Causing UPS Stress for Cervical Cancer Treatment Figure 2. Effect of RAMB treatment upon cervical cancer cell lines and primary human keratinocytes. Cultures of HPV-transformed cervical cancer cells (SiHa, CaSki and ME180) or primary human keratinocytes were treated with the indicated concentrations of RAMB1 (left panel) or RAMB4 (right panel) over a period of 48 hours. Cell viability was determined by XTT assay and plotted as a fraction of the untreated control cultures. doi:10.1371/journal.pone.0023888.g002 catalytic activities of the proteasomes, we tested for the ability of RAMB1 and RAMB4 (which induced a greater accumulation of polyubiquitinated protein than the other RAMBs, Figure 3) to inhibit specific catalytic sub-units within the 20S proteasome. Specifically the RAMBs were tested for their capacities to inhibit the chymotrypsin-like (CT-like), trypsin-like (T-like) and peptidyl- glutamyl peptide hydrolyzing-like (PGPH-like) activities in 20S purified proteasome pre-exposed to escalating doses up to 10 mM of RAMBs for a period of 30 minutes following addition of fluorogenic substrates [38]. The FDA licensed proteasome inhibitor Bortezomib was used as positive control. As shown in Figure 4 the profile of proteasome inhibition shows that unlike Bortezomib, RAMB1 and RAMB4 treatment failed to inhibit proteasomal functions when tested to concentrations up to 10 mM (similar results were obtained with the other compounds of the series, not shown). In order to assess whether the failure to inhibit proteasomal function in vitro can be recapitulated in the intact proteasome found in living cells, we utilized two different approaches. First, we utilized the ubiquitin-luciferase bioluminescent reporter 4XUb- FL, which resists cleavage by ubiquitin hydrolases [26], to transfect HeLa cells. Using Ub-FL and FL transfected HeLa cells we have monitored luciferase activity following exposure to RAMBs over a period of 6 hours. As shown in Figure 5 (left) unlike Bortezomib or our recently identified proteasome inhibitor RA1 [14], here used as proteasome inhibitors positive controls, RAMB1 or RAMB4 treatment induced a weaker stabilization of the Ub-FL reporter when tested at concentration up to 20 mM than seen for either RA-1 or Bortezomib. Quantification of Ub-FL/FL ratio in mock versus RAMBs exposed culture is provided in Figure 5 (middle panel). Next, the lack of inhibition of 20S proteasomal activity in RAMB-treated cells was confirmed by measuring the residual fluorogenic activity in 20S proteasome purified from CaSki cervical cancer cells pre-exposed to RAMB1 or RAMB4 for 4 hours. As shown in Figure 5 (right panel), unlike Bortezomib, RAMB1 treatment failed to inhibit the chymotryptic activity of proteasomes when tested to concentrations up to 20 mM. Taken together, this suggests that the loss of cell viability in cervical Figure 3. Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells. Left panel: immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 mM RAMBs. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. Right panel: Quantification of the Ubiquitin/GAPDH ratios. doi:10.1371/journal.pone.0023888.g003 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23888