摘要
目的探讨5-氟尿嘧啶(5-fluorouracil,5-FU)对乳腺癌细胞MDA-MB-231增殖的抑制作用,并在抑制caspases的条件下,对5-FU诱导的乳腺癌细胞的死亡形式进行探讨。方法 MTT法检测不同浓度的5-FU(0,4,8,16,24,32μmol/L)对乳腺癌细胞增殖的抑制作用,筛选适宜的药物浓度。在适宜药物浓度下实验分为对照组、z-VAD-fmk组、5-FU组、5-FU+zVAD-fmk组,MTT法检测乳腺癌细胞存活率;PI单染流式细胞术检测乳腺癌细胞的死亡率;DAPI染色检测乳腺癌细胞核的变化;透射电镜观察乳腺癌细胞的死亡形式;Western blot检测坏死性凋亡蛋白RIP1表达的变化。结果 8μmol/L为5-FU的适宜浓度,该浓度下MDA-MB-231细胞在24 h,48 h,72 h的存活率分别为(68.94±1.17)%,(48.76±1.47)%和(44.15±2.41)%。MTT、PI、DAPI实验结果表明与对照组和5-FU组相比,5-FU+z-VAD-fmk组细胞存活率明显降低(P<0.05),细胞核浓染加深,核固缩现象显著。电镜结果显示5-FU组细胞发生凋亡,5-FU+z-VAD-fmk组细胞发生坏死性凋亡。Western blot结果表明:z-VAD-fmk联用时5-FU明显上调RIP1蛋白的表达。结论 5-FU可以诱导MDA-MB-231细胞发生凋亡;caspases抑制条件下5-FU通过激活RIP1诱导MDA-MB-231细胞发生坏死性凋亡。
Objective To explore the inhibitive effect of 5-fluorouracil（ 5-FU） on the proliferation of human breast cancer MDA-MB-231 cells and analyze the form of cell death in the condition of caspase inhibition. Methods MTT assay was used to detect the viability of breast cancer MDA-MB-231 cells after exposed to different concentrations（ 0,4,8,16,24,32 μmol / L） of 5-FU for screening the appropriate concentration of 5-FU. Then the experiment was divided into four groups： control group,z-VAD-fmk group,5-FU group,5-FU ＋ z-VAD-fmk group. The viability of breast cancer was evaluated by MTT assay,the cell death rate was assessed using flow cytometry with PI staining,DAPI staining was performed to demonstrate the cell nucleus changes,the form of cell death was observed under transmission electron microscopy,and Western blot was used to detect the expression of necroptosis-related protein RIP1. Results The appropriate concentration of 5-FU in MDA-MB-231 cells was 8 μmol / L,and the cell survival rates were（ 68. 94± 1. 17） %,（ 48. 76 ± 1. 47） % and（ 44. 15 ± 2. 41） % after exposed to 8 μmol / L 5-FU for 24,48,72 h. MTT,PI,DAPI results showed that compared to control group and 5-FU group,the cell survival was significantly decreased in 5-FU ＋ z-VAD-fmk group（ P〈0. 05）,and the hyperchromatic nuclei was gradually darkened and karyopyknosis was observed. The electron microscope results indicated that apoptosis occurred in 5-FU group,and the necroptosis happened in 5-FU ＋ z-VAD-fmk group. RIP1 protein was up-regulated clearly after the combined treatment with 5-Fu and z-VAD-fmk. Conclusion 5-FU can induce the apoptosis in MDA-MB-231 cells,and it can induce the necroptosis by activating RIP1 when caspases activation is blocked by z-VAD-fmk.
作者
张智瑞
孙小锦
崇殿龙
周灿
任凯
刘浩
ZHANG Zhirui SUN Xiaojin CHONG Dianlong ZHOU Can REN Kai LIU Hao(Faculty ofPharmacy, Bengbu Medical College, Anhui Engiering Technology Research Center of Biochemical Pharmaceuticals, Bengbu 233030, Chin)
出处
《山西医科大学学报》
CAS
2016年第9期808-812,共5页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(81372899)
安徽省高等学校省级自然科学研究项目重大项目(KJ2016SD39)
