蔓荆子总黄酮抑制NCI-H446细胞系肺癌干细胞自我更新

Inhibition of Viticis Fructus total flavonoids on self-renewal of lung cancer stem cells derived from NCI-H446 cell line

目的观察蔓荆子总黄酮(Viticis Fructus total flavonoids,VFTF)抑制人小细胞肺癌NCI-H446细胞系肺癌干细胞(lung cancer stem cells,LCSCs)自我更新能力的作用。方法体外培养NCI-H446细胞系细胞,免疫磁珠分选和干细胞条件培养基悬浮培养得到和扩增LCSCs;肿瘤球形成法检测自我更新能力,Western blotting分析自我更新相关转录因子Bmi1和磷酸化Akt(p-Akt)蛋白表达。结果 VFTF显著抑制LCSCs自我更新能力,呈浓度依赖性(P<0.05);与亲本细胞比较,LCSCs细胞Bmi1和p-Akt蛋白表达水平增高;VFTF能有效下调LCSCs细胞Bmi1和p-Akt蛋白表达;PI3K特异性阻断剂LY294002增强VFTF抑制LCSCs自我更新能力的作用。结论 VFTF抑制源自NCI-H446细胞系LCSCs自我更新能力的作用与其下调p-Akt蛋白表达和抑制细胞自我更新相关转录因子Bmi1有关。

Objective To examine whether and how Fiticis Fructus total flavonoids （VFTF） inhibit the capacity of self-renewal in lung cancer stem cells （LCSCs） derived from human small cell lung cancer NCI-H446 cell line. Methods NCI-H446 cell line was cultured in vitro. LCSCs were obtained and amplified by Magnetically activated cell sorting system （MACS） and suspended culture with serum-free conditioned medium. The capacity of self-renewal was detected by tumor sphere-forming assay. The protein expression levels of the self-renewal associated transcription factors, Bmil and p-Akt, were analyzed using Western blotting. Results VFTF significantly suppressed the capacity of self-renewal in LCSCs, in a concentration-dependent manner （P 〈 0.05）. The expression levels of Bmil and p-Akt were elevated in LCSCs, compared with parental cells. VFTF effectively down-regulated the expression levels of Bmil and p- Akt in LCSCs. In addition, LY294002, a PI3K specific inhibitor, enhanced the inhibition of VFTF on the capacity of self-renewal in LCSCs. Conclusion VFTF could inhibit the self-renewal capacity of LCSCs derived from NCI-H446 cell line, which is associated with down-regulation of the expression of p-Akt and self-renewal associated transcription factors Bmi1.