吴茱萸碱抑制Wnt通路诱导人胰腺癌细胞凋亡和自噬的作用研究

摘要

Objective To investigate the effect of evodiamine (Evo) on inducing apotosis and autophagy in human pan-creatic cancer cells BxPC-3 and PANC-1. Methods MTS assay and flow cytometry were performed to detect the effect of Evo on cell proliferation andapotosis after the BxPC-3 and PANC-1 cells were dealt with different concentrations of Evo. The expressions of Bcl-2,Bax, LC3-Ⅰ, LC3-Ⅱand downstream molecules of Wnt pathway (c-Myc)and Cyclin D1 were detected by western blot. Results The MTS experiment showed that the survival rates of BxPC-3 and PANC-1 cells dealt with 2μmol/L Evo were(68.13±0.79)%and (57.19±1.47)%respectively, which were significantly lower than BxPC-3 and PANC-1 cells without intervention(t=31.45,21.65,P<0.05). The survival rates of BxPC-3 and PANC-1 cells dealt with 4μmol/L Evo were (55.07 ±1.97)% and (50.32 ±0.86)% respectively, which were significantly lower than BxPC-3 and PANC-1 cells dealt with 2 μmol/L Evo (t=6.13,4.02,P<0.05 ). The apoptosis rates of BxPC-3 and PANC-1 cells dealt with Evo were significantly higher than BxPC-3 and PANC-1 cells without intervention[(11.37±0.36)% vs (4.6±0.90)%, (11.36±0.32)% vs (4.0%±0.86) %], the differences were statistically significant(t=8.20,10.14,P<0.05 ). The Bcl-2 expressions of BxPC-3 and PANC-1 cells dealt with Evo were decreased compared with BxPC-3 and PANC-1 cells without intervention while the Bax expression were increased.The LC3-II/LC3-I of BxPC-3 and PANC-1 cells dealt with Evo were 2.5 times and 6.4 times of control group respectively. The expressions of c-Myc and Cyclin D1 were decreased in BxPC-3 and PANC-1 cells dealt with Evo. Conclusions Evo can induce apotosis and autophagy of human pancreatic cancer cells via downregulating the activation of the Wnt pathway.%目的探讨吴茱萸碱诱导人胰腺癌细胞株BxPC-3和PANC-1凋亡和自噬的作用机制。方法不同浓度吴茱萸碱干预胰腺癌细胞株BxPC-3和PANC-1后，用甲基三氯硅烷（MTS）法检测吴茱萸碱对胰腺癌细胞增殖的影响，并用流式细胞术检测细胞凋亡情况。用蛋白质印记试验（Western blot）检测凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、自噬相关蛋白胞浆型（LC3-Ⅰ）和膜型（LC3-Ⅱ），以及Wnt通路下游靶分子c-Myc、细胞周期素D1（Cyclin D1）的表达水平。结果 MTS实验结果显示2μmol/L的吴茱萸碱干预48 h后的胰腺癌细胞株BxPC-3和PANC-1的存活率分别为（68.13±0.79）％和（57.19±1.47）％，明显低于未干预的胰腺癌细胞株，差异均有统计学意义（t分别=31.45、21.65，P均＜0.05）。4μmol/L的吴茱萸碱干预48 h后的胰腺癌细胞株BxPC-3和PANC-1的存活率分别为（55.07±1.97）％和（50.32±0.86）％，也低于2μmol/L吴茱萸碱干预的胰腺癌细胞株，差异均有统计学意义（t分别=6.13、4.02，P均＜0.05）。2μmol/L吴茱萸碱干预的BxPC-3和PANC-1胰腺癌细胞株凋亡率分别为（11.37±0.36）％和（11.36±0.32）％，明显高于未干预的胰腺癌细胞株BxPC-3和PANC-1凋亡率（4.60±0.90）％和（4.00±0.86）％，两组之间，差异有统计学意义（t分别=8.27、10.14，P均＜0.05）。2μmol/L吴茱萸碱干预的BxPC-3和PANC-1胰腺癌细胞株较未干预的胰腺癌细胞株凋亡蛋白Bcl-2表达明显下降， Bax表达明显增加；LC3-Ⅱ/LC-3Ⅰ比值分别为对照组的2.5倍和6.4倍；Wnt通路下游靶分子c-Myc和Cyclin D1的表达均明显降低。结论吴茱萸碱能够通过抑制Wnt通路的活性诱导胰腺癌细胞凋亡和自噬。