小白菊内酯体外杀伤小鼠乳腺癌肿瘤干细胞的研究

Lethal Effects of Parthenolide on Mouse Breast Cancer CSC in vitro

目的:探讨小白菊内酯(PTL)在体外对小鼠乳腺癌肿瘤干细胞(CSC)的影响。方法:无血清微球体悬浮培养乳腺癌细胞系4T1细胞,以富集乳腺癌CSC,以第10代微球体细胞作为实验细胞,实验分为对照组、5-FU组、PTL组、5-FU+PTL组、PTL+NAC(N-乙酰半胱氨酸)组。首先按不同分组加入药物,然后以无血清培养基培养,7天后观察细胞的成球情况,并收集细胞检测CD44^+CD24^(-/low)细胞的含量、无荧光染色侧群(SP)细胞比例、耐药基因MDR1、BCRP mRNA表达。结果:无血清悬浮培养可以富集4T1细胞系中的CSC,CD44^+CD24^(-/low)细胞比例可达(68.9±3.78)%。药物处理后,对照组、5-FU组、PTL+NAC组细胞可形成明显的细胞球,PTL组形成的细胞球很少,5-FU+PTL组无存活细胞。对照组、5-FU组、PTL组、PTL+NAC组CD44^+CD24^(-/low)细胞含量分别为(71.2±2.3)%、(75.6±3.1)%、(10.3±1.9)%、(58.1±2.6)%;无荧光染色细胞(SP)细胞百分比分别为(56.7±314)%、(62.0±2.7)%、(8.1±1.1)%、(51.5±2_3)%,PTL组明显低于其它3组,具有统计学差异(P<0.01)。5-FU组、PTL组、PTL+NAC组MDR1和BCRP与对照组相比较,基因相对表达量分别为1.16±0.21、1.09±0.13、0.22±0.15和0.27±0.11、0.89±0.18、0.93±0.14,PTL组明显低于其它3组,具有统计学差异(P<0.01)。结论:PTL可以靶向作用于乳腺癌CSC,改变CSC的增殖状态,使培养细胞中CSC含量明显降低。抗氧化剂NAC可以明显拮抗PTL这种作用。

Objective： To investigate the lethal effects of Parthenolide （PTL） on cancer stem cells （CSC） of mouse breast cancer in vitro. Methods： Serum-free mammosphere suspension culture fluid was used to culture 4T1 cell lines in a manner designed to enrich for the breast cancer CSC. The 10th generation mammosphere cells were used as the experimental cells. The experimental cells were divided into 5 groups： the control group, the 5-FU group, the PTL group, the 5-FU＋PTL group and the PTL＋NAC （N-Acetylcysteine） group. The experimental cells were treated with the appropriate drugs, and then the cells were cultured using serum-free medium. Seven days later, the mammosphere forming conditions were observed and mammosphere cells were collected for testing the CD44 ＋ CD24-/Iow cell content, detecting the non-fluorescent dye staining side population （SP） cells ratio, and evaluating the MDR1, BCRP and mRNA expression levels. Results： The CSC in the 4T1 cell line were enriched by the serum-free suspension culture, and the CD44＋CD24-/Iow cell ratio reached 68.9＋3.78%. After treatment with different drugs, the cells of the control, the 5-FU and the PTL＋NAC groups formed visible mammospheres. Nevertheless, fewer mammospheres were formed in the cells of the PTL group, and no cells survived in the 5-FU＋PTL group. In the control, 5-FU, PTL, and PTL＋NAC groups, the CD44＋CD24-/Iow cell contents were 71.2±2.3%, 75.6±3.1%, 10.3±1.9% and 58.1±2.6% respectively, and the percentage of the non-fluorescent staining （SP） cells were 56.7±3.4%, 62.0± 2.7%, 8.1±1.1% and 51.5±2.3%, respectively. The cell contents were more significantly decreased in the PTL group than in the other three groups （P〈0.01）, with a statistical significance when comparing the differences among the groups （P〈0.01）. Compared with the cells of the control group, the relative expression of MDR1 and BCRP genes was 1.16±0.21 and 1.09±0.13 in the cells of the 5-FU group, respectively; 0.22＋0.15 and 0.27±0.11 in the cells of the PTL group, respectively; and 0.89±0.18 and 0.93±0.14 in the cells of the PTL＋NAC group, respectively. Conclusion： PTL can target the CSC of breast cancer, change the state of CSC, and significantly reduce the CSC content of the cultured mammosphere cells. The antioxidant NAC can apparently antagonize this effect of PTL in vitro.