放射治疗诱导的多倍体肿瘤细胞在三阴性乳腺癌复发中作用的研究

Study on the role of radiation-induced polyploid tumor cells in recurrence of triple negative breast cancer

目的分析放射治疗诱导的多倍体肿瘤细胞在三阴性乳腺癌(TNBC)复发中的潜在作用。方法将培养的人TNBC细胞系MDA-MB-231分为3组。在6MV-X射线模式下对其中两组的MDA-MB-231细胞分别给予7 Gy、14 Gy的放射剂量, 继续孵育3 d后, 收集的细胞分别为7 Gy组和14 Gy组, 未经放射处理的细胞作为对照组。镜下观察细胞的形态变化, 流式细胞术鉴定细胞DNA倍性, 二苯基四氮唑溴盐(MTT)法检测细胞增殖, Annexin-V/碘化丙啶染液(PI)双染流式细胞术检测细胞凋亡, Transwell试验检测细胞迁移及侵袭的能力。Western blot检测细胞性骨髓细胞瘤病病毒癌基因(C-myc)、大分子B淋巴细胞瘤蛋白(Bcl-xl)、髓细胞白血病-1蛋白(Mcl-1)、存活素、β-连环素、波形蛋白的表达水平。结果 7 Gy组和14 Gy组细胞体积明显增大, 多倍体细胞亚群(DNA含量>4 N)比例均明显高于对照组, 且随着放射剂量的增加, 14 Gy组多倍体细胞亚群比例显著高于7 Gy组, 差异均具有统计学意义(P<0.05)。与对照组相比, 14 Gy组多倍体MDA-MB-231细胞增殖受到抑制(P<0.05)。7 Gy组和14 Gy组多倍体MDA-MB-231细胞总凋亡及晚期凋亡均较对照组显著减少, 差异有统计学意义(P<0.05)。7 Gy组和14 Gy组多倍体MDA-MB-231细胞的迁移与侵袭能力明显下降, 14 Gy组多倍体MDA-MB-231细胞的迁移与侵袭能力弱于7 Gy组, 差异有统计学意义(P<0.05)。放射诱导的多倍体MDA-MB-231细胞Bcl-xl、Mcl-1和存活素的表达上调, C-myc、β-连环素和波形蛋白表达下调, 差异有统计学意义(P<0.05)。结论放射诱导的多倍体MDA-MB-231细胞增殖、迁移及侵袭能力降低, 但多倍体肿瘤细胞的凋亡率降低、抗凋亡相关蛋白表达上调。这些特性提示多倍体肿瘤细胞是三阴性乳腺癌放射治疗后复发的潜在危险因素。

Objective To investigate the potential role of radiotherapy-induced polyploid tumor cells in triple negative breast cancer(TNBC)recurrence.Methods The cultured human TNBC cell line MDA-MB-231 was divided into three groups.Two groups of MDA-MB-231 cells were given radiation dose of 7 Gy and 14 Gy respectively under 6MV-X-ray mode.After further incubation for 3 days,the collected cells were divided into the 7 Gy group and 14 Gy group,and the cells without radiotherapy were selected as the control group.Cell morphology was observed under microscope,DNA ploidy was identified by flow cytometry,cell proliferation was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide(MTT)assay,cell apoptosis was detected by Annexin-V/propidium iodide(PI)double staining flow cytometry,cell migration and invasion were detected by Transwell assay.The expression levels of C-myc,Bcl-xl,Mcl-1,Survivin,β-catenin and Vimentin was detected by Western blot.Results The cell volume of 7 Gy group and 14 Gy group was significantly increased,and the proportion of polyploid cell subsets(DNA content>4N)was significantly higher than that of the control group.With the increase of radiotherapy dose,the proportion of polyploid cell subsets in 14 Gy group was significantly higher than that in 7 Gy group,with statistically significant differences(P<0.05).Compared with the control group,the proliferation of polyploid MDA-MB-231 cells in 14 Gy group was inhibited(P<0.05).Compared with the control group,the total apoptosis and late apoptosis of polyploid MDA-MB-231 cells in both groups were significantly reduced(P<0.05).The migration and invasion abilities of polyploid MDA-MB-231 cells in both groups were significantly reduced,and the migration and invasion abilities of polyploid MDA-MB-231 cells in 14 Gy group were weaker than those in 7Gy group,the differences were statistically significant(P<0.05).The expressions of Bcl-xl,Mcl-1 and Survivin were up-regulated and the expressions of C-myc,β-catenin and Vimentin were down-regulated in radiotherapy induced polyploid MDA-MB-231 cells,the differences were statistically significant(P<0.05).Conclusions The proliferation,migration and invasion of polyploid MDA-MB-231 cells induced by induced radioactivity are reduced,but the apoptosis rate of polyploid tumor cells is decreased and the expression of anti-apoptosis related proteins is up-regulated.These characteristics suggest that polyploid tumor cells may be a potential risk factor for recurrence of triple negative breast cancer after radiotherapy.