吴茱萸碱对人甲状腺癌细胞增殖和凋亡的影响及其机制

Effects of evodiamine on proliferation and apoptosis of human thyroid cancer cells and its mechanism

目的探讨吴茱萸碱对人甲状腺癌TPC-1细胞体外增殖和凋亡的影响及其机制。方法2019年5-8月采用MTT法检测不同浓度吴茱萸碱在24、48和72 h对TPC-1细胞生存活性的影响;采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)双染流式细胞仪分析检测细胞凋亡率;实时荧光定量PCR和蛋白质印迹法检测不同浓度的吴茱萸碱诱导TPC-1细胞48 h后,细胞中存活蛋白、胱天蛋白酶-3(caspase-3)和叉头框蛋白3(Foxp3)mRNA和蛋白的表达情况。结果TPC-1细胞经吴茱萸碱处理后,细胞增殖能力明显下降(P<0.05);Annexin V-FITC/PI双染法检测结果显示,在1、3、6、8μmol/L吴茱萸碱作用下,其凋亡率为(7.93±1.02)%、(12.34±2.52)%、(18.92±3.14)%、(23.12±4.25)%,对照组为(5.61±0.83)%,凋亡率随着吴茱萸碱浓度增加而增加(F=22.551,P<0.001)。同时,吴茱萸碱可明显下调TPC-1细胞内存活蛋白、Foxp3蛋白的表达(F=107.406,P<0.001;F=51.725,P<0.001),上调caspase-3蛋白的表达(F=79.895,P<0.001)。结论吴茱萸碱能够抑制甲状腺癌癌细胞的增殖并诱导其凋亡,其机制可能与其抑制存活蛋白、Foxp3,上调caspase-3的表达有关。

Objective To investigate the effects of evodiamine on the proliferation and apoptosis of human thyroid cancer cell line TPC-1 cell in vitro and its mechanism.Methods The effects of different concentrations of evodiamine on the survival activity of TPC-1 cells at 24,48 and 72 hours were detected by methyl thiazolyl tetrazolium(MTT)assay from May to August 2019.The apoptosis rate was detected by Annexin V-FITC/PI double staining flow cytometry.Quantitative reverse transcription PCR and Western blotting were used to detect the mRNA and protein expressions of survivin,caspase-3 and forkhead box protein 3(Foxp3)in TPC-1 cells induced by different concentrations of evodiamine for 48 h.Results After treatment with evodiamine,the proliferation of TPC-1 cells decreased significantly(P<0.05).The result of Annexin V-FITC/PI double staining showed that the cell apoptosis rates were(7.93±1.02)%,(12.34±2.52)%,(18.92±3.14)%,and(23.12±4.25)%in evodiamine with 1μmol/L,3μmol/L,6μmol/L and 8μmol/L,and(5.61±0.83)%in the control group.The apoptosis rate increased with the increase of evodiamine concentration(F=22.551,P<0.001).Mean-while,evodiamine could significantly down-regulate the expression of survivin and Foxp3 protein in TPC-1 cells(F=107.406,P<0.001;F=51.725,P<0.001),and up-regulate the expression of caspase-3 protein(F=79.895,P<0.001).Conclusions Evodiamine can inhibit the proliferation and induce apoptosis of thyroid cancer cells.Its mechanism may be related to inhibiting survivin,Foxp3 and up-regu-lating the expression of caspase-3.