RESUMO
The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , Cadeias Pesadas de Clatrina/genética , Endocitose/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Adulto , Idoso , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Proteínas de Transporte/metabolismo , Cromatografia Líquida , Cadeias Pesadas de Clatrina/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Proteoma/genética , Proteoma/metabolismo , Espectrometria de Massas em TandemRESUMO
Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.