| [Background and objectives! Laryngeal carcrinoma is the second morbidity malignant tumor of head and neck, which accounts for about 20%of all head and neck cancers. Pathologically, over 90% is laryngeal squamous cell carcinoma (LSCC). The early laryngeal carcinomas have relatively good therapeutic effects, with a 5-year survival rate of 80%-95%, and the function of larynx can be conserved:while the advanced cancers have poor therapeutic effects, and the 5-year survival rate is less than 60%. In clinical practice,40%of patients presented with advanced stageâ…¢orâ…£. Therefore, the effective early diagnosis is crucial to cure the laryngeal carcinoma. So it is an essential task to screen and identify specific biomarkers for detection or diagnosis of LSCC.[Methods] In this study, proteomic methods were used to compare four pairs of the tumor and normal laryngeal tissue samples derived from patients with LSCC. With a chemically defined serum-free medium, the primary organ cultures were established from the paired tissue samples first. The conditional medium (CM) were then collected from the tissue cultures. Total proteins in the CM samples were separated by 10% SDS-PAGE, and then mass spectrometry (MS) was used to identify the proteins. A protein database of the CM for LSCC and normal laryngeal tissue was set up and analyzed by Biological Network Gene Ontology tool. Then we selected three proteins from the CM proteins identified in blood plasma samples and tissues. Enzyme linked immunosorbent assay (ELISA) technique was applied to detected plasma concentration of human kallikerin 6 (KLK6) protein. Immunohistochemisry (IHC) was used to detect the expression of KLK6, DJ-1 and 14-3-3sigma protein in LSCC tissues and adjacent normal laryngeal tissues.[Results] Totally,982 proteins were identified in the CM. Among them,213 proteins were only detected in the CM of normal laryngeal tissues,48 proteins were only detected in the CM of LSCC. By Biological Network Gene Ontology tool analysis, it showed that the CM proteins participate the processing of proteolysis, extracellular matrix organization, glucose metabolic process and immune response. Function analysis showed that in the CM of normal laryngeal tissues, proteins mainly take part in immune response and cytoskeleton constitution. These secreted proteins may play a role in the immune reaction and serine proteinase inhibitor. Whereas, the CM proteins of LSCC involved in epithelia cell proliferation and transforming growth factor-beta production. ELISA results showed that the plasma level of KLK6 protein in LSCC patients was higher than that of head and neck benign tumor patients and normal control groups (P<0.001).There was significant difference of plasma concentration of KLK6 between patients in pre-operative and post-operative LSCC groups (P<0.001). Comparing with groups of lymph node metastasis and groups of without lymph node metastasis, there was also difference (P<0.05). IHC results demonstrated that the positive expression rate of KLK.6 and DJ-1 protein in LSCC were higher than that of normal tissues (P<0.001). The 14-3-3sigma protein expression in LSCC were much lower than that in normal tissues (P<0.001).[Conclusions] We discovered a novel method to detect free proteins released into blood by laryngeal carcinoma. These data provided a new way to analyze tumor markers of LSCC in the peripheral blood for early detection and therapy. KLK6, DJ-1,14-3-3sigma proteins may play an important role in the pathogensis and development of LSCC.
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