Presentation Authors: Dongbo Xu*, Li Wang, Yue Wu, Gary Smith, Eric Kauffman, James Mohler, David Goodrich, Qiang Li, Buffalo, NY
Introduction: Previous studies demonstrated ERCC2 helicase domain mutations confer nucleotide excision repair (NER) deficiency and drive cisplatin sensitivity in muscle invasive bladder cancer (MIBC). We sought to investigate the role of spironolactone (SP), a NER inhibitor in potentiating cisplatin response in NER proficient bladder cancer cells and patient derived organoids.
Methods: KE1 (ERCC2 mutant, NER defective, cisplatin sensitive) bladder cells and parental KU1919 cells (ERCC2 wild-type, NER proficient, cisplatin resistant) were used. Cell viability was examined using CellTiter-glo. IC50 concentrations were calculated using a four parameter sigmoidal model and plots generated by GraphPad Prism 7. Drug combination indexes (CI) of cisplatin and SP were calculated using Chou-Talalay method. DNA damage response and apoptosis signaling was examined by Western blot of gamma-H2AX, P53, cleaved Caspase-3 and cleaved PARP.
Results: KU1919 cells are more sensitive to SP (IC50 = 29.2 vs IC50 = 37.4 Î¼M). Combinations of cisplatin and SP (5 Î¼M:40 Î¼M) resulted in strong synergy in KU1919 cells (CI = 0.29), whereas mild synergy of cisplatin and SP (1 Î¼M:40 Î¼M) was observed in KE1 cells (CI = 0.66). In a patient derived organoid model, 10 ÂµM SP alone exhibited no cytotoxicity, and combination with 10 Î¼MSP and 25 Î¼Mcisplatin significantly decreased cell survival compared with 25 Î¼M cisplatin alone (17.9% vs 32.1%, p < 0.01). Following 24 hours 5 Î¼M cisplatin treatment, we observed an increased DNA damage response genes (gamma-H2AX, P53) and activation of apoptosis (cleaved Caspase-3 and cleaved PARP) in KE1 cell, but not in KU1919 cells. The combination of SP and cisplatin induced activation of apoptosis (cleaved Caspase-3 and cleaved PARP expression) in KU1919 cells.
Conclusions: Targeting NER pathway by spironolactone in bladder cancer cells enhances cisplatin-induced DNA damage signaling and subsequent cell apoptosis. These findings provide a proof of concept and rational to combine NER inhibitors with platinum based chemotherapy in systemic treatment of bladder cancer.
Source of Funding: Friends of Urology at Roswell Park Comprehensive Cancer Center