RESUMO
Cell-free DNA is present in different biological fluids and when released by tumor cells may contribute to pro-tumor events such as malignant transformation of cells adjacent to the tumor and metastasis. Thus, this study analyzed the effect of tumor cell-free DNA, isolated from the blood of prostate cancer patients, on non-tumor prostate cell lines (RWPE-1 and PNT-2). To achieve this, we performed cell-free DNA quantification and characterization assays, evaluation of gene and miRNA expression profiling focused on cancer progression and EMT, and metabolomics by mass spectrometry and cellular migration. The results showed that tumor-free cell DNA was able to alter the gene expression of MMP9 and CD44, alter the expression profile of nine miRNAs, and increased the tryptophan consumption and cell migration rates in non-tumor cells. Therefore, tumor cell-free DNA was capable of altering the receptor cell phenotype, triggering events related to malignant transformation in these cells, and can thus be considered a potential target for cancer diagnosis and therapy.
Assuntos
Transformação Celular Neoplásica/genética , DNA Tumoral Circulante/efeitos adversos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , DNA Tumoral Circulante/análise , DNA Tumoral Circulante/isolamento & purificação , Progressão da Doença , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , Triptofano/metabolismoRESUMO
Patients with oral squamous cell carcinoma (OSCC) present significant alterations in their saliva proteome. We have used the shotgun Phage Display (PD) technology to identify candidate proteins that were upregulated in saliva of OSCC by selecting ligands to salivary proteins from a single-chain variable fragment (scFv) PD combinatorial library. After two selection cycles, the highly reactive clone scFv-D09 was able to distinguish saliva of OSCC patients from healthy subjects by enzyme-linked immunosorbent assay (ELISA) with sensitivity and specificity of 96.67%. Additionally, the scFv-D09 clone presented a positive immunostaining for invasive malignant epithelial cells in the connective tissue, keratin pearls in the OSCC, and ducts of salivary glands. We have further identified the target protein as the tropomyosin alpha-4 chain (TPM4) by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, and its binding to the scFV-D09 was demonstrated by bioinformatics. Briefly, we have identified TPM4 as upregulated salivary protein in patients with OSCC, which plays a central role in stabilizing cytoskeleton actin filaments, probably linked with tumor tissue remodeling. Long-term longitudinal studies are needed to validate TPM4 as a potential marker of a malignant process.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Proteínas de Neoplasias/genética , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/química , Tropomiosina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Biologia Computacional/métodos , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas de Neoplasias/metabolismo , Estrutura Secundária de Proteína , Saliva/química , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Sensibilidade e Especificidade , Tropomiosina/metabolismo , Regulação para CimaRESUMO
BACKGROUND: The identification of combinatorial antibodies against many different targets in oncology, autoimmune, inflammatory and infectious diseases has uncovered novel strategies to control and prevent diseases' onset and progression, and represents the fastest growing market for the pharmaceutical industry. Phage Display has been successfully used in the identification of unknown targets, which combines shotgun approaches with high throughput selection schemes. METHODS: This specific review covers many aspects of combinatorial phage display technology starting from antibody selection strategies to its redesign for application purposes. Emphasis is specifically directed to how these biotherapeutics function on specific targets with an interactome view, especially within complex diseases. CONCLUSION: Novel combinatorial antibodies will lead to improved interventions in cancer, autoimmune and infectious diseases; however, the very large genetic diversity associated with environmental variations highlight the importance of the personalized medicine using a system's biology approach. Therefore, combined therapies are expected in the near future.