15-氧代绣线菊内酯对转移性肾癌细胞系ACHN恶性表型的抑制作用

Inhibitory effect of 15-oxospiramilactone on metastatic renal cell carcinoma ACHN cells

目的探讨15-氧代绣线菊内酯对转移性肾癌细胞的体外抗肿瘤效应及其机制。方法采用不同浓度15-氧代绣线菊内酯处理ACHN细胞株,MTT比色法、划痕损伤实验、膜联蛋白A5-绿色荧光素/碘化丙啶(Annexin V-FITC/PI)染色以及流式细胞术分别观察ACHN细胞株增殖、迁移、细胞凋亡的变化,RT-PCR和/或Western blot检测Wnt信号通路靶基因表达。结果 15-氧代绣线菊内酯能明显抑制肾癌ACHN细胞株的增殖,其半数抑制浓度为0.987μmol/L。15-氧代绣线菊内酯呈浓度依赖性降低细胞迁移距离(P<0.05)、诱导细胞凋亡发生(P<0.05)、减少G0/G1期细胞、增加G2/M期细胞(P<0.05)。15-氧代绣线菊内酯能够显著抑制Axin2、LEF1、NKD1、Cyclin D1和生存素等Wnt靶基因以及Cdc25和Cdc2等细胞周期相关基因的表达。结论 15-氧代绣线菊内酯能在体外细胞水平显著抑制转移性肾癌ACHN细胞的增殖和迁移作用,诱导细胞凋亡,使细胞停滞于G2/M期;其对肾癌恶性表型的影响是通过抑制Wnt信号通路来实现的,可能是一种潜在的治疗转移性肾癌的药物。

Objective To investigate the effects of 15-oxospiramilactone on the proliferation and migration of human metastatic renal cell carcinoma cell line ACHN. Methods The inhibitory effect of 15-oxospiramilactone on the proliferation and migration of ACHN cells was detected by MTT and cell migration assay, respectively. The apoptosis of ACHN cells and its relationship with cell cycles were determined by Annexin V-FITC/PI assay and flow cytometry. Protein and mRNA expressions of Wnt target genes were detected by Western blot and qRT-PCR. Results 15-Oxospira- milactone significantly inhibited the growth of ACHN cells with a half dose of inhibition concentration （IC50） of 0. 987 μmol/L 15-Oxospiramilactone concentration-dependently decreased the migration distance,induced the apoptosis, reduced the number of cells in the G0/G1 phase and increased that in the G2/M phase（P〈0. 05）. 15-Oxospiramilactone inhibited the expressions of Axin2, LEF1, NKD1, Cyelin D1 and survivin,which were related to the Wnt target genes,Cdc25 and Cdc2 cell cycle genes. Conclusion 15-Oxospiramilactone can significantly inhibit the proliferation and migration of metastatic renalcell carcinoma ACHN cells in vitro, induce cell apoptosis and arrest the cell cycles at C2/M phase via the inhibition of Wnt/β-catenin signaling. 15-oxospiramilactone may be a potential compound for treating metastatic renal cell carcinoma.