Discovery of Novel Small Molecule Inhibitors of VEGF Expression in Tumor Cells Using a Cell-Based High Throughput Screening Platform
Fig 2
Structure and biological activity of a representative HTS hit, PTC-858.
(A) Chemical structure of PTC-858 (6-bromo-1-(1H-pyrrol-1-yl)-2,3,4,9-tetrahydro-1H-carbazole). (B) Inhibition of VEGF production in HeLa cells by PTC-858. Results are expressed as percent inhibition of hypoxia-induced VEGF production relative to vehicle-treated controls from a representative study. Cytotoxicity was performed in parallel to the VEGF ELISA using a Celltiter Glo assay kit (Promega). (C) Western blot analysis of effects of PTC-858 on membrane bound VEGF expression in HT-1080 cells. The same amount of protein for each cell lysate was loaded for western blot analysis. β-actin was used as an internal loading control. (D) PTC-858 preferentially inhibits reporter gene expression under the control of VEGF UTRs compared to the control UTRs derived from the vector. The stable cell lines B9 and B12 used in this study were generated in HEK293 cells transfected with the constructs shown in the diagrams on the top of the graphs. The activity of luciferase was measured with the substrate Bright-Glow (Promega). (E) PTC-858 has no dose-dependent effects on VEGF mRNA expression. Endogenous VEGF and β-actin message levels in the HeLa cells were determined using real-time PCR (pre-designed primer sets purchased from Applied Biosystems). Results shown in the graph from a typical study were normalized to the internal control of β-actin. Assay was done in triplicate and average VEGF mRNA levels from DMSO treated samples were arbitrarily set at 1.