穿心莲内酯通过抑制Fas/FasL信号轴降低子宫内膜癌Ishikawa/DPP细胞对顺铂的耐药性

Andrographolide reduces cisplatin resistance of endometrial cancer Ishikawa/DPP cells by inhibiting the Fas/FasL signaling axis

目的:探讨穿心莲内酯(Andro)调节脂肪酸合成酶(Fas)/脂肪酸合成酶配体(FasL)信号轴对子宫内膜癌Ishikawa细胞顺铂(DDP)耐药性的影响。方法:采用0、5、10、20μg/mL DDP分别处理Ishikawa细胞和顺铂耐药的Ishikawa/DPP细胞,0、5、10、25、50μmol/L Andro处理Ishikawa/DDP细胞,MTT法检测细胞增殖情况并为后续实验选择合适的给药剂量。将Ishikawa/DDP细胞随机分为对照组、DDP组(DDP干预)、Andro组(DDP、Andro干预)、pcDNA3.1-NC组(转染pcDNA3.1+DDP、Andro干预)、pcDNA3.1-Fas/FasL组(转染pcDNA3.1-Fas/FasL+DDP、Andro干预),24 h后,采用qPCR法检测Fas、FasL mRNA的表达,平板克隆形成实验、Transwell实验和流式细胞术分别检测细胞克隆能力、细胞迁移与侵袭和细胞凋亡,WB法检测增殖细胞核抗原(PCNA)、BAX、Bcl-2、MMP-2、PD-L1、多药耐药蛋白-1(MDR-1)及Fas、FasL蛋白表达。结果:DDP以剂量依赖的方式抑制Ishikawa和Ishikawa/DPP细胞增殖,并且与Ishikawa细胞比较,Ishikawa/DPP细胞对DDP的敏感性更低(均P<0.05);Andro以剂量依赖性的方式抑制Ishikawa/DPP细胞的增殖(均P<0.05)。Ishikawa/DPP细胞中Fas、FasL的表达水平均高于Ishikawa细胞(均P<0.05)。选取20μg/mL DDP和25μmol/L Andro为干预剂量,干预时间24h。与对照组比较,DDP组Ishikawa/DPP细胞中PD-L1、MDR-1、Fas、FasL mRNA及蛋白表达水平显著升高(P<0.05),而克隆形成率、迁移与侵袭细胞数、凋亡率差异均无统计学意义(均P>0.05);与DDP组比较,Andro组Ishikawa/DPP细胞中Fas、FasL mRNA表达水平、细胞克隆形成率、迁移与侵袭细胞数、PCNA、Bcl-2、MMP-2、PD-L1、MDR-1、Fas、FasL蛋白表达水平显著降低,BAX蛋白表达水平及凋亡率显著升高(P<0.05或P<0.01),pcDNA3.1-NC组与Andro组类似;与pcDNA3.1-NC组比较,pcDNA3.1-Fas/FasL组Ishikawa/DPP细胞上述指标变化均被逆转(P<0.05)。结论:Andro可能通过抑制Fas/FasL信号轴来抑制Ishikawa/DPP细胞增殖、迁移与侵袭,促进凋亡,从而降低细胞对DDP的耐药性。

Objective:To investigate the effect of andrographolide(Andro)regulation of fatty acid synthase(Fas)/fatty acid synthase ligand(FasL)signaling axis on drug resistance of endometrial cancer Ishikawa cells.Methods:Ishikawa cells and cisplatin-resistant Ishikawa/DPP cells were treated respectively with 0,5,10 and 20μg/mL DDP,and Ishikawa/DDP cells were treated with 0,5,10,25 and 50μmol/L Andro.Cell proliferation was detected by MTT and the appropriate dose of administration was selected for subsequent experiments.Ishikawa/DDP cells were randomly divided into the control group,the DDP group(intervention with DDP),the Andro group(intervention with DDP and Andro),the pcDNA3.1-NC group(transfection with pcDNA3.1 and intervention with DDP and Andro),and the pcDNA3.1-Fas/FasL group(transfection with pcDNA3.1-Fas/FasL and intervention with DDP and Andro).After 24 h of treatment,qPCR was used to detect the mRNA expression of Fas and FasL.Plate cloning assay;Transwell assay and flow cytometry were used to detect cell cloning ability,cell migration,invasion and apoptosis,respectively.The expressions of proliferating cell nuclear antigen(PCNA),BAX,Bcl-2,MMP-2,PD-L1,multidrug resistance protein-1(MDR-1)and Fas and FasL proteins were detected by WB method.Results:DDP inhibited the proliferation of Ishikawa and Ishikawa/DPP cells in a dose-dependent manner.In addition,the sensitivity of Ishikawa/DPP cells to DDP was lower than that of Ishikawa cells(all P<0.05).Andro inhibited the proliferation of Ishikawa/DPP cells in a dose-dependent manner(all P<0.05).The expression levels of Fas and FasL in Ishikawa/DPP cells were higher than those in Ishikawa cells(all P<0.05).20μg/mL DDP and 25μmol/L Andro were selected as the intervention doses,and the intervention time was 24 h.Compared with the control group,the mRNA and protein expression levels of PD-L1,MDR-1,Fas,FasL in Ishikawa/DPP cells in the DDP group increased significantly(P<0.05),but there were no significant differences in clone formation rate,number of migrating and invading cells and apoptosis rate(all P>0.05).Compared with the DDP group,the mRNA expression levels of Fas and FasL,the cell clone formation rate,the number of migrating and invading cells,the expression levl of PCNA,Bcl-2,MMP-2,PD-L1,MDR-1,Fas and FasL protein decreased significantly,while the expression level of BAX protein and the apoptosis rate increased significantly(P<0.05 or P<0.01).The pcDNA3.1-NC group was similar to the Andro group.Compared with the pcDNA3.1-NC group,the changes of Ishikawa/DPP cells in the pcDNA3.1-Fas/FasL group were reversed(P<0.05).Conclusion:Andro may inhibit the proliferation,migration and invasion of Ishikawa/DPP cells by inhibiting the Fas/FasL signaling axis,promote the apoptosis,and thereby reduce the resistance of cells to DDP.