Abstract
BackgroundMore effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer.MethodsViability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs.ResultsCombined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762.ConclusionsAZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.
Highlights
More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide
The combination of gemcitabine and AZD7762 significantly reduced the viability of Urothelial carcinoma cell line (UCC) In MTT assays, up to 40 nM AZD7762 had little effect of its own, but enhanced the effects of gemcitabine on the viability of four different urothelial carcinoma cell lines
We investigated whether the combination of gemcitabine and AZD7762 affects the clonogenicity of urothelial carcinoma cells
Summary
More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. Bladder cancer is a major cause of morbidity and mortality worldwide, with about 380,000 new cases and 150,000 deaths per year [1]. ATR is activated by recruitment to tracts of single strand DNA (ssDNA), and the ATR-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage [11]. These kinases subsequently activate the downstream serine/threonine kinases, checkpoint kinase 1 and 2 (CHK1 and CHK2). It remains unknown to which extent AZD7762 may promote chemotherapy-induced tumour cell death in urothelial cancer cell lines (UCCs)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have