蛇床子素对B-ALL 697细胞的抗肿瘤作用及其机制

Antitumor effec t of osthole on human B-cell acute lymphoblastic leukemia 697 cells and its mechanism

目的 :探讨蛇床子素对人急性B淋巴细胞白血病(B-cell acute lymphoblastic leukemia,B-ALL)697细胞的抗肿瘤作用及其可能的作用机制。方法 :不同浓度(8、16、32、64和128μmol/L)蛇床子素处理B-ALL697细胞后,采用CCK-8法检测细胞的增殖抑制率。8和32μmol/L蛇床子素处理B-ALL 697细胞后,采用FCM法检测细胞凋亡率,应用实时荧光定量PCR和蛋白质印迹法检测凋亡相关分子Bcl-2和Bax mRNA及蛋白的表达。8和32μmol/L蛇床子素单独或联合自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)处理B-ALL 697细胞后,进行单丹磺酰戊二胺(monodansylcadaverine,MDC)染色,FCM法检测细胞内平均荧光强度(meanuorescence intensity,MFI),以此反映B-ALL 697细胞的自噬情况;应用实时荧光定量PCR和蛋白质印迹法检测自噬相关分子Beclin 1 mRNA及蛋白的表达。结果 :8、16、32、64和128μmol/L蛇床子素处理组B-ALL 697细胞增殖被明显抑制,且呈剂量和时间依赖性(P值均<0.05)。8和32μmol/L蛇床子素处理组B-ALL 697细胞凋亡率明显上升(P值均<0.01),凋亡相关分子Bax mRNA及蛋白表达水平明显升高,Bcl-2 mRNA及蛋白表达水平明显下降(P值均<0.05)。8和32μmol/L蛇床子素处理组B-ALL697细胞中MDC MFI明显增加(P值均<0.01),自噬相关分子Beclin 1 mRNA及蛋白表达水平均明显升高(P值均<0.05);3-MA联合蛇床子素处理组细胞内MDC MFI无明显变化,Beclin 1 mRNA及蛋白表达水平均无明显变化(P值均> 0.05)。结论 :蛇床子素通过抑制B-ALL 697细胞增殖,诱导细胞凋亡和自噬,发挥抗肿瘤作用。促凋亡机制可能与Bax表达上调和Bcl-2表达下调有关,Beclin 1参与了细胞自噬过程。

Objective: To investigate the antitumor effect of osthole on human B-cell acute lymphoblastic leukemia(B-ALL) 697 cells and its possible mechanism.Methods: After B-ALL 697 cells were treated with different concentrations(8, 16, 32, 64 and 128 μmol/L) of osthole, the inhibition rate of cell proliferation was detected by CCK-8 assay. After B-ALL 697 cells were treated with 8 and 32 μmol/L osthole, the apoptosis was detected by FCM, the expressions of apoptosis-associated molecule Bcl-2 and Bax were detected by real-time fluorescent quantitative PCR and Western blotting. After B-ALL 697 cells were treated with 8 and 32 μmol/L osthole alone or in combination with autophagy inhibitor 3-methyladenine(3-MA), the intracellular mean fluorescent intensity(MFI) was detected by FCM [stained by monodansylcadaverine(MDC)] to reflect the autophagy. The expressions of autophagy-associated molecule Beclin 1 mRNA and protein was detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The proliferation of B-ALL 697 cells in 8, 16, 32, 64 or 128 μmol/L osthole treatment group was significantly inhibited in a dose-and time-dependent manner(all P < 0.05). The apoptosis rate of B-ALL 697 cells in 8 or 32 μmol/L osthole treatment group was significantly increased(both P < 0.01), the expression levels of Bax mRNA and protein were increased(all P < 0.05), while the expression levels of Bcl-2 mRNA and protein were decreased(all P < 0.05). The MDC MFI of B-ALL 697 cells in 8 or 32 μmol/L osthole treatment group was increased(both P < 0.01), and the expression levels of Beclin 1 mRNA and protein were significantly increased(all P < 0.05). There was no significant change in MDC MFI and the expressions of Beclin 1 mRNA and protein after treatment with 3-MA in combination with osthole(all P > 0.05).Conclusion: Osthole inhibits the proliferation, and induces the apoptosis and autophagy of B-ALL 697 cells. The mechanism of promoting apoptosis may be related to the up-regulation of Bax expression and the down-regulation of Bcl-2 expression. Beclin 1 participates in the autophagy.