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1.
J Neuroinflammation ; 19(1): 58, 2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-35227277

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterised by the loss of upper and lower motor neurons. Increasing evidence indicates that neuroinflammation mediated by microglia contributes to ALS pathogenesis. This microglial activation is evident in post-mortem brain tissues and neuroimaging data from patients with ALS. However, the role of microglia in the pathogenesis and progression of amyotrophic lateral sclerosis remains unclear, partly due to the lack of a model system that is able to faithfully recapitulate the clinical pathology of ALS. To address this shortcoming, we describe an approach that generates monocyte-derived microglia-like cells that are capable of expressing molecular markers, and functional characteristics similar to in vivo human brain microglia. METHODS: In this study, we have established monocyte-derived microglia-like cells from 30 sporadic patients with ALS, including 15 patients with slow disease progression, 6 with intermediate progression, and 9 with rapid progression, together with 20 non-affected healthy controls. RESULTS: We demonstrate that patient monocyte-derived microglia-like cells recapitulate canonical pathological features of ALS including non-phosphorylated and phosphorylated-TDP-43-positive inclusions. Moreover, ALS microglia-like cells showed significantly impaired phagocytosis, altered cytokine profiles, and abnormal morphologies consistent with a neuroinflammatory phenotype. Interestingly, all ALS microglia-like cells showed abnormal phagocytosis consistent with the progression of the disease. In-depth analysis of ALS microglia-like cells from the rapid disease progression cohort revealed significantly altered cell-specific variation in phagocytic function. In addition, DNA damage and NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome activity were also elevated in ALS patient monocyte-derived microglia-like cells, indicating a potential new pathway involved in driving disease progression. CONCLUSIONS: Taken together, our work demonstrates that the monocyte-derived microglia-like cell model recapitulates disease-specific hallmarks and characteristics that substantiate patient heterogeneity associated with disease subgroups. Thus, monocyte-derived microglia-like cells are highly applicable to monitor disease progression and can be applied as a functional readout in clinical trials for anti-neuroinflammatory agents, providing a basis for personalised treatment for patients with ALS.


Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/patologia , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Humanos , Microglia/metabolismo , Monócitos/metabolismo , Doenças Neurodegenerativas/metabolismo , Fagocitose
2.
Nano Lett ; 20(1): 478-495, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31789044

RESUMO

Microglia-mediated neuroinflammation is one of the most significant features in a variety of central nervous system (CNS) disorders such as traumatic brain injury, stroke, and many neurodegenerative diseases. Microglia become polarized upon stimulation. The two extremes of the polarization are the neuron-destructive proinflammatory M1-like and the neuron-regenerative M2-like phenotypes. Thus, manipulating microglial polarization toward the M2 phenotype is a promising therapeutic approach for CNS repair and regeneration. It has been reported that nanoparticles are potential tools for regulating microglial polarization. Gold nanoclusters (AuNCs) could penetrate the blood-brain barrier and have neuroprotective effects, suggesting the possibility of utilizing AuNCs to regulate microglial polarization and improve neuronal regeneration in CNS. In the current study, AuNCs functionalized with dihydrolipoic acid (DHLA-AuNCs), an antioxidant with demonstrated neuroprotective roles, were prepared, and their effects on polarization of a microglial cell line (BV2) were examined. DHLA-AuNCs effectively suppressed proinflammatory processes in BV2 cells by inducing polarization toward the M2-like phenotype. This was associated with a decrease in reactive oxygen species and reduced NF-kB signaling and an improvement in cell survival coupled with enhanced autophagy and inhibited apoptosis. Conditioned medium from DHLA-AuNC-treated BV2 cells was able to enhance neurogenesis in both the neuronal cell line N2a and in an ex vivo brain slice stroke model. The direct treatment of brain slices with DHLA-AuNCs also ameliorated stroke-related tissue injury and reduced astrocyte activation (astrogliosis). This study suggests that by regulating neuroinflammation to improve neuronal regeneration, DHLA-AuNCs could be a potential therapeutic agent in CNS disorders.


Assuntos
Polaridade Celular/efeitos dos fármacos , Ouro , Nanopartículas Metálicas/química , Microglia/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Ácido Tióctico/análogos & derivados , Animais , Linhagem Celular Tumoral , Ouro/química , Ouro/farmacologia , Camundongos , Ácido Tióctico/química , Ácido Tióctico/farmacologia
3.
Am J Hum Genet ; 101(2): 255-266, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28777932

RESUMO

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.


Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 11/genética , Ciclina D1/genética , Reparo do DNA/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Elementos Facilitadores Genéticos/genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/genética
4.
J Cell Mol Med ; 23(12): 8151-8160, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31565865

RESUMO

Suppressor of morphogenesis in genitalia 1 (SMG1) and ataxia telangiectasia mutated (ATM) are members of the PI3-kinase like-kinase (PIKK) family of proteins. ATM is a well-established tumour suppressor. Loss of one or both alleles of ATM results in an increased risk of cancer development, particularly haematopoietic cancer and breast cancer in both humans and mouse models. In mice, total loss of SMG1 is embryonic lethal and loss of a single allele results in an increased rate of cancer development, particularly haematopoietic cancers and lung cancer. In this study, we generated mice deficient in Atm and lacking one allele of Smg1, Atm-/- Smg1gt/+ mice. These mice developed cancers more rapidly than either of the parental genotypes, and all cancers were haematopoietic in origin. The combined loss of Smg1 and Atm resulted in a higher level of basal DNA damage and oxidative stress in tissues than loss of either gene alone. Furthermore, Atm-/- Smg1gt/+ mice displayed increased cytokine levels in haematopoietic tissues compared with wild-type animals indicating the development of low-level inflammation and a pro-tumour microenvironment. Overall, our data demonstrated that combined loss of Atm expression and decreased Smg1 expression increases haematopoietic cancer development.


Assuntos
Dano ao DNA , Neoplasias Hematológicas/genética , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Heterozigoto , Estimativa de Kaplan-Meier , Longevidade/genética , Linfoma/genética , Linfoma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência
5.
Hum Mol Genet ; 26(1): 109-123, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007901

RESUMO

Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Ataxia Telangiectasia/complicações , Inflamação/etiologia , Doenças Neurodegenerativas/etiologia , Neurônios/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Betametasona/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Inflamação/patologia , Inflamação/prevenção & controle , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/prevenção & controle , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Ratos , Ratos Mutantes
6.
Acta Neuropathol ; 138(6): 1033-1052, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31463571

RESUMO

Glioblastomas (GBMs) are malignant central nervous system (CNS) neoplasms with a very poor prognosis. They display cellular hierarchies containing self-renewing tumourigenic glioma stem cells (GSCs) in a complex heterogeneous microenvironment. One proposed GSC niche is the extracellular matrix (ECM)-rich perivascular bed of the tumour. Here, we report that the ECM binding dystroglycan (DG) receptor is expressed and functionally glycosylated on GSCs residing in the perivascular niche. Glycosylated αDG is highly expressed and functional on the most aggressive mesenchymal-like (MES-like) GBM tumour compartment. Furthermore, we found that DG acts to maintain an MES-like state via tight control of MAPK activation. Antibody-based blockade of αDG induces robust ERK-mediated differentiation leading to reduced GSC potential. DG was shown to be required for tumour initiation in MES-like GBM, with constitutive loss significantly delaying or preventing tumourigenic potential in-vivo. These findings reveal a central role of the DG receptor, not only as a structural element, but also as a critical factor promoting MES-like GBM and the maintenance of GSCs residing in the perivascular niche.


Assuntos
Neoplasias Encefálicas/metabolismo , Distroglicanas/metabolismo , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral/fisiologia , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/cirurgia , Transformação Celular Neoplásica , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Glioma/irrigação sanguínea , Glioma/cirurgia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias
7.
Mol Pharm ; 16(6): 2532-2539, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31009228

RESUMO

Cancer stem cells (CSCs) are a subpopulation of tumor cells that exhibit self-renewal, differentiation, and tumorigenicity. CSCs are highly resistant to the conventional cancer treatment and have been associated with metastasis. Several studies have been shown that salinomycin (Sal) has the potential to target cancer stem cells evidenced by in vitro and in vivo tumor models. Here, salinomycin was conjugated with biocompatible gold nanoparticles (AuNPs) coated with poly(ethylene glycol) to improve its specificity in targeting breast cancer stem cells (BCSCs). BCSCs derived from CD24low/CD44high subpopulation showed high sensitivity to Sal-AuNP treatment. An in-depth analysis on the mechanism of action of Sal-AuNPs indicated ferroptosis, an iron-dependent cell death, was achieved as a result of iron accumulation and inhibition of antioxidant properties. This also led to the induction of oxidative stress, mitochondrial dysfunction, and lipid oxidation. Our findings suggest Sal-AuNP treatment is an efficient therapeutic avenue in eliminating cancer stem cells.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Piranos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Fluoresceínas/metabolismo , Glutationa/metabolismo , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
8.
Immunol Cell Biol ; 96(1): 8-20, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29359354

RESUMO

The phosphoinositide-3-kinase like kinases are a family of very large protein kinases. These PI3-kinase like kinase (PIKK) proteins have well-established roles in detection and repair of damage to the genome, regulation of the transcriptome and cellular metabolism. Recently there has emerged, evidence for links between these proteins and inflammation. While some of these links come from an increased understanding of the impacts of damage to the cell on inflammatory responses, others suggest that PIKK proteins also have direct roles in regulation of immune responses. Particularly evident is the link between DNA damage and innate immune response pathways. Here, we review recent findings on the PIKK family of proteins and how they impact on inflammation, particularly activation of the innate immune system.


Assuntos
Sistema Imunitário/metabolismo , Inflamação/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Dano ao DNA , Reparo do DNA , Humanos , Imunidade Inata , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais
9.
J Neurooncol ; 138(3): 509-518, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29564746

RESUMO

Glioblastoma (GBM) is a highly fatal disease with a 5 year survival rate of less than 22%. One of the most effective treatment regimens to date is the use of radiotherapy which induces lethal DNA double-strand breaks to prevent tumour growth. However, recurrence occurs in the majority of patients and is in-part a result of robust radioresistance mechanisms. In this study, we demonstrate that the multifunctional cytokine, interleukin-6 (IL-6), confers a growth advantage in GBM cells but does not have the same effect on normal neural progenitor cells. Further analysis showed IL-6 can promote radioresistance in GBM cells when exposed to ionising radiation. Ablation of the Ataxia-telangiectasia mutated serine/threonine kinase that is recruited and activated by DNA double-strand breaks reverses the effect of radioresistance and re-sensitised GBM to DNA damage thus leading to increase cell death. Our finding suggests targeting the signaling cascade of DNA damage response is a potential therapeutic approach to circumvent IL-6 from promoting radioresistance in GBM.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proliferação de Células/efeitos da radiação , Neoplasias do Sistema Nervoso Central/radioterapia , Glioblastoma/radioterapia , Interleucina-6/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Morte Celular/fisiologia , Morte Celular/efeitos da radiação , Linhagem Celular , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Neoplasias do Sistema Nervoso Central/metabolismo , Dano ao DNA/efeitos da radiação , Glioblastoma/metabolismo , Humanos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos da radiação , RNA Mensageiro/metabolismo , Tolerância a Radiação/fisiologia , Radiação Ionizante , Receptores de Interleucina-6/metabolismo
10.
Nucleic Acids Res ; 43(17): 8352-67, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26240375

RESUMO

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Reparo de DNA por Recombinação , Transdução de Sinais , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/química , Humanos , Fosforilação , Radiação Ionizante
11.
J Neurooncol ; 122(2): 263-71, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25648357

RESUMO

Glioblastoma multiforme (GBM) has nearly uniformly fatal with a median survival of less than 2 years. While there have not been any novel anti-GBM therapeutics approved for many years, there has been the gradual accumulation of clinical data suggesting that the widely used anti-convulsant agent, valproic acid (VPA) may significantly prolong survival in GBM patients. This pre-clinical study aimed to determine the potential clinical utility of VPA in the treatment of GBM. Primary GBM cells were treated with VPA as a monotherapy and in combination with temozolomide and irradiation. At clinically achievable concentrations, VPA was shown to be effective as a monotherapy agent in the five primary lines tested. VPA was then used as a sensitizing agent to in vitro radiation and showed significant augmentation of in vitro irradiation therapy. In addition, when VPA, radiation and temozolomide were combined an additive, rather than synergistic effect was noted. Gene expression profiling demonstrated close clustering of triple treated cells with VPA mono-treated cells while untreated cells clustered closer with TMZ-irradiation dual treated cells. These microarray data suggest a dominant role of VPA at the gene expression level when combining these different treatment options. Moreover, in an in vivo tumor transplantation model, we were able to demonstrate an increase in animal survival when cells were pre-treated with irradiation-VPA and when triple treated. These findings provide a significant rationale for the investigation of VPA in the treatment of GBM patients.


Assuntos
Antineoplásicos/administração & dosagem , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Radiossensibilizantes/administração & dosagem , Ácido Valproico/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimiorradioterapia/métodos , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/administração & dosagem , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise em Microsséries , Transplante de Neoplasias , Regiões Promotoras Genéticas , Temozolomida , Proteínas Supressoras de Tumor/genética
12.
Growth Factors ; 32(6): 254-64, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25410964

RESUMO

The Eph genes are the largest sub-family of receptor tyrosine kinases; however, it is most likely the least understood and the arena for many conflicting reports. In this tribute to Prof. Martin Lackmann and Prof. Tony Pawson, we utilized The Cancer Genome Atlas resources to shed new light on the understanding of this family. We found that mutation and expression analysis define two clusters of co-expressed Eph family genes that relate to aggressive phenotypes across multiple cancer types. Analysis of signal transduction pathways using reverse-phase protein arrays revealed a network of interactions, which associates cluster-specific Eph genes with epithelial-mesenchymal transition, metabolism, DNA-damage repair and apoptosis. Our findings support the role of the Eph family in modulating cancer progression and reveal distinct patterns of Eph expression, which correlate with disease outcome. These observations provide further rationale for seeking cancer therapies, which target the Eph/ephrin system.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/diagnóstico , Receptores da Família Eph/metabolismo , Animais , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Receptores da Família Eph/genética
13.
J Immunother Cancer ; 12(4)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38658031

RESUMO

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.


Assuntos
Inibidores de Checkpoint Imunológico , Morte Celular Imunogênica , Animais , Camundongos , Morte Celular Imunogênica/efeitos dos fármacos , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Linhagem Celular Tumoral , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/terapia
14.
Neuro Oncol ; 25(2): 248-260, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35608632

RESUMO

BACKGROUND: Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. METHODS: We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. RESULTS: We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. CONCLUSION: PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Fosfofrutoquinase-1 , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo
15.
STAR Protoc ; 3(4): 101747, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36201317

RESUMO

Microglia are implicated in most neurodegenerative diseases. Here, we present a robust and efficient protocol to differentiate monocyte-derived microglia-like cells (MDMi) from whole blood. The protocol consists of three parts. The first part will describe two methods for PBMC isolation. This will be followed by MDMi differentiation, and lastly, the characterization of MDMi by immunocytochemistry. MDMi can be used to investigate microglial-related responses in various age-related neurodegenerative diseases and can be applied to drug testing on a personalized basis. For complete details on the use and execution of this protocol, please refer to Quek et al. (2022).


Assuntos
Leucócitos Mononucleares , Monócitos , Humanos , Microglia , Diferenciação Celular
16.
Biomedicines ; 10(2)2022 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-35203541

RESUMO

Adult diffuse glioma, particularly glioblastoma (GBM), is a devastating tumor of the central nervous system. The existential threat of this disease requires on-going treatment to counteract tumor progression. The present outcome is discouraging as most patients will succumb to this disease. The low cure rate is consistent with the failure of first-line therapy, radiation and temozolomide (TMZ). Even with their therapeutic mechanism of action to incur lethal DNA lesions, tumor growth remains undeterred. Delivering additional treatments only delays the inescapable development of therapeutic tolerance and disease recurrence. The urgency of establishing lifelong tumor control needs to be re-examined with a greater focus on eliminating resistance. Early genomic and transcriptome studies suggest each tumor subtype possesses a unique molecular network to safeguard genome integrity. Subsequent seminal work on post-therapy tumor progression sheds light on the involvement of DNA repair as the causative contributor for hypermutation and therapeutic failure. In this review, we will provide an overview of known molecular factors that influence the engagement of different DNA repair pathways, including targetable vulnerabilities, which can be exploited for clinical benefit with the use of specific inhibitors.

17.
Artigo em Inglês | MEDLINE | ID: mdl-33737223

RESUMO

The ability of natural extracts to inhibit melanocyte activity is of great interest to researchers. This study evaluates and explores the ability of mutated Shiitake (A37) and wildtype Shiitake (WE) extract to inhibit this activity. Several properties such as total phenolic (TPC) and total flavonoid content (TFC), antioxidant activity, effect on cell and component profiling were conducted. While having no significant differences in total phenolic content, mutation resulted in A37 having a TFC content (1.04 ± 0.7 mg/100 ml) compared to WE (0.86 ± 0.9 mg/100 ml). Despite that, A37 extract has lower antioxidant activity (EC50, A37 = 549.6 ± 2.70 µg/ml) than WE (EC50 = 52.8 ± 1.19 µg/ml). Toxicity tests on zebrafish embryos show that both extracts, stop the embryogenesis process when the concentration used exceeds 900 µg/ml. Although both extracts showed pigmentation reduction in zebrafish embryos, A37 extract showed no effect on embryo heartbeat. Cell cycle studies revealed that WE significantly affect the cell cycle while A37 not. Further tests found that these extracts inhibit the phosphorylation of Glycogen synthase kinase 3 ß (pGSK3ß) in HS27 cell line, which may explain the activation of apoptosis in melanin-producing cells. It was found that from 19 known compounds, 14 compounds were present in both WE and A37 extracts. Interestingly, the presence of decitabine in A37 extract makes it very potential for use in the medical application such as treatment of melanoma, skin therapy and even cancer.


Assuntos
Antineoplásicos/farmacologia , Melanócitos/efeitos dos fármacos , Crista Neural/efeitos dos fármacos , Cogumelos Shiitake/química , Peixe-Zebra/embriologia , Animais , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Melanócitos/citologia , Melanoma/tratamento farmacológico
18.
Front Chem ; 8: 601649, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33520933

RESUMO

Malignant cancer is a devastating disease often associated with a poor clinical prognosis. For decades, modern drug discoveries have attempted to identify potential modulators that can impede tumor growth. Cancer stem cells however are more resistant to therapeutic intervention, which often leads to treatment failure and subsequent disease recurrence. Here in this study, we have developed a specific multi-target drug delivery nanoparticle system against breast cancer stem cells (BCSCs). Therapeutic agents curcumin and salinomycin have complementary functions of limiting therapeutic resistance and eliciting cellular death, respectively. By conjugation of CD44 cell-surface glycoprotein with poly(lactic-co-glycolic acid) (PLGA) nanoparticles that are loaded with curcumin and salinomycin, we investigated the cellular uptake of BCSCs, drug release, and therapeutic efficacy against BCSCs. We determined CD44-targeting co-delivery nanoparticles are highly efficacious against BCSCs by inducing G1 cell cycle arrest and limiting epithelial-mesenchymal transition. This curcumin and salinomycin co-delivery system can be an efficient treatment approach to target malignant cancer without the repercussion of disease recurrence.

19.
PeerJ ; 8: e9197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32509463

RESUMO

BACKGROUND: DNA double strand break repair is important to preserve the fidelity of our genetic makeup after DNA damage. Rad50 is one of the components in MRN complex important for DNA repair mechanism. Rad50 mutations can lead to microcephaly, mental retardation and growth retardation in human. However, Rad50 mutations in human and other organisms have never been gathered and heuristically compared for their deleterious effects. It is important to assess the conserved region in Rad50 and its homolog to identify vital mutations that can affect functions of the protein. METHOD: In this study, Rad50 mutations were retrieved from SNPeffect 4.0 database and literature. Each of the mutations was analyzed using various bioinformatic analyses such as PredictSNP, MutPred, SNPeffect 4.0, I-Mutant and MuPro to identify its impact on molecular mechanism, biological function and protein stability, respectively. RESULTS: We identified 103 mostly occurred mutations in the Rad50 protein domains and motifs, which only 42 mutations were classified as most deleterious. These mutations are mainly situated at the specific motifs such as Walker A, Q-loop, Walker B, D-loop and signature motif of the Rad50 protein. Some of these mutations were predicted to negatively affect several important functional sites that play important roles in DNA repair mechanism and cell cycle signaling pathway, highlighting Rad50 crucial role in this process. Interestingly, mutations located at non-conserved regions were predicted to have neutral/non-damaging effects, in contrast with previous experimental studies that showed deleterious effects. This suggests that software used in this study may have limitations in predicting mutations in non-conserved regions, implying further improvement in their algorithm is needed. In conclusion, this study reveals the priority of acid substitution associated with the genetic disorders. This finding highlights the vital roles of certain residues such as K42E, C681A/S, CC684R/S, S1202R, E1232Q and D1238N/A located in Rad50 conserved regions, which can be considered for a more targeted future studies.

20.
Nat Commun ; 11(1): 4709, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948765

RESUMO

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.


Assuntos
Reparo do DNA , Instabilidade Genômica , Glioblastoma/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Proteína BRCA1 , Neoplasias Encefálicas/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Glioblastoma/patologia , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Tolerância a Radiação , Radiação Ionizante , Transcriptoma
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