全合成Jaspine B诱导人乳腺癌MDA-MB-231细胞凋亡及DNA损伤

目的研究全合成Jaspine B对人乳腺癌MDA-MB-231细胞的增殖抑制及凋亡诱导作用,并探讨其对细胞DNA的损伤。方法酸性磷酸酶法(APA)检测细胞增殖,荧光显微镜、透射电镜(TEM)观察细胞形态结构,DNA琼脂糖凝胶电泳检测DNA片段化,流式细胞技术检测凋亡率及线粒体膜电位(ΔΨm),Western blot检测凋亡相关蛋白表达,单细胞凝胶电泳(SCGE)检测DNA损伤。结果Jaspine B抑制细胞增殖,24、48、72h IC50分别为2.61、1.07、0.77μmol·L-1,其作用在一定范围内呈浓度和时间依赖性;细胞发生凋亡形态改变,生化特征上出现DNA梯状改变;1.50μmol·L-1及3.00μmol·L-1Jaspine B作用24h,早期凋亡细胞率分别为7.60%及15.48%,相应浓度作用48h,早期凋亡细胞率分别增至21.48%及23.60%;Jaspine B可引起ΔΨm明显下降、Cyt C水平增加,促使Caspase-3酶原剪切活化;Jaspine B作用24h,细胞出现明显DNA损伤。结论全合成Jaspine B对人乳腺癌MDA-MB-231细胞抑制增殖、诱导凋亡并诱发DNA损伤,凋亡经由依赖胱天蛋白酶的内在途径发生。

Apoptosis and DNA damage in MDA-MB-231 cells induced by total synthetic Jaspine B

Aim To investigate the proliferation inhibiting,apoptosis inducing and DNA damaging effects on human breast cancer cell line MDA-MB231 by total synthetic Jaspine B.Methods After MDA-MB-231 cells were incubated with Jaspine B,cell viability was evaluated by acid phosphatase assay(APA);fluorescent microscope and transmission electron microscope(TEM) were used to observe morphological changes;DNA agarose gel electrophoresis was used to detect the DNA fragmentation;flow cytometry was used to determine the apoptotic rate and mitochondrial membrane potential(ΔΨm);apoptosis related protein expression was analyzed by Western blot;single cell gel electrophoresis(SCGE) was employed to detect the DNA damage effect.Results Jaspine B inhibited MDA-MB-231 cells proliferation significantly,somewhat in a dose-andtime dependent manner;IC50 at 24 h,48 h and 72 h were 2.61,1.07 and 0.77 μmol·L-1 respectively.Morphological changes and biochemical characteristic,DNA laddering of apoptosis were visualized.1.50 μmol·L-1 and 3.00 μmol·L-1 Jaspine B treated MDA-MB-231 cells for 24 h,early apoptosis rate were 7.60% and 15.48%,and when treatment time doubled to 48 h,early apoptosis rate increased to 21.48% and 23.60% respectively.Jaspine B caused ΔΨm disruption,enhanced Cyt C expression level and activated Caspase-3 cleaving to active forms.Exposed to Jaspine B for 24 h,DNA damage had been detected in MDA-MB-231 cells obviously.Conclusions Total synthetic Jaspine B inhibited proliferation and induced apoptosis and DNA damage in MDA-MB-231 cells;apoptosis was achieved through the Caspase-dependent intrinsic pathway.