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

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、20 μg/mL DDP分别处理Ishikawa 细胞和顺铂耐药的Ishikawa/DPP 细胞，0、5、 10、25、50 μmol/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20 μg/mL DDP 和25 μmol/L Andro 为干预剂量，干预时间24 h。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。

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

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 20 μg/mL DDP, and Ishikawa/DDP cells were treated with 0, 5, 10, 25 and 50 μmol/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). 20 μg/mL DDP and 25 μmol/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.