Departamento de Medicina Genomica y Toxicologia Ambiental, Instituto de Investigaciones Biomedicas (IIBO), Universidad Nacional Autonoma de Mexico (UNAM), 04510 Ciudad de Mexico (CDMX), Mexico, Mexico.
Unidad de Bioquimica, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran (INCMNSZ), 14080, Mexico, CDMX, Mexico.
BMC Cancer. 2019 Apr 15;19(1):356. doi: 10.1186/s12885-019-5563-y.
Glucocorticoid receptor (GR) activation has been associated with breast cancer cell survival in vitro. Glucocorticoid (GC)-dependent protection against tumor necrosis factor (TNF)-induced cell death has been well characterized in MCF7 luminal A breast cancer cells. The GR activates a variety of protective mechanisms, such as inhibitors of apoptosis proteins (IAPs). However, the relative contribution of the GR-dependent expression of IAPs in the protection of cell death has not, to our knowledge, been evaluated.
MCF7 cells were used for all experiments. GR was activated with cortisol (CORT) or dexamethasone (DEX) and inhibited with mifepristone (RU486). Cell viability was determined in real-time with the xCELLigence™ RTCA System and at specific endpoints using crystal violet stain. The mRNA levels of the eight members of the IAP family were measured by qRT-PCR. The protein levels of GR, PR, ERα, HER2, PARP1, c-IAP1 and XIAP were evaluated by Western blot analysis. The knockdown of c-IAP1 and XIAP was accomplished via transient transfection with specific siRNAs. GR activation was verified by a gene reporter assay. Via the cBioportal interphase we queried the mRNA levels of GR and IAPs in breast cancer tumors.
RU486 significantly inhibited the anti-cytotoxic effect of both GCs. PARP1 processing was diminished in the presence of both GCs. The combined treatments of GCs + TNF increased the relative mRNA levels of Survivin>c-IAP1 > NAIP>Apollon>XIAP>Ts-IAP > ML-IAP > c-IAP2. Additionally, GR mRNA content increased with the combined treatments of GCs + TNF. Sustained levels of the proteins c-IAP1 and XIAP were observed after 48 h of the combined treatments with GCs + TNF. With c-IAP1 and XIAP gene silencing, the GC-mediated protection was diminished. In the breast tumor samples, the GR mRNA was coexpressed with Apollon and XIAP with a Pearson coefficient greater than 0.3.
The effect of GCs against TNF-mediated cytotoxicity involves increased mRNA expression and sustained protein levels of c-IAP1 and XIAP. The antagonist effects of RU486 and the qRT-PCR results also suggest the role of the GR in this process. This finding may have clinical implications because the GR and IAPs are expressed in breast tumor samples.
糖皮质激素受体 (GR) 的激活与体外乳腺癌细胞的存活有关。糖皮质激素 (GC) 依赖性保护作用已在 MCF7 腔 A 乳腺癌细胞中得到充分表征，可抵抗肿瘤坏死因子 (TNF) 诱导的细胞死亡。GR 激活了多种保护机制，如凋亡抑制蛋白 (IAPs)。然而，我们尚未评估 GR 依赖性 IAP 表达在细胞死亡保护中的相对贡献。
所有实验均使用 MCF7 细胞。用皮质醇 (CORT) 或地塞米松 (DEX) 激活 GR，并用米非司酮 (RU486) 抑制。使用实时 xCELLigence RTCA 系统和特定终点的结晶紫染色法测定细胞活力。通过 qRT-PCR 测定 IAP 家族的 8 个成员的 mRNA 水平。通过 Western blot 分析评估 GR、PR、ERα、HER2、PARP1、c-IAP1 和 XIAP 的蛋白水平。通过瞬时转染特异性 siRNA 敲低 c-IAP1 和 XIAP。通过基因报告测定验证 GR 激活。通过 cBioportal 间查询乳腺癌肿瘤中 GR 和 IAP 的 mRNA 水平。
RU486 显著抑制了两种 GC 的抗细胞毒性作用。在存在两种 GC 的情况下，PARP1 处理减少。GC+TNF 的联合处理增加了 Survivin>c-IAP1>NAIP>Apollon>XIAP>Ts-IAP>ML-IAP>c-IAP2 的相对 mRNA 水平。此外，GR mRNA 含量随着 GC+TNF 的联合处理而增加。在用 GC+TNF 联合处理 48 小时后，观察到 c-IAP1 和 XIAP 的蛋白水平持续升高。通过 c-IAP1 和 XIAP 基因沉默，GC 介导的保护作用减弱。在乳腺癌样本中，GR mRNA 与 Apollon 和 XIAP 共表达，皮尔逊系数大于 0.3。
GC 对抗 TNF 介导的细胞毒性作用涉及 c-IAP1 和 XIAP 的 mRNA 表达增加和蛋白水平持续升高。RU486 的拮抗作用和 qRT-PCR 结果也表明了 GR 在这一过程中的作用。这一发现可能具有临床意义，因为 GR 和 IAPs 在乳腺癌样本中表达。
