表皮生长因子受体信号通路在MCF-7细胞对三苯氧胺产生耐药中的作用

Involvement of Epidermal Growth Factor Receptor Signaling Pathway in Tamoxifen Resistance of MCF-7 Cells

背景与目的:以三苯氧胺(tamoxifen,TAM)为代表的内分泌治疗是乳腺癌综合治疗的重要组成部分,研究内分泌治疗耐药的机制、寻找克服耐药的方法有十分重要的意义。表皮生长因子受体(epidermalgrowthfactorreceptor,EGFR)在多种肿瘤中表达。在乳腺癌组织中EGFR的阳性表达提示乳腺癌的预后较差。我们选择雌激素受体(estrogenreceptor,ER)阳性的乳腺癌细胞株MCF-7,研究EGFR信号转导通路在TAM产生耐药过程中的作用。方法:ER(+)乳腺癌细胞株MCF-7,持续给予TAM(10-7mol/L)处理形成耐药后,采用荧光定量逆转录-聚合酶链反应(fluorescentquantitationreversetranscription-polymerasechainreaction,fqRT-PCR)及Westernblot方法,分析EGFR信号通路成分与ER在处理前后的改变,并研究EGFR抑制剂AG1478对耐药细胞的作用。结果:MCF-7细胞给予TAM处理的早期表现为细胞生长的抑制,持续用药(6个月)后,细胞生长速度恢复到用药前水平,细胞生长抑制试验表明MCF-7细胞对TAM耐受。此时耐药MCF-7细胞中EGFRmRNA表达量增加了4.5倍,EGFR蛋白水平明显增加,其下游通路中的细胞外信号调节激酶磷酸化水平(phosphorylation-extracellularsignal-regulatedkinase,p-ERK1/2)也明显提高(P<0.05),提示EGFR信号转导通路的活性水平增高,而ER蛋白水平无明显变化。同时,耐药MCF-7细胞对EGFR抑制剂AG1478的敏感性增强。结论:ER+的乳腺癌细胞长期给予TAM处理,可通过EGFR信号转导通路的活性水平提高而对内分泌治疗产生耐药,同时给予EGFR抑制剂可提高内分泌治疗的效果。

BACKGROUND ＆ OBJECTIVE： Tamoxifen （TAM） is an important endocrine therapeutic drug used in combined treatment for breast cancer. However the drug resistant effect of TAM has become a major concern in clinical use. Epidermal growth factor receptor （EGFR） is expressed in many tumors and the expression of EGFR in breast cancer tissues is associated with poor prognosis. This study was to investigate EGFR signaling pathway in the drug resistant effect of TAM in breast cancer cells. METHODS, An estrogen receptor （ER） positive breast cancer cell line, MCF-7 was incubated with TAM （10-7 mol/L） for up to 6 months to produce drug resistant cells, mRNA and protein expression of EGFR signaling pathway related genes and ER gene was analyzed by fluorescent quantitation reverse transcription-polymerase chain reaction （fqRT-PCR） and Western blot. AG1478, an EGFR inhibitor, was also used to study the drug resistant effect of TAM. RESULTS, The proliferation of MCF-7 cells was inhibited in the early stage after TAM treatment. However the inhibitory effect vanished after continuous 6-moS}h treatment and the cell proliferation rate returned back to normal. The tolerance of MCF-7 cells to TAM was confirmed by cell growth inhibitory test. In TAM resistant MCF-7 cells, the expression of EGFR mRNA was increased by 4.5 times and the protein expression of EGFR, phosphorylation-extracellular signal-regulated kinase（p-ERK1/2） was also increased dramatically （P〈0.05）. While ER protein expression remained unchanged. Moreover the drug resistant cells were identified more sensitive to AG1478. CONCLUSION. In endocrine therapy, TAM resistant breast cancer cells are likely to be generated by the activation of EGFR signaling pathway after long-term TAM treatment, and EGFR inhibitors might increase the therapeutic effects in breast cancer treatment.