Causing UPS Stress for Cervical Cancer Treatment Figure 4. RAMB treatment fails to inhibit the 20S proteasome proteolytic activities. Purified 20S proteasomes were treated for 30 min with or without RAMB compounds or Bortezomib, here used as positive control, at the indicated concentrations and the specific fluorogenic substrates for chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolyzing-like hydrolytic proteasome capacities were subsequently added. A representative example of two independent experiments is shown. doi:10.1371/journal.pone.0023888.g004 cancer cells following RAMBs exposure occurs concomitantly with the accumulation of polyubiquitated proteins without the direct inhibition of 20S proteasomal activity. RAMB1 treatment induces aggresome formation and triggers heat-shock responses We and others have shown that inhibition of ubiquitin-mediated protein degradation via proteasomal inhibition triggers heat-shock and unfolded protein responses including formation of cytopro- tective structures called aggresomes as a mechanism to compen- sate for inhibition of proteasomal functions and increasing levels of UPS stress within cancer cells [4,6,39]. To test whether the rapid accumulation of poly-ubiquitinated protein upon RAMB1 treat- ment occurs concomitantly with heat-shock protein responses (UPR) we monitored the protein expression levels of Hsp90 in HeLa cervical cancer cells exposed to 10 mM RAMB1–3 for 8 hours. As shown in Figure 6A (left panel) immunoblot analysis of Hsp90 protein expression levels revealed that a strong pattern of accumulation of Hsp90 in RAMB1-treated versus mock-treated HeLa cell cultures (similar results were obtained with the other derivative of the series, not shown). A semi quantitative analysis of the Hsp90 protein levels show that the GAPDH-normalized levels of polyubiquitinated proteins are nearly 2-fold higher in treated versus non-treated cells is shown in Figure 6A (right panel). Next, we hypothesized that accumulation of poly-ubiquitinated proteins following RAMB compound exposure would lead to activation of alternative compensatory pathways to ubiquitin- mediated protein degradation, specifically to lysosomal pathway activation. To test this hypothesis we monitored the sub-cellular localization of ubiquitin by immunofluorescence microscopy analysis in HeLa cervical cancer cells exposed to 10 mM of RAMB1. As shown in Figure 6B immunofluorescence analysis of poly-ubiquitinated proteins in HeLa cells treated with RAMB1 reveals the presence of vimentin-caged, ubiquitin-positive, aggre- somes structure consistent with that previously described upon treatment with Bortezomib [6]. Taken together, these findings indicate that in RAMB1-treated cells the accumulation of poly- ubiquitinated results in similar cytoprotective responses as inhibition of proteasome catalytic activities by Bortezomib, but occurs through a mechanism independent from it. RAMB1 treatment leads to p53 stabilization, cyclin D1 destabilization and onset of apoptosis The E6 oncoprotein of HPV exerts its oncogenic activity by targeting p53 and other tumor suppressor proteins for rapid ubiquitin-mediated proteasomal degradation. This reduces the level of this key cellular cell cycle regulator without mutation of p53. To test whether the impairment of ubiquitin-mediated protein degradation following RAMBs treatment leads to stabilization of p53 as a potentially contributing mechanism initiating cell death, we examined the expression levels of p53 following 6 hours exposure to compounds 10 mM of RAMBs. As Figure 5. RAMB treatment fails to inhibit proteasomal activity in living cells. Left panel: Luciferase activity in lysates of Ub-FL or FL-vector transfected cells either with or without treatment with RAMB compounds or Bortezomib were quantified in Relative Luminescence Units (RLU) and expressed as % of control. Error bars are Standard Deviation (SD) for three independent experiments. Middle panel: Quantification of the Ub-FL/FL ratio. Right panel: Luminescence activity in cell lysates derived from CaSki cervical cancer cells with or without RAMB or Botezomib treatment quantified in Relative Luminescence Units (RLUs). *, P,0.05, **, P,0.02. doi:10.1371/journal.pone.0023888.g005 PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23888