Abstract
Procaspase-3-activating compound 1 (PAC-1) induces procaspase-3 activation via zinc chelation. However, whether PAC-1 employs other mechanisms remains unknown. Here we systematically screened for potent PAC-1 targets using 29 enhanced green fluorescent protein-labeled reporter cell lines and identified hypoxia-inducible factor 1α (HIF1α) and RAD51 pathways as PAC-1 targets. These results were verified in HepG2 cells and two other cancer cell lines. Mechanistically, PAC-1 specifically blocked HIF1α hydroxylation and upregulated HIF1α target genes. In addition, DNA damage, G1/S cell cycle arrest, and the inhibition of DNA synthesis were induced following PAC-1 administration. Interestingly, by using ferrozine-iron sequestration and iron titration assays, we uncovered the iron sequestering capacity of PAC-1. Additionally, the expression levels of iron shortage-related genes were also increased in PAC-1-treated cells, and iron (II) supplementation reversed all of the observed cellular responses. Thus, our results indicate that PAC-1 induces HIF1α stabilization and DNA damage by sequestering ferrous iron.
Highlights
Escaping apoptosis represents one of the hallmarks of cancer, and the induction of apoptotic cell death is a rational anticancer strategy
A 30 μM dose of procaspase-3activating compound 1 (PAC-1) induced a similar effect to the maximum effect observed with 100 μM of BP in hypoxia-inducible factor 1α (HIF1α) assays and approximately half that observed in the presence of 10 μM of camptothecin in RAD51 assays
During a comprehensive screening assay performed in our laboratory, PAC-1 was found to induce cellular responses, including the stabilization of HIF1α and the formation of RAD51 foci, in a concentration-dependent manner in enhanced green fluorescent protein (EGFP)-labeled reporter cell lines
Summary
Escaping apoptosis represents one of the hallmarks of cancer, and the induction of apoptotic cell death is a rational anticancer strategy. The discovery of procaspase-3activating compound 1 (PAC-1) may overcome this limitation. By activating procaspase-3 to generate caspase-3, the main apoptosis effector, PAC-1 bypasses the complex upstream pro-apoptotic signaling cascades and directly induces apoptotic cell death[2]. The first report describing PAC-1 did not address the mechanisms underlying procaspase-3 activation, and these still remain unclear to date[8]. Hergenrother and co-workers reported that PAC-1 activates procaspase-3 by chelating the zinc ions required for its activity[9]. This mechanism has been widely accepted, it might not account for the full function of PAC-1. The antitumor effect of PAC-1 has not been so far validated in humans
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