The Role Of GEF C3G In Malignant Behaviors Of Ovarian Cancer SKOV3Cells

Objective: To further evaluate the role of GEF C3G in malignantbehaviors of ovarian cancer SKOV3cells and its mechanism.Methods:1. In vitro experiments: the expression of p38MAPK wasdetected by Western blotting in SKOV3cells with C3G deficiency(SKOV3-C3Gi) and negative control for transfection (SKOV3-NT);Cellular apoptosis was evaluated by AnnexinV/PI staining and flowcytometry measurement; DNA damage was assessed by Comet assay;Invasive ability of tumor cells was evaluated by intravasation andextravasation assay.2. In vivo experiments：Subcutaneous xenograft andintraperitoneal implantation metastasis model in nude mice wereestablished to observe the tumorigenesis and metastasis potency of cancercells. Expression of Ki67was detected by immunohistochemistry insubcutaneous xenograft tissues.3. Detection of C3G in clinical samples：The expression of C3G was measured by western blotting in primaryovarian cancer tissue and corresponding metastatic focis isolated fromomentum and intestine.Results: Compared to SKOV3-NT cells, SKOV3-C3Gi cells showedno significant difference in p38MAPK expression (0.5±0.09vs.0.4±0.06, P>0.05); However, the proportion of early apoptotic cells (40.3%±8.8%vs.5.6%±1.5%, P<0.05) and the DNA damage (Comet area37.8±10.3vs.10.8±3.6, P<0.05) were increased significantly; Numbers of intravasatedcells (14.8±5.1vs.52.7±8.0, P<0.05) and extravasated cells (8.3±2.1vs.29.2±3.6, P<0.05) reduced significantly. The two aforementioned tumorcells were employed to establish subcutaneous model in nude mice. Thevolume and weight of xenograft of SKOV3-C3Gi cells were0.30±0.11cm3and0.27±0.10g respectively at the30th day after implantation. Both ofthem were lower than the SKOV3-NT cells xenograft (1.15±0.25cm3,P<0.05and0.73±0.12g, P<0.05). However, the positive rate of Ki67presented no significant difference in the two group (75.8%±9.5%vs.83.2%±12.7%, P>0.05). Interestingly, SKOV3-C3Gi cells formed lessimplantation metastatic foci on the surface of visceral organs thanSKOV3-NT cells, especially on the intestine at the5thweek after the tumorcells were injected intraperitoneally (22.0±5.6vs.59.2±2.7, P<0.05).Moreover, in samples from four EOC patients with both bowel and omentalinvolvement, the C3G expression was increased in intestinal metastaticfoci as compared with primary tumor and omental implants(P<0.05). Andthis was the case with Rap1expression and Rap-GTP level in the only onecase in which the assay was performed successfully.Conclusion: When C3G is knocked down in ovarian cancer SKOV3cells, the cellular proliferation show no significant change, while the apoptosis is increased, and the capabilities of invasion, tumorigensis inxenograft model and implantation metastasis on intestine are reduced. Thus,C3G and related signal pathway probably promote growth by inhibitingapoptosis and enhance cancer invasion to contribute to ovarian tumorgrowth and spreading on visceral organs, especially on the surface ofintestine.