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1.
Diabet Med ; 41(2): e15258, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37935454

RESUMO

AIMS: Maternally inherited diabetes and deafness (MIDD) is a rare form of adult-onset diabetes that can be difficult to diagnose due to its variable clinical phenotype. Transfer RNA-derived small fragments are a novel, emerging class of small non-coding RNAs (sncRNAs) that have significant potential as serum biomarkers due to their stress-induced generation, abundance, stability and ease of detection. METHODS: We investigated the levels of tiRNA 5'ValCAC (alone and in combination with miR-23b-3p) identified from small RNA sequencing studies in serum samples from healthy controls, type 1 diabetes, type 2 diabetes and MIDD subjects. RESULTS: Serum levels of 5'ValCAC were reduced in MIDD and type 2 diabetes subjects compared to controls. Type 2 diabetes subjects had higher serum levels of miR-23b-3p compared to all other subjects. Receiver Operating Characteristic analysis showed the potential of 5'ValCAC and miR-23b-3p as MIDD biomarkers, with the combination showing excellent separation from type 2 diabetes subjects. CONCLUSIONS: This is the first report showing altered serum levels of tiRNAs in diabetes subjects. The combined use of 5'ValCAC and miR-23b-3p as serum biomarkers could potentially differentiate between MIDD subjects and type 2 diabetes subjects.


Assuntos
Surdez , Diabetes Mellitus Tipo 2 , MicroRNAs , Doenças Mitocondriais , Adulto , Humanos , Diabetes Mellitus Tipo 2/genética , Biomarcadores , RNA de Transferência , MicroRNAs/genética
2.
Ann Oncol ; 34(3): 300-314, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36494005

RESUMO

BACKGROUND: New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles have failed to direct treatment strategies. We hypothesised that interrogation of the GBM tumour microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision immunotherapy treatment strategies. MATERIALS AND METHODS: A refined and validated microenvironment cell population (MCP) counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition around medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified three novel patient clusters, uniquely characterised by TME composition, functional orientation markers and immune checkpoint proteins. Validation was carried out in three independent GBM-RNA-seq datasets. Neoantigen, mutational and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal Glioma Longitudinal AnalySiS (GLASS) cohort and three immunotherapy clinical trial cohorts [treatment with neoadjuvant/adjuvant anti-programmed cell death protein 1 (PD-1) or PSVRIPO] were further interrogated to assess subtype alterations between primary and recurrent tumours and to assess the utility of TME classifiers as immunotherapy biomarkers. RESULTS: TMEHigh tumours (30%) displayed elevated lymphocyte, myeloid cell immune checkpoint, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 transcripts. TMEHigh/mesenchymal+ patients featured tertiary lymphoid structures. TMEMed (46%) tumours were enriched for endothelial cell gene expression profiles and displayed heterogeneous immune populations. TMELow (24%) tumours were manifest as an 'immune-desert' group. TME subtype transitions upon recurrence were identified in the longitudinal GLASS cohort. Assessment of GBM immunotherapy trial datasets revealed that TMEHigh patients receiving neoadjuvant anti-PD-1 had significantly increased overall survival (P = 0.04). Moreover, TMEHigh patients treated with adjuvant anti-PD-1 or oncolytic virus (PVSRIPO) showed a trend towards improved survival. CONCLUSIONS: We have established a novel TME-based classification system for application in intracranial malignancies. TME subtypes represent canonical 'termini a quo' (starting points) to support an improved precision immunotherapy treatment approach.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Microambiente Tumoral , Recidiva Local de Neoplasia , Imunoterapia/métodos , Neoplasias Encefálicas/tratamento farmacológico
3.
Pharmacogenomics J ; 21(4): 510-519, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33731881

RESUMO

Previous research has identified differences in mutation frequency in genes implicated in chemotherapy resistance between mucinous and non-mucinous colorectal cancers (CRC). We hypothesized that outcomes in mucinous and non-mucinous CRC may be influenced by expression of genes responsible for chemotherapy resistance. Gene expression data from primary tumor samples were extracted from The Cancer Genome Atlas PanCancer Atlas. The distribution of clinical, pathological, and gene expression variables was compared between 74 mucinous and 521 non-mucinous CRCs. Predictors of overall survival (OS) were assessed in a multivariate analysis. Kaplan-Meier curves were constructed to compare survival according to gene expression using the log rank test. The median expression of 5-FU-related genes TYMS, TYMP, and DYPD was significantly higher in mucinous CRC compared to non-mucinous CRC (p < 0.001, p = 0.003, p < 0.001, respectively). The median expression of oxaliplatin-related genes ATP7B and SRPK1 was significantly reduced in mucinous versus non-mucinous CRC (p = 0.004, p = 0.007, respectively). At multivariate analysis, age (odds ratio (OR) = 0.96, p < 0.001), node positive disease (OR = 0.49, p = 0.005), and metastatic disease (OR = 0.32, p < 0.001) remained significant negative predictors of OS, while high SRPK1 remained a significant positive predictor of OS (OR = 1.59, p = 0.037). Subgroup analysis of rectal cancers demonstrated high SRPK1 expression was associated with significantly longer OS compared to low SRPK1 expression (p = 0.011). This study highlights that the molecular differences in mucinous CRC and non-mucinous CRC extend to chemotherapy resistance gene expression. SRPK1 gene expression was associated with OS, with a prognostic role identified in rectal cancers.


Assuntos
Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Inativação Metabólica/genética , Idoso , ATPases Transportadoras de Cobre/genética , Feminino , Expressão Gênica/genética , Humanos , Masculino , Prognóstico , Proteínas Serina-Treonina Quinases/genética
4.
Ann Oncol ; 31(12): 1679-1692, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32918998

RESUMO

Glioblastoma represents the most common primary malignancy of the central nervous system in adults and remains a largely incurable disease. The elucidation of disease subtypes based on mutational profiling, gene expression and DNA methylation has so far failed to translate into improved clinical outcomes. However, new knowledge emerging from the subtyping effort in the IDH-wild-type setting may provide directions for future precision therapies. Here, we review recent learnings in the field, and further consider how tumour microenvironment differences across subtypes may reveal novel contexts of vulnerability. We discuss recent treatment approaches and ongoing trials in the IDH-wild-type glioblastoma setting, and propose an integrated discovery stratagem incorporating multi-omics, single-cell technologies and computational approaches.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Adulto , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Metilação de DNA , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Isocitrato Desidrogenase/genética , Mutação , Medicina de Precisão , Microambiente Tumoral
5.
Surg Oncol ; 34: 57-62, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32891354

RESUMO

BACKGROUND: Response to neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer is variable. Identification of biomarkers to predict response is desirable in order to provide prognostic information and targeted therapy. Several studies have investigated microsatellite instability (MSI) as a predictor of response to CRT with contradictory results. This study aims to clarify the effect of MSI status on response to CRT in locally advanced rectal cancer through systematic review and meta-analysis. METHODS: A systematic search of PubMed, Embase and Cochrane databases was performed for all studies relating to MSI and response to CRT in rectal cancer using the search algorithm (Microsatellite Instability) AND (Chemoradiotherapy) AND (Rectal Cancer). From each included study the number of patients with MSI tumors and Microsatellite Stable (MSS) tumors and the numbers achieving pathological complete response (pCR) were recorded. Pooled outcome measures were determined using a random effects model and the odds ratio estimated with variance and 95% confidence interval. RESULTS: Nine published studies were identified reporting data on MSI and its effect on outcome after CRT for locally advanced rectal cancer. Five studies describing 5,877 patients included data on MSI and the number of patients achieving pCR. There was no significant association between MSI and pCR (MSI Vs MSS: 10.1% Vs 6.6%, OR 1.38, 95% CI: 0.7-2.72, p = 0.35). CONCLUSION: This meta-analysis concludes that there appears to be no significant difference in pCR rate following CRT in patients with MSI versus MSS rectal tumors.


Assuntos
Quimiorradioterapia Adjuvante/métodos , Instabilidade de Microssatélites , Terapia Neoadjuvante/métodos , Neoplasias Retais/patologia , Humanos , Prognóstico , Neoplasias Retais/genética , Neoplasias Retais/terapia
6.
Br J Surg ; 106(6): 682-691, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30945755

RESUMO

BACKGROUND: Mucinous differentiation occurs in 5-15 per cent of colorectal adenocarcinomas. This subtype of colorectal cancer responds poorly to chemoradiotherapy and has a worse prognosis. The genetic aetiology underpinning this cancer subtype lacks consensus. The aim of this study was to use meta-analytical techniques to clarify the molecular associations of mucinous colorectal cancer. METHODS: This study adhered to MOOSE guidelines. Databases were searched for studies comparing KRAS, BRAF, microsatellite instability (MSI), CpG island methylator phenotype (CIMP), p53 and p27 status between patients with mucinous and non-mucinous colorectal adenocarcinoma. A random-effects model was used for analysis. RESULTS: Data from 46 studies describing 17 746 patients were included. Mucinous colorectal adenocarcinoma was associated positively with KRAS (odds ratio (OR) 1·46, 95 per cent c.i. 1·08 to 2·00, P = 0·014) and BRAF (OR 3·49, 2·50 to 4·87; P < 0·001) mutation, MSI (OR 3·98, 3·30 to 4·79; P < 0·001) and CIMP (OR 3·56, 2·85 to 4·43; P < 0·001), and negatively with altered p53 expression (OR 0·46, 0·31 to 0·67; P < 0·001). CONCLUSION: The genetic origins of mucinous colorectal adenocarcinoma are predominantly associated with BRAF, MSI and CIMP pathways. This pattern of molecular alterations may in part explain the resistance to standard chemotherapy regimens seen in mucinous adenocarcinoma.


Assuntos
Adenocarcinoma Mucinoso/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Adenocarcinoma Mucinoso/patologia , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilação de DNA , Humanos , Instabilidade de Microssatélites , Modelos Estatísticos , Mutação , Fenótipo , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética
7.
Cell Death Dis ; 7: e2087, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26844701

RESUMO

Colorectal cancer (CRC) is one of the most common cancers in the Western world. 5-Fluorouracil (5FU)-based chemotherapy (CT) remains the mainstay treatment of CRC in the advanced setting, and activates executioner caspases in target cells. Executioner caspases are key proteins involved in cell disassembly during apoptosis. Activation of executioner caspases also has a role in tissue regeneration and repopulation by stimulating signal transduction and cell proliferation in neighbouring, non-apoptotic cells as reported recently. Tissue microarrays (TMAs) consisting of tumour tissue from 93 stage II and III colon cancer patients were analysed by immunohistochemistry. Surprisingly, patients with low levels of active Caspase-3 had an increased disease-free survival time. This was particularly pronounced in patients who received 5FU-based adjuvant CT. In line with this observation, lower serum levels of active Caspase-3 were found in patients with metastasised CRC who revealed stable disease or tumour regression compared with those with disease progression. The role of Caspase-3 in treatment responses was explored further in primary human tumour explant cultures from fresh patient tumour tissue. Exposure of explant cultures to 5FU-based CT increased the percentage of cells positive for active Caspase-3 and Terminal Deoxynucleotidyl Transferase dUTP Nick end Labelling (TUNEL), but also the expression of regeneration and proliferation markers ß-Catenin and Ki-67, as well as cyclooxygenase-2 (COX-2). Of note, selective inhibition of Caspase-3 with Ac-DNLD-CHO, a selective, reversible inhibitor of Caspase-3, significantly reduced the expression of proliferation markers as well as COX-2. Inhibition of COX-2 with aspirin or celecoxib did not affect Caspase-3 levels but also reduced Ki-67 and ß-Catenin levels, suggesting that Caspase-3 acted via COX-2 to stimulate cell proliferation and tissue regeneration. This indicates that low levels of active Caspase-3 may represent a new predictor of CT responsiveness, and inhibition of Caspase-3, or antagonising downstream effectors of Caspase-3 paracrine signalling, such as COX-2 may improve patient outcomes following CT in advanced CRC.


Assuntos
Caspase 3/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Fluoruracila/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Caspase 3/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Análise Serial de Tecidos
8.
Biochim Biophys Acta ; 1863(4): 499-509, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26721623

RESUMO

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Gossipol/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-X/genética , Apoptose/genética , Autofagia/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Gossipol/farmacologia , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Proteína bcl-X/metabolismo
9.
Br J Cancer ; 114(2): 188-98, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26657652

RESUMO

BACKGROUND: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. METHODS: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. RESULTS: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. CONCLUSIONS: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Dacarbazina/análogos & derivados , Dipeptídeos/farmacologia , Glioblastoma/patologia , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Animais , Western Blotting , Caspase 8/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Contraste de Fase , Transplante de Neoplasias , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Radiat Oncol ; 10: 131, 2015 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-26071313

RESUMO

BACKGROUND: The mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance. METHODS: We obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined. RESULTS: We found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins. CONCLUSION: These data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may be a useful indicator of neoadjuvant radio chemotherapy resistance in rectal cancer. Moreover, as XIAP levels increase with radio chemotherapy it is possible that a subset of more resistant tumour cells survive this treatment and may be resistant to further adjuvant treatment. Patients with resistant tumours highly expressing XIAP may benefit from alternative treatment strategies, such as Smac mimetics post neoadjuvant radio chemotherapy.


Assuntos
Biomarcadores Tumorais/análise , Quimiorradioterapia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/análise , Proteínas Mitocondriais/análise , Terapia Neoadjuvante , Proteínas de Neoplasias/análise , Tolerância a Radiação/fisiologia , Neoplasias Retais/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/análise , Adulto , Idoso , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Proteína 3 com Repetições IAP de Baculovírus , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Proteínas Inibidoras de Apoptose/análise , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas Inibidoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Retais/patologia , Neoplasias Retais/terapia , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
12.
Cell Death Differ ; 22(9): 1502-16, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25633195

RESUMO

An accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) mediated via the activation of three transmembrane proteins IRE1, PERK and ATF6. Signalling through these proteins is aimed at enhancing the ER folding capacity and reducing the folding load. If these processes fail to re-establish protein homeostasis within the ER, then cell death prevails via apoptosis. How the shift from pro-survival to pro-apoptotic signalling is regulated remains unclear with both IRE1 and PERK signalling associated with pro-survival as well as pro-apoptotic signalling. To investigate the temporal activation of IRE1 and PERK in live cells and their relationship to cellular fate, we devised single cell reporters for both ER stress signalling branches. SH-SY5Y neural cells stably expressing these fluorescent protein reporter constructs to monitor IRE1-splicing activity and PERK-mediated ATF4-translation were imaged using single cell and high content time lapse live cell microscopy. We could correlate an early onset and attenuation of XBP1 splicing in the IRE1-reporter cells as cytoprotective. Indeed, silencing of IRE1 expression using shRNA inhibited splicing of XBP1 resulting in an early onset of cell death. In contrast, in the PERK-reporter cells, we observed that a slow rate of ATF4-translation and late re-initiation of general translation coincided with cells which were resistant to ER stress-induced cell death. Interestingly, whereas silencing of PERK did not affect overall levels of cell death in response to ER stress, it did increase sensitivity to ER stressors at early time points following treatment. Our results suggest that apoptosis activation in response to ER stress is not caused by a preferential activation of a single UPR branch, or by a switch from one branch to the other. Rather, our data indicated that the relative timing of IRE1 and PERK signalling determines the shift from cell survival to apoptosis.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , eIF-2 Quinase/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Análise de Célula Única/métodos , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box
13.
J Mol Med (Berl) ; 93(3): 315-26, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25388617

RESUMO

UNLABELLED: In locally advanced rectal cancer, neoadjuvant chemoradiotherapy is performed prior to surgery to downstage the tumour. Thirty to 40 % of patients do not respond. Defects in apoptotic machinery lead to therapy resistance; however, to date, no study quantitatively assessed whether B cell lymphoma 2 (BCL2)-dependent regulation of mitochondrial apoptosis, effector caspase activation downstream of mitochondria or a combination of both predicts patient responses. In a cohort of 20 rectal cancer patients, we performed protein profiling of tumour tissue and employed validated ordinary differential equation-based systems models of apoptosis signalling to calculate the ability of cancer cells to undergo apoptosis. Model outputs were compared to clinical responses. Systems modelling of BCL2-signalling predicted patients in the poor response group (p = 0.0049). Systems modelling also demonstrated that rectal cancers depended on BCL2 rather than B cell lymphoma-extra large (BCL(X)L) or myeloid cell leukemia 1 (MCL1) for survival, suggesting that poor responders may benefit from therapy with selective BCL2 antagonists. Dynamic modelling of effector caspase activation could not stratify patients with poor response and did not further improve predictive power. We deliver a powerful patient stratification tool identifying patients who will likely not benefit from neoadjuvant chemoradiotherapy and should be prioritised for surgical resection or treatment with BCL2 antagonists. KEY MESSAGES: Modelling BCL2-family proteins identifies patients unresponsive to therapy. Caspase activation downstream of mitochondria cannot identify these patients. Rectal tumours of poor responders are BCL2- but not BCL-XL-dependent. DR_MOMP allows clinicians to identify patients who would not benefit from therapy. DR_MOMP is also a useful patient stratification tool for BCL2 antagonists.


Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Retais/metabolismo , Adulto , Idoso , Apoptose , Quimiorradioterapia Adjuvante , Dano ao DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Terapia Neoadjuvante , Neoplasias Retais/terapia , Transdução de Sinais , Resultado do Tratamento
14.
Br J Cancer ; 111(12): 2275-86, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25375271

RESUMO

BACKGROUND: Glioblastoma (GBM), being a highly vascularised and locally invasive tumour, is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks. METHODS: Cell viability, endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures. RESULTS: An endothelial/GBM cell viability inhibitory effect, as well as an anti-angiogenic and anti-invasive response, to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020, P=0.030), anti-angiogenic (P=0.040, P<0.0001) and pro-apoptotic (P=0.0083, P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response, respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed. CONCLUSIONS: Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis, invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy.


Assuntos
Glioblastoma/tratamento farmacológico , Gossipol/farmacologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cell Death Dis ; 5: e1456, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25299781

RESUMO

Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Isquemia Encefálica/metabolismo , Glucose/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética
16.
Cell Death Dis ; 5: e1258, 2014 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-24874730

RESUMO

Our understanding of the mitochondrial or intrinsic apoptosis pathway and its role in chemotherapy resistance has increased significantly in recent years by a combination of experimental studies and mathematical modelling. This combined approach enhanced the quantitative and kinetic understanding of apoptosis signal transduction, but also provided new insights that systems-emanating functions (i.e., functions that cannot be attributed to individual network components but that are instead established by multi-component interplay) are crucial determinants of cell fate decisions. Among these features are molecular thresholds, cooperative protein functions, feedback loops and functional redundancies that provide systems robustness, and signalling topologies that allow ultrasensitivity or switch-like responses. The successful development of kinetic systems models that recapitulate biological signal transduction observed in living cells have now led to the first translational studies, which have exploited and validated such models in a clinical context. Bottom-up strategies that use pathway models in combination with higher-level modelling at the tissue, organ and whole body-level therefore carry great potential to eventually deliver a new generation of systems-based diagnostic tools that may contribute to the development of personalised and predictive medicine approaches. Here we review major achievements in the systems biology of intrinsic apoptosis signalling, discuss challenges for further model development, perspectives for higher-level integration of apoptosis models and finally discuss requirements for the development of systems medical solutions in the coming years.


Assuntos
Apoptose , Simulação por Computador , Resistência a Medicamentos , Modelos Biológicos , Biologia de Sistemas , Animais , Humanos
17.
Eur J Clin Microbiol Infect Dis ; 33(8): 1381-90, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24599709

RESUMO

Commensal bacteria in the colon may play a role in colorectal cancer (CRC) development. Recent studies from North America showed that Fusobacterium nucleatum (Fn) infection is over-represented in disease tissue versus matched normal tissue in CRC patients. Using quantitative real-time polymerase chain reaction (qPCR) of DNA extracted from colorectal tissue biopsies and surgical resections of three European cohorts totalling 122 CRC patients, we found an over-abundance of Fn in cancerous compared to matched normal tissue (p < 0.0001). To determine whether Fn infection is an early event in CRC development, we assayed Fn in colorectal adenoma (CRA) tissue from 52 Irish patients. While for all CRAs the Fn level was not statistically significantly higher in disease versus normal tissue (p = 0.06), it was significantly higher for high-grade dysplasia (p = 0.015). As a secondary objective, we determined that CRC patients with low Fn levels had a significantly longer overall survival time than patients with moderate and high levels of the bacterium (p = 0.008). The investigation of Fn as a potential non-invasive biomarker for CRC screening showed that, while Fn was more abundant in stool samples from CRC patients compared to adenomas or controls, the levels in stool did not correlate with cancer or adenoma tissue levels from the same individuals. This is the first study examining Fn in the colonic tissue and stool of European CRC and CRA patients, and suggests Fn as a novel risk factor for disease progression from adenoma to cancer, possibly affecting patient survival outcomes. Our results highlight the potential of Fn detection as a diagnostic and prognostic determinant in CRC patients.


Assuntos
Adenoma/microbiologia , Neoplasias Colorretais/microbiologia , Fusobacterium nucleatum/isolamento & purificação , Adenoma/genética , Adenoma/mortalidade , Adenoma/patologia , Idoso , Idoso de 80 Anos ou mais , Carga Bacteriana , Estudos de Coortes , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Progressão da Doença , Fezes/microbiologia , Feminino , Fusobacterium nucleatum/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Mutação de Sentido Incorreto , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética
18.
J Neurosci Res ; 92(6): 679-91, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24464409

RESUMO

The N-methyl-D-aspartate (NMDA) receptor, one of the ionotropic glutamate receptor, plays important physiological and pathological roles in learning and memory, neuronal development, acute and chronic neurological diseases, and neurogenesis. This work examines the contribution of the NR2B NMDA receptor subunit to adult neurogenesis/cell proliferation under physiological conditions and following an excitotoxic insult. We have previously shown in vitro that a discrete NMDA-induced, excitotoxic injury to the hippocampus results in an increase in neurogenesis within the dentate gyrus. Here we have characterized adult neurogenesis or proliferation, using BrdU, in an in vivo model of excitotoxic injury to the CA1 subfield of the hippocampus. We demonstrate a peak in neural stem cell proliferation/neurogenesis between 6 and 9 days after the excitotoxic insult. Treatment with ifenprodil, an NR2B subunit-specific NMDA receptor antagonist, without prior injury induction, also increased the number of BrdU-positive cells within the DG and posterior periventricle, indicating that ifenprodil itself could modulate the rate of proliferation. Interestingly, though, the increased level of cell proliferation did not change significantly when ifenprodil was administered following an excitotoxic insult. In conclusion, our results suggest and add to the growing evidence that NR2B subunit-containing NMDA receptors play a role in neural stem cell proliferation.


Assuntos
Proliferação de Células/efeitos dos fármacos , Hipocampo/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/fisiologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos
19.
Apoptosis ; 19(4): 629-42, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24213561

RESUMO

Glioblastoma (GBM) is the most aggressive form of primary brain tumour, with dismal patient outcome. Treatment failure is associated with intrinsic or acquired apoptosis resistance and the presence of a highly tumourigenic subpopulation of cancer cells called GBM stem cells. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising novel therapy for some treatment-resistant tumours but unfortunately GBM can be completely resistant to TRAIL monotherapy. In this study, we identified Mcl-1, an anti-apoptotic Bcl-2 family member, as a critical player involved in determining the sensitivity of GBM to TRAIL-induced apoptosis. Effective targeting of Mcl-1 in TRAIL resistant GBM cells, either by gene silencing technology or by treatment with R-roscovitine, a cyclin-dependent kinase inhibitor that targets Mcl-1, was demonstrated to augment sensitivity to TRAIL, both within GBM cells grown as monolayers and in a 3D tumour model. Finally, we highlight that two separate pathways are activated during the apoptotic death of GBM cells treated with a combination of TRAIL and R-roscovitine, one which leads to caspase-8 and caspase-3 activation and a second pathway, involving a Mcl-1:Noxa axis. In conclusion, our study demonstrates that R-roscovitine in combination with TRAIL presents a promising novel strategy to trigger cell death pathways in glioblastoma.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Ativação Enzimática , Inativação Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Purinas/farmacologia , Roscovitina
20.
Transl Psychiatry ; 3: e317, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24150226

RESUMO

Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer's and Huntington's disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKß, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on TMRM uptake were largely mediated by plasma membrane hyperpolarization, precluding a purely 'mitochondrial' mechanism of action. In line with a stabilizing effect of latrepirdine on plasma membrane potential, pretreatment with latrepirdine reduced spontaneous Ca(2+) oscillations as well as glutamate-induced Ca(2+) increases in primary neurons, and protected neurons against glutamate toxicity. In conclusion, our experiments demonstrate that latrepirdine is a potent activator of AMPK, and suggest that one of the main pharmacological activities of latrepirdine is a reduction in neuronal excitability.


Assuntos
Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Indóis/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nootrópicos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Cerebelo/citologia , Inativação Gênica , Transportador de Glucose Tipo 3/efeitos dos fármacos , Transportador de Glucose Tipo 3/metabolismo , Camundongos , Mitocôndrias/metabolismo , Neocórtex/citologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos
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