雌激素受体诱导乳腺癌细胞辐射抗性的机制研究

Mechanisms on radiation resistance induced by an estrogen receptor in breast cancer cells

目的探讨雌激素受体(ESR1)对乳腺癌细胞辐射抗性的影响及分子机制。方法在雌激素受体阴性乳腺癌细胞中转染ESR1过表达质粒(过表达对照vector组、过表达ESR1 OE组),在雌激素受体阳性乳腺癌细胞中通过慢病毒转染方法构建稳定敲低ESR1(敲低对照shNC组、敲低shESR1组)的细胞模型,采用实时荧光定量聚合酶链式反应(qPCR)和免疫印迹方法检测自噬相关基因mRNA(ATG5)和蛋白表达水平(LC3B-I、LC3B-II、P62、FIP200、ATG5、ATG7、ATG12、Beclin1、ULK1);在8 Gy X射线的电离辐射处理下,免疫印迹法检测自噬通量;采用流式细胞术检测细胞死亡;采用集落形成实验检测乳腺癌细胞辐射敏感性(未照射sham组、照射IR组、电离辐射联合铁死亡抑制剂处理Fer-1+IR组、电离辐射联合凋亡抑制剂处理Z-VAD+IR组、电离辐射联合自噬抑制剂处理CQ+IR组);用台盼蓝染色检测自噬基因敲低和过表达细胞模型以及雌激素受体抑制剂他莫昔芬(TAM)与电离辐射联合治疗下乳腺癌细胞的死亡率(他莫昔芬未处理TAM-组、他莫昔芬处理TAM+组)。结果在8 Gy X射线照射后24 h处理条件下,雌激素受体阳性乳腺癌ZR75细胞ESR1的敲低促进细胞死亡(t=3.49、3.13,P<0.05),而雌激素受体阴性乳腺癌MDA-MB-231细胞ESR1的过表达抑制细胞死亡(t=4.16、7.48,P<0.05);与单纯照射相比,自噬抑制剂氯喹的处理后增加了敲低ESR1的ZR75细胞集落形成数量(t=8.49,P<0.05),抑制自噬可以减少沉默ESR1所致的ZR75细胞死亡。在电离辐射处理下,MDA-MB-231细胞过表达ESR1促进了细胞保护性自噬;ZR75细胞敲低ESR1后细胞保护性自噬降低。在ZR75细胞中敲低ATG5发现自噬降低,细胞死亡增加(t=4.19、6.39,P<0.05),而在ZR75中过表达ATG5可以逆转因敲低ESR1导致的细胞死亡增加(t=1.70、4.65,P<0.05)。化疗药物他莫昔芬处理雌激素受体阳性乳腺癌细胞ZR75后发现自噬基因ATG5、ATG12的表达下降,自噬抑制,细胞死亡增加(t=18.70,P<0.05),电离辐射处理促进了这一进程(t=16.82,P<0.05)。结论雌激素受体ESR1通过增加ATG5自噬相关蛋白,促进雌激素受体阳性乳腺癌细胞的保护性自噬,从而导致雌激素受体阳性乳腺癌细胞的辐射抵抗,化疗药物他莫昔芬联合电离辐射治疗增加了雌激素受体阳性乳腺癌细胞的辐射敏感性。

Objective To explore the effects of estrogen receptorα(ERα)encoded by protein encoding gene ESR1 on the radiation resistance of breast cancer cells and their molecular mechanisms.Methods The ESR1 overexpression plasmid was transfected into estrogen receptor(ER)-negative breast cancer cells.Then,the shRNA-ESR1 vector was introduced into ER-positive cell to establish models with different phenotype.The ATG5 mRNA level and protein expression levels of LC3B-I,LC3B-II,P62,FIP200,ATG5,ATG7,ATG12,Beclin1,ULK1 were detected using qPCR and Western blot techniques.Cell death was measured using flow cytometry.The radiation sensitivity was determined through the colony formation assay.The mortality of breast cancer cells under the autophagy gene knockdown and overexpression or treated with estrogen receptor inhibitor(TAM)combined with ionizing radiation were detected by trypan blue staining.Results Under the condition of 8 Gy X-ray irradiation,the knockdown of ESR1 in ER-positive ZR75 breast cancer cells promoted cell death(t=3.49,3.13,P<0.05),while the overexpression of ESR1 in ER-negative MDA-MB-231 breast cancer cells inhibited cell death(t=4.16,7.48,P<0.05).Compared to the control group,the treatment with chloroquine increased the number of formed colonies of ESR1 knockdown ZR75 cells(t=8.49,P<0.05),and inhibiting autophagy could reduce the death of ZR75 cells caused by ESR1 silencing.Under the treatment with ionizing radiation,the overexpression of ESR1 in MDA-MB-231 cells promoted protective autophagy,which,however,was reduced after ESR1 knockdown in ZR75 cells.Furthermore,it was observed that the knockdown of ATG5 in ZR75 cells was associated with reduced autophagy and an increase in cell death(t=4.19,6.39,P<0.05).In contrast,the overexpression of ATG5 in ZR75 cells reversed the increase in cell death caused by ESR1 knockdown(t=1.70,4.65,P<0.05).After the treatment of ER-positive ZR75 breast cancer cells with TAM,the expressions of ATG5 and ATG12 decreased,suggesting inhibited autophagy and an increase in cell death(t=18.70,P<0.05).Furthermore,these processes were promoted by ionizing radiation(t=16.82,P<0.05).Conclusions The estrogen receptor encoded by ESR1 promotes protective autophagy of ER-positive breast cancer cells by increasing ATG5,further leading to radiation resistance in ER-positive breast cancer cells.Treatment with tamoxifen combined with ionizing radiation can increase the radiation sensitivity of ER-positive breast cancer cells.