槲皮素抑制磷脂酰肌醇-3激酶／Akt通路的体外抗肿瘤作用研究

Quercetin inhibits leukemia cells by regulating PI3k/Akt pathway in vitro

目的 研究槲皮素对白血病NB4细胞的抗肿瘤效应和磷脂酰肌醇-3激酶（PI3K）／Akt、bcl-2及Bax在槲皮素诱导NB4细胞凋亡中的作用，探讨其可能的作用机制。方法 采用四甲基偶氮唑蓝（MTT）法检测细胞增殖活性，Hoechst 33258荧光染色及流式细胞术观察槲皮素对NB4细胞凋亡的影响，Western blot检测槲皮素对NB4细胞相关蛋白的表达变化，激光共聚焦显微技术检测细胞凋亡时Akt的胞核、胞质分布变化。结果 槲皮素（20～160 μmol／L）能明显抑制NB4细胞增殖，具有剂量-时间依赖性。20、40、80 μmol／L槲皮素作用24 h后，其总凋亡率分别为（9.25±0.11）%、（20.83±2.10）%、（41.43±2.90）%，与空白对照组的（2.17±1.12）%比较差异有统计学意义（t值分别为4.14、6.56、7.02，P＜0.05），细胞凋亡呈剂量依赖性并伴随明显的凋亡细胞形态学改变，而槲皮素的凋亡诱导效应可能与其诱导NB4细胞周期阻滞于G2／M期有关。槲皮素对PI3K／Akt表达水平显著降低，导致凋亡抑制因子bc1-2表达受阻，同时Bax表达增加，但对总Akt蛋白没有明显影响，Akt在胞核表达减少，胞质表达增多，与空白对照相比差异具有统计学意义（F＝15.12，P＜0.05）。结论 槲皮素能够通过多条信号途径促进白血病NB4细胞发生凋亡，发挥较强的抗白血病效应，因而PI3K／Akt通道有望成为白血病诊治的新靶标。

Objective To study the anti-tumor activity of quercetin in NB4 leukemia cells and the roles of PI3K/Akt, bcl-2, and Bax on the quercetin-induced apoptosis, and to investigate the potential underlying mechanism. Methods MTT assay was used to monitor cell proliferation, Hoechst 33258 fluorescent staining and flow cytometry were employed to detect apoptosis in NB4 cells. Western blot was used to detect the expression changes of related proteins in quercetin treated NB4 cells. Confocal laser microscopy was used to test the distributional variation of Akt between cytoplasm and nucleus. Results Quercetin significantly inhibited the NB4 cell proliferation in a dose- and time-dependent manner （20-160 μmol/L）. In addition, treated by 20, 40 80 μmol/L quercetin, the rates of apoptosis were （9.25±0.11） %, （20.83±2.10） % and （41.43±2.90） %, there were statistical difference compared with blank cells （t were 4.14, 6.56 and 7.02, all P 〈 0.05）. This was concentration dependent and accompanied by morphological changes characteristic of apoptosis. Further, quercetin induced a G2/M arrest, which might account for its cytotoxic effects. Quercetin decreased PI3k/Akt expression and caused an inhibition of the anti-apoptotic protein bcl-2, while increasing the expression of Bax. Quercetin had no effects on total Akt, but it promoted Akt translocation from cell nucleus to the cytoplasm （F = 15.12, P 〈 0.05）. Conclusion Quercetin induces the leukemia NB4 cell apoptosis by affecting multiple signal pathways and plays a strong anti-leukemia effect. In addition, our results suggest that PI3K/Akt pathway could be a novel target for the leukemia chemotherapy.