三苯氧胺诱导人乳腺癌MCF-7细胞株耐药及自噬的关系研究

摘要

Background and purpose: Tamoxifen (TAM), the pioneering selective estrogen receptor modulator (SERM), which blocks estrogen action by binding to the ER in breast cancers, has been used ubiquitously for endocrine therapy for the hormone-sensitive breast cancer. Intrinsic and acquired resistance to TAM limits the clinical use of TAM to a narrow therapeutic window and they are big challenges to the drug therapy for breast cancer patients. Better understanding of the TAM resistant mechanisms is therefore of considerable clinical significance. In our study, we induced breast cancer cell MCF-7 resistant to TAM in vitro and tried to explore the changes of autophagic levels and the expression of ERK (extracellular signal-regulated kinase, one of MAPK family proteins) and Phospho-ERKl/2 in TAM resistant cells compared with MCF-7 cells. Methods: Stepwise TAM selection was used to establish the TAM resistant TR5 subline. Autophagic vacuoles in cells were observed by means of transmission electron microscopy. The growth of the two cell lines were measured by CCK8, and the expression of LC3 H, ERK1/2 and Phospho-ERKl/2 were measured by Western blot. Results: The TAM resistant cell line TR5 we established can be resistant to 5 umol/ L TAM. The number of autophagic vacuoles and the expression of LC3 D protein in TR5 were obviously higher than that in MCF-7. There was no significant difference in the protein expression level of ERK 1/2 between MCF-7 and TR5 cells, but the level of Phospho-ERKl/2 was markedly higher in TR5 cells than that in MCF-7 cells. Conclusion: TAM resistant MCF-7 cells have relatively high autophagic level and MAPK pathway is involved in facilitating TAM resistance.%背景与目的:三苯氧胺(tamoxifen)作为第一代选择性雌激素受体调节剂(selective estrogen receptor modulator,SERM)被广泛地应用于激素敏感型乳腺癌的内分泌一线治疗.三苯氧胺耐药的发生严重限制了临床治疗,是乳腺癌患者用药面临的重大难题,明确其耐药机制对乳腺癌的治疗有重要临床意义.本研究通过体外诱导人乳腺癌细胞MCF-7三苯氧胺耐药,探讨细胞产生三苯氧胺耐药时自噬水平的变化与MAPK家族蛋白细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)蛋白表达量及磷酸化水平的变化.方法:浓度递增筛选法诱导MCF-7细胞耐药,透射电镜观察MCF-7细胞与耐药细胞内的自噬泡数量,CCK8法检测细胞增殖状态,应用Western blot检测LC3II、ERK1/2、Phospho-ERK1/2蛋白的表达情况.结果:诱导的三苯氧胺耐药细胞株TR5达到5 μmol/L的耐药浓度.TR5细胞内的自噬泡数量与LC3II表达量明显高于MCF-7细胞.ERK蛋白在两种细胞中的表达量差异无统计学意义,但其在TR5中的磷酸化水平比MCF-7细胞高.结论:MCF-7三苯氧胺耐药细胞有较高的自噬水平,MAPK信号通路参与了三苯氧胺耐药.