Department of Pharmaco-Biology, Post-Graduate School of Clinical Pathology, University of Calabria, Rende, Italy.
Cell Cycle. 2012 Aug 1;11(15):2911-21. doi: 10.4161/cc.21336.
Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5'-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.
睾丸生殖细胞肿瘤是男性中最常见的肿瘤，也是研究最少的肿瘤。我们使用 TCAM2 细胞系专注于研究人类精原细胞瘤。通过 ERβ，10 nM 雌二醇（E2）能够诱导 PTEN 基因表达和启动子转录激活。瞬时转染、ChIP 和 EMSA 实验证实了 PTEN 基因启动子的 5'-侧翼区对 E2 有反应。ERβ与 PTEN 启动子上的 Sp1 结合减少了细胞存活。存在 ERβ或 PTEN 是诱导 E2 诱导细胞存活丧失所必需的，表明它们在这种作用中具有合作关系。E2 和 DPN 下调了 pAKT 和 AKT 的表达，而已知的凋亡标志物似乎没有变化。PI3K/AKT 通路的抑制也会导致自噬：E2 和 DPN 增强了自噬相关标志物的表达，如 PI3III、Beclin 1、AMBRA 和 UVRAG。MDC 和 TEM 检测证实了 E2 诱导的自噬。没有 DNA 片段化、caspase 9 和 PARP1 裂解表明可能发生坏死性凋亡和/或副凋亡。FACS 分析、LDH 测定和 RIP1 表达证实了这一假说。我们的研究揭示了一种独特的机制，即通过 ERβ/PTEN 信号通路通过自噬和坏死性凋亡诱导 TCAM2 中的细胞死亡。这些数据支持人类精原细胞瘤对雌激素的依赖性，提出了 ERβ 配体用于治疗这种病理状况。
