阿霉素通过循环肿瘤细胞介导三阴性乳腺癌转移

Doxorubicin Mediates Triple Negative Breast Cancer Metastasis Through Circulating Tumor Cells

目的观察阿霉素(doxorubicin,DOX)对三阴性乳腺癌(triplenegativebreastcancer,TNBC)细胞上皮细胞-间充质表型转化(epithelial-mesenchymaltransition,EMT)以及肿瘤转移的影响。方法体外实验,采用Westernblot、PCR、划痕实验、Transwell实验检测DOX对不同TNBC细胞(MDA-MB-231、MDA-MB-468、4T1)生物学行为及EMT相关标志物表达的影响。体内实验,将成功构建的稳定表达绿色荧光蛋白(GFP)的小鼠4T1(4T1-GFP)细胞,接种于BALB/c小鼠乳腺脂肪垫内,复制TNBC转移模型,并给予DOX处理,应用免疫磁珠分选结合GFP荧光方法检测模型小鼠外周血中循环肿瘤细胞(circulatingtumorcells,CTCs)的数量,并观察肿瘤原发灶和转移灶的变化。结果低浓度DOX可以增强人源MDA-MB-231、MDA-MB-468细胞和鼠源4T1细胞的迁移与侵袭能力,促进间质表型标志物Vimentin、N-cadherin的表达,抑制上皮表型标志物E-cadherin的表达。DOX抑制了4T1-GFPTNBC小鼠转移模型原发瘤生长,但增加了其外周血CTCs的数量和肺转移灶的数量。结论低浓度DOX可促进TNBC细胞的EMT转化,进而形成CTCs,促进乳腺癌的侵袭和转移。

Objective To observe the effect of doxorubicin(DOX)on the epithelial-mesenchymal transition(EMT)and tumor metastasis of triple negative breast cancer(TNBC)cells.Methods In vitro,Western blotting,real-time quantitative RT-PCR,wound-healing assay and transwell assay were used to detect the effect of DOX on the biological behavior and EMT-related markers of different TNBC cells(MDA-MB-231,MDA-MB-468,4 T1).In vivo,a mouse breast cancer transplantation model was conducted by inoculating 4 T1-GFP cells,which is a green fluorescent protein(GFP)positive 4 T1 mice cell,in BALB/c mice mammary fat pad DOX treatment was applied.CTCs in peripheral blood was isolated and counted by magnetic bead separation combined with GFP fluorescence method,and primary tumors and metastases were observed as well.Results Low concentration of DOX promoted the migration and invasion of human MDA-MB-231 and MDA-MB-468 cells and mouse 4 T1 cells,increased the expression of mesenchymal phenotypic markers vimentin and N-cadherin,and inhibited the expression of epithelial phenotypic marker E-cadherin.In the 4 T1-GFP TNBC mouse model,DOX increased the number of CTCs in peripheral blood and lung metastatic tumors,while inhibited the growth of primary tumors.Conclusion Low concentration of DOX may promote the transformation of EMT in TNBC cells,increase the number of CTCs in peripheral blood,which maybe results in the metastasis of breast cancer.