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目的通过药物诱导雌激素受体(ER)阴性的乳腺癌细胞株MDA-MB-435恢复ER的表达,探讨其对内分泌治疗的敏感性。方法采用HDAC抑制剂古曲抑菌素(TSA)联合DNMT抑制剂5-AZA-CdR(AZA)作用于MDA-MB-435细胞;以RT-PCR方法检测细胞ERα及其下游基因产物孕激素受体(PR)、雌激素调节蛋白(pS2)的mRNA水平;以水溶性四氮唑盐-8(WST-8)方法检测诱导前后细胞增殖水平。将药物诱导后的MDA-MB-435细胞接种裸小鼠,观察其在体内增殖能力的改变。结果药物诱导后,MDA-MB-435细胞恢复ERα表达,ERα下游基因产物PR和pS2的表达增加。2．5μmol／L AZA和100 ng／ml TSA诱导MDA-MB-435细胞后,其ERα表达最强。诱导后的MDA- MB-435细胞在不同浓度的雌激素中表现出不同的增殖能力。与未经诱导的细胞相比,诱导后的MDA-MB-435细胞增殖能力下降(P<0．01);经诱导的细胞联合4-羟基三苯氧胺后,其增殖能力进一步下降(P<0．01);而单用4-羟基三苯氧胺不能抑制未经诱导的细胞的增殖(P>0．05)。在裸鼠体内,诱导处理后的MDA-MB-435移植瘤较未经处理的细胞增殖能力下降(P<0．01)。去除雌激素后,诱导后肿瘤细胞在体内增殖能力进一步受到抑制(P<0．01)。结论通过TSA和AZA联合诱导作用,MDA-MB-435细胞株中沉默的ER恢复表达ERα,并且该ERα是具有功能的。经过诱导的细胞株在体内及体外对雌激素均恢复反应。联合应用HDAC抑制剂以及DNMT1抑制剂可以恢复乳腺癌细胞MDA-MB-435对内分泌治疗的敏感性,这为ER阴性的乳腺癌患者治疗开辟一条新的思路,具有重要的临床意义。
OBJECTIVE: To investigate the expression of ER in drug-induced estrogen receptor (ER) -negative breast cancer cell line MDA-MB-435 and investigate its sensitivity to endocrine therapy. Methods HDAC inhibitor TSA and DNMT inhibitor 5-AZA-CdR (AZA) were used in MDA-MB-435 cells. The levels of ERα and its downstream progesterone receptor (PR) and estrogen regulatory protein (pS2) mRNA levels were measured. The proliferation of cells before and after induction was detected by water-soluble tetrazolium salt-8 (WST-8) The drug-induced MDA-MB-435 cells were inoculated with nude mice to observe their changes in proliferative capacity in vivo. Results After drug induction, the expression of ERα was restored in MDA-MB-435 cells and the expression of PR and pS2 in ERα gene was increased. The expression of ERα was the strongest in MDA-MB-435 cells induced by 2.5μmol / L AZA and 100 ng / ml TSA. The induced MDA-MB-435 cells showed different proliferative capacities in different concentrations of estrogen. Compared with non-induced cells, MDA-MB-435 cells had a decreased ability to proliferate (P <0.01). The proliferation of the cells induced by 4-hydroxy tamoxifen decreased further (P <0.01). 01); whereas 4-hydroxy tamoxifen alone failed to suppress the proliferation of non-induced cells (P> 0.05). In nude mice, MDA-MB-435 xenografted tumor cells were less proliferative than untreated cells (P <0.01). After estrogen removal, the proliferation of tumor cells in vivo was further inhibited (P <0.01). Conclusion Silencing of ER in MDA-MB-435 cell line restored ERα expression by TSA and AZA co-induction and this ERα is functional. After induction of cell lines in vivo and in vitro estrogen recovery response. The combined application of HDAC inhibitor and DNMT1 inhibitor can restore the sensitivity of breast cancer cell MDA-MB-435 to endocrine therapy, which opens up a new idea for the treatment of ER-negative breast cancer patients and has important clinical significance.