title: AZD1775 induces toxicity through double-stranded DNA breaks independently of chemotherapeutic agents in p53-mutated colorectal cancer cells.
abstract: 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 IC50from 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.
authors.affiliation: a School of Medicine, University of Leeds , Leeds , UK.
authors.forename: Peter John
authors.initials: PJ
authors.lastname: Webster
authors.affiliation: a School of Medicine, University of Leeds , Leeds , UK.
authors.forename: Anna Tiffany
authors.identifier: http://orcid.org/0000-0002-7566-0121
authors.initials: AT
authors.lastname: Littlejohns
authors.affiliation: a School of Medicine, University of Leeds , Leeds , UK.
authors.forename: Hannah Jane
authors.initials: HJ
authors.lastname: Gaunt
authors.affiliation: b Department of Hepatobiliary and Transplant Surgery , St. James's University Hospital , Leeds , UK.
authors.forename: K Raj
authors.initials: KR
authors.lastname: Prasad
authors.affiliation: a School of Medicine, University of Leeds , Leeds , UK.
authors.forename: David John
authors.initials: DJ
authors.lastname: Beech
authors.affiliation: a School of Medicine, University of Leeds , Leeds , UK.|c Department of Colorectal Surgery , St. James's University Hospital , Leeds , UK.
authors.forename: Dermot Anthony
authors.initials: DA
authors.lastname: Burke
mesh_terms: D000970:Antineoplastic Agents / Q000276:immunology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D005472:Fluorouracil / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D011744:Pyrimidinones; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; D016159:Tumor Suppressor Protein p53; C505407:CASP3 protein, human; D053148:Caspase 3; C549567:adavosertib; D005472:Fluorouracil
keywords: DNA damage; cell cycle; checkpoint kinase; colorectal cancer; mitosis