新型吴茱萸碱衍生物抗小细胞肺癌活性效应的初步评价

Antitumor activity of 2 new evodiamine derivatives against small-cell lung cancer cells in vitro

目的研究吴茱萸碱(evodiamine, EVO)的两种衍生物:硝基吴茱萸碱衍生物(10-nitro evodiamine derivatives, END)与氨基吴茱萸碱衍生物(10-amino evodiamine derivatives, EAD)对小细胞肺癌细胞株H1688的抗癌活性及机制。方法 H1688细胞分别用不同浓度的吴茱萸碱EVO、END、EAD处理24、48、72 h后用MTT法检测其存活率;10μmol/L的EVO、END、EAD处理H1688细胞24 h后,流式细胞术检测其凋亡与周期;同样浓度处理48 h后提取总蛋白,Western blot检测Caspase 12、Caspase 9、Caspase 3、细胞色素C(cytochome C, Cyt C)的表达。结果 MTT结果显示,0.625μmol/L的END或EAD处理24 h后,即可观察到药物对H1688的抑制作用,20μmol/L EVO、END和EAD处理24、48、72 h后,与0μmol/L组存活率相比显著降低。END和EAD处理组H1688存活率分别为16.45%和61.20%(P_(END)=0.000,P_(EAD)=0.046, 24 h)、9.10%和43.37%(P_(END)=0.000,P_(EAD)=0.000, 48 h)、5.24%和29.55%(P_(END)=0.000,P_(EAD)=0.000, 72 h);而EVO处理组存活率为60.81%(P=0.038,24 h)、21.99%(P=0.000,48 h)、18.51%(P=0.000,72 h),其中20μmol/L END对H1688抑制效果最好。10μmol/L的END、EAD处理H1688 24 h后,细胞凋亡率明显升高,凋亡率分别为54.98%(P=0.000)和19.73%(P=0.029),且分别有79.78%(P=0.001)和77.72%(P=0.001)的细胞被阻滞在G_2/M期。END、EAD处理48 h后,与Control组相比,H1688细胞中Caspase 9(P_(END)=0.011,P_(EAD)=0.026)、Caspase 12(P_(END)=0.031,P_(EAD)=0.018)、Caspase 3(P_(END)=0.002,P_(EAD)=0.043)、Cyt C(P_(END)=0.023,P_(EAD)=0.028)等凋亡相关蛋白均上调。与EVO相比,END对Caspase 3(P=0.026)和Cyt C(P=0.002)作用都明显强于EVO,而EAD对Caspase家族与Cyt C的作用则与EVO类似。结论 EVO的两种衍生物END、EAD都对小细胞肺癌细胞株H1688有体外抗肿瘤作用,这种作用呈时间、浓度依赖性,END体外抗肿瘤效果更好。

Objective To investigate the antitumor activity and the underlying mechanisms of 2 new derivatives of evodiamine(EVO),namely nitro evodiamine derivatives(END)and amino evodiamine derivatives(EAD),in cultured small-cell lung cancer(SCLC)cells.Methods Human SCLC cell line H1688 was treated with EVO,END,or EAD at different concentrations for 24,48,or 72 h,and the changes in the cell viability were assessed using MTT assay.The effects of treatment with 10μmol/L EVO,END or EAD for 24 h on cell apoptosis and cell cycle were investigated using flow cytometry.Western blotting was performed to detect the changes in the expressions of Caspase-9,Caspase-12,Caspase-3,and cytochrome C(Cyt C)in the cells following treatment with 10μmol/L EVO,END or EAD for 48 h.Results MTT assay showed that treatment with END or EAD for 24 h at the concentration as low as 0.625μmol/L was sufficient to reduce the viability of H1688 cells.Treatment of the cells with 20μmol/L END or EAD significantly lowered the cell viability to 16.45%(P=0.000)and 61.20%(P=0.046)at 24 h,to 9.10%(P=0.000)and 43.37%(P=0.000)at 48 h,and to 5.24%(P=0.000)and 29.55%(P=0.000)at 72 h,respectively;the viability of EVO-treated cells was 60.81%(P=0.038)at 24 h,21.99%(P=0.000)at 48 h,and 18.51%(P=0.000)at 72 h.Treatments of the cells with 10μmol/L END and EAD for 24 h both induced obvious cell apoptosis with apoptotic rates of 54.98%(P=0.000)and 19.73%(P=0.029),respectively,and caused cell cycle arrest in G2/M phase in 77.78%(P=0.001)and 77.72%(P=0.001)of the cells,respectively.Treatments with 10μmol/L END and EAD for 48 h both significantly upregulated the expression of Caspase-9(P=0.011,0.026),Caspase-12(P=0.031,0.018),Caspase-3(P=0.002,0.043)and Cyt C(P=0.023,0.028)in H1688 cells.END produced stronger effects than EVO in up-regulating caspase-3(P=0.026)and Cyt C(P=0.002);the effects of EAD on caspase family and Cyt C were similar to those of EVO.Conclusion Both of the two derivatives of EVO,END and EAD,and particularly the former,have concentration-and time-dependent antitumor effects against H1688 cells in vitro.Both END and EAD can cause cell cycle arrest in G2/M phase and induce apoptosis of H1688 cells via the mitochondrial pathway.