Dexamethasone inhibits TNF-alpha-induced apoptosis and IAP protein downregulation in MCF-7 cells

Abstract

Exposure of human mammary carcinoma cell line MCF-7 to TNF-alpha leads to apoptotic cell death within 24 h. In search for apoptosis-preventing signals, we identified glucocorticoids as potent death-preventing compounds. Ten nM dexamethasone provided a significant protective effect whereas 100 nM dexamethasone roughly blocked 80 - 90% of TNF-alpha-induced apoptosis. Surprisingly, dexamethasone exerted a protective effect even when supplied several hours after TNF-alpha. This points to a powerful inhibition of even advanced apoptotic processes by dexamethasone. To further pinpoint the anti-apoptotic glucocorticoid action, we investigated the expression levels of several members of the inhibitors of apoptosis (IAPs) family of proteins in response to TNF-alpha and dexamethasone. IAP proteins directly block caspase protease activities including caspase-3, caspase-7, and caspase-9. Exposure of MCF-7 cells to TNF caused an extensive downregulation of cIAP1, cIAP2, and XIAP protein levels. The decline of the IAP protein levels temporally paralleled the appearance of apoptotic DNA fragments which started 12 - 14 h following TNF-alpha addition and maximal effects were seen within 24 h. Coincubation of cells with TNF-alpha and dexamethasone potently blocked cIAP1, cIAP2, and XIAP downregulation. TNF-alpha-mediated IAP protein downregulation was not affected by proteasome inhibitors like lactacystin, ALLN or ALLM, whereas it was blocked by the broad-spectrum caspase inhibitor Z-VAD-fmk which also prevented TNF-alpha-induced apoptotic cell death. These data suggest that inhibition of IAP downregulation mediated by a caspase proteolytic activity constitutes the anti-apoptotic action of glucocorticoids in MCF-7 carcinoma cells.

Figure 1

Dexamethasone blocks TNF-α-induced apoptotic DNA fragmentation and the appearance of cells exhibiting apoptotic morphology in MCF-7 cells. (A) MCF-7 cells were exposed for 24 h to (A) different concentrations of TNF-α in the absence or presence of either 10 nM or 100 nM dexamethasone, (B) various concentrations of TNF-α or 50 ng ml⁻¹ TNF-α (TNF) plus 50 or 100 nM dexamethasone (50 Dex or 100 Dex, respectively) or 100 nM dexamethasone (Dex), (C,D) either 50 ng ml⁻¹ TNF-α or 50 ng ml⁻¹ TNF-α plus 100 nM dexamethasone. DNA fragmentation was determined by using the diphenylamine reaction (A,C) or apoptotic cells exhibiting characteristic chromatin condensation were counted by fluorescence microscopy (B,D). Data are means±standard deviations of four individual experiments.

Figure 2

Time dependency of dexamethasone protection. MCF-7 cells were cultured as described in the experimental section and stimulated for 24 h with 50 ng ml⁻¹ TNF-α. 100 nM dexamethasone were added at various times in relation to TNF-α application starting 6 h before the apoptotic inducer till 14 h after TNF-α application (TNF-α was added at time point 0) DNA fragmentation was determined by using the diphenylamine assay (A) while apoptotic cells exhibiting characteristic chromatin condensation were counted by fluorescence microscopy (B) Values are means±standard deviation out of three independent experiments.

Figure 3

Effects of TNF-α and dexamethasone on Bcl-2-like protein expression and mitochondrial cytochrome c release. (A) MCF-7 cells were incubated with 50 ng ml⁻¹ TNF-α, 100 nM dexamethasone plus 50 ng ml⁻¹ TNF-α, 100 nM dexamethasone (Dex), or vehicle (control) for the times indicated. The determination of Bid cleavage was done by Western blot analysis whereas for the determination of cytochrome c release MCF-7 cells were harvested by trypsinization, and a cytosolic extract was prepared as described in the method section. The samples were then subjected to Western blot analysis for cytochrome c. (B) Bak, Bax, Bad, Bcl-2, and Bcl-x_L protein levels were determined by Western blot analysis. Values are representative of three individual experiments.

Figure 4

TNF-α mediates cIAP1, cIAP2, and XIAP protein cleavage concomitant with apoptotic cell death induction: inhibition by dexamethasone. Cells were treated for the times indicated with 50 ng ml⁻¹ TNF-α (TNF-α), 100 nM dexamethasone (Dex), 50 ng ml⁻¹ TNF-α plus 100 nM dexamethasone (TNF-α+Dex), or vehicle as a control (co). Proteins were normalized to 50 μg lane⁻¹ and cell lysates were subjected to Western blot analysis for cIAP1 (A), cIAP2 (B), XIAP (C), and GAPDH (D) proteins followed by ECL detection. Each blot is representative of three similar experiments.

Figure 5

TNF-α and LPS mediate caspase-dependent cIAP1 protein cleavage concomitant with apoptotic cell death induction in glomerular endothelial cells. Bovine glomerular endothelial cells were treated for the times indicated with 30 ng ml⁻¹ LPS, 30 ng ml⁻¹ LPS plus 100 μM Z-Asp-CH₂-DCB (Z-Asp), 25 ng ml⁻¹ TNF-α (TNF-α), or 25 ng ml⁻¹ TNF-α plus 100 μM Z-Asp-CH₂-DCB (Z-Asp), or vehicle as a control (co). Proteins were normalized to 50 μg lane⁻¹ and cell lysates were subjected to Western blot analysis for cIAP1 protein followed by ECL detection. The blot is representative of three similar experiments.

Figure 6

IAP protein cleavage is blocked by the caspase protease inhibitor Z-VAD-fmk. (A) MCF-7 cells were treated for 24 h with 50 ng ml⁻¹ TNF-α, 50 ng ml⁻¹ TNF-α plus 100 μM Z-Asp-CH₂-DCB or 100 μM Z-VAD-fmk or vehicle (control) as indicated. DNA fragmentation was quantified by using the diphenylamine assay. (B–E) Cells were exposed for various times to 50 ng ml⁻¹ TNF-α or to 50 ng ml⁻¹ TNF-α plus 100 μM Z-Asp-CH₂-DCB or 100 μM Z-VAD-fmk, or vehicle as a control (co) as indicated. Subsequently, proteins were normalized to 50 μg lane⁻¹ and cell lysates were subjected to Western blot analysis for cIAP1 using the antibody from R&D Systems or from Santa Cruz (B and C, respectively), cIAP2 (D) or XIAP proteins (E) followed by ECL detection and video densitometry. Each blot is representative of three similar experiments.

Figure 7

Effect of proteasome inhibitors on TNF-α-induced apoptosis and IAP protein cleavage in MCF-7 cells. (A) Cells were exposed for 24 h to 50 ng ml⁻¹ TNF-α (TNF) plus 5 μM or 10 μM lactacystine, 10 μM lactacystine (Lacta) or vehicle (control) as indicated. DNA cleavage was determined by the diphenylamine assay. Values are means±standard deviation out of four experiments. (B) MCF-7 cells were exposed for various times to 50 ng ml⁻¹ TNF-α or to 50 ng ml⁻¹ TNF-α plus 10 μM lactacystine or different concentrations of ALLN or ALLM, or vehicle as a control (co) as indicated. Subsequently, proteins were normalized to 50 μg lane⁻¹ and cell lysates were subjected to Western blot analysis for cIAP1, cIAP2 or XIAP proteins followed by ECL detection and video densitometry. Each blot is representative of three similar experiments.