Department of Sciences, Section Biomedical Sciences and Technology, University Roma Tre, Rome, Italy.
J Cell Physiol. 2018 May;233(5):3713-3722. doi: 10.1002/jcp.26251. Epub 2017 Nov 24.
17β-estradiol (E2) regulates diverse physiological effects including cell proliferation through the estrogen receptor α (ERα), which as a transcription factor drives gene transcription and as an extra-nuclear localized receptor triggers the membrane-dependent activation of diverse kinase cascades. E2 also modifies ERα intracellular levels via diverse intracellular mechanisms. In this way, the E2-acivated ERα integrates signaling cascades with the modulation of receptor intracellular concentration and with the induction of DNA synthesis and ultimately drives cell proliferation. In turn, E2 signaling deregulation can cause many diseases including breast cancer (BC). Recently, we performed a Western blotting (WB)-based screen to identify novel pathways affecting ERα intracellular levels and BC cell proliferation. However, because WB lacks high throughput potential, a high-content method to detect all aspects of E2:ERα signaling (nuclear and extra-nuclear receptor activity, ERα levels, E2-induced DNA synthesis) is desirable. Here, we set up a rapid way to measure E2:ERα signaling in 96-well plate format. To demonstrate its robustness, we also challenged 4OH-tamoxifen resistant (Tam-Res) BC cells with a library of anti-cancer drugs and identified methotrexate (MTX) as a molecule inducing ERα degradation and preventing BC cell proliferation. Overall, our research provides a high-content technique to study the physiology of E2:ERα signaling in cells and further suggests a possible anti-ERα and anti-proliferative use for MTX in Tam-Res BCs.
17β-雌二醇（E2）通过雌激素受体α（ERα）调节多种生理效应，包括细胞增殖，作为转录因子，它驱动基因转录，作为核外定位的受体，触发多种激酶级联的膜依赖性激活。E2 还通过多种细胞内机制来改变 ERα 的细胞内水平。通过这种方式，E2 激活的 ERα 将信号级联与受体细胞内浓度的调节以及 DNA 合成的诱导结合起来，最终驱动细胞增殖。反过来，E2 信号失调会导致许多疾病，包括乳腺癌（BC）。最近，我们进行了基于 Western blot（WB）的筛选，以鉴定影响 ERα 细胞内水平和 BC 细胞增殖的新途径。然而，由于 WB 缺乏高通量的潜力，因此需要一种高内涵的方法来检测 E2：ERα 信号的各个方面（核和核外受体活性、ERα 水平、E2 诱导的 DNA 合成）。在这里，我们建立了一种在 96 孔板格式中测量 E2：ERα 信号的快速方法。为了证明其稳健性，我们还用一组抗癌药物对 4-羟基他莫昔芬耐药（Tam-Res）BC 细胞进行了挑战，并确定甲氨蝶呤（MTX）是一种诱导 ERα 降解并阻止 BC 细胞增殖的分子。总的来说，我们的研究提供了一种高内涵技术，用于研究细胞中 E2：ERα 信号的生理学，并进一步表明 MTX 在 Tam-Res BC 中可能具有抗 ERα 和抗增殖作用。
