异嗪皮啶对人乳腺癌干细胞凋亡相关基因Bcl-2与Caspase家族表达的影响

目的: 研究草珊瑚中异嗪皮啶成分对乳腺癌干细胞中凋亡相关基因通路Bcl-2、Caspase-3、Caspase-8基因表达影响，进而明确其抑制增殖、迁移和促凋亡的机制。方法: 无血清培养法诱导MDA-MB-231细胞株富集乳腺癌干细胞，流式细胞仪分选CD44⁺ /CD24 ^-/low干细胞群。各组分别给予0、17、50、150、450 μmol/L异嗪皮啶，CCK-8法观察给药后24、48、72 h的细胞活力；细胞划痕实验法检测给药后细胞痕道宽度变化值并判断其对细胞迁移能力的影响；Annexin V-FITC/PI染色法检测给药后细胞总凋亡率变化；实时荧光定量PCR测定各组Bcl-2、Caspase-3、Caspase-8基因的mRNA水平；Western blot检测上述基因的蛋白水平。结果: 与对照组比较，50、150和450 μmol/L异嗪皮啶组24、48、72 h的细胞活力降低，细胞迁移能力下降。与对照组比较，50、150和450 μmol/L异嗪皮啶组细胞总凋亡率高于对照组，Bcl-2基因mRNA和蛋白表达水平降低，Caspase-3和Caspase-8基因mRNA和蛋白表达水平升高。以上结果均呈明显剂量效应关系。结论: 异嗪皮啶能通过下调Bcl-2基因表达与激活Caspase基因家族诱导乳腺癌干细胞的凋亡，同时能抑制其增殖与迁移。

Inhibition of isofraxidin from sarcandra glabra on the breast cancer stem cells via Bcl-2 and Caspase signaling pathway

AIM: To investigate the effects of isofraxidin from sarcandra glabra on the expression levels of Bcl-2, Caspase-3 and Caspase-8 which are related with the apoptosis in breast cancer stem cells. METHODS: The breast cancer stem-like cells were enriched from MDA-MB-231 cell lines using the serum-free culture method, and the CD44⁺/CD24 ^-/low subpopulation was sorted using the flow cytometry. After 0, 17, 50, 150 and 450 μmol/L isofraxidin were administrated to each group, the cell viability were detected using CCK-8 reagent, the width of cell wound was continuously recorded via microscopic photograph, and the total cell apoptosis rates were recorded using Annexin V-FITC/PI staining. The mRNA and protein levels of Bcl-2, Caspase-3 and Caspase-8 were measured using Real-time quantitative PCR and Western blot, respectively. RESULTS: Significantly lower rates of proliferation were observed in 50, 150, and 450 μmol/L isofraxidin group at 24, 48 and 72 h. 50, 150 and 450 μmol/L isofraxidin given for 48 h results in the decline of cell migration and increase in the rate of apoptotic. For the levels of mRNA and protein, Bcl-2 gene of 50, 150 and 450 μmol/L group was higher than that of the control, nevertheless, caspase-3 and caspase-8 gene of these groups were lower than those of the control. All the data showed a clear-cut dose-effect relationship. CONCLUSION: The up-regulation of Bcl-2 and the down-regulation of Casepase-3/8 genes are related with the apoptosis, proliferation inhibition and reduced migration of breast cancer stem cells after isofraxidin treatment.