The aberrant activity of deubiquitinating enzymes (DUBs) is linked to carcinogenesis and chemoresistance. RA-9 is a potent, cell-permeable inhibitor of 19S regulatory particle (RP)-associated DUBs. It blocks ubiquitin-dependent protein degradation without affecting the activity of 20S proteasome. Treatment of ES-2 cells with RA-9 (5 - 40 μM) resulted in a concentration-dependent accumulation of poly-ubiquitinated proteins in cells. RA-9 treatment (0.05 - 50 μM, 100 - 300s) dose- and time-dependently reduced the activity of 19S RP-associated DUB in ES-2 cells. The exposure of cisplatin-sensitive ovarian cancer cell lines TOV-21G and ES-2 with increasing concentrations of RA-9 (10 - 30 μM) for 48 hours decreased cell viability in a dose-dependent fashion. RA-9 exposure (1.25 μM and 5 μM) for 18 hours also led to a concentration-dependent increase in the fraction of G2-M cell cycle phase in ES-2 cells. Treatment of ES-2 cell lysates with 5 μM RA-9 for 24 hours resulted in time-dependent accumulation of the cleaved formed of PARP staring at 8-hour post-treatment. In NCr nu/nu mice inoculated with ES-2 ovarian cancer cells, a significant reduction in tumor burden was observed in the group treated with RA-9 (i.p., 5 mg/kg, one-day on, two-day off schedule) compared to the controls by day 5 of treatment[1].
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