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Vascular calcification (VC) is a cardiovascular disease characterized by calcium salt deposition in vascular smooth muscle cells (VSMCs). Standard in vitro models used in VC investigations are based on VSMC monocultures under static conditions. Although these platforms are easy to use, the absence of interactions between different cell types and dynamic conditions makes these models insufficient to study key aspects of vascular pathophysiology. The present study aimed to develop a dynamic endothelial cell-VSMC co-culture that better mimics the in vivo vascular microenvironment. A double-flow bioreactor supported cellular interactions and reproduced the blood flow dynamic. VSMC calcification was stimulated with a DMEM high glucose calcification medium supplemented with 1.9 mM NaH2PO4/Na2HPO4 (1:1) for 7 days. Calcification, cell viability, inflammatory mediators, and molecular markers (SIRT-1, TGFß1) related to VSMC differentiation were evaluated. Our dynamic model was able to reproduce VSMC calcification and inflammation and evidenced differences in the modulation of effectors involved in the VSMC calcified phenotype compared with standard monocultures, highlighting the importance of the microenvironment in controlling cell behavior. Hence, our platform represents an advanced system to investigate the pathophysiologic mechanisms underlying VC, providing information not available with the standard cell monoculture.
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Diferenciação Celular , Técnicas de Cocultura , Músculo Liso Vascular , Miócitos de Músculo Liso , Calcificação Vascular , Humanos , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Células Cultivadas , Sobrevivência Celular , Fator de Crescimento Transformador beta1/metabolismo , Sirtuína 1/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Reatores BiológicosAssuntos
Técnicas de Cocultura , Células Endoteliais , Músculo Liso Vascular , Miócitos de Músculo Liso , Calcificação Vascular , Humanos , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/fisiopatologia , Calcificação Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Músculo Liso Vascular/patologia , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células CultivadasRESUMO
Introduction: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare incurable neurodegenerative disease caused by mutations in the SACS gene, which codes for sacsin, a large protein involved in protein homeostasis, mitochondrial function, cytoskeletal dynamics, autophagy, cell adhesion and vesicle trafficking. However, the pathogenic mechanisms underlying sacsin dysfunction are still largely uncharacterized, and so attempts to develop therapies are still in the early stages. Methods: To achieve further understanding of how processes are altered by loss of sacsin, we used untargeted proteomics to compare protein profiles in ARSACS fibroblasts versus controls. Results: Our analyses confirmed the involvement of known biological pathways and also implicated calcium and lipid homeostasis in ARSACS skin fibroblasts, a finding further verified in SH-SY5Y SACS -/- cells. Validation through mass spectrometry-based analysis and comparative quantification of lipids by LC-MS in fibroblasts revealed increased levels of ceramides coupled with a reduction of diacylglycerols. Discussion: In addition to confirming aberrant Ca2+ homeostasis in ARSACS, this study described abnormal lipid levels associated with loss of sacsin.
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Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.
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Complemento C3 , Fator H do Complemento , Camundongos Knockout , Fator H do Complemento/metabolismo , Fator H do Complemento/genética , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Estudo de Prova de Conceito , Camundongos Endogâmicos C57BLRESUMO
CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.
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Autofagia , Modelos Animais de Doenças , Lipofuscinoses Ceroides Neuronais , Fenótipo , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Autofagia/fisiologia , Autofagia/efeitos dos fármacos , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Animais Geneticamente Modificados , Trealose/farmacologiaRESUMO
BACKGROUND: Primary sclerosing cholangitis (PSC) is a rare chronic inflammatory liver disease characterized by biliary strictures and cholestasis. Due to the lack of effective serological indicators for diagnosis and prognosis, in the present study, we examined the potentiality of the saliva proteome to comprehensively screen for novel biomarkers. METHODS: Saliva samples of PSC patients and healthy controls were processed and subsequently analyzed using a liquid chromatography-tandem mass spectrometry technique. A bioinformatic approach was applied to detect the differentially expressed proteins, their related biological functions and pathways, and the correlation with the clinical evidence in order to identify a possible marker for the PSC group. RESULTS: We identified 25 differentially expressed proteins in PSC patients when compared to the healthy control group. Among them, eight proteins exhibited area under the curve values up to 0.800, suggesting these saliva proteins as good discriminators between the two groups. Multiple positive correlations were also identified between the dysregulated salivary proteins and increased serum alkaline phosphatase levels and the presence of ulcerative colitis. Pathway analysis revealed significant enrichments in the immune system, neutrophil degranulation, and in the interleukine-17 signaling pathway. CONCLUSION: We demonstrated the potentiality of saliva as a useful biofluid to obtain a fingerprint of the pathology, suggesting disulfide-isomerase A3 and peroxiredoxin-5 as the better discriminating proteins in PSC patients. Hence, analysis of saliva proteins could become, in future, a useful tool in the screening of patients with suspected PSC.
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Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-ß (TGF-ß)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-ß. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-ß-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.
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Curcumina , Fator de Crescimento Transformador beta , Humanos , Fator de Crescimento Transformador beta/metabolismo , Curcumina/farmacologia , Proteômica , Fígado/metabolismo , Fibrose , Anti-InflamatóriosRESUMO
Vascular calcification is a systemic disease characterized by calcium salt deposition within vascular walls. Here, we present a protocol for establishing an advanced dynamic in vitro co-culture system using endothelial and smooth muscle cells to replicate vascular tissue complexity. We describe steps for cell culture and seeding in a double-flow bioreactor that recreates the action of blood in humans. We then detail the induction of calcification, setting up of the bioreactor, followed by cell viability assessment and calcium quantification.
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Plastic use dramatically increased over the past few years. Besides obvious benefits, the consequent plastic waste and mismanagement in disposal have caused ecological problems. Plastic abandoned in the environment is prone to segregation, leading to the generation of microplastics (MPs) and nanoplastics (NPs), which can reach aquatic and terrestrial organisms. MPs/NPs in water can access fish's bodies through the gills, triggering an inflammatory response in loco. Furthermore, from the gills, plastic fragments can be transported within the circulatory system altering blood biochemical parameters and hormone levels and leading to compromised immunocompetence and angiogenesis. In addition, it was also possible to observe an unbalanced ROS production, damage in vascular structure, and enhanced thrombosis. MPs/NPs led to cardiotoxicity, pericardial oedema, and impaired heart rate in fish cardiac tissue. MPs/NPs effects on aquatic organisms pose serious health hazards and ecological consequences because they constitute the food chain for humans. Once present in the mammalian body, plastic particles can interact with circulating cells, eliciting an inflammatory response, with genotoxicity and cytotoxicity of immune cells, enhanced haemolysis, and endothelium adhesion. The interaction of MPs/NPs with plasma proteins allows their transport to distant organs, including the heart. As a consequence of plastic fragment internalisation into cardiomyocytes, oxidative stress was increased, and metabolic parameters were altered. In this scenario, myocardial damage, fibrosis and impaired electrophysiological values were observed. In summary, MPs/NPs are an environmental stressor for cardiac function in living organisms, and a risk assessment of their influence on the cardiovascular system certainly merits further analysis.
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Vascular calcification is a systemic disease contributing to cardiovascular morbidity and mortality. The pathophysiology of vascular calcification involves calcium salt deposition by vascular smooth muscle cells that exhibit an osteoblast-like phenotype. Multiple conditions drive the phenotypic switch and calcium deposition in the vascular wall; however, the exact molecular mechanisms and the connection between vascular smooth muscle cells and other cell types are not fully elucidated. In this hazy landscape, effective treatment options are lacking. Due to the pathophysiological complexity, several research models are available to evaluate different aspects of the calcification process. This review gives an overview of the in vitro cell models used so far to study the molecular processes underlying vascular calcification. In addition, relevant natural and synthetic compounds that exerted anticalcifying properties in in vitro systems are discussed.
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The overall increase in cardiovascular diseases and, specifically, the ever-rising exposure to cardiotoxic compounds has greatly increased in vivo animal testing; however, mainly due to ethical concerns related to experimental animal models, there is a strong interest in new in vitro models focused on the human heart. In recent years, human pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) emerged as reference cell systems for cardiac studies due to their biological similarity to primary CMs, the flexibility in cell culture protocols, and the capability to be amplified several times. Furthermore, the ability to be genetically reprogrammed makes patient-derived hiPSCs, a source for studies on personalized medicine. In this mini-review, the different models used for in vitro cardiac studies will be described, and their pros and cons analyzed to help researchers choose the best fitting model for their studies. Particular attention will be paid to hiPSC-CMs and three-dimensional (3D) systems since they can mimic the cytoarchitecture of the human heart, reproducing its morphological, biochemical, and mechanical features. The advantages of 3D in vitro heart models compared to traditional 2D cell cultures will be discussed, and the differences between scaffold-free and scaffold-based systems will also be spotlighted.
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Primary Sjögren's syndrome (pSS) is a complex autoimmune disorder that particularly affects the salivary and lachrymal glands, generally causing a typical dryness of the eyes and of the mouth. The disease encompasses diverse clinical representations and is characterized by B-cell polyclonal activation and autoantibodies production, including anti-Ro/SSA. Recently, it has been suggested that autoantibody profiling may enable researchers to identify susceptible asymptomatic individuals in a pre-disease state. In this pilot study, we used mass spectrometry to analyze and compare the salivary proteomics of patients with established pSS and patients with pre-clinical SS, identifying a common protein signature in their salivary fluid. We found that several inflammatory, immunity-related, and typical acinar proteins (such as MUC5B, PIP, CST4, and lipocalin 1) were differently expressed in pSS and in pre-clinical SSA+ carriers, compared to healthy controls. This suggests that saliva may closely reflect exocrine gland inflammation from the early phases of the disease. This study confirms the value of salivary proteomics for the identification of reliable biomarkers for SS that could be identified, even in a preclinical phase of the disease.
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Síndrome de Sjogren , Biomarcadores/metabolismo , Humanos , Projetos Piloto , Proteômica/métodos , Saliva/metabolismo , Síndrome de Sjogren/diagnósticoRESUMO
TGF-ß is a cytokine implicated in multiple cellular responses, including cell cycle regulation, fibrogenesis, angiogenesis and immune modulation. In response to pro-inflammatory and chemotactic cytokines and growth factors, cholangiocytes prime biliary damage, characteristic of cholangiopathies and pathologies that affect biliary tree. The effects and signaling related to TGF-ß in cholangiocyte remains poorly investigated. In this study, the cellular response of human cholangiocytes to TGF-ß was examined. Wound-healing assay, proliferation assay and cell cycle analyses were used to monitor the changes in cholangiocyte behavior following 24 and 48 h of TGF-ß stimulation. Moreover, proteomic approach was used to identify proteins modulated by TGF-ß treatment. Our study highlighted a reduction in cholangiocyte proliferation and a cell cycle arrest in G0/G1 phase following TGF-ß treatment. Moreover, proteomic analysis allowed the identification of four downregulated proteins (CaM kinase II subunit delta, caveolin-1, NipSnap1 and calumin) involved in Ca2+ homeostasis. Accordingly, Gene Ontology analysis highlighted that the plasma membrane and endoplasmic reticulum are the cellular compartments most affected by TGF-ß. These results suggested that the effects of TGF-ß in human cholangiocytes could be related to an imbalance of intracellular calcium homeostasis. In addition, for the first time, we correlated calumin and NipSnap1 to TGF-ß signaling.
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BACKGROUND: Atherosclerosis is a chronic inflammatory disease. The balance between pro- and anti-inflammatory factors, acting on the arterial wall, promotes less or more coronary plaque macro-calcification, respectively. We investigated the association between monocyte phenotypic polarization and CTCA-assessed plaque dense-calcium volume (DCV) in patients with stable coronary artery disease (CAD). METHODS: In 55 patients, individual DCV component was assessed by quantitative CTCA and normalized to total plaque volume. Flow cytometry expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1 and CXCR4 was quantified. Adhesion molecules and cytokines were measured by ELISA. RESULTS: DCV values were significantly associated, by multiple regression analysis, with the expression (RFI) of CCR5 (p = 0.04), CX3CR1 (p = 0.03), CCR2 (p = 0.02), CD163 (p = 0.005) on all monocytes, and with the phenotypic M2-like polarization ratio, RFI CCR5/CD11b (p = 0.01). A positive correlation with the increased expression of chemokines receptors CCR2, CCR5 and CX3CR1 on subsets Mon1 was also present. Among cytokines, the ratio between IL-10 and IL-6 was found to be strongly associated with DCV (p = 0.009). CONCLUSIONS: The association between DCV and M2-like phenotypic polarization of circulating monocytes indicates that plaque macro-calcification in stable CAD may be partly modulated by an anti-inflammatory monocyte functional state, as evidenced by cell membrane receptor patterns.
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Primary Sjögren's syndrome (pSS) is a complex heterogeneous disease characterized by a wide spectrum of glandular and extra-glandular manifestations. In this pilot study, a SWATH-MS approach was used to monitor extracellular vesicles-enriched saliva (EVs) sub-proteome in pSS patients, to compare it with whole saliva (WS) proteome, and assess differential expressed proteins between pSS and healthy control EVs samples. Comparison between EVs and WS led to the characterization of compartment-specific proteins with a moderate degree of overlap. A total of 290 proteins were identified and quantified in EVs from healthy and pSS patients. Among those, 121 proteins were found to be differentially expressed in pSS, 82% were found to be upregulated, and 18% downregulated in pSS samples. The most representative functional pathways associated to the protein networks were related to immune-innate response, including several members of S100 protein family, annexin A2, resistin, serpin peptidase inhibitors, azurocidin, and CD14 monocyte differentiation antigen. Our results highlight the usefulness of EVs for the discovery of novel salivary-omic biomarkers and open novel perspectives in pSS for the identification of proteins of clinical relevance that could be used not only for the disease diagnosis but also to improve patients' stratification and treatment-monitoring. Data are available via ProteomeXchange with identifier PXD025649.
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Vesículas Extracelulares/metabolismo , Redes Reguladoras de Genes , Proteoma/genética , Saliva/metabolismo , Síndrome de Sjogren/genética , Adulto , Idoso , Anexina A2/genética , Anexina A2/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biomarcadores/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Vesículas Extracelulares/química , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Projetos Piloto , Mapeamento de Interação de Proteínas , Proteoma/classificação , Proteoma/metabolismo , Proteômica/instrumentação , Proteômica/métodos , Resistina/genética , Resistina/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Saliva/química , Serpinas/genética , Serpinas/metabolismo , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologiaRESUMO
Cell signalling is tightly regulated by post-translational modification of proteins. Among them, phosphorylation is one of the most interesting and important. Identifying phosphorylation sites on proteins is challenging and requires strategies for pre-separation and enrichment of the phosphorylated species. We applied four different methods for phospho-enrichment involving TiO2 and IMAC matrix to human melanoma cell lysates of starved A375 induced for 1 h with 1% FBS. Comparison of protocol efficiency was evaluated through peptide concentration, sulphur and phosphorus content and peptide analysis by LC-MS in the collected fractions. Our results underlined that each single method is not sufficient for a comprehensive phosphoproteome analysis. In fact, each methodology permits to identify only a fraction of the phosphoproteome contained in a whole cell lysate. The selection of the most efficient protocols and a combination of two phospho-enrichment methods allowed the assessment of this workflow able to pinpoint the main actors in the phospho-proteome cascade of A375 human melanoma cells treated with Vemurafenib.
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Melanoma , Proteômica , Cromatografia Líquida , Humanos , Melanoma/tratamento farmacológico , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteoma/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND AND AIMS: Atherosclerosis is an inflammatory disease with long-lasting activation of innate immunity and monocytes are the main blood cellular effectors. We aimed to investigate monocyte phenotype (subset fraction and marker expression) at different stages of coronary atherosclerosis in stable coronary artery disease (CAD) patients. METHODS: 73 patients with chronic coronary syndrome were evaluated by CT coronary angiography (CTCA) and classified by maximal diameter stenosis of major vessels into three groups of CAD severity: CAD1 (no CAD/minimal CAD, n° = 30), CAD2 (non-obstructive CAD, n° = 21), and CAD3 (obstructive CAD, n° = 22). Flow cytometry for CD14, CD16, and CCR2 was used to quantify Mon1, Mon2, and Mon3 subsets. Expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, and CXCR4 was also measured. Adhesion molecules and cytokines were quantified by ELISA. RESULTS: Total cell count and fraction of Mon2 were higher in CAD2 and CAD3 compared to CAD1. By multivariate regression analysis, Mon2 cell fraction and Mon2 expression of CX3CR1, CD18, and CD16 showed a statistically significant and independent increase, parallel to stenosis severity, from CAD1 to CAD2 and CAD3 groups. A similar trend was also present for CX3CR1 and HLA-DR expressions on total monocyte population. A less calcified plaque composition was associated to a higher Mon2 expression of CD16 and higher TNF-α levels. IL-10 levels were lower at greater stenosis severity, while the IFN-γ/IL-10 ratio, a marker of a systemic pro-inflammatory imbalance, was directly correlated to stenosis degree and number of noncalcified plaques. CONCLUSIONS: The results of this study suggest that a specific pattern of inflammation-correlated monocyte marker expression is associated to higher stenosis severity and less calcified lesions in stable CAD. The clinical trial Identifier is NCT04448691.
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Antígenos CD/sangue , Angiografia Coronária , Citocinas/sangue , Citometria de Fluxo , Antígenos HLA-DR/sangue , Monócitos/metabolismo , Receptores de Quimiocinas/sangue , Idoso , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico por imagem , Estudos Transversais , Feminino , Humanos , Masculino , Índice de Gravidade de DoençaRESUMO
Primary sclerosing cholangitis is a complex pathological condition, characterized by chronic inflammation and fibrosis of the biliary epithelium. Without proper clinical management, progressive bile ducts and liver damage lead to cirrhosis and, ultimately, to liver failure. The known limited role of current drugs for treating this cholangiopathy has driven researchers to assess alternative therapeutic options. Some herbal remedies and their phytochemicals have shown anti-fibrotic properties in different experimental models of hepatic diseases and, occasionally, in clinical trials in primary sclerosing cholangitis patients; however their mechanism of action is not completely understood. This review briefly examines relevant studies focusing on the potential anti-fibrotic properties of Silybum marianum, Curcuma longa, Salvia miltiorrhiza, and quercetin. Each natural product is individually reviewed and the possible mechanisms of action discussed.
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Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.
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Antineoplásicos/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Rabdomiossarcoma/patologia , Quinases da Família src/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Camundongos , Reação em Cadeia da Polimerase , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Glioblastoma (GB) is the most common and aggressive primary tumor of the central nervous system. The current standard of care for GB consists of surgical resection, followed by radiotherapy combined with temozolomide chemotherapy. However, despite this intensive treatment, the prognosis remains extremely poor. Therefore, more effective therapies are urgently required. Recent studies indicate that SRC family kinases (SFKs) could represent promising molecular targets for GB therapy. Here, we challenged four GB cell lines with a new selective pyrazolo[3,4-d]pyrimidine derivative SFK inhibitor, called SI221. This compound exerted a significant cytotoxic effect on GB cells, without significantly affecting non-tumor cells (primary human skin fibroblasts), as evaluated by MTS assay. We also observed that SI221 was more effective than the well-known SFK inhibitor PP2 in GB cells. Notably, despite the high intrinsic resistance to apoptosis of GB cells, SI221 was able to induce this cell death process in all the GB cell lines, as observed through cytofluorimetric analysis and caspase-3 assay. SI221 also exerted a long-term inhibition of GB cell growth and was able to reduce GB cell migration, as shown by clonogenic assay and scratch test, respectively. Moreover, through in vitro pharmacokinetic assays, SI221 proved to have a high metabolic stability and a good potential to cross the blood brain barrier, which is an essential requirement for a drug intended to treat brain tumors. Therefore, despite the need of developing strategies to improve SI221 solubility, our results suggest a potential application of this selective SFK inhibitor in GB therapy.