酒精对乳腺癌细胞表皮生长因子受体-钙激活中性蛋白酶通路及细胞迁移的影响

背景与目的：酒精(ethyl alcohol，EtOH)可促进乳腺癌的恶性演进，但其信号机制尚未完全明了。本研究通过观察EtOH对乳腺癌细胞钙激活中性蛋白酶(calcium-activated neutral protease，CANP)-周期蛋白E/局部黏着斑激酶(focal adhesion kinase，FAK)信号通路和细胞迁移的影响，以及表皮生长因子受体(epidermal growth factor receptor，EGFR)在其中的介导作用，探讨EtOH促进乳腺癌细胞迁移的信号机制。方法：以人乳腺肿瘤细胞系MCF-7为模型细胞(MCF-10A乳腺上皮细胞为对照)；采用蛋白[质]印迹法(Western blot)分析周期蛋白E和FAK蛋白剪切反应；通过伤口愈合实验观察细胞迁移效应；采用EGFR抑制剂(EGFR inhibitor， EGFR-I)或CANP抑制剂Calpeptin抑制EGFR或CANP活性，观察抑制剂对EtOH诱导CANP1大亚基、周期蛋白E/FAK蛋白剪切及细胞迁移的影响。结果：以EtOH(0.3%)处理模型细胞可观察到周期蛋白E和FAK出现明显蛋白剪切且该效应呈剂量和时间依赖性，还观察到EtOH刺激细胞迁移活动(+47.30%，P<0.05)，但EtOH对MCF-10A细胞迁移无明显影响；以Calpeptin(10μmol/L)预处理模型细胞，可见EtOH(0.3%)或EGF(10 ng/mL)诱导的周期蛋白E/FAK蛋白剪切效应受到明显抑制；EGFR-I(3μmol/L)可显著抑制EtOH诱导的CANP1大亚基和周期蛋白E/FAK蛋白剪切及细胞迁移效应(-53.00%，P<0.01)。结论：EtOH可通过EGFR激活乳腺癌细胞CANP信号活动并促进细胞迁移，EGFR-CANP通路参与介导EtOH与EGF之间的串话，提示EGFR-CANP信号通路中的关键分子可为防止乳腺癌转移的潜在药物靶点。

Impacts of ethanol on the epidermal growth factor receptor (EGFR) -calpain signaling and migration in breast cancer cells

Background and purpose:Ethanol has been reported to stimulate progression of breast cancer, yet the underlying mechanism is not fully understood. This study aimed to investigate effects of ethyl alcohol (EtOH) on the calcium-activated neutral protease (CANP)-cyclin E/focal adhesion kinase (FAK) signaling and cell migration in breast cancer cells, as well as the role of epidermal growth factor receptor (EGFR) in the EtOH-stimulated effects, in order to assess the signaling mechanism(s) underlying how EtOH enhances cancer progression.Methods:Human breast cancer cell line MCF-7 was employed as a model system, with MCF-10A mammary epithelial cells as control. In vitro wound healing assay was carried out to evaluate EtOH-induced cell migration. The effects of EtOH or epidermal growth factor on the proteolysis of cyclin E/FAK were detected by Western blot. EGFR inhibitor (EGFR-I) and a speciifc inhibitor for CANP, Calpeptin, were applied to pretreat cultured cells to explore their inlfuences on the cell migration and cyclin E/FAK proteolysis triggered by EtOH.Results:Treatment of model cells with EtOH (0.3%) stimulated significant proteolysis of cyclin E/FAK in a dose-/time-dependent manner and increased migration (+47.30%,P<0.05) in MCF-7 breast cancer cells, but had no signiifcant effect on migration in MCF-10A cells. Pretreatment with Calpeptin (10 μmol/L) signiifcantly reduced EtOH (0.3%)- or EGFR (10 ng/mL)-induced cyclin E/FAK truncation. EGFR-I (3 μmol/L) pro-foundly reduced EtOH-indcued CANP dependent proteolysis of CANP1 and cyclin E/FAK as well as cell migration (-53.00%,P<0.01).Conclusion:EtOH signiifcantly stimulates activation of CANP via EGFR pathway, resulting in proteolysis of cyclin E/FAK and migration in MCF-7 breast cancer cells, suggesting EGFR-CANP signaling to be a potential target for suppression of metastasis in breast cancer.