[Effects and mechanisms of platelet-activating factor on the invasiveness of ovarian cancer cells in vitro]

Abstract

Objective: To investigate the effects and possible mechanisms of platelet-activating factor (PAF) on the invasion of ovarian cancer cells and to provide a potential target for ovarian cancer therapy.

Methods: (1)Serous type ovarian cancer cell line OVCA429 with platelet-activating factor receptor (PAFR) positive and mucinous type cell line RMUG-L (PAFR negative) were treated with 100 nmol/L of the PAF, cell invasion ability was determined by transwell cell migration assay. (2) For determination of the optimal PAF concentration, ovarian cancer cell OVCA429 was treated by 0, 0.1, 1, 10, 100, and 1000 nmol/L of PAF for 10 minutes or 24 hour, respectively. To observe the different time point of protein changes, OVCA429 were treated by 100 nmol/L of PAF for 0, 5 minutes, 10 minutes, 30 minutes, 1 hour or 12 hours, respectively. The total proteins of treated cells were extracted according to standard protocol. The expression of p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), transcription factor response element-binding protein (CREB), phosphorylated CREB (p-CREB) and matrix metalloproteinase-2 (MMP-2) were detected by western blot. (3) To verify the pathway involved in the PAF induction of the cancer cell invasion, we repeated the experiments by adding the inhibitors when treating cells with PAF. The inhibitors used were as follows, PAFR inhibitor-WEB2076 (50 µmol/L), p-p38 MAPK inhibitor-SB203580 (10 µmol/L), CREB binding protein (CBP)-CREB interaction inhibitor-217505(25 µmol/L). All treated cells were divided into 6 groups: control group, PAF group, PAF + WEB2076 group, PAF + SB203580 group, PAF + 217505 group and PAF + SB203580 + 217505 group.

Results: (1) By transwell assay, 100 nmol/L of PAF could improve the invasion ability of OVCA429 cell significantly [PAF: (118 ± 23) cells vs. control: (36 ± 8) cells, P < 0.01], while the same treatment had no effect on RMUG-L cells [PAF: (45 ± 13) cells vs. control: (53 ± 9) cells, P > 0.05]. (2) Even a very low concentration of PAF (0.1 nmol/L) could increase the expression of p-CREB and MMP-2, while the most effective concentration of PAF was 100 nmol/L. The highest p-CREB protein expression was detected at 10 minutes after administration of 100 nmol/L PAF, as well as the expression of p-p38 MAPK protein. Even 12 hours after treatment the p-p38 MAPK protein could be detected, while there was no significant difference in the expression of CREB (P > 0.05). (3) As compared with PAF group, both in PAF + WEB2076 group and PAF + SB203580 group, the expressions of p-p38 MAPK, p-CREB and MMP-2 protein were decreased significantly; in PAF + 217505 group, although the expression of p-p38 MAPK and p-CREB protein was significantly higher than the control group, the expression of MMP-2 protein was significantly lower; in PAF + SB203580 + 217505 group, the expression of these three proteins were also significantly lower, but there was no significant difference as compared with that in the PAF + WEB2076 or PAF + SB203580 group.

Conclusion: PAF could induce MMP-2 expression and contributed to PAFR positive ovarian cancer cell invasion via activation of CREB by phosphorylating of p38 MAPK.