Causing UPS Stress for Cervical Cancer Treatment Figure 7. Stabilization of p53, reduction in cyclin D1 and onset of apoptosis following RAMB1 treatment. A. Immunoblot analysis of p53 expression level in CaSki cells treated with the indicated concentration of RAMB1 over a period of 8 hours. Equal loading is each lane was verified by amido black staining. B. Top left panel: Immunoblot analysis of cyclin D1 expression level in CaSki cells with or without 10 mM RAMB1 for a period up to 8 hours. Protein loading was examined using an antibody against b-actin. Top right panel: Quantification of the cyclin D1/b-actin ratio. Bottom left panel: Immunoblot analysis of cyclin D1 expression level in CaSki cells with or without RAMB1, at the indicated concentration for a period of 8 hours. Equal protein loading in each lane was verified by using an antibody against b-actin. Bottom right panel: Quantification of the cyclin D1/b- actin ratio. C. Left panel. HeLa cells were treated with 10 mM of RAMB1 for 8 hours and analyzed by flow cytometry after staining for annexin V binding and 7-AAD incorporation. Middle panel: Immunoblot analysis of full length and cleaved PARP in HeLa cells treated 10 mM of RAMBs over a period of 8 hours. Protein loading was assessed using an antibody against b-actin. Right panel: Quantification of cleaved PARP/b-actin ratio. doi:10.1371/journal.pone.0023888.g007 cells as compared to mock-treated cultures when used at concentrations as low as 0.62 mM. We next quantified the impact of RAMB1 treatment on the reduction of colony number and size in 16102 HeLa cervical cancer cells treated with or without 0.15 or 0.5 mM RAMB1. As shown in Figure 8B (left panel) RAMB1 exposure resulted in 40% and 60% reduction in colony number when exposed to 0.15 or 0.3 mM RAMB1 respectively. RAMB1 treatment also resulted in 50% and 80% reduction in colony size in mock versus RAMB1 treated HeLa cells as shown in Figure 8B (middle panel). A representative example of crystal violet staining in HeLa cervical cancer cells following RAMB1 exposure is given in Figure 8B (right panel). The increase in the steady-levels of Hsp90 and formation of aggresome-like structure following RAMB1 treatment suggest compensatory activation of lysosomal pathway of protein degrada- tion (Fig. 6). Therefore, we hypothesized that RAMB1 treatment would result in synergistic cell killing when combined with an inhibitor of this lysosomal pathway. To test this hypothesis, we compared the effect of combined treatment with RAMB1 and the lysosome inhibitor Chloroquine on the viability of HeLa cervical cancer cells. Consistent with our hypothesis, submaximal doses of RAMB1 and Chloroquine act synergistically to cause enhanced cytotoxicity in HeLa cervical cancer cells with a Combination Index (CI) of 0.48 observed using 1.45 mM RAMB1 and 7.23 mM Chloroquine (Figure 7C). Similar results were obtained with SiHa and CaSki cell lines (data not shown). Taken together, this suggest that the combinatorial approach of inhibiting ubiquitin-dependent protein degradation upstream of proteasome and lysosomal pathway could lead to development of combination therapeutic strategies where the side effects of each individual compounds can be reduced without compromising the anti-cancer activity of the treatments. Discussion Numerous naturally-derived compounds are either FDA approved or currently under evaluation for the treatment of human malignances. Among these natural compounds with PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23888