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AZD1775 induces toxicity through double-stranded DNA breaks independently of chemotherapeutic agents in p53-mutated colorectal cancer cells

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posted on 09.11.2017 by Peter John Webster, Anna Tiffany Littlejohns, Hannah Jane Gaunt, K. Raj Prasad, David John Beech, Dermot Anthony Burke

AZD1775 is a small molecule WEE1 inhibitor used in combination with DNA-damaging agents to cause premature mitosis and cell death in p53-mutated cancer cells. Here we sought to determine the mechanism of action of AZD1775 in combination with chemotherapeutic agents in light of recent findings that AZD1775 can cause double-stranded DNA (DS-DNA) breaks. AZD1775 significantly improved the cytotoxicity of 5-FU in a p53-mutated colorectal cancer cell line (HT29 cells), decreasing the IC50 from 9.3 μM to 3.5 μM. Flow cytometry showed a significant increase in the mitotic marker pHH3 (3.4% vs. 56.2%) and DS-DNA break marker γH2AX (5.1% vs. 50.7%) for combination therapy compared with 5-FU alone. Combination therapy also increased the amount of caspase-3 dependent apoptosis compared with 5-FU alone (4% vs. 13%). The addition of exogenous nucleosides to combination therapy significantly rescued the increased DS-DNA breaks and caspase-3 dependent apoptosis almost to the levels of 5-FU monotherapy. In conclusion, AZD1775 enhances 5-FU cytotoxicity through increased DS-DNA breaks, not premature mitosis, in p53-mutated colorectal cancer cells. This finding is important for designers of future clinical trials when considering the optimal timing and duration of AZD1775 treatment.

Funding

This research was supported by the following funding: David Beech: British Heart Foundation (FS/14/22/30734). David Beech: British Heart Foundation (FS/13/23/30122). David Beech: Medical Research Council (G1002076). Peter Webster: Leeds Teaching Hospitals Charitable Foundation (9R01/14–06).

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