Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.
Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.
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DOI：
10.3892/ijo.2016.3461
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发表时间：
2016-06
影响因子：
5.2
通讯作者：
Gonzalez-Cadavid NF
中科院分区：
文献类型：
--
作者：
Gelfand R;Vernet D;Bruhn K;Vadgama J;Gonzalez-Cadavid NF
酗酒与乳腺癌的发生和发展相关，适度的慢性乙醇摄入是一个风险因素。酒精致癌作用所涉及的机制尚不清楚，但据推测可能是由乙醛介导的。我们使用永生化的正常人乳腺上皮细胞系MCF - 12A，以体内适用的乙醇浓度来确定短期或长期暴露于乙醇或乙醛是否会诱导其致癌转化和/或上皮 - 间质转化（EMT）的发生。将MCF - 12A细胞培养物用25 mM乙醇或2.5 mM乙醛培养1周，或用较低浓度（乙醇为1.0 - 2.5 mM，乙醛为1.0 mM）培养4周。在4周的培养中，还对细胞进行了锚定非依赖性测试，包括从2.5 mM乙醇培养物中分离软琼脂筛选细胞（SASC）。通过免疫细胞荧光、流式细胞术、蛋白质印迹、DNA微阵列、逆转录/聚合酶链反应以及微小RNA分析对细胞进行了分析。我们发现，短期暴露于乙醇（一般而言，不是乙醛）与金属硫蛋白家族基因、酒精代谢基因以及提示EMT启动的基因的转录上调相关，但没有相关的表型变化。长期暴露于较低浓度的乙醇或乙醛会在单层培养物和SASC中引起明显的EMT变化，这通过细胞表型、mRNA表达和微小RNA表达的变化得以证明。这表明低浓度的乙醇在很少或没有乙醛介导的情况下，会诱导正常乳腺上皮细胞发生EMT以及一些致癌转化的特征，比如锚定非依赖性。
Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0–2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.