Jiang Wei, Wang Yi-sheng, Cong Qing, Li Ming-jiang, Ye Bin, Xu Cong-jian
Department of Gynecology, Fudan University, Shanghai, China.
Zhonghua Fu Chan Ke Za Zhi. 2011 Dec;46(12):931-5.
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.
(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.
(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.
PAF could induce MMP-2 expression and contributed to PAFR positive ovarian cancer cell invasion via activation of CREB by phosphorylating of p38 MAPK.
探讨血小板活化因子（PAF）对卵巢癌细胞侵袭的影响及其可能机制，为卵巢癌治疗提供潜在靶点。
（1）用100 nmol/L的PAF处理血小板活化因子受体（PAFR）阳性的浆液性卵巢癌细胞系OVCA429和PAFR阴性的黏液性细胞系RMUG-L，采用Transwell细胞迁移实验检测细胞侵袭能力。（2）为确定PAF的最佳浓度，分别用0、0.1、1、10、100和1000 nmol/L的PAF处理卵巢癌细胞OVCA429 10分钟或24小时。为观察蛋白质变化的不同时间点，分别用100 nmol/L的PAF处理OVCA429 0、5分钟、10分钟、30分钟、1小时或12小时。按照标准方案提取处理后细胞的总蛋白。采用蛋白质印迹法检测p38丝裂原活化蛋白激酶（p38 MAPK）、磷酸化p38 MAPK（p-p38 MAPK）、转录因子反应元件结合蛋白（CREB）、磷酸化CREB（p-CREB）和基质金属蛋白酶-2（MMP-2）的表达。（3）为验证PAF诱导癌细胞侵袭所涉及的途径，在用PAF处理细胞时添加抑制剂重复实验。所用抑制剂如下，PAFR抑制剂-WEB2076（50 μmol/L）、p-p38 MAPK抑制剂-SB203580（10 μmol/L）、CREB结合蛋白（CBP）-CREB相互作用抑制剂-217505（25 μmol/L）。所有处理后的细胞分为6组：对照组、PAF组、PAF + WEB2076组、PAF + SB203580组、PAF + 217505组和PAF + SB203580 + 217505组。
（1）通过Transwell实验，100 nmol/L的PAF可显著提高OVCA429细胞的侵袭能力[PAF组：（118±23）个细胞 vs. 对照组：（36±8）个细胞，P < 0.01]，而相同处理对RMUG-L细胞无影响[PAF组：（45±13）个细胞 vs. 对照组：（53±9）个细胞，P > 0.05]。（2）即使是非常低浓度的PAF（0.1 nmol/L）也可增加p-CREB和MMP-2的表达，而PAF的最有效浓度为100 nmol/L。给予100 nmol/L PAF后10分钟检测到p-CREB蛋白表达最高，同时p-p38 MAPK蛋白表达也最高。即使处理12小时后仍可检测到p-p38 MAPK蛋白，而CREB的表达无显著差异（P > 0.05）。（3）与PAF组相比，PAF + WEB2076组和PAF + SB203580组中p-p38 MAPK、p-CREB和MMP-2蛋白的表达均显著降低；在PAF + 217505组中，虽然p-p38 MAPK和p-CREB蛋白的表达显著高于对照组，但MMP-2蛋白的表达显著降低；在PAF + SB203580 + 217505组中，这三种蛋白的表达也显著降低，但与PAF + WEB2076组或PAF + SB203580组相比无显著差异。
PAF可诱导MMP-2表达，并通过磷酸化p38 MAPK激活CREB促进PAFR阳性卵巢癌细胞的侵袭。
