Effects of HLEC on the secreted proteins of epithelial ovarian cancer cells prone to metastasize to lymph nodes.

OBJECTIVE: To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer (EOC) cells (SKOV3-PM4) with directional highly lymphatic metastasis. METHODS: Supernatants of four groups of cultured cells, namely, SKOV3 (A), SKOV3+HLEC (B), SKOV3-PM4 (C), SKOV3-PM4+HLEC (D), were collected, and their proteins were detected by antibody arrays and iTRAQ-2D-LC-MALDI-TOF/TOF/MS. Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA. RESULTS: Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C (compared with group A), whereas IGFBP7 and SPARC were downregulated in group D (compared with group C). Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other. Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers, benign tumors, and control groups. Two proteins were upegulated in the first group. VEGFA in the control group was downregulated. For IGFBP, upregulation in the control group and down-regulation in the first group were also observed. CONCLUSION: The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors. The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.

[Screening of differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines using SELDI-TOF-MS technology].

BACKGROUND & OBJECTIVE: The ovarian serous papillary adenocarcinoma cell line SKOV3 and its subclones SKOV3-pm2 and SKOV3-pm3 are cell models to investigate the molecular mechanism of invasion and metastasis of ovarian cancers. This study was to screen differentially expressed proteins between ovarian carcinoma cell lines with directional (SKOV3-pm2 and SKOV3-pm3) and non-directional (SKOV3) highly lymphatic metastasis potentials using time-of-flight mass spectrometry technology and protein chips. METHODS: The lymphatic metastasis rates of the three cell lines were detected in animal models. Proteins in endochylema and supernatants of the three cell lines were screened using surface-enhanced laser desorption and ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Each sample was examined using weak cation exchange (CM-10) protein chip assay and immobilized metal affinity capture (IMAC-3) SELDI ProteinChip array. Detected protein peaks were filtrated and analyzed using Ciphergen proteinchip software 3.2.0 and Biomarker Wizard software. Differentially expressed proteins were defined as those whose absolute ratio values were greater than 0.5. RESULTS: Lymphatic metastasis rates in SKOV3, SKOV3-pm2 and SKOV3-pm3 cell xenografts in nude mice were 20%, 90% and 100%, respectively (P<0.05). Proteins in endochylema whose mass-to-charge ratio (m/z) were 6971, 7475, 9089, 9453, 10103, 11655, and the protein in supernatants whose m/z was 4746 were differentially expressed in SKOV3, SKOV3-pm2 and SKOV3-pm3 cells. CONCLUSION: Combined with weak cation exchange protein chip assay and immobilized metal affinity capture SELDI ProteinChip array, SELDI-TOF-MS technology can be used to screen and identify differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines.

[Establishment of human ovarian carcinoma cell lines with directional highly lymphatic metastasis and study of their biological characteristics].

OBJECTIVE: To establish a human ovarian carcinoma cell line with directional highly lymphatic metastasis and to study their biological characteristics. METHODS: The clone cells of ovarian carcinoma, SKOV3, were inoculated into the hind foot pad of nude mice. The cancer cells of lymph node metastatic foci were transplanted into nude mice again when the metastatic nude of mice were observed. After repetition of this procedure for 3 cycles, the metastatic rate and the metastatic paths were observed in nude mice of every passage. We used limited dilution method to separate and select colonial cells with directional highly lymphatic metastatic potentials from the lymphatic metastasis of human ovarian carcinoma cell line SKOV3. The cells with biological characteristics were assayed by growth curve, HE staining, karyotype analysis, nude mice transplantation and immunohistochemistry, respectively. RESULTS: We established a series of cell lines from lymph node metastasis and designated them as SKOV3-PM1, SKOV3-PM2 and SKOV3-PM3 cell strain. When the cells of SKOV3-PM3 were injected into the hind foot pad of nude mice, they produced 100% (10/10) spontaneous lymphatic metastasis. The lymphatic metastatic rates (26/10) were stable and higher than the mother cell line (1/10, P < 0.01). The metastatic paths were single, mostly to lymphatic nodes. The proliferation ability was increased in cancer cells of every passage in vivo and in vitro after passage of cancer cells of lymphatic metastatic foci for 3 cycles in nude mice. Cytogenetics study showed the karyotypes of SKOV3-PM3 had modes from 83 to 89, and the SKOV3 from 91 to 105. In comparison of SKOV3 with SKOV3-PM3, the number of chromosomes was significantly different (P < 0.05). Immunocytochemical study demonstrated all cell lines were still polygonal epithelial cells. The expression of epithelial membranes antigen (EMA) was positive, exhibiting features of human carcinoma. The cell of SKOV3-PM3 grew more quickly than SKOV3, their cell population doubling time being 22.7 hours and 49.6 hours, respectively (P < 0.05). Flow cytometry revealed the proportion of cells in DNA synthesis and mitosis was higher in SKOV3-PM1 (24.2%), SKOV3-PM2 (29.4%), and SKOV3-PM3 (36.7%) than in SKOV3 (21.5%; P < 0.05). CONCLUSIONS: The ovarian carcinoma sublines with directional highly lymphatic metastasis potential have been established successfully. The cells could provide a good experimental material for further investigation of the mechanism of metastasis and invasion of ovarian carcinoma.