RESUMO
Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.
Assuntos
Bloqueadores do Receptor Tipo 2 de Angiotensina II , Compostos Benzidrílicos , Neoplasias Encefálicas , Reposicionamento de Medicamentos , Glioblastoma , Isoquinolinas , Receptor Tipo 2 de Angiotensina , Analgésicos/farmacologia , Angiotensina II/química , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/uso terapêutico , Apoptose , Compostos Benzidrílicos/química , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Conformação Proteica em alfa-Hélice , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
The role of astrocytes in the progression of Alzheimer's disease (AD) remains poorly understood. We assessed the consequences of ablating astrocytic proliferation in 9 months old double transgenic APP23/GFAP-TK mice. Treatment of these mice with the antiviral agent ganciclovir conditionally ablates proliferating reactive astrocytes. The loss of proliferating astrocytes resulted in significantly increased levels of monomeric amyloid-ß (Aß) in brain homogenates, associated with reduced enzymatic degradation and clearance mechanisms. In addition, our data revealed exacerbated memory deficits in mice lacking proliferating astrocytes concomitant with decreased levels of synaptic markers and higher expression of pro-inflammatory cytokines. Our data suggest that loss of reactive astrocytes in AD aggravates amyloid pathology and memory loss, possibly via disruption of amyloid clearance and enhanced neuroinflammation.
Assuntos
Doença de Alzheimer/patologia , Astrócitos/patologia , Proliferação de Células/fisiologia , Memória Espacial/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Memória de Curto Prazo/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Cancer occupies a high rank in the global morbidity and mortality scale with glioblastoma multiforme (GBM) accounting for almost 80% of all primary tumors of the brain. Despite the increasing availability of targeted and immunotherapeutic agents, chemotherapy still plays an important role in the treatment of neoplastic diseases. Limitations to the effective use of chemotherapy such as low aqueous solubility and high toxicity have directed the scientific community's interest to the development of new therapeutic agents with enhanced efficacy and limited toxicity. Supramolecular chemistry has offered an alternative way on the design and development of new therapeutic agents as a result of their unique properties. Supramolecules can be used as drug carriers since their cavities can host a wide range of small drugs and surpass in this way the drawbacks of current therapeutic agents. Herein, we present the principles that should be followed for the encapsulation of small drugs in supramolecules with enhanced physicochemical properties and increased efficacy against glioblastoma multiforme.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Portadores de Fármacos , Glioblastoma , Temozolomida , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Solubilidade , Temozolomida/química , Temozolomida/farmacocinética , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. Maximal surgical resection followed by radiotherapy and concomitant chemotherapy with temozolomide remains the first-line therapy, prolonging the survival of patients by an average of only 2.5 months. There is therefore an urgent need for novel therapeutic strategies to improve clinical outcomes. Reactive oxygen species (ROS) are an important contributor to GBM development. Here, we describe the rational design and synthesis of a stable hybrid molecule tethering two ROS regulating moieties, with the aim of constructing a chemopreventive and anticancer chemical entity that retains the properties of the parent compounds. We utilized the selective AT1R antagonist losartan, leading to the inhibition of ROS levels, and the antioxidant flavonoid quercetin. In GBM cells, we show that this hybrid retains the binding potential of losartan to the AT1R through competition-binding experiments and simultaneously exhibits ROS inhibition and antioxidant capacity similar to native quercetin. In addition, we demonstrate that the hybrid is able to alter the cell cycle distribution of GBM cells, leading to cell cycle arrest and to the induction of cytotoxic effects. Last, the hybrid significantly and selectively reduces cancer cell proliferation and angiogenesis in primary GBM cultures with respect to the isolated parent components or their simple combination, further emphasizing the potential utility of the current hybridization approach in GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Losartan , Quercetina/farmacologia , Temozolomida/farmacologiaRESUMO
Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10-500â¯ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods' reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.
Assuntos
Antineoplásicos Alquilantes/sangue , Neoplasias Encefálicas/tratamento farmacológico , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Monitoramento de Medicamentos/métodos , Espectrometria de Massas em Tandem , Temozolomida/sangue , Administração Oral , Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Encefálicas/sangue , Calibragem , Precipitação Química , Cromatografia Líquida de Alta Pressão/normas , Cromatografia de Fase Reversa/normas , Monitoramento de Medicamentos/normas , Humanos , Limite de Detecção , Valor Preditivo dos Testes , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normas , Temozolomida/administração & dosagemRESUMO
The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Calixarenos/química , Glioblastoma/tratamento farmacológico , Nanocápsulas/química , Temozolomida/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Estabilidade de Medicamentos , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , Temozolomida/química , Temozolomida/farmacocinéticaRESUMO
Appropriate post-translational processing of collagen requires prolyl hydroxylation, catalyzed by collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase, and is essential for normal cell function. Here we have investigated the expression, transcriptional regulation, and function of the collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase families in melanoma. We show that the collagen prolyl 3-hydroxylase family exemplified by Leprel1 and Leprel2 is subject to methylation-dependent transcriptional silencing in primary and metastatic melanoma consistent with a tumor suppressor function. In contrast, although there is transcriptional silencing of P4HA3 in a subset of melanomas, the collagen prolyl 4-hydroxylase family members P4HA1, P4HA2, and P4HA3 are often overexpressed in melanoma, expression being prognostic of worse clinical outcomes. Consistent with tumor suppressor function, ectopic expression of Leprel1 and Leprel2 inhibits melanoma proliferation, whereas P4HA2 and P4HA3 increase proliferation, and particularly invasiveness, of melanoma cells. Pharmacological inhibition with multiple selective collagen prolyl 4-hydroxylase inhibitors reduces proliferation and inhibits invasiveness of melanoma cells. Together, our data identify the collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase families as potentially important regulators of melanoma growth and invasiveness and suggest that selective inhibition of collagen prolyl 4-hydroxylase is an attractive strategy to reduce the invasive properties of melanoma cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Prolil Hidroxilases/genética , Neoplasias Cutâneas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Colágeno/metabolismo , Metilação de DNA/genética , Humanos , Melanoma/patologia , Processamento de Proteína Pós-Traducional/genética , Valores de Referência , Neoplasias Cutâneas/patologiaRESUMO
Despite emergence of new systemic therapies, metastatic melanoma remains a challenging and often fatal form of skin cancer. The renin-angiotensin system (RAS) is a major physiological regulatory pathway controlling salt-water equilibrium, intravascular volume and blood pressure. Biological effects of the RAS are mediated by the vasoactive hormone angiotensin II (AngII) via two receptor subtypes, AT1R (encoded by AGTR1) and AT2R (encoded by AGTR2). We report decreasing expression and increasing CpG island methylation of AGTR1 in metastatic versus primary melanoma and detection in serum of methylated genomic DNA from the AGTR1 CpG island in metastatic melanoma implying that AGTR1 encodes a tumour suppressor function in melanoma. Consistent with this hypothesis, antagonism of AT1R using losartan or shRNA-mediated knockdown in melanoma cell lines expressing AGTR1 resulted in acquisition of the ability to proliferate in serum-free conditions. Conversely, ectopic expression of AGTR1 in cell lines lacking endogenous expression inhibits proliferation irrespective of the presence of AngII implying a ligand-independent suppressor function for AT1R. Treatment of melanoma cell lines expressing endogenous AT2R with either AngII or the AT2R-selective agonist Y6AII induces proliferation in serum-free conditions whereas the AT2R-specific antagonists PD123319 and EMA401 inhibit melanoma growth and angiogenesis and potentiate inhibitors of BRAF and MEK in cells with BRAF V600 mutations. Our results demonstrate that the RAS has both oncogenic and tumour suppressor functions in melanoma. Pharmacological inhibition of AT2R may provide therapeutic opportunities in melanomas expressing this receptor and AGTR1 CpG island methylation in serum may serve as a novel biomarker of metastatic melanoma.
Assuntos
Proliferação de Células , Melanoma/patologia , Melanoma/terapia , Terapia de Alvo Molecular , Sistema Renina-Angiotensina/fisiologia , Amidas/farmacologia , Amidas/uso terapêutico , Angiotensina II/farmacologia , Angiotensina II/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Embrião não Mamífero , Fumaratos/farmacologia , Fumaratos/uso terapêutico , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Melanoma/genética , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Metástase Neoplásica , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Glioblastoma multiforme (GBM) remains a cancer with a poor prognosis and few effective therapeutic options. Successful medical management of GBM is limited by the restricted access of drugs to the central nervous system (CNS) caused by the blood brain barrier (BBB). We previously showed that a subset of GBM are arginine auxotrophic because of transcriptional silencing of ASS1 and/or ASL and are sensitive to pegylated arginine deiminase (ADI-PEG20). However, it is unknown whether depletion of arginine in peripheral blood in vivo has therapeutic activity against intracranial disease. In the present work, we describe the efficacy of ADI-PEG20 in an intracranial model of human GBM in which tumour growth and regression are assessed in real time by measurement of luciferase activity. Animals bearing intracranial human GBM tumours of varying ASS status were treated with ADI-PEG20 alone or in combination with temozolomide and monitored for tumour growth and regression. Monotherapy ADI-PEG20 significantly reduces the intracranial growth of ASS1 negative GBM and extends survival of mice carrying ASS1 negative GBM without obvious toxicity. The combination of ADI-PEG20 with temozolomide (TMZ) demonstrates enhanced effects in both ASS1 negative and ASS1 positive backgrounds.Our data provide proof of principle for a therapeutic strategy for GBM using peripheral blood arginine depletion that does not require BBB passage of drug and is well tolerated. The ability of ADI-PEG20 to cytoreduce GBM and enhance the effects of temozolomide argues strongly for its early clinical evaluation in the treatment of GBM.
Assuntos
Argininossuccinato Sintase/genética , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Hidrolases/farmacologia , Polietilenoglicóis/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Arginina/metabolismo , Argininossuccinato Sintase/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Understanding the mechanisms of neurodegeneration is crucial for development of therapies to treat neurological disorders. S100 proteins are extensively expressed in the injured brain but S100's role and signalling in neural cells remain elusive. We recently demonstrated that the S100A4 protein protects neurons in brain injury and designed S100A4-derived peptides mimicking its beneficial effects. Here we show that neuroprotection by S100A4 involves the growth factor family receptor ErbB4 and its ligand Neuregulin 1 (NRG), key regulators of neuronal plasticity and implicated in multiple brain pathologies. The neuroprotective effect of S100A4 depends on ErbB4 expression and the ErbB4 signalling partners ErbB2/Akt, and is reduced by functional blockade of NRG/ErbB4 in cell models of neurodegeneration. We also detect binding of S100A4 with ErbB1 (EGFR) and ErbB3. S100A4-derived peptides interact with, and signal through ErbB, are neuroprotective in primary and immortalized dopaminergic neurons, and do not affect cell proliferation/motility - features which make them promising as potential neuroprotectants. Our data suggest that the S100-ErbB axis may be an important mechanism regulating neuronal survival and plasticity.