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Broadband Coherent Anti-Stokes Raman Scattering (BCARS) is a valuable spectroscopic imaging tool for visualizing cellular structures and lipid distributions in biomedical applications. However, the inevitable biological changes in the samples (cells/tissues/lipids) introduce spectral variations in BCARS data and make analysis challenging. In this work, we conducted a systematic study to estimate the biological variance in BCARS data of two commonly used cell lines (HEK293 and HepG2) in biomedical research. The BCARS data were acquired from two different experimental setups (Leibniz Institute of Photonics Technology (IPHT) in Jena and Politecnico di Milano (POLIMI) in Milano) to evaluate the reproducibility of results. Also, spontaneous Raman data were independently acquired at POLIMI to validate those results. First, Kramers-Kronig (KK) algorithm was utilized to retrieve Raman-like signals from the BCARS data, and a pre-processing pipeline was subsequently used to standardize the data. Principal component analysis - Linear discriminant analysis (PCA-LDA) was performed using two cross-validation (CV) methods: batch-out CV and 10-fold CV. Additionally, the analysis was repeated, considering different spectral regions of the data as input to the PCA-LDA. Finally, the classification accuracies of the two BCARS datasets were compared with the results of spontaneous Raman data. The results demonstrated that the CH band region (2770-3070 cm-1) and spectral data in the 1500-1800 cm-1 region have significantly contributed to the classification. A maximum of 100% balanced accuracies were obtained for the 10-fold CV for both BCARS setups. However, in the case of batch-out CV, it is 92.4% for the IPHT dataset and 98.8% for the POLIMI dataset. This study offers a comprehensive overview for estimating biological variance in biomedical applications. The insights gained from this analysis hold promise for improving the reliability of BCARS measurements in biomedical applications, paving the way for more accurate and meaningful spectroscopic analyses in the study of biological systems.
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The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
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Mitocôndrias/química , Proteoma/fisiologia , Proteômica/normas , Linhagem Celular , Cromatografia Líquida , Humanos , Itália , Proteínas Mitocondriais/análise , Mapas de Interação de Proteínas/fisiologia , Espectrometria de Massas em TandemRESUMO
We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.
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Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Ferro/metabolismo , Proteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas , Neoplasias da Mama/genética , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Eritropoetina/sangue , Feminino , Ferritinas/metabolismo , Hepcidinas/metabolismo , Humanos , Interleucina-6/sangue , Pessoa de Meia-Idade , Análise Serial de Proteínas , Proteínas/genética , Receptores da Transferrina/metabolismo , Adulto JovemRESUMO
Sequencing data show that both specific genes and a number of signaling pathways are recurrently mutated in various types of lymphoma. DNA sequencing analyses of lymphoma have identified several aberrations that might affect the interaction between malignant cells and the tumor microenvironment. Microenvironmental functions are essential to lymphoma; they provide survival and proliferation signals and license immune evasion. It is plausible that interventions that aim to destroy tumor-microenvironment interactions may improve responses to therapeutics. Accordingly, the identification of extrinsic factors and their downstream intracellular signaling targets has led to much progress in understanding tumor-microenvironment interactions. Lymphoma cells are differently influenced by cells' interactions with components of their microenvironment; these cell extrinsic factors include soluble and immobilized factors, the extracellular matrix, and signals presented by neighboring cells. Soluble factors, which are often cell-secreted autocrine and paracrine factors, comprise a significant fraction of targetable molecules. To begin to understand how intercellular communication is conducted in lymphoma, a first order of study is deciphering the soluble factors secreted by malignant cells and microenvironmental cells. These soluble factors are shed into the interstitial fluid in lymphoma and can be conveniently explored using mass spectrometry. Protein components can be detected and quantified, thus enabling the routine navigation of the soluble part of the microenvironment. Elucidating functional and signaling states affords a new paradigm for understanding cancer biology and devising new therapies. This review summarizes knowledge in this field and discusses the utility of studying tumor-secreted factors.
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Exossomos/metabolismo , Linfoma/etiologia , Linfoma/metabolismo , Animais , Transporte Biológico , Comunicação Celular , Líquido Extracelular/metabolismo , Humanos , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Linfoma/diagnóstico , Linfoma/patologia , Proteoma , Proteômica/métodos , Transdução de Sinais , Microambiente TumoralRESUMO
OBJECTIVE: Hepcidin-25 production is stimulated by systemic inflammation, and it interferes with iron utilization, leading to anemia. This study aimed to investigate the relationships between the plasma levels of hepcidin, interleukin-6 (IL-6), erythropoietin (EPO) and erythroferrone (ERFE) in patients with benign breast disease or cancer. METHODS: Plasma samples from a cohort of 131 patients (47 with benign breast disease and 84 with breast cancer) were subjected to the evaluation of hepcidin, IL-6, EPO and ERFE using SELDI-TOF-MS or immunoassays. RESULTS: An elevated hepcidin was observed in malignant breast tumors compared to benign ones. No correlation was observed between hepcidin and IL-6, EPO or ERFE. CONCLUSION: Since the study included a cohort of patients (87%) with breast cancers smaller than 2 cm, these results may support our previous evidence about the potential role of hepcidin in breast cancer disease.
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Neoplasias da Mama/sangue , Eritropoetina/sangue , Hepcidinas/sangue , Interleucina-6/sangue , Hormônios Peptídicos/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Primary effusion lymphoma (PEL) is a rare B-cell neoplasm in which tumor cells are consistently infected by Kaposi's sarcoma-associated herpesvirus and usually grow in body cavities without tumor mass formation. To detect new proteins related to pathogenesis, four established cell lines from PEL (CRO-AP2, CRO-AP3, CRO-AP5, and CRO-AP6) were characterized by proteomics analysis of the secretome. The secretomes were analyzed using two complementary mass spectrometry platforms: liquid chromatography-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight-based approaches. Among 266 proteins identified from the proteomics analysis, 139 were considered as predicted secreted. Twenty proteins were specifically secreted by PEL cell lines after comparison with secretomes of human cell lines representative of diverse solid tumors and leukemias. More important, 27 additional proteins were shared by all CRO-AP PEL cell lines. The presence of these proteins was confirmed by IHC in CRO-AP cell lines and in six other PEL cell lines, four PEL clinical samples, and three extracavitary Kaposi's sarcoma-associated herpesvirus-positive solid lymphomas included for comparative analysis. Functional classification showed that PEL cell secretomes were enriched in proteins specifically involved in inflammation/immune response, growth/cell cycle, and mRNA processing, in addition to structural/matrix proteins and proteins with enzymatic activity.
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Biomarcadores Tumorais/isolamento & purificação , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/metabolismo , Sarcoma de Kaposi/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Biologia Computacional , Humanos , Linfoma de Efusão Primária/patologia , Proteoma/química , Sarcoma de Kaposi/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies.
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Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Receptores ErbB/sangue , Neoplasias Pulmonares/diagnóstico , Idoso , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Detecção Precoce de Câncer , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Over recent years, analyses of secretomes (complete sets of secreted proteins) have been reported in various organisms, cell types, and pathologies and such studies are quickly gaining popularity. Fungi secrete enzymes can break down potential food sources; plant secreted proteins are primarily parts of the cell wall proteome; and human secreted proteins are involved in cellular immunity and communication, and provide useful information for the discovery of novel biomarkers, such as for cancer diagnosis. Continuous development of methodologies supports the wide identification and quantification of secreted proteins in a given cellular state. The role of secreted factors is also investigated in the context of the regulation of major signaling events, and connectivity maps are built to describe the differential expression and dynamic changes of secretomes. Bioinformatics has become the bridge between secretome data and computational tasks for managing, mining, and retrieving information. Predictions can be made based on this information, contributing to the elucidation of a given organism's physiological state and the determination of the specific malfunction in disease states. Here we provide an overview of the available bioinformatics databases and software that are used to analyze the biological meaning of secretome data, including descriptions of the main functions and limitations of these tools. The important challenges of data analysis are mainly related to the integration of biological information from dissimilar sources. Improvements in databases and developments in software will likely substantially contribute to the usefulness and reliability of secretome studies. This article is part of a Special Issue entitled: An Updated Secretome.
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Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Biologia de Sistemas/métodos , Animais , Bases de Dados de Proteínas , Humanos , SoftwareRESUMO
Investigation of cell signaling pathways in 16 clear cell renal cell carcinomas to identify groups based on commonly shared phosphorylation-driven signaling networks. Using laser capture microdissection and reverse-phase protein arrays, we profiled 75 key nodes spanning signaling pathways important in tumorigenesis. Analysis revealed significantly different (P < 0.05) signaling levels for 27 nodes between two groups of samples, designated A (4 samples; high EGFR, RET, and RASGFR1 levels, converging to activate AKT/mTOR) and B (12 samples; high ERK1/2 and STAT phosphorylation). Group B was further partitioned into groups C (7 samples; elevated expression of LC3B) and D (5 samples; activation of Src and STAT). Network analysis indicated that group A was characterized by signaling pathways related to cell cycle and proliferation, and group B by pathways related to cell death and survival. Homogeneous clear cell renal cell carcinomas could be stratified into at least two major functional groups.
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Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Transdução de Sinais , Adulto , Idoso , Carcinoma de Células Renais/patologia , Feminino , Dosagem de Genes , Humanos , Neoplasias Renais/patologia , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Fosforilação , Análise Serial de Proteínas , Adulto JovemRESUMO
We reported that the clinical efficacy of dendritic cell-based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B(+) killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.
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Anticorpos/uso terapêutico , Proteínas de Choque Térmico HSP110/imunologia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Animais , Reações Antígeno-Anticorpo , Linhagem Celular Tumoral , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP110/genética , Humanos , Imuno-Histoquímica , Linfoma não Hodgkin/genética , Camundongos , Camundongos SCID , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis.
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Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.
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Neoplasias , Vacúolos , Humanos , Vacúolos/fisiologia , Citoplasma , Doxorrubicina , Microscopia Confocal , TomografiaRESUMO
Iron chelators, such as deferoxamine, exert an anticancer effect by altering the activity of biomolecules critical for regulation of the cell cycle, cell metabolism, and apoptotic processes. Thus, iron chelators are sometimes used in combination with radio- and/or chemotherapy in the treatment of cancer. The possibility that deferoxamine could induce a program of senescence similar to radio- and/or chemotherapy, fostering adaptation in the treatment of cancer cells, is not fully understood. Using established biochemical techniques, biomarkers linked to lipid composition, and coherent anti-Stokes Raman scattering microscopy, we demonstrated that hepatocellular carcinoma-derived HepG2 cells survive after deferoxamine treatment, acquiring phenotypic traits and representative hallmarks of senescent cells. The results support the view that deferoxamine acts in HepG2 cells to produce oxidative stress-induced senescence by triggering sequential mitochondrial and lysosomal dysfunction accompanied by autophagy blockade. We also focused on the lipidome of senescent cells after deferoxamine treatment. Using mass spectrometry, we found that the deferoxamine-induced senescent cells presented marked remodeling of the phosphoinositol, sulfatide, and cardiolipin profiles, which all play a central role in cell signaling cascades, intracellular membrane trafficking, and mitochondria functions. Detection of alterations in glycosphingolipid sulfate species suggested modifications in ceramide generation, and turnover is frequently described in cancer cell survival and resistance to chemotherapy. Blockade of ceramide generation may explain autophagic default, resistance to apoptosis, and the onset of senescence.
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Desferroxamina , Sulfoglicoesfingolipídeos , Humanos , Desferroxamina/farmacologia , Desferroxamina/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Sulfoglicoesfingolipídeos/farmacologia , Células Hep G2 , Quelantes de Ferro/farmacologia , Quelantes de Ferro/metabolismo , Mitocôndrias/metabolismo , Senescência CelularRESUMO
Anticancer therapy screening in vitro identifies additional treatments and improves clinical outcomes. Systematically, although most tested cells respond to cues with apoptosis, an appreciable portion enters a senescent state, a critical condition potentially driving tumor resistance and relapse. Conventional screening protocols would strongly benefit from prompt identification and monitoring of therapy-induced senescent (TIS) cells in their native form. We combined complementary all-optical, label-free, and quantitative microscopy techniques, based on coherent Raman scattering, multiphoton absorption, and interferometry, to explore the early onset and progression of this phenotype, which has been understudied in unperturbed conditions. We identified TIS manifestations as early as 24 hours following treatment, consisting of substantial mitochondrial rearrangement and increase of volume and dry mass, followed by accumulation of lipid vesicles starting at 72 hours. This work holds the potential to affect anticancer treatment research, by offering a label-free, rapid, and accurate method to identify initial TIS in tumor cells.
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Neoplasias , Humanos , Prevenção Secundária , Apoptose , Sinais (Psicologia) , Imagem MolecularRESUMO
Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and tissues. In narrowband CARS, two spatiotemporally superimposed picosecond pulses, pump and Stokes, illuminate the sample to interrogate a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite recent technological advancements, BCARS microscopes still struggle to image biological samples over the entire Raman-active region (400-3100 cm-1). Here, we demonstrate a robust BCARS platform that answers this need. Our system is based on a femtosecond ytterbium laser at a 1035 nm wavelength and a 2 MHz repetition rate, which delivers high-energy pulses used to produce broadband Stokes pulses by white-light continuum generation in a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20 fs duration, with narrowband pump pulses, we generate a CARS signal with a high (<9 cm-1) spectral resolution in the whole Raman-active window, exploiting both the two-color and three-color excitation mechanisms. Aided by an innovative post-processing pipeline, our microscope allows us to perform high-speed (≈1 ms pixel dwell time) imaging over a large field of view, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, paving the way for applications in histopathological settings.
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Luz , Microscopia , Animais , Camundongos , Análise Espectral Raman/métodos , Microscopia Óptica não Linear , LasersRESUMO
In principle, targeted therapies have optimal activity against a specific subset of tumors that depend upon the targeted molecule or pathway for growth, survival, or metastasis. Consequently, it is important in drug development and clinical practice to have predictive biomarkers that can reliably identify patients who will benefit from a given therapy. We analyzed tumor cell-line secretomes (conditioned cell media) to look for predictive biomarkers; secretomes represent a potential source for potential biomarkers that are expressed in intracellular signaling and therefore may reflect changes induced by targeted therapy. Using Gene Ontology, we classified by function the secretome proteins of 12 tumor cell lines of different histotypes. Representations and hierarchical relationships among the functional groups differed among the cell lines. Using bioinformatics tools, we identified proteins involved in intracellular signaling pathways. For example, we found that secretome proteins related to TGF-beta signaling in thyroid cancer cells, such as vasorin, CD109, and ßIG-H3 (TGFBI), were sensitive to RPI-1 and dasatinib treatments, which have been previously demonstrated to be effective in blocking cell proliferation. The secretome may be a valuable source of potential biomarkers for detecting cancer and measuring the effectiveness of cancer therapies.
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Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Neoplasias/química , Neoplasias/metabolismo , Proteoma/análise , Proteoma/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Análise por Conglomerados , Biologia Computacional , Dasatinibe , Bases de Dados de Proteínas , Humanos , Espaço Intracelular , Modelos Biológicos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/química , Neoplasias/tratamento farmacológico , Fosfotirosina/análise , Fosfotirosina/química , Pirimidinas/farmacologia , Transdução de Sinais , Tiazóis/farmacologia , Fator de Crescimento Transformador betaRESUMO
BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors to develop in the digestive tract. These tumors are highly resistant to conventional chemotherapy and only the introduction of imatinib mesylate has improved the prognosis of patients. However, Response Evaluation Criteria in Solid Tumors are inappropriate for assessing tumor response, and the histological/pathological response to imatinib is variable, heterogeneous, and does not associate with clinical response. The effects of imatinib on responding GISTs are still being explored, and few studies correlate the clinical response with the histological response after pharmacological treatment. Recently, apoptosis and autophagy were suggested as possible alternative mechanisms of pharmacological response. METHODS: Here, we used a proteomic approach, combined with other analyses, to identify some molecular stromal components related to the response/behavior of resected, high-risk GISTs after neoadiuvant imatinib therapy. RESULTS: Our proteomic results indicate an elevated concentration of Stem Cell Growth Factor (SCGF), a hematopoietic growth factor having a role in the development of erythroid and myeloid progenitors, in imatinib-responsive tumor areas. SCGFα expression was detected by mass spectrometry, immunohistochemistry and/or western blot and attributed to acellular matrix of areas scored negative for KIT (CD117). RT-PCR results indicated that GIST samples did not express SCGF transcripts. The recently reported demonstration by Gundacker et al. 1 of the secretion of SCGF in mature pro-inflammatory dendritic cells would indicate a potential importance of SCGF in tissue inflammatory response. Accordingly, inflammatory infiltrates were detected in imatinib-affected areas and the CD68-positivity of the SCGF-positive and KIT-negative areas suggested previous infiltration of monocytes/macrophages into these regions. Thus, chronic inflammation subsequent to imatinib treatment may determine monocyte/macrophage recruitment in imatinib-damaged areas; these areas also feature prominent tumor-cell loss that is replaced by dense hyalinization and fibrosis. CONCLUSIONS: Our studies highlight a possible role of SCGFα in imatinib-induced changes of GIST structure, consistent with a therapeutic response.
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Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/metabolismo , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Piperazinas/uso terapêutico , Proteômica/métodos , Pirimidinas/uso terapêutico , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Regulação Neoplásica da Expressão Gênica , Glicosilação , Fatores de Crescimento de Células Hematopoéticas/genética , Humanos , Mesilato de Imatinib , Imuno-Histoquímica , Lectinas Tipo C/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
BACKGROUND: TPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1. RESULTS: Dasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130CAS, Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin ß1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed. CONCLUSIONS: All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.
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Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Indóis/antagonistas & inibidores , Indóis/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Linhagem Celular Tumoral , Dasatinibe , Humanos , Integrina beta1/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor EphA2/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Myotonic Dystrophy type 2 (DM2) is caused by a DNA microsatellite expansion within the Zinc Finger Protein 9 gene leading to an abnormal splicing pattern largely responsible for the pathological condition. To better define the functional changes occurring in human DM2 myotubes we performed a quantitative proteome comparison between myotubes of DM2 and control patients using two-dimensional gel electrophoresis followed by mass spectrometry. Our results indicate that the proteins, altered in DM2 cultures, belong to two major functional categories: i) mitochondrial components, with a reduction of EFTu, HSP60, GRP75 and Dienoyl-CoA-Isomerase, an enzyme involved in fatty acids degradation; ii) the ubiquitin proteasome system with increase of the 26S proteasome regulatory subunit 13 and a reduction of Proteasome subunit Alfa6 and of Rad23B homolog. Altered ubiquitin-proteasomal activity is supported by a global reduction of cytosolic ubiquitinated proteins. Although future work is required to clarify how these changes affect the degradation machinery and mitochondrial function and to evaluate if these changes also occur in the biopsies of DM2 patients, these results identify the mitochondrial proteins and the ubiquitin-proteasomal system as candidates potentially relevant to DM2 pathogenesis.
Assuntos
Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Miotônica/metabolismo , Proteoma/análise , Western Blotting , Fracionamento Celular , Eletroforese em Gel Bidimensional , Humanos , Hibridização In Situ , Espectrometria de Massas , Mitocôndrias/genética , Distrofia Miotônica/genéticaRESUMO
The coatomer protein complex zeta 1 (COPZ1) represents a non-oncogene addiction for thyroid cancer (TC); its depletion impairs the viability of thyroid tumor cells, leads to abortive autophagy, ER stress, UPR and apoptosis, and reduces tumor growth of TC xenograft models. In this study we investigated the molecular pathways activated by COPZ1 depletion and the paracrine effects on cellular microenvironment and immune response. By comprehensive and target approaches we demonstrated that COPZ1 depletion in TPC-1 and 8505C thyroid tumor cell lines activates type I IFN pathway and viral mimicry responses. The secretome from COPZ1-depleted cells was enriched for several inflammatory molecules and damage-associated molecular patterns (DAMPs). Moreover, we found that dendritic cells, exposed to these secretomes, expressed high levels of differentiation and maturation markers, and stimulated the proliferation of naïve T cells. Interestingly, T cells stimulated with COPZ1-depleted cells showed increased cytotoxic activity against parental tumor cells. Collectively, our findings support the notion that targeting COPZ1 may represent a promising therapeutic approach for TC, considering its specificity for cancer cells, the lack of effect on normal cells, and the capacity to prompt an anti-tumor immune response.