多柔比星通过Stat3-Oct-4/CD44途径诱导三阴性乳腺癌4T1细胞干性的产生

Doxorubicin induces stemness of mouse TNBC 4T1 cells through Stat3-Oct-4/CD44 signaling pathway

目的:观察多柔比星对三阴性乳腺癌细胞干性的诱导作用并研究其作用机制。方法:用不同终浓度(0、0.05、0.1和0.5μmol/L)多柔比星培养细胞,观察细胞形态及存活情况,筛选出最适多柔比星浓度(0.1μmol/L)用于后续实验。小鼠乳腺癌4T1细胞和人乳腺癌MDA-MB-468细胞经多柔比星(0.1μmol/L)持续刺激4周后分别命名为4T1-DOX和MDA-MB-468-DOX细胞。通过成球实验观察细胞的干性,免疫荧光检测CD133的表达,流式细胞术检测CD44的表达,Western blot检测Stat3、p-Stat3和Oct-4的表达。结果:成球实验发现4T1-DOX细胞成球能力较4T1细胞显著增强,MDA-MB-468-DOX细胞成球能力较MDA-MB-468细胞也显著增强(P <0.05);免疫荧光结果显示CD133在4T1-DOX细胞的表达比4T1细胞明显增高(P <0.05);流式细胞术结果表明CD44在4T1-DOX中表达较4T1细胞显著增高(P <0.05);Western blot结果显示Oct-4及p-Stat3在4T1-DOX细胞中的蛋白水平增高(P <0.05),而总Stat3的表达量未见显著改变。以上结果表明在0.1μmol/L多柔比星持续诱导下4T1细胞干性特征明显增强。加入Stat3抑制剂WP1066后,CD44、Oct-4和p-Stat3的蛋白水平均下降(P <0.05)。结论:多柔比星通过激活Stat3-Oct-4信号通路,使三阴性乳腺癌细胞成球能力增强,4T1细胞干性标志物CD133及CD44表达增多,诱导乳腺癌细胞干性的产生。因此,Stat3-Oct-4通路可能成为三阴性乳腺癌治疗的新靶标。

AIM:To investigate the stemness of mouse triple-negative breast cancer(TNBC)4T1 cells induced by doxorubicin(DOX)and the underlying mechanism.METHODS:The 4T1 cells and MDA-MB-468 cells were treated with DOX at different concentrations(0,0.05,0.1 and 0.5 μmol/L)for 24 h,and the shape and viability of the cells were observed.The concentration of DOX at 0.1 μmol/L was chosen as the optimal concentration for the following experiments.The 4T1 cells and MDA-MB-468 cells resistant to DOX were established by continuous stimulation with DOX for 4 weeks,and named as 4T1-DOX and MDA-MB-468-DOX.Sphere formation assay was used to detect the stemness of 4T1 cells and MDA-MB-468 cells.The expression of CD133 was observed by immunofluorescence staining.The expression of CD44 was analyzed by flow cytometry.The protein levels of Stat3,phosphorylated Stat3(p-Stat3)and Oct-4 were determined by Western blot.RESULTS:The sphere formation ability of the 4T1-DOX cells was stronger than that of the 4T1 control cells.The 4T1-DOX cells expressed high levels of the stemness markers CD133 and CD44 as compared with the 4T1 cells(P<0.05).Furthermore,the 4T1-DOX cells exhibited enhanced activation of Stat3(p-Stat3)and increased expression of Oct-4(P<0.05),while the expression of total Stat3 had no obvious variation.In addition,when activation of Stat3 was inhibited by WP1066,the protein levels of p-Stat3,Oct-4 and CD44 were down-regulated(P<0.05).Furthermore,inhibition of Stat3 phosphorylation reduced the sphere formation ability of the 4T1-DOX cells(P<0.05).CONCLUSION:DOX induces the stemness of mouse TNBC 4T1 cells through Stat3-Oct-4 signaling pathway.