![Effects of BIBF 1000 on stroma-derived interleukin-6 (IL-6) release and related inhibition of MAPK phosphorylation. (A-B) Effects of BIBF 1000 on bone marrow stroma cell (BMSC)–derived IL-6 release: BIBF 1000 (0.5 μM) significantly inhibited basal IL-6 release from BMSCs and IL-6 secretion induced by VEGF121, VEGF165, bFGF, and TGF-β (105.3 ± 28.1 versus 37.3 ± 16.1 [vehicle control versus BIBF 1000]; 169.2 ± 23.4 versus 45.3 ± 16.6 [VEGF121 versus VEGF121 + BIBF 1000]; 172.0 ± 22.3 versus 60.1 ± 21.9 [VEGF165 versus VEGF165 + BIBF 1000]; 185.1 ± 17.3 versus 44.7 ± 14.3 [bFGF versus bFGF + BIBF 1000]; 499.5 ± 33.8 versus 195.7 ± 36.7 3 [TGF-β versus TGF-β + BIBF 1000], respectively; P < .001). In contrast, IL-6 release on exposure to TNF-α or IL-1β was not significantly reduced (754.7 ± 122.6 versus 547.4 ± 83.0 [TNF-α versus TNF-α + BIBF 1000]; 1881.7 ± 122.3 versus 1855.8 ± 122.9 [IL-1β versus IL-1β + BIBF 1000]). IL-6 concentrations were determined in triplicates from supernatants of BMSC cultures derived from different patients with myeloma (n = 4). BMSCs were starved prior to incubation with either VEGF121, VEGF165, bFGF, TGF-β, TNF-α, or IL-1β ± BIBF 1000 and kept in serum-free conditions for 72 hours. Data are presented as means ± SE. The Wilcoxon test was used to identify differences between controls and corresponding BIBF 1000–treated BMSCs (means ± SE). (C) Inhibition of MAPK phosphorylation by BIBF 1000 in BMSCs: subconfluent BMSCs were starved for 4 hours in serum-free medium and subsequently exposed for 2 hours to either VEGF (50 ng/mL), bFGF (10 ng/mL), TGF-β (10 ng/mL), TNF-α (10 ng/mL) or IL-1β (10 ng/mL) in the absence or presence of BIBF 1000 (1, 5, or 10 μM), to BIBF alone (10 μM) or to the MAPK-inhibitor PD98059 (20 μM). Proteins were extracted, boiled, and separated by SDS-electrophoresis. Subsequent immunoblotting was performed using a rabbit polyclonal p-MAPK (p44/42) antibody and a corresponding goat anti–rabbit HRP–conjugated secondary antibody. Notably, inhibition of MAPK phosphorylation by BIBF 1000 was most effective in BMSCs exposed to VEGF, bFGF, or TGF-β. The results shown are representative of 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/107/5/10.1182_blood-2004-11-4250/2/zh80050692090002.jpeg?Expires=1720000833&Signature=syfx04uQXJ~XGdP8LnCGUdX4VAGxn15JVXSH~ojBqDcgUcSBnhxSWfR9Mvn1RgZTHxiM~YEFeds2AXFqQNTyDSp6BiZd6HFdhrKZwlcIhVIy3q8YPXSTsUxzq3X3ALdfumIlqJJbHZS~dHn2H~OTOebwbqvMYYBAp5vdm-8dSKLYh5vY-p~zgjN-1veRIGZPTLvfzpKuWiHGh6CHHwhbOL4TzLUt77~houTeCTVhiAQbKqpCkCkGVC60smHxjpVRkNgZSzSYGS9dhQWp29Rn8aRo10dXzrjLxcA4shN4X40nQDb-EK8Wp0izHZfaRs-ofJY4uyepkKTWzLQ5o4uFqw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effects of BIBF 1000 on stroma-derived interleukin-6 (IL-6) release and related inhibition of MAPK phosphorylation. (A-B) Effects of BIBF 1000 on bone marrow stroma cell (BMSC)–derived IL-6 release: BIBF 1000 (0.5 μM) significantly inhibited basal IL-6 release from BMSCs and IL-6 secretion induced by VEGF121, VEGF165, bFGF, and TGF-β (105.3 ± 28.1 versus 37.3 ± 16.1 [vehicle control versus BIBF 1000]; 169.2 ± 23.4 versus 45.3 ± 16.6 [VEGF121 versus VEGF121 + BIBF 1000]; 172.0 ± 22.3 versus 60.1 ± 21.9 [VEGF165 versus VEGF165 + BIBF 1000]; 185.1 ± 17.3 versus 44.7 ± 14.3 [bFGF versus bFGF + BIBF 1000]; 499.5 ± 33.8 versus 195.7 ± 36.7 3 [TGF-β versus TGF-β + BIBF 1000], respectively; P < .001). In contrast, IL-6 release on exposure to TNF-α or IL-1β was not significantly reduced (754.7 ± 122.6 versus 547.4 ± 83.0 [TNF-α versus TNF-α + BIBF 1000]; 1881.7 ± 122.3 versus 1855.8 ± 122.9 [IL-1β versus IL-1β + BIBF 1000]). IL-6 concentrations were determined in triplicates from supernatants of BMSC cultures derived from different patients with myeloma (n = 4). BMSCs were starved prior to incubation with either VEGF121, VEGF165, bFGF, TGF-β, TNF-α, or IL-1β ± BIBF 1000 and kept in serum-free conditions for 72 hours. Data are presented as means ± SE. The Wilcoxon test was used to identify differences between controls and corresponding BIBF 1000–treated BMSCs (means ± SE). (C) Inhibition of MAPK phosphorylation by BIBF 1000 in BMSCs: subconfluent BMSCs were starved for 4 hours in serum-free medium and subsequently exposed for 2 hours to either VEGF (50 ng/mL), bFGF (10 ng/mL), TGF-β (10 ng/mL), TNF-α (10 ng/mL) or IL-1β (10 ng/mL) in the absence or presence of BIBF 1000 (1, 5, or 10 μM), to BIBF alone (10 μM) or to the MAPK-inhibitor PD98059 (20 μM). Proteins were extracted, boiled, and separated by SDS-electrophoresis. Subsequent immunoblotting was performed using a rabbit polyclonal p-MAPK (p44/42) antibody and a corresponding goat anti–rabbit HRP–conjugated secondary antibody. Notably, inhibition of MAPK phosphorylation by BIBF 1000 was most effective in BMSCs exposed to VEGF, bFGF, or TGF-β. The results shown are representative of 3 independent experiments.
