RESUMO
Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.
Assuntos
Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Papillomavirus Humano , Infecções por Papillomavirus/complicações , Papillomaviridae/genética , TecnologiaRESUMO
Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture-based LC-MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2-PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.
Assuntos
Mucoproteínas , Neoplasias , Proteínas Oncogênicas , Isomerases de Dissulfetos de Proteínas , Cromatografia Líquida , Humanos , Simulação de Acoplamento Molecular , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismo , Mapas de Interação de Proteínas , Espectrometria de Massas em TandemRESUMO
Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to tumorigenesis. Screening for the most common DNA point mutations, especially in such genes as TP53, BRCA1 and BRCA2, EGFR, KRAS, or BRAF, is crucial to determine predisposition risk for cancer or to predict response to therapy. In this review, we briefly depict how these genes are involved in cancer, followed by a description of the most common techniques routinely applied for their analysis, including high-throughput next-generation sequencing technology and less expensive low-throughput options, such as real-time PCR, restriction fragment length polymorphism, or high resolution melting analysis. We then introduce benefits of electrochemical biosensors as interesting alternatives to the standard methods in terms of cost, speed, and simplicity. We describe most common strategies involved in electrochemical biosensing of point mutations, relying mostly on PCR or isothermal amplification techniques, and critically discuss major challenges and obstacles that, until now, prevented their more widespread application in clinical settings.
Assuntos
Técnicas Biossensoriais , Neoplasias , Humanos , Mutação Puntual , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Predisposição Genética para DoençaRESUMO
Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.
Assuntos
Lipidômica , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Lipidômica/métodos , Análise de Injeção de Fluxo , Plasma/química , Lipídeos/análiseRESUMO
The TGF-ß signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-ß signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-ß effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-ß treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-ß treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-ß have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-ß treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-ß have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.
Assuntos
Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Ácido Araquidônico , Linhagem Celular Tumoral , Movimento Celular/genética , Ciclo-Oxigenase 2/genética , Transição Epitelial-Mesenquimal/genética , Prostaglandinas E , Fator de Crescimento Transformador beta/genética , Vinculina/genéticaRESUMO
Fully acetylated deoxyfluorinated hexosamine analogues and non-fluorinated 3,4,6-tri-O-acylated N-acetyl-hexosamine hemiacetals have previously been shown to display moderate anti-proliferative activity. We prepared a set of deoxyfluorinated GlcNAc and GalNAc hemiacetals that comprised both features: O-acylation at the non-anomeric positions with an acetyl, propionyl and butanoyl group, and deoxyfluorination at selected positions. Determination of the in vitro cytotoxicity towards the MDA-MB-231 breast cancer and HEK-293 cell lines showed that deoxyfluorination enhanced cytotoxicity in most analogues. Increasing the ester alkyl chain length had a variable effect on the cytotoxicity of fluoro analogues, which contrasted with non-fluorinated hemiacetals where butanoyl derivatives had always higher cytotoxicity than acetates. Reaction with 2-phenylethanethiol indicated that the recently described S-glyco-modification is an unlikely cause of cytotoxicity.
Assuntos
GalactosaminaRESUMO
DNA methylation, i.e., addition of methyl group to 5'-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.
Assuntos
Técnicas Biossensoriais/métodos , 5-Metilcitosina/metabolismo , Animais , Metilação de DNA/genética , Metilação de DNA/fisiologia , Epigênese Genética/genética , HumanosRESUMO
Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.
Assuntos
Anticorpos Monoclonais/farmacologia , Cádmio/química , Európio/química , Ouro/química , Prata/química , Anticorpos Monoclonais/química , Humanos , Immunoblotting , Lasers , Limite de Detecção , Espectrometria de Massas , Nanopartículas Metálicas/química , Pontos Quânticos/química , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.
Assuntos
Neoplasias da Mama , Neoplasias do Endométrio , Cromatografia Líquida , Neoplasias do Endométrio/tratamento farmacológico , Feminino , Humanos , Estatmina/genética , Tamoxifeno/efeitos adversos , Espectrometria de Massas em TandemRESUMO
One recently discussed general mechanism affecting gene expression is 3'-untranslated region (3'UTR) length. Events such as shortening, translocation or loss of 3'UTRs are observed within oncogenes and are proposed to associate with increased expression. Thus, increased efforts are being made to understand constitutive and differential transcript 3'end formation. Investigation of AGR2 mRNA revealed a direct impact of its 3'UTR length on AGR2 expression. In silico analyses identified several regulatory sequences within the distal part of AGR2 mRNA that may regulate 3'UTR length and associated protein levels. Short 3'UTRs were observed in a panel of AGR2-positive cancer cell lines and in human breast cancer specimens, in which more extensive 3'UTR shortening correlated with increased AGR2 protein levels. AGR2 is an important member of PI3K/AKT signalling pathway, which along with the proposed involvement of mTOR in the regulation of alternative polyadenylation, prompted us to study the role of mTOR in relation to AGR2 mRNA 3'UTR shortening. A direct impact of mTOR signalling on AGR2 3'UTR shortening associated with increased protein synthesis was found, which led to the identification of a novel molecular mechanism involved in upregulation of AGR2 levels in mTOR-activated cells via modulating the 3'UTR length of AGR2 mRNA.
Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica/genética , Complexos Multiproteicos/genética , Proteínas/genética , Proteínas/metabolismo , Serina-Treonina Quinases TOR/genética , Células A549 , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Clonagem Molecular , Feminino , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , Mucoproteínas , Proteínas Oncogênicas , Poliadenilação/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) strains, especially HPV 16 and HPV 18, is associated with the onset of various malignant diseases, including cervical carcinoma in women. HPV DNA testing is thus being implemented as a complementary method to standard cytological examination, mainly due to its increased sensitivity. AIM: This review outlines the role of HPV in cervical carcinogenesis, with a focus on the formation of cervical intraepithelial neoplasias (CIN1-3) and the molecular mechanism underlying cellular transformation. Current biomarkers used to screen premalignant lesions are described, including mRNA transcripts of the E6 and E7 genes, protein p16 (a cyclin-dependent kinase inhibitor that regulates cell cycle progression from G1 to S phase), altered DNA methylation patterns, and actions of specific microRNAs (short (18-22 bp), non-coding, single-stranded RNA molecules that regulate gene expression at the post-transcriptional level). This review also describes the advantages and drawbacks of commercial HPV tests, and depicts novel methods for more cost-effective and faster HPV diagnostics based on optical or electrochemical detection. CONCLUSION: Although great progress has been made, the incidence and mortality rates of cervical malignancies remain relatively high, especially in developing countries. Incorporation of HPV testing into routine screening programs could help to decrease mortality rates; however, the cost of such testing must be reduced if it is to compete with current cytology-based examinations.Key words: HPV - cervical carcinoma - HPV testing - nucleic acid hybridization - mRNA - DNA methylation - microRNA This work was supported by MEYS-NPS I-LO1413 and GACR 17-08971S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 25. 9. 2017Accepted: 26. 1. 2018.
Assuntos
Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Displasia do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Feminino , Testes de DNA para Papilomavírus Humano , Humanos , Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/etiologia , Displasia do Colo do Útero/etiologiaRESUMO
BACKGROUND: Due to the irreplaceable role of chemotherapy in cancer treatment, research has focused on improving the efficacies of individual drugs and minimizing, or completely suppressing, their negative side effects. Based on long-term experience and the results of clinical trials, the selection of appropriate treatment is currently based on classical clinical diagnostic criteria, such as tumor size, grade, and the presence or absence of standard markers. However, complications arise due to variability between patients and tumor heterogeneity. Characterization of intratumoral heterogeneity and acquisition of more reliable drug performance indicators should improve personalized therapy. Development and selection of suitable models are therefore important issues in cancer research focused on predicting sensitivity to therapy. AIM: This work provides an overview of various chemosensitivity tests that have been previously employed and those that are currently used. Great emphasis is placed on comparing 2D and 3D cell culture models, since their importance and popularity are increasing. Particular attention is paid to in vivo systems, which have significantly improved recently and are tested in clinical trials to predict responses to therapy. CONCLUSION: This work provides a brief overview of chemosensitivity tests, focusing on the importance of individual tests and their application in decision-making and patient stratification to improve the clinical responses of patients and the development of targeted personalized therapy.Key words: cell culture techniques - personalized medicine - drug screening - biological models - tumor cell lines - carcinoma - cytotoxicity assays This work was supported by the project MEYSNPS I-LO1413 and GACR 17-05838S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 21. 9. 2017Accepted: 20. 12. 2017.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias/tratamento farmacológico , Humanos , Medicina de PrecisãoRESUMO
Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.
Assuntos
Fatores de Despolimerização de Actina/biossíntese , Neoplasias da Mama/genética , Moléculas de Adesão Celular/biossíntese , Proteínas de Ligação a RNA/biossíntese , Estatmina/biossíntese , Trombospondinas/biossíntese , Fatores de Despolimerização de Actina/genética , Adulto , Idoso , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Prognóstico , Proteômica , Proteínas de Ligação a RNA/genética , Receptores de Estrogênio/genética , Estatmina/genética , Trombospondinas/genéticaRESUMO
Tamoxifen treatment in breast cancer patients is associated with increased risk of endometrial malignancies. Significantly, higher AGR2 expression was found in endometrial cancers that developed in women previously treated with tamoxifen compared to those who had not been exposed to tamoxifen. An association of elevated AGR2 level with myometrial invasion occurrence and invasion depth was also found. In vitro analyses identified a stimulatory effect of AGR2 on cellular proliferation. Although adverse tamoxifen effects on endometrial cells remain elusive, our work identifies elevated AGR2 as a candidate tamoxifen-dependent mechanism of action responsible for increased incidence of endometrial cancer.
Assuntos
Adenocarcinoma/induzido quimicamente , Adenocarcinoma/metabolismo , Antineoplásicos Hormonais/toxicidade , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Neoplasias do Endométrio/induzido quimicamente , Endométrio/efeitos dos fármacos , Proteínas/metabolismo , Tamoxifeno/toxicidade , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Células MCF-7 , Mucoproteínas , Invasividade Neoplásica , Proteínas Oncogênicas , Proteínas/genética , Interferência de RNA , Estudos Retrospectivos , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Transfecção , Regulação para CimaRESUMO
BACKGROUND: During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. METHODS: The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-ß alone or to combined treatment with TGF-ß and the Erk1/2 inhibitor PD98059 or the TGF-ß receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay. RESULTS: Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-ß-mediated inhibition of AGR2. CONCLUSION: Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Caderinas/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Mucoproteínas , Proteínas Oncogênicas , Vimentina/metabolismoRESUMO
RATIONALE: The goal of this work is the comparison of differences in the lipidomic compositions of human cell lines derived from normal and cancerous breast tissues, and tumor vs. normal tissues obtained after the surgery of breast cancer patients. METHODS: Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC/ESI-MS) using the single internal standard approach and response factors is used for the determination of relative abundances of individual lipid species from five lipid classes in total lipid extracts of cell lines and tissues. The supplementary information on the fatty acyl composition is obtained by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters. Multivariate data analysis (MDA) methods, such as nonsupervised principal component analysis (PCA), hierarchical clustering analysis (HCA) and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples and the correlation of similarity between cell lines and tissues either for tumor or normal samples. RESULTS: MDA methods are used for differentiation of sample groups and also for identification of the most up- and downregulated lipids in tumor samples in comparison to normal samples. Observed changes are subsequently generalized and correlated with data from tumor and normal tissues of breast cancer patients. In total, 123 lipid species are identified based on their retention behavior in HILIC and observed ions in ESI mass spectra, and relative abundances are determined. CONCLUSIONS: MDA methods are applied for a clear differentiation between tumor and normal samples both for cell lines and tissues. The most upregulated lipids are phospholipids (PL) with a low degree of unsaturation (e.g., 32:1 and 34:1) and also some highly polyunsaturated PL (e.g., 40:6), while the most downregulated lipids are PL containing polyunsaturated fatty acyls (e.g., 20:4), plasmalogens and ether lipids. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Neoplasias da Mama/química , Mama/química , Cromatografia Líquida/métodos , Lipídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Análise Multivariada , Análise de Componente PrincipalRESUMO
Expression of the AGR2 oncogene was shown to be associated with estrogen receptor positive tumors. This gene contributes to enhanced cellular proliferation, drug resistance, metastasis development and may also serve as a predictor of poor prognosis. However, our analysis of AGR2 expression in a subset of estrogen-receptor negative tumors revealed that AGR2 could also be upregulated in hormone-independent manner. AGR2 expression was shown to be significantly increased in HER2 positive breast tumors on both the mRNA and the protein level. Moreover, in a subset of estrogen- and progesterone-receptor negative and simultaneously HER2-positive cases, increased AGR2 expression significantly correlated with worse patient prognosis. Subsequent analysis of independent data sets either collected in our institute or obtained from Oncomine cancer microarray database confirmed all these findings.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Proteínas/metabolismo , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Mucoproteínas , Proteínas Oncogênicas , Prognóstico , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Septic acute kidney injury affects 40-50% of all septic patients. Molecular differences between septic patients with and without acute kidney injury (AKI) are only poorly understood. Here, we investigated gene expression changes that differentiated the subjects who developed septic AKI from those who did not and coupled this approach with traditional parameters of renal physiology. METHODS: In 15 anesthetized, mechanically ventilated and instrumented pigs, progressive sepsis was induced either by peritonitis or by continuous intravenous infusion of Pseudomonas aeruginosa. Animals received standard intensive care including goal-directed hemodynamic management. Analyses were performed on kidneys from sham operated animals, septic pigs without AKI, and pigs with septic AKI. Before, and at 12, 18 and 22 h of progressive sepsis, systemic and renal hemodynamics, cortex microcirculation and plasma IL-6 and TNF-α were measured. At 22 h whole kidney expression of pre-selected genes was analyzed by quantitative Real Time PCR. RESULTS: Animals with septic AKI had systemic hemodynamic phenotype (normo- or hyperdynamic) comparable with non-AKI subjects, but demonstrated higher plasma levels of cytokines, an increase in renal vascular resistance and early fall in cortical microcirculatory blood flow. The genes whose expression discriminated septic AKI from non-AKI included Toll like receptor 4 (up-regulated 2.7-fold, P = 0.04); Cyclooxygenase-2 (up-regulated 14.6-fold, P = 0.01), Angiotensin II Receptor (up-regulated 8.1-fold, P = 0.01), Caspase 3 (up-regulated 5.1-fold, P = 0.02), Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Alpha (down-regulated 2-fold, P = 0.02). CONCLUSIONS: In this preliminary experimental study, kidney gene expression was profoundly different in animals that developed septic AKI as opposed to septic animals that did not. The biological functions of the genes differentially expressed support a role of inflammatory overstimulation coupled with metabolic and apoptotic molecular responses in early septic AKI. Cyclooxygenase-2 and angiotensin type 2 receptor-dependent downstream mechanisms appear fruitful targets for future mechanistic research.
Assuntos
Injúria Renal Aguda/genética , Expressão Gênica , Sepse/complicações , Injúria Renal Aguda/microbiologia , Injúria Renal Aguda/fisiopatologia , Animais , Caspase 3/genética , Ciclo-Oxigenase 2/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Hemodinâmica , Interleucina-6/sangue , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Receptores de Angiotensina/genética , Sepse/fisiopatologia , Suínos , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: Derivatives of D-glucosamine and D-galactosamine represent an important family of the cell surface glycan components and their fluorinated analogs found use as metabolic inhibitors of complex glycan biosynthesis, or as probes for the study of protein-carbohydrate interactions. This work is focused on the synthesis of acetylated 3-deoxy-3-fluoro, 4-deoxy-4-fluoro and 3,4-dideoxy-3,4-difluoro analogs of D-glucosamine and D-galactosamine via 1,6-anhydrohexopyranose chemistry. Moreover, the cytotoxicity of the target compounds towards selected cancer cells is determined. RESULTS: Introduction of fluorine at C-3 was achieved by the reaction of 1,6-anhydro-2-azido-2-deoxy-4-O-benzyl-ß-D-glucopyranose or its 4-fluoro analog with DAST. The retention of configuration in this reaction is discussed. Fluorine at C-4 was installed by the reaction of 1,6:2,3-dianhydro-ß-D-talopyranose with DAST, or by fluoridolysis of 1,6:3,4-dianhydro-2-azido-ß-D-galactopyranose with KHF2. The amino group was introduced and masked as an azide in the synthesis. The 1-O-deacetylated 3-fluoro and 4-fluoro analogs of acetylated D-galactosamine inhibited proliferation of the human prostate cancer cell line PC-3 more than cisplatin and 5-fluorouracil (IC50 28 ± 3 µM and 54 ± 5 µM, respectively). CONCLUSION: A complete series of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine is now accessible by 1,6-anhydrohexopyranose chemistry. Intermediate fluorinated 1,6-anhydro-2-azido-hexopyranoses have potential as synthons in oligosaccharide assembly.