| Part I:Establishing an ovarian cancer-derived secretory/releasing proteomeBackground:Ovarian cancer is the most lethal gynecological malignancy worldwide. Apart from that of lung cancer, ovarian cancer has the highest death-to-incidence ratio of all cancer types in women. The five-year survival rate for patients diagnosed with early stage disease is remarkably higher than that for patients with late stage disease; nevertheless most cases are diagnosed when it has progressed to an advanced stage. These facts highlight the need for diagnostic biomarkers to allow early detection of this malignancy. Circulating tumor markers are useful to diagnosis, prognosis and treatment for the disease, yet there are no accurate diagnostic markers for ovarian cancer in the clinic.Objective:To obtain an ovarian cancer-derived secretory/releasing proteome. to validate candidate ovarian cancer biomarkers in plasma, and to develop a candidate multianalyte panel for ovarian cancer diagnosis.Experimental design:Based on the novel model previously developed in this laboratory. primary organ cultures were established with the tumor tissues and corresponding normal tissues obtained from six ovarian cancer patients in this study. Then the serum-free conditioned medium (CM) samples were collected for proteomic analysis. The total proteins from the CM samples were separated by SDS-PAGE, and then identified by liquid chromatography-tandem mass spectrometry. Subsequently, the basic properties of the CM protein database were analyzed with GOFFA, PANTHER and MetaCore tools. After that, candidate proteins were selected and their levels in the plasma samples from 59 ovarian cancer patients,51 patients with benign pelvic mass and 58 healthy women were measured by double antibody sandwich enzyme-linked immunosorbent assay. Multianalyte panels were eventually modeled utilizing the binary logistic regression method.Results:In total,1129 proteins were identified in the CM from all the organ cultures, making up an ovarian cancer-derived secretory/releasing proteome. Gene Ontology analysis showed ’extracellular protein’ and’plasma membrane protein’ accounted for 38.3%of the total CM proteins. Moreover, the total CM proteins were enriched in the biological processes including "extracellular matrix protein-mediated signaling’ ’proteolysis’, ’protein folding’, ’glycolysis’. ’cell structure’ and immunity (?) reactions, etc. For validation, six CM proteins (NIDI, TIMP2, VCAN. B2M. TF an; CA125) were selected. The plasma levels of all six proteins were significantly differen in the healthy cohort and the cancer cohort (P<0.05 for all six markers). With regard t NIDI. VCAN and CA125, there was a considerable difference between the benign cohoi and the health cohort (P<0.00l for all three markers). Meanwhile, there was significant difference between the cancer cohort and the benign cohort regarding B2M TF and CA125 (P<0.001 for all three markers). Based on the data presented above multianalyte panels were further developed. A three-protein marker panel (consists o NIDI, TIMP2 and CA125) was applied to distinguish ovarian cancer cases from healthy cases, achieving an accuracy, sensitivity and specificity of 98.3%,96.6%and 100.0% respectively. In addition, a five-protein marker panel (consists of NIDI. TIMP2. B2M TF and CA125) was applied to the discrimination of ovarian cancer cases ant non-ovarian cancer cases with 89.9%accuracy.81.4%sensitivity and 94.5%specificity The preliminary results indicated that both panels had a greater discriminating powe than CA125. regarding ovarian cancer diagnosis.Conclusions:This ovarian cancer-derived secretory/releasing protein database provides credible repertoire of potential biomarkers for this malignant disease. Additiona studies will be required to validate these panels in independent sample sets, and to searel for more candidate biomarkers in this high-capacity, promising protein database. Part II:Study on the function of ENO1 in non-small-cell lung cancerBackground:In previous lung cancer-derived secretory/releasing proteome. it highlighted that the levels of six glycolytic proteins were increased concordantly in the serum-free media of tumor tissues. Among them, a-enolase (ENOI) was then validated as a candidate biomarker of lung cancer, with its elevated level in the tumor tissue and the plasma from non-small-cell lung cancer (NSCLC) patients. To date, there are no reports as to the biological function of ENO1 in NSCLC.Objective:This study aimed to explore the biological effect of ENO1 in NSCLC cells.Experimental design:The ENO1 protein levels in human NSCLC cell lines (i.e. A549, H520. HI299 and PG), immortalized human bronchial epithelial cell lines (i.e. M-BE and Y-BE) and the mouse embryonic fibroblasts N1H/3T3 were analyzed by Western blot. Following stable cctopic ENOI expression or transient ENO1 knockdown, the proliferative and migratory abilities of NSCLC cells (i.e. A549 and H520) were detected by CCK-8 proliferation assay, colony formation assay and scrath assay, respectively. In addition, the levels of Cyclin Bl and Cyclin Dl in ENOI-genetically altered A549 cell were examined by Western blot.Results:Compared to that inⅢ/3T3 cells, the level of ENOI protein was generally increased in NSCLC cell lines and immortalized human bronchial epithelial cell lines: while its over-expression in NSCLC cell lines was remarkable. There was no alteration of ENOI protein expression in serial passages of M-BE cells. However, ENOI protein levels were relatively higher in the median passage and the later passage of Y-BE cells. Ectopic ENOI expression stimulated the proliferation of A549 cells; while ENOI knockdown inhibited this cell behavior. Additionally. ENOI had no effect on the prolierative ability of H520 cells. Furthemore. there was no changes of Cyclin Bl and Cyclin Dl levels in ENOl-genetically altered A549 cells.Conclusions:ENOl had the growth-promoting capability in A549 cells, not H520 cells, possibly independent of cell cycle aberration. It proposed that ENOI might function in the development of NSCLC subtypes via mediating distinct cellular behaviors. |
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