线粒体通路在大戟大枣汤对乳腺癌MCF-7细胞增殖抑制及凋亡诱导中的调控作用

Role of mitochondrial pathway on proliferation inhibition and apoptosis induction in breast cancer cells induced by decoction of Euphorbia fischeriana Steud.and Ziziphus jujuba Mil.

目的探讨大戟大枣汤(DEFSJ)对人乳腺癌MCF-7细胞增殖抑制及诱导凋亡的作用及其相关机制。方法SPF级SD大鼠每天2次分别ig给予溶剂(生理盐水)、DEFSJ 2.5,5.0和10.0 g·kg^-1,共3 d。末次给药1 h后,腹主动脉采血,分离制备DEFSJ含药血清(DEFSJ-CS)。乳腺癌MCF-7细胞分别用对照血清、DEFSJ-CS 2.5,5.0和10.0 g·kg^-1及紫杉醇8 mg·L^-1处理48 h。用甲基纤维素克隆形成实验检测细胞克隆能力;JC-1染色后激光共聚焦显微镜检测细胞线粒体膜电位;流式细胞仪检测细胞内活性氧(ROS)含量;逆转录聚合酶链反应(RT-PCR)法检测Bcl-2,Bax和Bim mRNA表达水平;免疫荧光法检测Bcl-2,Bax和Bim蛋白表达水平。结果与含溶剂(生理盐水)血清对照组相比,DEFSJ-CS 2.5,5.0和10.0 g·kg^-1组及紫杉醇组人乳腺癌MCF-7细胞克隆形成率显著降低(P<0.05,P<0.01);MCF-7细胞线粒体膜电位显著降低(P<0.01);细胞内ROS含量显著升高(P<0.01);Bcl-2基因和蛋白的表达下调,Bax和Bim基因和蛋白的表达上调(P<0.05,P<0.01)。结论细胞内线粒体通路参与调控DEFSJ抑制MCF-7细胞增殖并诱导细胞凋亡的作用。

OBJECTIVE To explore the effect of decoction of Euphorbia fischeriana Steud.and Ziziphus jujuba Mil.(DEFSJ)on proliferation inhibition and apoptosis induction in breast cancer MCF-7 cells,and reveal the underlying mechanism.METHODS SPF-grade SD rats were given DEFSJ 0(the same amount of normal saline used as vehicle control),2.5,5.0 and 10.0 g·kg^-1 by ig twice a day for 3 d.Blood was collected from the abdominal aorta and centrifuged to prepare the DEFSJ containing serum(DEFSJ-CS).MCF-7 cells were treated with DEFSJ-CS(2.5,5.0 and 10.0 g·kg^-1)and paclitaxol(Taxol,Tax 8 mg·L^-1)for 48 h.The cloning ability of cells was detected via methyl cellulose cloning experiments.Mitochondrial membrane potential was detected by laser confocal microscope with JC-1 staining.The intracellular reactive oxygen species(ROS)was detected by flow cytometry.The mRNA expressions of Bcl-2,Bax and Bim was detected by reverse transcription-polymerase chain reaction(RT-PCR).The protein expressions Bcl-2,Bax and Bim were detected by immunofluorescence assay.RESULTS In human breast cancer MCF-7 cells treated with DEFSJ-CS(2.5,5.0 and 10.0 g·kg^-1)and Tax as compared with the vehicle control group,the colony-forming efficiency of cells was significantly reduced(P<0.05,P<0.01),the mitochondrial membrane potential of cells was significantly inhibited(P<0.01),the intracellular ROS content of cells was significantly increased(P<0.01),the expression of Bcl-2 gene and protein was down-regulated,while the expressions of Bax and Bim gene and protein were up-regulated(P<0.05,P<0.01).CONCLUSION The intracellular mitochondrial pathway is involved in the regulatory effect of DEFSJ on the proliferation inhibition and apoptosis induction of MCF-7 cells.