RESUMO
BACKGROUND/AIMS: Aquaporin-3 (AQP3) is an aquaglyceroporin and peroxiporin that plays a crucial role in skin barrier homeostasis. Dysregulated AQP3 expression has been observed in different inflammatory skin conditions. Hidradenitis Suppurativa (HS) is an autoinflammatory keratinization disease that typically appears between 10 and 21 years of age, characterized by alteration of skin barrier homeostasis. METHODS: To evaluate in vitro the role of AQP3 in the development of HS, we performed real-time PCR and Western blot to analyze gene and protein levels in human keratinocyte cell lines knock-out (KO) for NCSTN and PSENEN genes, simulating genetic-associated HS. Additionally, we investigated the impact of Glyceryl Glucoside (GG) on biological processes by performing MTT, scratch, proliferation assays and proteome studies. RESULTS: We detected a significant decrease of the levels of AQP3 gene and protein in KO cell lines. GG effectively elevated the levels of mRNA and protein, significantly decreased the hyperproliferation rate, and enhanced cell migration in our in vitro model of genetic Hidradenitis Suppurativa. Pathway enrichment analysis further confirmed GG's role in the migration and proliferation pathways of keratinocytes. CONCLUSION: Our results suggest that AQP3 may act as a new novel actor in HS etio-pathogenesis, and GG could be further explored as potential treatment option for managing HS in patients.
Assuntos
Aquaporina 3 , Movimento Celular , Proliferação de Células , Glucosídeos , Hidradenite Supurativa , Queratinócitos , Humanos , Aquaporina 3/metabolismo , Aquaporina 3/genética , Hidradenite Supurativa/metabolismo , Hidradenite Supurativa/patologia , Hidradenite Supurativa/tratamento farmacológico , Hidradenite Supurativa/genética , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Queratinócitos/citologia , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Linhagem CelularRESUMO
Among gynecological cancers, endometrial cancer is the most common in developed countries. Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that contain proteins involved in immune response and apoptosis. A deep proteomic approach can help to identify dysregulated extracellular matrix (ECM) proteins in EVs correlated to key pathways for tumor development. In this study, we used a proteomics approach correlating the two acquisitions-data-dependent acquisition (DDA) and data-independent acquisition (DIA)-on EVs from the conditioned medium of four cell lines identifying 428 ECM proteins. After protein quantification and statistical analysis, we found significant changes in the abundance (p < 0.05) of 67 proteins. Our bioinformatic analysis identified 26 pathways associated with the ECM. Western blotting analysis on 13 patients with type 1 and type 2 EC and 13 endometrial samples confirmed an altered abundance of MMP2. Our proteomics analysis identified the dysregulated ECM proteins involved in cancer growth. Our data can open the path to other studies for understanding the interaction among cancer cells and the rearrangement of the ECM.
Assuntos
Neoplasias do Endométrio , Proteínas da Matriz Extracelular , Matriz Extracelular , Vesículas Extracelulares , Proteômica , Humanos , Feminino , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Proteômica/métodos , Vesículas Extracelulares/metabolismo , Matriz Extracelular/metabolismo , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/metabolismo , Pessoa de Meia-Idade , Biologia Computacional/métodos , Metaloproteinase 2 da Matriz/metabolismoRESUMO
Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.
Assuntos
Neoplasias do Endométrio , Fosfoproteínas , Humanos , Feminino , Fosfoproteínas/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Cromatografia de Afinidade/métodos , ProteomaRESUMO
Endometrial cancer (EC) is the most frequent gynecologic cancer in postmenopausal women. Pathogenetic mechanisms that are related to the onset and progression of the disease are largely still unknown. A multi-omics strategy can help identify altered pathways that could be targeted for improving therapeutical approaches. In this study we used a multi-omics approach on four EC cell lines for the identification of common dysregulated pathways in type 1 and 2 ECs. We analyzed proteomics and metabolomics of AN3CA, HEC1A, KLE and ISHIKAWA cell lines by mass spectrometry. The bioinformatic analysis identified 22 common pathways that are in common with both types of EC. In addition, we identified five proteins and 13 metabolites common to both types of EC. Western blotting analysis on 10 patients with type 1 and type 2 EC and 10 endometria samples confirmed the altered abundance of NPEPPS. Our multi-omics analysis identified dysregulated proteins and metabolites involved in EC tumor growth. Further studies are needed to understand the role of these molecules in EC. Our data can shed light on common pathways to better understand the mechanisms involved in the development and growth of EC, especially for the development of new therapies.
Assuntos
Neoplasias do Endométrio , Multiômica , Humanos , Feminino , Neoplasias do Endométrio/metabolismo , Metabolômica , Biologia ComputacionalRESUMO
Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Endométrio/metabolismo , Proteoma/metabolismo , Adulto , Antígenos de Diferenciação/metabolismo , Regulação para Baixo/fisiologia , Endométrio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Oncogenes/fisiologia , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Regulação para Cima/fisiologiaRESUMO
Recurrent acute otitis media (RAOM) in children is clinically defined as the occurrence of at least three episodes of acute otitis media over a course of 6 months. A further common pathological condition of interest in the context of pediatric otolaryngology is adenotonsillar hypertrophy (ATH), a common cause of obstructive sleep apnea syndrome. Aimed at unraveling the differential modulation of proteins in the two pathologies and at understanding the possible pathways involved in their onset, we analyzed the proteomic profile of the adenoids from 14 RAOM and ATH patients by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The 2-DE coupled with MS allowed us to identify 23 spots with significant (p-value < 0.05) changes in protein amount, recognizing proteins involved in neutrophil degranulation and glycolysis pathways.
Assuntos
Otite Média/etiologia , Otite Média/metabolismo , Proteoma , Proteômica , Suscetibilidade a Doenças , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Glicólise , Humanos , Espectrometria de Massas , Redes e Vias Metabólicas , Otite Média/patologia , Proteômica/métodos , Recidiva , Transdução de SinaisRESUMO
Gynecologic cancers are an important cause of worldwide mortality. The interstitium consists of solid and fluid phases, situated between the blood vessels and cells. The interstitial fluid (IF), or fluid phase, is an extracellular fluid bathing and surrounding the tissue cells. The TIF (tumor interstitial fluid) is a dynamic fluid rich in lipids, proteins and enzyme-derived substances. The molecules found in the IF may be associated with pathological changes in tissues leading to cancer growth and metastatization. Proteomic techniques have allowed an extensive study of the composition of the TIF as a source of biomarkers for gynecologic cancers. In our review, we analyze the composition of the TIF, its formation process, the sampling methods, the consequences of its accumulation and the proteomic analyses performed, that make TIF valuable for monitoring different types of cancers.
Assuntos
Líquido Extracelular/metabolismo , Neoplasias dos Genitais Femininos/metabolismo , Biomarcadores Tumorais/metabolismo , Fenômenos Biofísicos , Feminino , Neoplasias dos Genitais Femininos/patologia , Humanos , Padrões de Prática Médica , Microambiente TumoralRESUMO
Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p < 0.05) protein spots, 12 of which were found to be associated with the metabolic processes of the leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes.
Assuntos
Leiomioma/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Regulação para Cima , Neoplasias Uterinas/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Espectrometria de Massas/métodos , Miométrio/metabolismoRESUMO
A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia.
Assuntos
Astenozoospermia/metabolismo , Proteoma , Sêmen/metabolismo , Estudos de Casos e Controles , Eletroforese em Gel Bidimensional , Humanos , Masculino , Espectrometria de MassasRESUMO
Subunit T of the native muscle troponin complex is a recognised substrate of transglutaminase both in vitro and in situ with formation of isopeptide bonds. Using a proteomic approach, we have now determined the precise site of in vitro labelling of the protein. A preparation of troponin purified from ether powder from mixed rabbit skeletal muscles was employed as transglutaminase substrate. The only isoform TnT2F present in our preparation was recognised as acyl-substrate by human type 2 transglutaminase which specifically modified glutamine 13 in the N-terminal region. During the reaction, the troponin protein complex was polymerized. Results are discussed in relation to the structure of the troponin T subunit, in the light of the role of troponins in skeletal and cardiac muscle diseases, and to the rules governing glutamine side chain selection by tissue transglutaminase.
Assuntos
Glutamina/química , Transglutaminases/química , Troponina T/química , Sequência de Aminoácidos , Animais , Cálcio/química , Sequência Conservada , Eritrócitos/enzimologia , Proteínas de Ligação ao GTP , Humanos , Dados de Sequência Molecular , Músculo Esquelético , Proteína 2 Glutamina gama-Glutamiltransferase , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Coelhos , Análise de Sequência de Proteína , Espermina/químicaRESUMO
Endometrial cancer (EC) is the most common gynecologic malignancy, and it arises in the inner part of the uterus. Identification of serum biomarkers is essential for diagnosing the disease at an early stage. In this study, we selected 44 healthy controls and 44 type I EC at tumor stage 1, and we used the Immuno-oncology panel and the Target 96 Oncology III panel to simultaneously detect the levels of 92 cancer-related proteins in serum, using a proximity extension assay. By applying this methodology, we identified 20 proteins, associated with the outcome at binary logistic regression, with a p-value below 0.01 for the first panel and 24 proteins with a p-value below 0.02 for the second one. The final multivariate logistic regression model, combining proteins from the two panels, generated a model with a sensitivity of 97.67% and a specificity of 83.72%. These results support the use of the proposed algorithm after a validation phase.
RESUMO
Endometrial cancers (ECs) are mostly adenocarcinomas arising from the inner part of the uterus. The identification of serum biomarkers, either soluble or carried in the exosome, may be useful in making an early diagnosis. We used label-free quantification mass spectrometry (LFQ-MS)-based proteomics to investigate the proteome of exosomes in the albumin-depleted serum from 12 patients with EC, as compared to 12 healthy controls. After quantification and statistical analysis, we found significant changes in the abundance (p < 0.05) of 33 proteins in EC vs. control samples, with a fold change of ≥1.5 or ≤0.6. Validation using Western blotting analysis in 36 patients with EC as compared to 36 healthy individuals confirmed the upregulation of APOA1, HBB, CA1, HBD, LPA, SAA4, PF4V1, and APOE. A multivariate logistic regression model based on the abundance of these proteins was able to separate the controls from the EC patients with excellent sensitivity levels, particularly for stage 1 ECs. The results show that using LFQ-MS to explore the specific proteome of serum exosomes allows for the identification of biomarkers in EC. These observations suggest that PF4V1, CA1, HBD, and APOE represent biomarkers that are able to reach the clinical stage, after a validation phase.
RESUMO
Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.
Assuntos
Anti-Infecciosos , Dermocidinas , Hidradenite Supurativa , Criança , Humanos , Anti-Infecciosos/metabolismo , Hidradenite Supurativa/genética , Hidradenite Supurativa/metabolismo , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Pele/metabolismo , Masculino , FemininoRESUMO
The exact role of malignant ascites in the development of intraperitoneal metastases remains unclear, and the mechanisms by which extracellular vesicles (EVs) promote tumor progression in the pre-metastatic niche have not been fully discovered. In this study, we characterized ascites from high-grade epithelial ovarian cancer patients. Small-EVs (30-150 nm) were isolated from two sources-the bulk ascites and the ascitic fluid-derived tumor cell cultures-and assessed with a combination of imaging, proteomic profiling, and protein expression analyses. In addition, Gene Ontology and pathway analysis were performed using different databases and bioinformatic tools. The results proved that the small-EVs derived from the two sources exhibited significantly different stiffness and size distributions. The bulk ascitic fluid-derived small-EVs were predominantly involved in the complement and coagulation cascade. Small-EVs derived from ascites cell cultures contained a robust proteomic profile of extracellular matrix remodeling regulators, and we observed an increase in transforming growth factor-ß-I (TGFßI), plasminogen activator inhibitor 1 (PAI-1), and fibronectin expression after neoadjuvant chemotherapy. When measured in the two sources, we demonstrated that fibronectin exhibited opposite expression patterns in small-EVs in response to chemotherapy. These findings highlight the importance of an ascites cell isolation workflow in investigating the treatment-induced cancer adaption processes.
Assuntos
Vesículas Extracelulares , Neoplasias Ovarianas , Ascite/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Neoplasias Ovarianas/genética , ProteômicaRESUMO
Endometrial cancer is the most common gynecologic malignancy arising from the endometrium. Identification of serum biomarkers could be beneficial for its early diagnosis. We have used 2D-Difference In Gel Electrophoresis (2D-DIGE) coupled with Mass Spectrometry (MS) procedures to investigate the serum proteome of 15 patients with endometrial cancer and 15 non-cancer subjects. We have identified 16 proteins with diagnostic potential, considering only spots with a fold change in %V ≥ 1.5 or ≤0.6 in intensity, which were statistically significant (p < 0.05). Western blotting data analysis confirmed the upregulation of CLU, ITIH4, SERPINC1, and C1RL in endometrial and exosome cancer sera compared to those of control subjects. The application of the logistic regression constructed based on the abundance of these four proteins separated the controls from the cancers with excellent levels of sensitivity and specificity. After a validation phase, our findings support the potential of using the proposed algorithm as a diagnostic tool in the clinical stage.
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Uterine leiomyoma presents the highest incidence among benign tumors of the female reproductive tract. The present study compared the proteome of leiomyoma treated with ulipristal acetate with that of untreated leiomyoma to investigate protein expression patterns in relation to oxidative stress. Paired tissue samples from seven treated and untreated leiomyomas were collected and the proteome was analyzed by twodimensional gel electrophoresis (2DE). Western blotting was used to validate the results of 2DE, and mass spectrometry was used to identify proteins. The tissue expression of 30 proteins was markedly affected by treatment with ulipristal acetate. Bioinformatics analysis revealed that several of the differentially expressed proteins were involved in the degradation of hydrogen peroxide and the synthesis of reactive oxygen species. The present study suggested the involvement of oxidative stress as a novel mechanism of action of ulipristal acetate. These findings require further investigations to understand the role of ulipristal acetate in the treatment of the leiomyoma.
Assuntos
Redes Reguladoras de Genes/efeitos dos fármacos , Leiomioma/tratamento farmacológico , Norpregnadienos/administração & dosagem , Proteômica/métodos , Neoplasias Uterinas/tratamento farmacológico , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Leiomioma/metabolismo , Espectrometria de Massas , Norpregnadienos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Mapas de Interação de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Uterinas/metabolismoRESUMO
Uterine leiomyomas are benign smooth muscle cell tumors originating from the myometrium. The present study focused on leiomyoma and myometrium phosphoproteome enrichment by using immobilized metal affinity chromatography (IMAC). The phosphoproteome was analyzed by twodimensional gel electrophoresis coupled with mass spectrometry. Western blotting was used for data validation. The results from IMAC identified 26 proteins significantly differentially phosphorylated in leiomyomas compared with normal myometrium. Three upregulated proteins (peroxiredoxin 2, protein disulfide isomerase family A member 3 and peroxiredoxin 4) were further validated by western blotting. Ingenuity pathway analysis revealed that four phosphoproteins were involved in the inhibition of oxidative stress and synthesis of reactive oxygen species. The present results demonstated for the first time an association between oxidative stress and phosphorylation in leiomyoma development.
Assuntos
Leiomioma/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Neoplasias Uterinas/genética , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leiomioma/patologia , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miométrio/metabolismo , Miométrio/patologia , Neoplasias/genética , Neoplasias/patologia , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio , Neoplasias Uterinas/patologiaRESUMO
Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the Nglycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose6P to mannose1P, which results in low concentration levels of guanosine 5'diphosphoDmannose (GDPMan), a nucleotideactivated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDPManloaded poly (D,Llactidecoglycolide) (PLGA) nanoparticles (NPs), which were used to treat CDGIa fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of αmannosidase, ßglucoronidase and ßgalactosidase in defective and normal fibroblasts. GDPMan (30 µg/ml GDPMan PLGA NPs) was incubated for 48 h with the cells and the specific activities of αmannosidase and ßgalactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of ßglucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDGIa fibroblasts. The glycosylation process of fibroblasts was also analysed by twodimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDPMan PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.
Assuntos
Defeitos Congênitos da Glicosilação/tratamento farmacológico , Portadores de Fármacos , Guanosina Difosfato Manose , Nanopartículas , Fosfotransferases (Fosfomutases)/deficiência , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Células Cultivadas , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Fibroblastos , Glicosilação/efeitos dos fármacos , Guanosina Difosfato Manose/química , Guanosina Difosfato Manose/farmacologia , Humanos , Nanopartículas/química , Nanopartículas/uso terapêutico , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologiaRESUMO
Uterine leiomyomas are benign smooth muscle cell tumors originating from the myometrium. In this study we focus on leiomyoma and normal myometrium phosphoproteome, to identify differentially phosphorylated proteins involved in tumorigenic signaling pathways, and in anti-apoptotic processes and cell survival. We obtained paired tissue samples of seven leiomyomas and adjacent myometria and analyzed the phosphoproteome by two-dimensional gel electrophoresis (2-DE) combined with immobilized metal affinity chromatography (IMAC) and Pro-Q Diamond phosphoprotein gel stain. We used mass spectrometry for protein identification and Western blotting for 2-DE data validation. Quantities of 33 proteins enriched by the IMAC approach were significantly different in the leiomyoma if compared to the myometrium. Bioinformatic analysis revealed ten tumorigenic signaling pathways and four phosphoproteins involved in both the inhibition of apoptosis and cell survival. Our study highlights the involvement of the phosphoproteome in leiomyoma growth. Further studies are needed to understand the role of phosphorylation in leiomyoma. Our data shed light on mechanisms that still need to be ascertained, but could open the path to a new class of drugs that not only can block the growth, but could also lead to a significant reduction in tumor size.
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Uterine leiomyomas are benign smooth muscle cell tumors that originate from the myometrium. In this study we focus on dysregulated chaperones associated with cell proliferation and apoptosis. Paired tissue samples of 15 leiomyomas and adjacent myometria were obtained and analyzed by two-dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification and western blotting for 2-DE data validation. The values of 6 chaperones were found to be significantly different in the leiomyoma when compared with the myometrium. A total of 4 proteins were upregulated in the leiomyoma and 2 proteins were downregulated. Calreticulin and 78 kDa glucose-regulated protein were further validated by western blotting because the first is considered a marker of cell proliferation, while the second protects against apoptotic cell death. In addition, we also validated the two downregulated proteins heat shock protein ß-1 and heat shock 70 kDa protein 1A. Our study shows the existence of a dysregulation of chaperone proteins associated with leiomyoma development. Functional studies are needed to ascertain the role of these chaperones in the leiomyoma. This may be crucial for the further development of specific inhibitors against the activity of these proteins in order to block the growth of the leiomyoma.