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Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.
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Agaricales , Glioblastoma , Toxinas Biológicas , Antineoplásicos Alquilantes , Linhagem Celular Tumoral , Metilases de Modificação do DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Humanos , Ribonucleases , Temozolomida/farmacologiaRESUMO
BACKGROUND: Gliomas represent about 80% of primary brain tumours and about 30% of malignant ones, which today don't have a resolution therapy because of their variability. A valid model for the study of new personalized therapies can be represented by primary cultures from patient's tumour biopsies. METHODS: In this study we consider 12 novel cell lines established from patients' gliomas and immunohistochemically and molecularly characterized according to the newly updated 2016 CNS Tumour WHO classification. RESULTS: Eight of these lines were glioblastoma cells, two grade III glioma cells (anaplastic astrocytoma and oligo astrocytoma) and two low grade glioma cells (grade II astrocytoma and oligodendroglioma). All cell lines were analysed by immunohistochemistry for specific glioma markers, respectively VIMENTIN, GFAP, IDH1R132, and ATRX. The methylation status of the MGMT gene promoter was also determined in all lines. The comparison of the immunohistochemical characteristics and of the MGMT methylation status of the lines with the tissues of origin shows that the cells in culture maintain the same characteristics. CONCLUSIONS: Human cancer cell lines represent a support in the knowledge of tumour biology and in drug discovery through its facile experimental manipulation. TRIAL REGISTRATION: NCT04180046.
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Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Neoplasias do Sistema Nervoso Central/patologia , Metilação de DNA , Glioma/patologia , Mutação , Regiões Promotoras Genéticas , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Sistema Nervoso Central/classificação , Neoplasias do Sistema Nervoso Central/genética , Feminino , Glioma/classificação , Glioma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Células Tumorais Cultivadas , Organização Mundial da SaúdeRESUMO
Huntington's disease (HD) is the most common neurodegenerative disorder for which no effective cure is yet available. Although several agents have been identified to provide benefits so far, the number of therapeutic options remains limited with only symptomatic treatment available. Over the past few years, we have demonstrated that sphingolipid-based approaches may open the door to new and more targeted treatments for the disease. In this study, we investigated the therapeutic potential of stimulating sphingosine-1-phosphate (S1P) receptor 5 by the new selective agonist A-971432 (provided by AbbVie) in R6/2 mice, a widely used HD animal model. Chronic administration of low-dose (0.1 mg/kg) A-971432 slowed down the progression of the disease and significantly prolonged lifespan in symptomatic R6/2 mice. Such beneficial effects were associated with activation of pro-survival pathways (BDNF, AKT and ERK) and with reduction of mutant huntingtin aggregation. A-971432 also protected blood-brain barrier (BBB) homeostasis in the same mice. Interestingly, when administered early in the disease, before any overt symptoms, A-971432 completely protected HD mice from the classic progressive motor deficit and preserved BBB integrity. Beside representing a promising strategy to take into consideration for the development of alternative therapeutic options for HD, selective stimulation of S1P receptor 5 may be also seen as an effective approach to target brain vasculature defects in the disease.
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Barreira Hematoencefálica/efeitos dos fármacos , Proteína Huntingtina/genética , Doença de Huntington/tratamento farmacológico , Agregação Patológica de Proteínas/tratamento farmacológico , Receptores de Lisoesfingolipídeo/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/genética , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Agregação Patológica de Proteínas/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Lisoesfingolipídeo/agonistasRESUMO
In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.
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Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Lactonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sesquiterpenos/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma , Humanos , Lactonas/química , Estrutura Molecular , Sesquiterpenos/químicaRESUMO
Given the ongoing pandemic, there is a need to identify SARS-CoV-2 and differentiate it from other respiratory viral infections in various critical settings. Since its introduction, rapid antigen testing is spreading worldwide, but diagnostic accuracy is extremely variable and often in disagreement with the manufacturer's specifications. Our study compared the clinical performances of two microfluidic rapid antigen tests towards a molecular assay, starting from positive samples. A total of 151 swabs collected at the Microbiology and Virology Laboratory of A.O. "SS Antonio e Biagio e C. Arrigo" (Alessandria, Italy) for the diagnosis of SARS-CoV-2 were simultaneously tested to evaluate accuracy, specificity, and agreement with the RT-qPCR results. Both assays showed an overall agreement of 100% for negative specimens, while positive accuracy comprised between 45.10% and 54.90%. According to the manufacturer's instructions, the greatest correlation between the antigenic and molecular assays was observed for the subset with high viral load (18/19, 94.74%), while it dramatically decreased for other subsets. Moreover, the ability to differentiate between SARS-CoV-2 and Flu provides an added value and could be addressed in an epidemic context. However, an in-house validation should be performed due to differences observed in performance declared by manufacturers and those actually obtained.
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p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.
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Neoplasias Encefálicas , Glioblastoma , Apoptose , Neoplasias Encefálicas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Ácidos Cumáricos/farmacologia , Ácidos Cumáricos/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêuticoRESUMO
Glioblastoma multiforme (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population; its high recurrence rate and resistance to current therapeutics urgently demand a better therapy. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. UPS deregulation is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, the UPS represents a potentially valuable target for GBM treatment. Using an integrated approach that includes proteomics, transcriptomics and metabolic profiling, we identify praja2, a RING E3 ubiquitin ligase, as the key component of a signaling network that regulates GBM cell growth and metabolism. Praja2 is preferentially expressed in primary GBM lesions expressing the wild-type isocitrate dehydrogenase 1 gene (IDH1). Mechanistically, we found that praja2 ubiquitylates and degrades the kinase suppressor of Ras 2 (KSR2). As a consequence, praja2 restrains the activity of downstream AMP-dependent protein kinase in GBM cells and attenuates the oxidative metabolism. Delivery in the brain of siRNA targeting praja2 by transferrin-targeted self-assembling nanoparticles (SANPs) prevented KSR2 degradation and inhibited GBM growth, reducing the size of the tumor and prolonging the survival rate of treated mice. These data identify praja2 as an essential regulator of cancer cell metabolism, and as a potential therapeutic target to suppress GBM growth.
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Neoplasias Encefálicas , Glioblastoma , Adulto , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , UbiquitinaRESUMO
Glioblastoma is a brain tumour, characterised by recurrent or innate resistance to conventional chemoradiotherapy. Novel natural molecules and phyto-extracts have been proposed as adjuvants to sensitise the response to Temozolomide (TMZ). In this study, we investigated the effect of GS extract on human glioblastoma cells U87Mg. According to the IC50-values, GS extract displayed a significant cytotoxicity. This was confirmed by cell growth inhibition and alteration in metabolic activity evaluated by cell count and MTT assay. GS induced reduction in Pro-caspase 9, 3, but not PARP cleavage nor DNA fragmentation. Thus, in GS-induced cytotoxicity, cell death is not associated with apoptosis. In this context, short-term treatment of U87Mg cells with GS extract (1 mg/mL) reduced the phosphorylation levels of mTOR and of its downstream target P70 S6 kinase, highlighting the role of GS extract into autophagy induction. The activation of autophagic flux by GS extract was confirmed by Western blot analysis, which revealed the reduction in p62 and the concomitant increase in LC3B II/I ratio. Immunofluorescence evidenced the accumulation of LC3B puncta in U87Mg cells pretreated with autophagy inhibitor Bafilomycin A1. Furthermore, as main key regulators of type II programmed cell death, p53, p21 and CDK4 were also investigated and were inhibited by GS treatment. In conclusion, GS extract could be considered as an autophagy inducer in glioblastoma cells U87Mg.
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Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.
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Glioblastoma/tratamento farmacológico , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chenopodium quinoa/enzimologia , Humanos , Sementes/enzimologia , Temozolomida/farmacologiaRESUMO
Although several agents have been identified to provide therapeutic benefits in Huntington disease (HD), the number of conventionally used treatments remains limited and only symptomatic. Thus, it is plausible that the need to identify new therapeutic targets for the development of alternative and more effective treatments is becoming increasingly urgent. Recently, the sphingosine-1-phosphate (S1P) axis has been reported to be a valid potential novel molecular target for therapy development in HD. Modulation of aberrant metabolism of S1P in HD has been proved to exert neuroprotective action in vitro settings including human HD iPSC-derived neurons. In this study, we investigated whether promoting S1P production by stimulating Sphingosine Kinase 1 (SPHK1) by the selective activator, K6PC-5, may have therapeutic benefit in vivo in R6/2 HD mouse model. Our findings indicate that chronic administration of 0.05 mg/kg K6PC-5 exerted an overall beneficial effect in R6/2 mice. It significantly slowed down the progressive motor deficit associated with disease progression, modulated S1P metabolism, evoked the activation of pro-survival pathways and markedly reduced the toxic mutant huntingtin (mHtt) aggregation. These results suggest that K6PC-5 may represent a future therapeutic option in HD and may potentially counteract the perturbed brain function induced by deregulated S1P pathways.
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A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.
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Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Doenças Neurodegenerativas/diagnóstico , Imagem Óptica/métodos , Albumina Sérica/farmacocinética , Animais , Modelos Animais de Doenças , Fluoresceína-5-Isotiocianato/farmacocinética , Infusões Intravenosas , Camundongos , Doenças Neurodegenerativas/patologia , PermeabilidadeRESUMO
Alterations of lipid metabolism have been frequently associated with Huntington's disease (HD) over the past years. HD is the most common neurodegenerative disorder, with a complex pathogenic profile, typically characterized by progressive striatal and cortical degeneration and associated motor, cognitive and behavioral disturbances. Previous findings from our group support the idea that disturbed sphingolipid metabolism could represent an additional hallmark of the disease. Although such a defect represents a common biological denominator among multiple disease models ranging from cells to humans through mouse models, more efforts are needed to clearly define its clinical significance and the role it may play in the progression of the disease. In this study, we provided the first evidence of a defective de novo biosynthetic pathway of sphingolipids in multiple HD pre-clinical models. qPCR analysis revealed perturbed gene expression of sphingolipid-metabolizing enzymes in both early and late stage of the disease. In particular, reduction in the levels of sptlc1 and cerS1 mRNA in the brain tissues from manifest HD mice resulted in a significant decrease in the content of dihydroSphingosine, dihydroSphingosine-1-phospahte and dihydroCeramide [C18:0] as assessed by mass spectrometry. Moreover, in vitro studies highlighted the relevant role that aberrant sphingolipid metabolism may have in the HD cellular homeostasis. With this study, we consolidate the evidence of disturbed sphingolipid metabolism in HD and demonstrate for the first time that the de novo biosynthesis pathway is also significantly affected in the disease. This finding further supports the hypothesis that perturbed sphingolipid metabolism may represent a crucial factor accounting for the high susceptibility to disease in HD.
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Blood-brain barrier (BBB) breakdown, due to the concomitant disruption of the tight junctions (TJs), normally required for the maintenance of BBB function, and to the altered transport of molecules between blood and brain and vice-versa, has been suggested to significantly contribute to the development and progression of different brain disorders including Huntington's disease (HD). Although the detrimental consequence the BBB breakdown may have in the clinical settings, the timing of its alteration remains elusive for many neurodegenerative diseases. In this study we demonstrate for the first time that BBB disruption in HD is not confined to established symptoms, but occurs early in the disease progression. Despite the obvious signs of impaired BBB permeability were only detectable in concomitance with the onset of the disease, signs of deranged TJs integrity occur precociously in the disease and precede the onset of overt symptoms. To our perspective this finding may add a new dimension to the horizons of pathological mechanisms underlying this devastating disease, however much remains to be elucidated for understanding how specific BBB drug targets can be approached in the future.
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Barreira Hematoencefálica/patologia , Doença de Huntington/patologia , Envelhecimento/patologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Claudina-5/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Camundongos , PermeabilidadeRESUMO
Huntington's disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.