Fig. 1

Gossypol disrupts the binding of mouse double minute 2 (MDM2) RING protein to vascular endothelial growth factor (VEGF) 3′UTR to inhibit the expression of MDM2 and VEGF in human breast cancer cell lines. a Protein-RNA binding assay to detect the effect of gossypol on the interaction between MDM2 protein and VEGF mRNA. MCF-7 and MDA-MB-468 cells were treated with or without gossypol before harvesting. Following co-immunoprecipitation with anti-MDM2, VEGF mRNA was detected by quantitative RT-PCR analysis. b UV crosslinking and RNA binding assay for testing the effect of gossypol on the interaction between MDM2 RING protein and VEGF 3′UTR (labeled with ³²P). Controls include VEGF non-3′UTR probe, competitor (25-fold molar excess of unlabeled VEGF 3′UTR), and control small-molecule compound (no MDM2 inhibition). c Western blot assays showing the dose–response and time-course of MDM2 and VEGF inhibition by gossypol. MCF-7 and MDA-MB-468 cells were treated with different concentrations of gossypol for 24 h and 10 μM gossypol for different times, respectively. The expression of MDM2, p53 and VEGF were detected by western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for equal protein loading. d Comparison of the effects of gossypol (labeled MX11) and other compounds (labeled MX2, MX3, MX25 and MX28) on MDM2 and VEGF expression in MDA-MB-468 cells. e, f Determination of gossypol binding to MDM2 RING protein (e) or VEGF 3′UTR (f) as detected by isothermal titration calorimetry. The upper box is raw heating power over time and the lower box is a fit of integrated energy values, normalized for each injection