Abstract:
The present study investigated the mechanism of polyphyllin A(PPA) in inhibiting gastric cancer(GC) cells. GC cells(SGC7901 and MGC803 cell lines) were treated with PPA at different concentrations. The effect of PPA on the proliferation of GC cells was detected by MTT assay, real-time cell analysis(RTCA) assay, and clone-forming assay, respectively. Reactive oxygen species(ROS) of GC cells was detected by flow cytometry. The change of mitochondrial membrane potential was detected by JC-1 assay. The expression and phosphorylation levels of apoptosis-related proteins(caspase-9, caspase-3, and PARP) and proteins related to the signaling pathway(ETS-1, CIP2 A, and Akt) were detected by Western blot. The binding sites of PPA to ETS-1 were analyzed by molecular docking. The affinity of PPA and ETS-1 was detected by drug affinity responsive target stability(DARTS) assay. PPA had a significant inhibitory effect on the proliferation and colony formation of GC cells at a low concentration. The PPA groups showed increased ROS and decreased mitochondrial membrane potential. PPA down-regulated the precursor expression of caspase-9 and caspase-3 and promoted the cleavage of PARP, suggesting that PPA induced the apoptosis of GC cells through the mitochondrial pathway. PPA significantly reduced expression levels of CIP2 A and the phosphorylation of downstream Akt. Molecular docking showed that PPA bound to the ETS domain of ETS-1, the transcription factor of CIP2 A, and formed hydrogen bonds with Pro319 and Asp317. DARTS assay further confirmed that PPA significantly prevented the hydrolysis of ETS-1 by pronase, which was inductive of the direct binding effect of PPA and ETS-1. PPA inhibits the proliferation and induces the apoptosis of GC cells by directly targeting ETS-1 to down-regulate the ETS-1/CIP2 A/Akt signaling pathway.
摘要:
本研究探讨了多叶黄素A(PPA)抑制胃癌(GC)细胞的机制。用不同浓度的PPA处理GC细胞(SGC7901和MGC803细胞系)。MTT法检测PPA对GC细胞增殖的影响,实时细胞分析(RTCA)和克隆形成试验,分别。流式细胞仪检测GC细胞的活性氧(ROS)。用JC-1法检测线粒体膜电位的变化。凋亡相关蛋白(caspase-9,caspase-3和PARP)和与信号通路相关的蛋白(ETS-1,CIP2A,和Akt)通过蛋白质印迹检测。通过分子对接分析PPA与ETS-1的结合位点。PPA和ETS-1的亲和力通过药物亲和响应靶稳定性(DARTS)测定来检测。低浓度PPA对GC细胞的增殖和集落形成有明显的抑制作用。PPA组显示ROS增加和线粒体膜电位降低。PPA下调caspase-9和caspase-3的前体表达,促进PARP的裂解,提示PPA通过线粒体途径诱导GC细胞凋亡。PPA显着降低CIP2A的表达水平和下游Akt的磷酸化。分子对接表明PPA与ETS-1的ETS结构域结合,ETS-1是CIP2A的转录因子,并与Pro319和Asp317形成氢键。DARTS试验进一步证实,PPA显著阻止了链霉蛋白酶对ETS-1的水解,这是PPA和ETS-1直接结合作用的诱导作用。PPA通过直接靶向ETS-1下调ETS-1/CIP2A/Akt信号通路抑制GC细胞增殖并诱导其凋亡。