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doi: 10.1242/10.1242/jcs.00097

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Intrinsic actions of IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelial cells: inhibition or accentuation of attachment and survival is dependent upon the presence of fibronectin

Division of Surgery, Department of Hospital Medicine, Bristol Royal Infirmary, Bristol, BS2 8HW, UK

View larger version (35K): [in a new window]   Fig. 1. The effects of increasing doses of an RGD-containing fibronectin fragment and IGFBPs on the adhesion of Hs578T cells to ECM. The graphs show the percentage of cells attached to ECM gel. Cells were treated with (A) RGD (0-100 µg/ml), RGD (10 µg/ml) or RGE (10µg/ml) and RGD (10 µg/ml) with (B) IGFBP-2 (50-800 ng/ml), (C) IGFBP-3 (0-100 ng/ml), (D) IGFBP-5 (0-100 ng/ml) or (E) IGFBP-4 (100 ng/ml) and (F) IGFBP-1 (100 ng/ml) or IGFBP-6 (100 ng/ml). Where * is considered statistically significant relative to the control in all experiments as defined by Bonferroni/Dunn post-hoc test. Data represent the mean of three experiments each in triplicate±standard error of the mean (s.e.m.).

View larger version (33K): [in a new window]   Fig. 2. The effects of the IGFBPs on the adhesion of Hs578T cells to laminin, collagen type IV and fibronectin. The graphs show the percentage of cells attached. Cells were treated with RGD (10 µg/ml), IGFBP-1 (100 ng/ml), IGFBP-2 (100 ng/ml) IGFBP-3 (100 ng/ml), IGFBP-4 (100 ng/ml), IGFBP-5 (100 ng/ml) and IGFBP-6 (100 ng/ml) before plating on either laminin (5 µg/ml) (A-C), collagen type IV (0.25 µg/ml) (D-F) or fibronectin (0.25 µg/ml) (G-I). * indicates a statistically significant difference relative to the control in each experiments as defined by Bonferroni/Dunn post-hoc test. Data represent the mean of three experiments each in triplicate±s.e.m.

View larger version (21K): [in a new window]   Fig. 3. The effects of IGF-I on the actions of IGFBP-3 and -5 on the adhesion of Hs578T cells to ECM. The graphs show the percentage of cells attached. Cells were treated with (A) RGD (10 µg/ml), IGFBP-3 (100 ng/ml), IGF-I (50 ng/ml) or IGF-I and IGFBP-3 together or (B) RGD, IGFBP-5 (100 ng/ml), IGF-I or IGF-I and IGFBP-5 together. * indicates statistically significant differences as defined by Bonferroni/Dunn post-hoc test for RGD<CT; IGFBP-5>CT; IGFBP-3<CT; IGFBP-3+IGF-I>IGFBP-3; IGFBP-5+IGF-I<IGFBP-5. The data represent the mean of three experiments each in triplicate±s.e.m.

View larger version (22K): [in a new window]   Fig. 4. The effects of an RGD-containing fibronectin fragment or soluble thrombospondin on the actions of IGFBP-3 and -5 on the attachment of Hs578T cells to ECM. The graphs show the percentage of cells attached. Cells were treated with either (A) RGD (10 µg/ml), IGFBP-3 (100 ng/ml) or IGFBP-3 and RGD, (B) RGD, IGFBP-5 (100 ng/ml) or IGFBP-5 and RGD, (C) RGD, IGFBP-3, IGFBP-5, Thrombospondin (TSP) (0.01 µg/ml), IGFBP-3 and TSP, or IGFBP-5 and TSP, or (D) RGD, IGFBP-5, TSP or IGFBP-5 and TSP where * RGD<CT, IGFBP-3<CT; IGFBP-5>CT, IGFBP-5+RGD<IGFBP-5 is considered statically significant as defined by Bonferroni/Dunn post-hoc test. Data represent the mean of three experiments each in triplicate±s.e.m.

View larger version (18K): [in a new window]   Fig. 5. The effects an RGD-containing fibronectin fragment on the actions of IGFBP-3 and -5 on C2-induced apoptosis. The graphs show the percentage of dead cells. Cells were treated with either (A) IGFBP-3 (100 ng/ml) or (B) IGFBP-5 (100 ng/ml) ± a non-apoptotic dose of RGD (10 µg/ml) for 24 hours followed by a co-incubation with an apoptotic dose of C2-ceramide. * indicates a statistically significant difference relative to the control in each experiments as defined by Bonferroni/Dunn post hoc test. Data represent the mean of three experiments each in triplicate±s.e.m.

View larger version (20K): [in a new window]   Fig. 6. The effects of growing Hs578T cells on fibronectin on the ability of IGFBP-5 and -3 to modulate C2-induced death. The graphs show the percentage of dead cells. Cells were seeded on plastic (dark grey bars) or on fibronectin (light grey bars) coated wells for 24 hours prior to being switched to SFM for a further 24 hours before a pre-incubation with either (A) IGFBP-5 (100 ng/ml) or (B) IGFBP-3 (100 ng/ml) for 24 hours followed by a co-incubation with an apoptotic dose of ceramide. * is considered statically significant where on plastic C2>CT; IGFBP-5+C2<C2 and IGFBP-3+C2>C2, and on fibronectin IGFBP-5>CT, IGFBP-3+C2<C2. Data represent the mean of three experiments each in triplicate±s.e.m.