Relationship between raf kinase inhibitor protein and metastasis of ovarian carcinoma

Abstract

To investigate the relationship between raf kinase inhibitor protein (RKIP), a novel metastasis suppressor gene, and metastasis of ovarian carcinoma. Immunohistochemistry, RT-PCR, and western blot analysis were performed to examine the expression of RKIP in clinical samples of ovarian tumors and five human ovarian carcinoma cell lines. Stable cell lines over-expressed or deleted of RKIP were cloned to investigate the function of RKIP in ovarian cancer cells. The recombinant plasmids expressing sense (ss) or antisense (as) RKIP cDNA or empty vector was transfected into ovarian cancer cell line SKOV3 by lipofectamine. The expression level of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in ovarian cancer cells were detected by western blot analysis. Assays of cell proliferation, soft-agar colony formation, cell adhesion, and cell invasion in vitro were used to examine the malignant phenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of RKIP on cell cycle distribution before and after transfection. (1) The expression levels of RKIP protein in ovarian carcinoma tissues from patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2) RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0.35, 0.34; while the expression level of phosphorylation ERK in ssRKIP #1 and ssRKIP #4 cells were 0. 48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P < 0.01). (4)Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 +/- 5.7, 106.0 +/- 9.2) were decreased compared with that in the empty vector-transfected cells (158.3 +/- 14.6, P < 0.01). (5)Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3 +/- 0.8)%, (64.1 +/- 0.9)%] were decreased compared with that in the non-transfected cells [(100.0 +/- 1.1)%, P < 0.01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 +/- 5, 25 +/- 4) were decreased compared with that in the non-transfected cells (68 +/- 5, P < 0.01). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells. patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2) RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0.35, 0.34; while the expression level of phosphorylation ERK in ssRKIP #1 and ssRKIP #4 cells were 0.48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P < 0.01). (4) Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 +/- 5.7, 106.0 +/- 9.2) were decreased compared with that in the empty vector-transfected cells (158.3 +/- 14.6, P < 0.01). (5) Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3 +/- 0.8)%, (64.1 +/- 0.9)%] were decreased compared with that in the non-transfected cells [(100.0 +/- 1.1)%, P < 0.01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 +/- 5, 25 +/- 4) were decreased compared with that in the nontransfected cells (68 +/- 5, P < 0.01). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells.