Abstract
BackgroundGlucocorticoid 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.MethodsMCF7 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.ResultsRU486 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.ConclusionsThe 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.
Highlights
Glucocorticoid receptor (GR) activation has been associated with breast cancer cell survival in vitro
The MCF7 control cells grew at a rate of 0.025 normalized cell index (NCI)/h (100%); with the GCs treatments, growth decreased to one-half of the control growth (0.009 (38%) and 0.014 (55%) for CORT and DEX, respectively)
The presence of cortisol and dexamethasone inhibits Poly polymerase-1 (PARP1) processing in tumor necrosis factor (TNF)-treated MCF7 cells and suggests the expression of inhibitor of apoptosis proteins To establish whether the cytotoxic TNF effect resulted in cell death, we evaluated the cleavage of the enzyme PARP1, a marker of cell damage
Summary
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 relative contribution of the GR-dependent expression of IAPs in the protection of cell death has not, to our knowledge, been evaluated. Due to the relevance of CORT and DEX in cancer, their effects on cancer cell proliferation and death have been extensively studied [8]. GC treatment induced the downregulation of the nuclear factor-kappa beta (NF-κB)-dependent expression of first apoptosis signal receptor (FAS) [10]. We tested the protective effect of CORT as well as DEX, and we evaluated the relative participation of c-IAP1 and XIAP in the interference of GC-mediated cell death with transient RNAi
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