低剂量力达霉素对小鼠P19胚胎癌细胞的生长抑制作用及其机制

Inhibitory effect of low-dose lidamycin on growth of P19 embryonal carcinoma cells and its mechanism

目的:探讨低剂量力达霉素对小鼠P19胚胎癌细胞生长的影响及其可能的作用机制,为以肿瘤干细胞为靶点的肿瘤治疗提供依据。方法:体外培养P19细胞,实验分非加药组、阿霉素组和力达霉素组。MTT法检测力达霉素处理P19细胞后的剂量曲线和时间曲线。流式细胞术进一步确定力达霉素和阿霉素处理过的P19细胞的非凋亡剂量,并检测其细胞周期改变。Rea-l time PCR、Western blotting和免疫荧光化学技术检测力达霉素和阿霉素处理过的P19细胞中转录因子Oct4 mRNA和蛋白质的表达水平。结果:MTT法分析结果显示,力达霉素和阿霉素分别处理P19细胞后的半数有效抑制浓度(IC50)为1×10-11mol·L-1和0.5×10-6mol·L-1,从0.001 nmol·L-1到0.1 nmol·L-1浓度范围内的力达霉素处理细胞120 h后,仍可有效抑制小鼠P19胚胎癌细胞生长。流式细胞术分析表明,非凋亡剂量0.01 nmol·L-1力达霉素阻滞P19细胞生长发生在G1期,而0.5μmol·L-1阿霉素阻滞P19细胞生长则发生在G2期。Rea-l time PCR、Western blotting和免疫荧光化学实验均揭示低剂量力达霉素下调P19细胞中的Oct4表达,而阿霉素无此作用。结论:低剂量力达霉素抑制P19细胞生长可能与其下调了胚胎干细胞基因Oct4有关。

Objective To investigate the inhibitory effect of low-dose lidamycin on the growth of P19 cells,in order to provide a reliable evidence for targeting cancer stem cells in cancer therapy.Methods MTT assay was used to measure the dose curve and time curve after P19 cells were treated with lidamycin.Flow cytometry was employed to examine the apoptosis and cell cycle of P19 cells treated with lidamycin and adriamycin.The Oct4 expressions at mRNA and protein levels were tested by Real-time PCR,Western blotting and immunocytochemistry when the P19 cells were exposed to lidamycin and adriamycin.Results After exposure of the cells to lidamycin and adiramycin,the IC50 of lidamycin and adiramycin in P19 cells were 1×10^-11 mol·L^-1 and 0.5×10-9 mol·L^-1,respectively.Low doses of lidamycin range from 0.001 to 0.1 nmol·L^-1 were still effective to inhibit P19 embryonal carcinoma cell growth in 120 h.Flow cytometry indicated that low-dose lidamycin induced G1 stage arrest in P19 cells without apoptosis and adiamycin induced G2 stage arrest.Real-time-PCR,Western blotting and immunochemistry showed that the expression levels of Oct4 were decreased in all test when the P19 cells were exposed to lidamycin,but not to adiamycin.Conclusion The inhibitory effect of low-dose lidamycin on P19 cell growth may be related to down-regulation of embryonic stem cell-like gene Oct4.