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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Mol Cancer Ther. 2018 Oct 23;18(1):3–16. doi: 10.1158/1535-7163.MCT-18-0863

Figure 2. Inhibition of VEGFA translation by PTC299 is coupled with inhibition of de novo pyrimidine synthesis.

Figure 2.

(A) Transcriptome profiling of PTC299-resistant HT1080 cells using microarrays. Highlighted on the left volcano plot are those genes with greater than 2-fold changes in expression with a false discovery rate (FDR) less than 0.05; the heat maps of the expression of these genes are shown on the right. (B) PTC299 selectively decreases pyrimidine nucleotides in HT1080 cells. HT1080 cells were treated with PTC299 (100 nM) or 0.5% DMSO control for 8 hours. Data are representative of two independent studies. (C) PTC299 inhibits de novo pyrimidine nucleotide synthesis. HT1080 cells in log phase growth were treated with compounds (100 nM) or vehicle control (0.5% DMSO) in the presence of 1 mM 15N-glutamine for 8 hours. 15N-labelled CTP, UTP and UMP were quantified with LC/MS. Data are representative of two independent studies. (D) PTC299 acts upstream of UMPS. Cells were treated with 1 μM PTC299 or MP-pyrazofurin or combination of the two for 8 hours. Peak area ratio of orotic acid (normalized to an internal control) for each treatment was determined and average ± SD was shown in the picture (n = 2). MP-pyrazofurin is a UMPS inhibitor; (E) Inhibition of VEGFA production in HT1080 cells by PTC299 is completely blocked by addition of exogenous uridine into the cell culture. Cells were treated with PTC299 (100 nM) or vehicle (0.5% DMSO) in the presence of 100 μM various nucleosides (indicated in the figure) for 48 hours. Data are mean ± SD, n = 2. (F) Inhibition of VEGFA production in HT1080 cells by PTC299 is dose-dependently blocked by addition of exogenous uridine. Assays were done in triplicate, and data represent average inhibition against DMSO control after VEGFA levels normalized to viable cell number measured by Celltiter-Glo. Data represent the mean ± SD, n = 2. (G) Cell cycle arrest in S-phase induced by PTC299 is completely blocked by addition of exogenous uridine or cytidine into the cell culture. HT1080 Cells were treated with PTC299 (100 nM) or vehicle (0.5% DMSO) in the presence of 100 μM of the indicated nucleosides for 24 hours. (H) A scheme showing de novo and salvage pathways of pyrimidine nucleotide synthesis. CDA: cytidine deaminase; UPP: Uridine phosphorylase; UCK: uridine cytidine kinase; CAD: Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; UMPS: UMP synthase.