a more immature, less differentiated phenotype. Gene and microRNA arrays demonstrated that holoclones downregulated a number of stem cell differentiation regulators while displaying enhanced regulation of G2 to M transition and the mitotic spindle checkpoint components of the cell cycle. PC3 holoclones displayed pronounced downregulation of known regulators of osteoblast differentiation from mesenchymal stem cells and Epithelial Mesenchymal Transition. CONCLUSIONS: Our results suggest that some PCa cells retain the ability to transition to a more immature state in which differentiation and metastatic mechanisms are suppressed. The highlighting of osteoblast differentiation regulators in this mechanism is particularly notable, considering the propensity of PCa to metastasise to bone.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neoplásicas/patologia , Osteoblastos/citologia , Neoplasias da Próstata/patologia , Transcriptoma , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo
13.
Microrna ; 4(1): 57-63, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25882990

RESUMO

BACKGROUND: Atherosclerosis-related cardiovascular disease and osteoporosis (OP) occur concurrently and may share a common pathogenesis. Aberrant expression of miR-21 and vitamin D deficiency have been independently linked to the pathogenesis of atherosclerosis and OP. OBJECTIVES: To examine the relationship between miR-21 expression and vitamin D in aorta and bone in atherosclerotic disease. METHODS: Aorta, internal mammary artery (IMA) and sternal bone samples were collected from patients undergoing coronary artery bypass graft (CABG) surgery. Bone density was measured by dual x-ray absorbtiometry (DXA). MiR-21 was quantified using a two-step reverse transcription-polymerase chain reaction. RESULTS: Ten patients were included for analysis; 5 were vitamin D deficient (<25nmol/L). MiR-21 was expressed at a greater level in aorta compared with the IMA (p = 0.003), and sternal bone (p = 0.002). Expression of miR-21 between the IMA and bone was similar (p = 0.7). A positive correlation between the magnitude of difference (fold-difference) of miR-21 expression between aorta and IMA and CRP (correlation coefficient 0.9, p = 0.009) was found. Vitamin D deficient patients had greater expression of miR-21 in aorta compared with non-deficient patients (p = 0.03). Increasing CRP and vitamin D deficiency were independent predictors of miR-21 expression in aorta. The lower the difference in miR-21 expression between aorta and bone, the lower the bone density. CONCLUSION: In atherosclerosis, miR-21 is increased in the aorta and associated with vitamin D deficiency. Vitamin D deficiency may influence aberrant miR-21 expression in vasculature and bone contributing to the concurrent development of atherosclerosis and osteoporosis.


Assuntos
Doença da Artéria Coronariana/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Isquemia Miocárdica/genética , Deficiência de Vitamina D/genética , Adulto , Idoso , Aorta/patologia , Densidade Óssea , Osso e Ossos/patologia , Ponte de Artéria Coronária , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/patologia , Feminino , Humanos , Masculino , Artéria Torácica Interna/patologia , Pessoa de Meia-Idade , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/patologia
14.
PLoS One ; 9(6): e100816, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24977712

RESUMO

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.


Assuntos
Cistadenocarcinoma Seroso/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Fator 88 de Diferenciação Mieloide/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Receptor 4 Toll-Like/genética , Idoso , Antineoplásicos Fitogênicos/farmacologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/diagnóstico , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/mortalidade , Feminino , Genótipo , Humanos , Imuno-Histoquímica , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/mortalidade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Paclitaxel/farmacologia , Fenótipo , Prognóstico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-22969748

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs approximately 22 nucleotides in length that function as regulators of gene expression. Dysregulation of miRNAs has been associated with initiation and progression of oncogenesis in humans. Our group has previously described a unique miRNA expression signature, including the MIR200 family member MIR141, which can differentiate papillary thyroid cancer (PTC) cell lines from a control thyroid cell line. An investigation into the expression of MIR141 in a series of archival thyroid malignancies [n = 140; classic PTC (cPTC), follicular variant PTC, follicular thyroid carcinoma, Hashimoto thyroiditis (HT), or control thyrocytes] was performed. Each cohort had a minimum of 20 validated samples surgically excised within the period 1980-2009. A subset of the HT and cPTC cohorts (n = 3) were also analyzed for expression of TGFßR1, a key member of the TGFß pathway and known target of MIR141. Laser capture microdissection was used to specifically dissect target cells from formalin-fixed paraffin-embedded archival tissue. Thyrocyte- and lymphocyte-specific markers (TSHR and LSP1, respectively), confirmed the integrity of cell populations in the HT cohort. RNA was extracted and quantitative RT-PCR was performed using comparative CT (ΔΔCT) analysis. Statistically significant (p < 0.05) differential expression profiles of MIR141 were found between tissue types. HT samples displayed significant downregulation of MIR141 compared to both cPTC and control thyrocytes. Furthermore, TGFßR1 expression was detected in cPTC samples but not in HT thyrocytes. It is postulated that the downregulation of this miRNA is due, at least in part, to its involvement in regulating the TGFß pathway. This pathway is exquisitely involved in T-cell autoimmunity and has previously been linked with HT. In conclusion, HT epithelium can be distinguished from cPTC epithelium and control epithelium based on the relative expression of MIR141.

16.
J Ovarian Res ; 5(1): 2, 2012 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-22260314

RESUMO

BACKGROUND: Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease. METHODS: Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples. RESULTS: Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component. CONCLUSION: We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

17.
Urol Oncol ; 30(6): 900-5, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21396847

RESUMO

OBJECTIVE: To examine the role of RET in renal malignancy, in particular papillary renal cell carcinoma (RCC). MATERIALS AND METHODS: A cohort of 111 archival renal samples was used consisting of 94 renal cancers (66 papillary RCC, 18 conventional clear cell carcinoma, 10 chromophobe RCC), 4 benign oncocytomas, and 13 normal kidney tissues. RET protein expression was examined by immunohistochemistry and expression levels were correlated with clinicopathologic and patient survival data. RESULTS: Positive RET staining was seen in 34/66 (52%) papillary RCCs, 4/10 (40%) chromophobe carcinomas, 4/4 (100%) oncocytomas, and 11/13 (85%) normal kidney samples. All 18 cases of conventional clear cell carcinoma had negative RET staining. RET expression was associated with low Fuhrman nuclear grade. CONCLUSIONS: RET protein may be contributing in part to an adaptation of a papillary growth pattern in certain renal malignancies. Given the possible therapeutic benefit of small molecule inhibitors of RET activation, further work needs to be done to highlight the functional relevance of RET protein expression in papillary RCC.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Carcinoma de Células Renais/patologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-ret/análise
18.
Histopathology ; 59(6): 1240-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22175903

RESUMO

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2. METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients. CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma/metabolismo , Proteínas de Membrana/biossíntese , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Adulto , Idoso , Carcinoma/patologia , Humanos , Imuno-Histoquímica , Microdissecção e Captura a Laser , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos
19.
PLoS One ; 6(10): e26125, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22022533

RESUMO

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.


Assuntos
Degranulação Celular , Neovascularização Patológica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Adesividade Plaquetária , Transdução de Sinais , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Apirase/farmacologia , Ácido Araquidônico/farmacologia , Degranulação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Fragmentos de Peptídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores de Trombina/antagonistas & inibidores , Receptores de Trombina/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos
20.
Appl Immunohistochem Mol Morphol ; 18(6): 512-7, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20661134

RESUMO

Prostate cancer (CaP) is a significant cause of illness and death in males. Current detection strategies do not reliably detect the disease at an early stage and cannot distinguish aggressive versus nonaggressive CaP leading to potential overtreatment of the disease and associated morbidity. Zinc-α-2-glycoprotein (ZAG) and proteasome subunit ß-Type 6 (PSMB-6) were found to be up-regulated in the serum of CaP patients with higher grade tumors after 2-dimensional difference gel electrophoresis analysis. The aim of this study was to investigate if ZAG and PSMB-6 were also overexpressed in prostatic tumor tissue of CaP patients. Immunohistochemical analysis was performed on CaP tissue microarrays with samples from 199 patients. Confirmatory gene expression profiling for ZAG and PSMB-6 were performed on 4 cases using Laser Capture Microdissection and TaqMan real-time polymerase chain reaction. ZAG expression in CaP epithelial cells was inversely associated with Gleason grade (benign prostatic hyperplasia>G3>G4/G5). PSMB-6 was not expressed in either tumor or benign epithelium. However, strong PSMB-6 expression was noted in stromal and inflammatory cells. Our results indicate ZAG as a possible predictive marker of Gleason grade. The inverse association between grade and tissue expression with a rising serum protein level is similar to that seen with prostate-specific antigen. In addition, the results for both ZAG and PSMB-6 highlight the challenges in trying to associate the protein levels in serum with tissue expression.


Assuntos
Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Plasma Seminal/metabolismo , Análise Serial de Tecidos , Adulto , Idoso , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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