PAFR对卵巢癌细胞顺铂敏感性的影响及机制研究

The effect of PAFR on cisplatin sensitivity in ovarian cancer cells and its mechanism

背景与目的:目前卵巢癌的治疗方式仍是手术及术后辅助铂类药物为主的化疗,但复发率高,容易耐药。前期研究已证实血小板活化因子受体(platelet-activating factor receptor,PAFR)在上皮性卵巢癌中高表达,且能促进卵巢癌细胞增殖、侵袭及转移。探索顺铂(cisplatin,CDDP)作用后卵巢癌细胞中PAFR的表达变化及其对卵巢癌细胞CDDP敏感性的影响,并对其机制进行初步探讨,旨在为卵巢癌靶向治疗及克服CDDP耐药提供新方法。方法:采用蛋白质印迹法(Western blot)及实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测不同浓度的CDDP作用于卵巢癌细胞株(SKOV-3和CAOV-3)不同时间后各组细胞PAFR的表达情况。采用Western blot及免疫荧光法验证CDDP作用于卵巢癌细胞后核因子κB(nuclear factor Kappa-B,NF-κB)/p65及缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的表达。采用Western blot及RTFQ-PCR检测小RNA干扰沉默NF-κB及HIF-1α后PAFR的表达情况。采用细胞增殖和凋亡实验检测抑制PAFR表达后对卵巢癌细胞CDDP敏感性的影响。采用Western blot验证CDDP和(或)PAFR抑制剂作用细胞后下游信号通路关键分子P70S6K/AKT/ERK的变化情况。结果:CDDP能够引起卵巢癌细胞中PAFR表达升高,并呈现剂量及时间依赖性(P<0.01)。CDDP能够引起转录因子NF-κB及HIF-1α的核聚集,沉默NF-κB及HIF-1α后,CDDP诱导的PAFR表达下降。PAFR特异性小分子拮抗剂WEB2086或RNA干扰抑制PAFR表达均能显著提高卵巢癌细胞对于CDDP的敏感性,即细胞增殖能力明显降低(P<0.01),而凋亡率明显升高(P<0.01)。CDDP作用于卵巢癌细胞后,表达升高的PAFR能够激活下游的AKT及ERK信号通路分子。结论:CDDP作用于卵巢癌细胞后能够引起转录因子NF-κB及HIF-1α的核聚集从而上调PAFR的表达。抑制PAFR表达能够增加卵巢癌细胞对CDDP的敏感性,可能成为卵巢癌靶向治疗的新方法。

Background and purpose:The current treatment of ovarian cancer is surgery and adjuvant platinum-based chemotherapy.However,relapse and drug resistance are common.We have demonstrated the platelet-activating factor receptor(PAFR)is highly expressed in epithelial ovarian cancer,promoting ovarian cancer cell proliferation and invasion.The objective was to explore the effect of PAFR expression on cisplatin(CDDP)in ovarian cancer cells to provide novel theoretical basis for ovarian cancer therapy.Methods:The upregulation of PAFR in CDDP-treated ovarian cancer cells was observed using Western blot and real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR).The role of nuclear factor Kappa-B(NF-κB)/p65 and hypoxia inducible factor-1α(HIF-1α)in modulating PAFR expression was assessed using Western blot,siRNA and immunofluorescence.The effect of PAFR on CDDP sensitivity was observed using a pharmacological inhibitor and siRNA knockdown.Results:CDDP induced dose-and timedependent upregulation of PAFR in two ovarian cancer cell lines(P<0.01).The downregulation of PAFR by CDDP correlated with the inhibitions of NF-κB and HIF-1αwhich were accumulated by CDDP in the nucleus.Inhibition of PAFR expression by PAFR specific small molecule antagonist WEB2086 or RNA interference could significantly improve the sensitivity of ovarian cancer cells to CDDP.The cell proliferation ability decreased significantly(P<0.01),while the apoptotic rate increased significantly(P<0.01).Increased expression of PAFR activated downstream AKT and ERK pathways in CDDP-treated cells.Conclusion:CDDP induces upregulation of PAFR by accumulating NF-κB and HIF-1αin the nucleus.PAFR inhibition may modulate the CDDP sensitivity in ovarian cancer cells,which is a novel and promising therapeutic target for sensitizing ovarian cancer cells to CDDP.