Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT

Abstract

Background: The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death.

Results: Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected.

Conclusion: NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.

Figure 1

Dexamethasone inhibits in a dose-dependent manner the TNF-α mediated cytotoxicity in MCF-7 cells: (A) MCF-7 cells were treated with different concentrations of TNF-α and cell survival was determined at the indicated times (24, 48, 72 and 96 h). Values are mean ± SD from three independent experiments performed in triplicate. a indicates p < 0.001 with respect to control value. b indicates p < 0.001 with respect to the other values of the same group: (B) Cell survival of cultures treated with TNF-α (10 ng ml-¹) for 48 h in the presence of different concentrations of Dex. Values are mean ± SD from three independent experiments performed in triplicate. * indicates p < 0.05 vs TNF + vehicle, ** indicates p < 0.001 vs. TNF + vehicle.: (C) Micrograph of sub-confluent MCF-7 cells treated for 48 h with 10 ng ml^-1 of TNF-α in the presence (right panel) or absence of Dex 10 μM (left panel) (Scale bar: 25 μm).

Figure 2

The PI3K/Akt pathway does not participate in the dexamethasone-mediated protection from TNF-α-dependent cell death in MCF-7 cells: (A) Cell cultures were treated with TNF-α (10 ng ml^-1), dexamethasone (Dex) (10 μM) or both for 20 min: Western blotts of whole-cell extracts were performed with anti-phosphorylated Akt (pAkt) specific antibodies (upper panel): After stripping, membranes were re-blotted against total Akt (tAkt) (lower panel): (B) Parental cells and cells from ΔP85-expressing MCF-7 clones (A6, A8 and A10) were treated with TNF-α (10 ng ml-¹) for 20 min and total (tAkt) and phosphorylated (pAkt) Akt were determined as in A: IκB protein degradation (C) and NF-κB nuclear translocation (D) were determined by Western blot and EMSA respectively in parental and ΔP85 expressing cell clone A6 treated with TNF-α (10 ng ml^-1) for 20 min. Each blot is representative of three independent experiments. Below the blots in A, B, C, and D the bar graphs indicate the relative density of each lane with respect to control, which has an arbitrary value of 1: (E) Dexamethasone protection against TNF-α-mediated cytotoxicity was evaluated in parental MCF-7 cells (MCF-7) and three independent clones expressing the ΔP85 protein (A6, A8 and A10): Cell viability of clones incubated either with TNF-α (10 ng ml^-1) alone or with TNF-α and Dex (10 μM) for 48 hrs was determined. Values are mean ± SD from three independent experiments performed in triplicate. * indicates p < 0.01 vs TNF.

Figure 3

Inhibition of NF-κB activity abrogates the dexamethasone protection from TNF-α mediated cell death: (A) MCF-7 cells were treated as in figure 2A and DNA NF-κB binding was evaluated in nuclear extracts by EMSA: (B) Wild type MCF-7 cells and MCF-7 clones 1 and 8 expressing a dominant negative IκB protein (dnIκB) or the empty vector (GFP) were treated with TNF-α (10 ng ml^-1) for 20 min: After cell lysis, endogenous (wtIκB) and recombinant (dnIκB) proteins were detected in whole cell extract by Western blot: Total Akt (tAkt) is shown as loading control: (C) Indicated cell cultures were treated as in 2A: Then, NF-κB nuclear translocation was evaluated by EMSA. Each blot is representative of three independent experiments. Below the blots in A, B, and C the bar graphs indicate the relative density of each lane with respect to control, which has an arbitrary value of 1. In C the first bar graph represents the relative densities for dnIκBα, while the second bar graph represents the relative densities for wtIκBα: (D) The indicated cell clones were incubated with TNF-α (10 ng ml^-1) alone or TNF-α (10 ng ml^-1) and Dex (10 μM) for 48 hrs and cell viability was determined. Values are mean ± SD from three independent experiments performed in triplicate. * indicates p < 0.01 with respect to MCF-7 survival after TNF-α treatment.

Figure 4

The dominant negative IκB mutant protein stimulates the dexamethasone induced downregulation of IAPs: MCF-7 parental cells and Clone 1 of the dnIκB expressing cells were treated for 20 min with TNF-α (10 ng ml^-1) alone (A and C) or in combination with Dex (10 μM) (B and D): Protein expression of XIAP (A and B) or c-IAP (C and D) at different times was determined by Western blot: Actin was used as loading control. Each blot is representative of three independent experiments. Below the blots the bar graphs indicate the relative density of each lane with respect to the respective control, which has an arbitrary value of 1.