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Temozolomide (TMZ) is the standard of care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug resistance mediated by a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), which eliminates the TMZ-induced DNA lesions. Here we report the development of an iron oxide nanoparticle (NP) system for targeted delivery of siRNAs to suppress the TMZ-resistance gene (MGMT). We show that our NP is able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially-passaged patient-derived xenografts. The treatment with sequential administration of this NP and TMZ resulted in increased apoptosis of GBM stem-like cells, reduced tumor growth, and significantly-prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.
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Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Glicoproteínas de Membrana/genética , Oncogenes/genética , Isoformas de RNA/genética , Splicing de RNA , Receptor trkB/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Células Cultivadas , Perfilação da Expressão Gênica , Ontologia Genética , Glioma/metabolismo , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de RNA/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/genéticaRESUMO
Glioblastoma (GBM) remains incurable, and recurrent tumors rarely respond to standard-of-care radiation and chemo-therapies. Therefore, strategies that enhance the effects of these therapies should provide significant benefits to GBM patients. We have developed a nanoparticle delivery vehicle that can stably bind and protect nucleic acids for specific delivery into brain tumor cells. These nanoparticles can deliver therapeutic siRNAs to sensitize GBM cells to radiotherapy and improve GBM treatment via systemic administration. We show that nanoparticle-mediated knockdown of the DNA repair protein apurinic endonuclease 1 (Ape1) sensitizes GBM cells to radiotherapy and extend survival in a genetic mouse model of GBM. Specific knockdown of Ape1 activity by 30% in brain tumor tissue doubled the extended survival achieved with radiotherapy alone. Ape1 is a promising target for increasing the effectiveness of radiotherapy, and nanoparticle-mediated delivery of siRNA is a promising strategy for tumor specific knockdown of Ape1.
Assuntos
Neoplasias Encefálicas/radioterapia , Reparo do DNA , Portadores de Fármacos/química , Glioblastoma/radioterapia , Nanopartículas/química , RNA Interferente Pequeno/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/efeitos da radiação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Modelos Animais de Doenças , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Camundongos , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi/métodosRESUMO
Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anticancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, suboptimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O6-methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O6-benzylguanosine (DABGS) to achieve high drug loading, and polyethylene glycol (PEG) to ameliorate water solubility and maintain NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated Temozolomide (TMZ) toxicity in vitro, demonstrating potential as a new treatment option for glioblastomas (GBMs).
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Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Guanosina/química , Concentração de Íons de Hidrogênio , Polímeros/química , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Humanos , Microscopia Eletrônica de Transmissão , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , TemozolomidaRESUMO
The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.
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Biomarcadores Tumorais/metabolismo , Quitosana/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Ácido Hialurônico/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Alicerces Teciduais/química , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Quitosana/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Glioblastoma/metabolismo , Humanos , Ácido Hialurônico/química , FenótipoRESUMO
Human tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is an attractive cancer therapeutic because of its ability to induce apoptosis in tumor cells while having a negligible effect on normal cells. However, the short serum half-life of TRAIL and lack of efficient in vivo administration approaches have largely hindered its clinical use. Using nanoparticles (NPs) as carriers in gene therapy is considered as an alternative approach to increase TRAIL delivery to tumors as transfected cells would be induced to secrete TRAIL into the tumor microenvironment. To enable effective delivery of plasmid DNA encoding TRAIL into glioblastoma (GBM), we developed a targeted iron oxide NP coated with chitosan-polyethylene glycol-polyethyleneimine copolymer and chlorotoxin (CTX) and evaluated its effect in delivering TRAIL in vitro and in vivo. NP-TRAIL successfully delivers TRAIL into human T98G GBM cells and induces secretion of 40 pg mL(-1) of TRAIL in vitro. Transfected cells show threefold increased apoptosis as compared to the control DNA bound NPs. Systemic administration of NP-TRAIL-CTX to mice bearing T98G-derived flank xenografts results in near-zero tumor growth and induces apoptosis in tumor tissue. Our results suggest that NP-TRAIL-CTX can potentially serve as a targeted anticancer therapeutic for more efficient TRAIL delivery to GBM.
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Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Nanopartículas/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Quitosana/administração & dosagem , Feminino , Terapia Genética/métodos , Meia-Vida , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Plasmídeos/administração & dosagem , Polietilenoglicóis/administração & dosagem , Polietilenoimina/administração & dosagem , Venenos de Escorpião/administração & dosagem , Transfecção/métodos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Medulloblastoma (MB) and ependymoma (EP) are the most common pediatric brain tumors, afflicting 3000 children annually. Radiotherapy (RT) is an integral component in the treatment of these tumors; however, the improvement in survival is often accompanied by radiation-induced adverse developmental and psychosocial sequelae. Therefore, there is an urgent need to develop strategies that can increase the sensitivity of brain tumors cells to RT while sparing adjacent healthy brain tissue. Apurinic endonuclease 1 (Ape1), an enzyme in the base excision repair pathway, has been implicated in radiation resistance in cancer. Pharmacological and specificity limitations inherent to small molecule inhibitors of Ape1 have hindered their clinical development. Here we report on a nanoparticle (NP) based siRNA delivery vehicle for knocking down Ape1 expression and sensitizing pediatric brain tumor cells to RT. The NP comprises a superparamagnetic iron oxide core coated with a biocompatible, biodegradable coating of chitosan, polyethylene glycol (PEG), and polyethyleneimine (PEI) that is able to bind and protect siRNA from degradation and to deliver siRNA to the perinuclear region of target cells. NPs loaded with siRNA against Ape1 (NP:siApe1) knocked down Ape1 expression over 75% in MB and EP cells, and reduced Ape1 activity by 80%. This reduction in Ape1 activity correlated with increased DNA damage post-irradiation, which resulted in decreased cell survival in clonogenic assays. The sensitization was specific to therapies generating abasic lesions as evidenced by NP:siRNA not increasing sensitivity to paclitaxel, a microtubule disrupting agent. Our results indicate NP-mediated delivery of siApe1 is a promising strategy for circumventing pediatric brain tumor resistance to RT.
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Neoplasias Encefálicas , Reparo do DNA , Ependimoma , Raios gama , Meduloblastoma , Nanopartículas , Radiossensibilizantes/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Ependimoma/metabolismo , Ependimoma/patologia , Ependimoma/radioterapia , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Meduloblastoma/radioterapia , Proteínas de Neoplasias/biossínteseRESUMO
BACKGROUND: CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. METHODS: We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). RESULTS: Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. CONCLUSIONS: Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. GENERAL SIGNIFICANCE: MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status.
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BACKGROUND: Periostin is a secreted matricellular protein critical for epithelial-mesenchymal transition and carcinoma metastasis. In glioblastoma, it is highly upregulated compared with normal brain, and existing reports indicate potential prognostic and functional importance in glioma. However, the clinical implications of periostin expression and function related to its therapeutic potential have not been fully explored. METHODS: Periostin expression levels and patterns were examined in human glioma cells and tissues by quantitative real-time PCR and immunohistochemistry and correlated with glioma grade, type, recurrence, and survival. Functional assays determined the impact of altering periostin expression and function on cell invasion, migration, adhesion, and glioma stem cell activity and tumorigenicity. The prognostic and functional relevance of periostin and its associated genes were analyzed using the TCGA and REMBRANDT databases and paired recurrent glioma samples. RESULTS: Periostin expression levels correlated directly with tumor grade and recurrence, and inversely with survival, in all grades of adult human glioma. Stromal deposition of periostin was detected only in grade IV gliomas. Secreted periostin promoted glioma cell invasion and adhesion, and periostin knockdown markedly impaired survival of xenografted glioma stem cells. Interactions with αvß3 and αvß5 integrins promoted adhesion and migration, and periostin abrogated cytotoxicity of the αvß3/ß5 specific inhibitor cilengitide. Periostin-associated gene signatures, predominated by matrix and secreted proteins, corresponded to patient prognosis and functional motifs related to increased malignancy. CONCLUSION: Periostin is a robust marker of glioma malignancy and potential tumor recurrence. Abrogation of glioma stem cell tumorigenicity after periostin inhibition provides support for exploring the therapeutic impact of targeting periostin.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Moléculas de Adesão Celular/metabolismo , Glioma/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/prevenção & controle , Adesão Celular , Moléculas de Adesão Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Glioma/mortalidade , Glioma/patologia , Glioma/prevenção & controle , Humanos , Integrinas/metabolismo , Estimativa de Kaplan-Meier , Gradação de Tumores , Invasividade Neoplásica , Regulação para CimaRESUMO
Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O(6)-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Guanina/análogos & derivados , Magnetismo , Nanopartículas , Animais , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Guanina/administração & dosagem , Guanina/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , OxirreduçãoRESUMO
Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.
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Alginatos/farmacologia , Proliferação de Células/efeitos dos fármacos , Quitosana/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Alicerces Teciduais/química , Alginatos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Quitosana/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glioblastoma , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Humanos , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Células-Tronco Neoplásicas/citologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors. RESULTS: We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations. CONCLUSIONS: Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.
Assuntos
Neoplasias Encefálicas/genética , Heterogeneidade Genética , Glioblastoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Oligodendroglioma/genética , Neoplasias Encefálicas/patologia , Amplificação de Genes , Glioblastoma/patologia , Humanos , Dados de Sequência Molecular , Oligodendroglioma/patologia , Mutação Puntual , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Salivares Ricas em Prolina/genética , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genéticaRESUMO
Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.
Assuntos
Alquilantes/síntese química , DNA/química , Adenina/análogos & derivados , Adenina/química , Alquilantes/química , Alquilantes/toxicidade , Animais , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Glicosilases/química , DNA Glicosilases/metabolismo , Metilação de DNA , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , TermodinâmicaRESUMO
Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O(6)-methylguanine (O(6)-meG) mediates GBM cell killing. Moreover, low or absent expression of O(6)-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O(6)-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O(6)-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O(6)-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O(6)-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.
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Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3- and 18-month-old mice into 3- and 20-month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3-month (37.5 vs. 83 days) and 20-month (38 vs. 67 days) hosts, indicating that age-dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF-1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age-dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age-dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.
Assuntos
Envelhecimento/patologia , Neoplasias Encefálicas/patologia , Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Fator 1 Induzível por Hipóxia/genética , Idoso , Envelhecimento/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Transformação Celular Neoplásica , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Feminino , Instabilidade Genômica , Glioma/tratamento farmacológico , Glioma/genética , Glioma/mortalidade , Humanos , Hipóxia/genética , Hipóxia/mortalidade , Hipóxia/patologia , Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Células-Tronco Neurais/patologia , Células-Tronco Neurais/transplante , Taxa de SobrevidaRESUMO
Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Glioma/tratamento farmacológico , Glioma/enzimologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Neoplasias Encefálicas/genética , Glioma/genética , Humanos , Terapia de Alvo Molecular , O(6)-Metilguanina-DNA Metiltransferase/genéticaRESUMO
Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Doxorrubicina/administração & dosagem , Compostos Férricos/administração & dosagem , Glioma/tratamento farmacológico , Nanopartículas Metálicas/administração & dosagem , Animais , Linhagem Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacocinética , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Compostos Férricos/química , Concentração de Íons de Hidrogênio , Nanopartículas Metálicas/química , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoimina/química , Ratos , Solubilidade , Distribuição TecidualRESUMO
Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers radiation resistance in human cells. Here we examined the association between Ap endo activity and response to radiotherapy in pediatric ependymomas, tumors for which treatment options are limited and survival rates are only about 50%. We assayed Ap endo activity in 36 ependymomas and expression of Ape1/Ref-1, the predominant Ap endo activity in humans, in 44 tumors by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity or expression with progression-free survival or with overall survival. Activity varied 13-fold and was not associated with tumor or patient characteristics. In univariate models with Ap endo activity entered as a continuous variable, the hazard ratio for progression increased by a factor of 2.18 for every 0.01 unit increase in activity (p ≤ 0.003) in 24 grade II ependymomas. Risk for death increased by a factor of 1.89 (p ≤ 0.02) in the same population. The fraction of Ape1/Ref-1 immunopositive cells varied widely within individual tumors and was not associated with either progression-free or with overall survival. Suppressing Ap endo activity in pediatric ependymoma cells significantly increased radiation sensitivity, suggesting that the association of activity with radiation response reflected, at least in part, repair of radiation-induced DNA lesions. Our data indicate that Ap endo activity is predictive of outcome following radiotherapy, and suggest that Ape1/Ref-1 promotes radiation resistance in pediatric ependymomas. Our findings support the use of inhibitors of Ap endo activity to overcome resistance.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/radioterapia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Ependimoma/enzimologia , Ependimoma/radioterapia , Adolescente , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Criança , Intervalo Livre de Doença , Ependimoma/imunologia , Ependimoma/mortalidade , Feminino , Humanos , Lactente , Masculino , Tolerância a RadiaçãoRESUMO
BACKGROUND: Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro. RESULTS: To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells. CONCLUSIONS: Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change.
Assuntos
Transição Epitelial-Mesenquimal , Glioblastoma/patologia , Invasividade Neoplásica , Proteínas Nucleares/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Linhagem Celular Tumoral , HumanosRESUMO
Concurrent treatment with the methylating agent temozolomide during radiotherapy has yielded the first significant improvement in the survival of adult glioblastomas (GBM) in the last three decades. However, improved survival is observed in a minority of patients, most frequently those whose tumors display CpG methylation of the O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) promoter, and adult GBMs remain invariably fatal. Some, although not all, preclinical studies have shown that temozolomide can increase radiosensitivity in GBM cells that lack MGMT, the sole activity in human cells that removes O(6)-meG from DNA. Here, we systematically examined the temozolomide dose dependence of radiation killing in established GBM cell lines that differ in ability to remove O(6)-meG or tolerate its lethality. Our results show that minimally cytotoxic doses of temozolomide can produce dose-dependent radiosensitization in MGMT-deficient cells, MGMT-proficient cells, and MGMT-deficient cells that lack mismatch repair, a process that renders cells tolerant of the lethality of O(6)-meG. In cells that either possess or lack MGMT activity, radiosensitization requires exposure to temozolomide before but not after radiation and is accompanied by formation of double-strand breaks within 45 minutes of radiation. Moreover, suppressing alkyladenine-DNA glycosylase, the only activity in human cells that excises 3-methyladenine from DNA, reduces the temozolomide dose dependence of radiosensitization, indicating that radiosensitization is mediated by 3-methyladenine as well as by O(6)-meG. These results provide novel information on which to base further mechanistic study of radiosensitization by temozolomide in human GBM cells and to develop strategies to improve the outcome of concurrent temozolomide radiotherapy.