Makoto Isono, Michèle J. Hoffmann, Maria Pinkerneil, Akinori Sato, Martin Michaelis, Jindrich Cinatl Jr., Günter Niegisch and Wolfgang A. Schulz
Institution
Department of Urology, Medical Faculty, Heinrich-Heine-University
Country
Germany
Year
2017
Journal
Journal of Experimental & Clinical Cancer Research
Abstract
Background: More 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.
Methods: Viability, 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.
Results: Combined 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.
Conclusions: AZD7762 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.