Effect of soluble factors derived from ZR 75.30 breast cancer cells on endothelial activation

Abstract

In this study, we analyzed soluble factors secreted by two Estrogen Receptor Positive (ER-α) human breast cancer cell lines, ZR 75.30 (luminal B) and MCF7 (luminal A), and evaluated their effect on endothelial activation. The composition of tumoral soluble factors (TSFs) was analyzed by ELISA (Bio-Plex). TSFs from ZR 75.30 cells expressed higher levels of TNF, IFN-γ, IL-6, and IL-8 compared to TSFs from MCF-7 cells. TSFs from ZR 75.30 cells induced a pro-adhesive phenotype in human umbilical vein endothelial cells (HUVECs), as characterized by increased monocytic cell adhesion, adhesion molecule expression and NF-κB activation and decreased IκB-α expression. Conversely, TSFs from MCF-7 cells exerted none of these effects on HUVECs. We then added TNF, IFN-γ, IL-6 or IL-8 alone or in combination with TSFs from MCF-7 cells to HUVECs. Only the combinations that included TNF induced endothelial activation. A neutralizing antibody against IL-1β (this cytokine was not measured in the ELISA) had a modest blocking effect on cellular adhesion or the expression of adhesion molecules induced by TSFs from ZR 75.30 cells in HUVECs. However neutralizing antibodies against TNF, IFN-γ, IL-6 or IL-8 had no effect. Our results suggest that although TNF is an inducer of endothelial cell activation, it is not the only molecule that is responsible for this effect in TSFs from ZR 75.30 cells.

Keywords: TNF; Tumoral soluble factors; breast cancer; endothelial activation; endothelial cell adhesion molecules.

Figure 1

Endothelial activation induced by TSFs from ZR 75.30 and MCF-7 cells. A: Induction of pro-adhesive phenotype in HUVECs by TSFs derived from breast cancer cells (MCF-7 or ZR 75.30) after 3 h of treatment, followed by 3 h of co-incubation with U937 cells. B: Western blot of adhesion molecules after 6 h of treatment. C: Western blot of IκB-α after 20 min of treatment. D: Activation of NF-κB in nuclear protein extracts from HUVECs treated for 20 min was analyzed by EMSA. HUVECs were treated with TNF (10 ng/ml) (C [+]), LPS (10 ng/ml), polymyxin-D (10 μg/ml) or TSFs from MCF-7 or ZR 75.30 cells (5 μg/ml). C (-): untreated cells.

Figure 2

Endothelial activation induced by TNF, IFN-γ, IL-6 or IL-8. (A) Induction of pro-adhesive phenotype in HUVECs by recombinant cytokines alone or in combination (All), as in Figure 1A. (B) Same as (A) but with different concentrations of the recombinant cytokines, at logarithmic intervals. (C) Western blot of of adhesion molecules after 6 h of treatment (upper 4 lanes) and IκB-α after 20 min of treatment (lower 2 lanes. (D) Activation of NF-κB in nuclear protein extracts from HUVECs treated for 20 min was analyzed by EMSA. HUVECs were treated with TNF (1.3 ng/ml), IFN-γ (2.4 ng/ml), IL-6 (2.8 ng/ml), or IL-8 (1.2 ng/ml). Legends as in Figure 1.

Figure 3

Endothelial activation by TSFs of MCF-7 supplemented with one or all of the following recombinant cytokines: TNF, IFN-γ, IL-6, or IL-8. A: Adhesion Assay, as in Figure 1A. B: Western blot of adhesion molecules after 6 h of treatment. C: Western blot of IκB-α after 20 min of treatment. D: Activation of NF-κB in nuclear protein extracts from HUVECs treated for 20 min was analyzed by EMSA. Legends as in Figure 1. Cytokine concentrations as in Figure 2.

Figure 4

Endothelial activation by TSFs from ZR 75.30 treated with neutralizing antibodies against one or all of the following cytokines TNF, IFN-γ, IL-6, or IL-8. A: Upper graph: Induction of pro-adhesive phenotype in HUVECs by depleted TSFs from ZR 75.30 cells, using individually antibodies or their combination, as in Figure 1A. Lower part: Western blot of adhesion molecules after 6 h of treatment. HUVECs were treated with TSFs (5 μg/ml), and/or anti-TNF (1.4 ng/ml), anti-IFN-γ (2.2 ng/ml), anti-IL-6 (3.0 ng/ml), and/or anti-IL-8 (1.4 ng/ml) antibodies. B: Induction of pro-adhesive phenotype in HUVECs by depleted TSFs from ZR 75.30 cells, using neutralizing antibodies against IL-1β. Legends as in Figure 1, anti-TNF, anti-IFN-γ, anti-IL-6, anti-IL-8 and anti-IL-1β indicated addition of corresponding neutralizing antibodies.