Causing UPS Stress for Cervical Cancer Treatment Figure 8. RAMB1 treatment prevents anchorage-dependent colony formation and synergizes with Chloroquine to kill cervical cancer cells. A. Equal numbers of SiHa and Caski cells (103) were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentrations over a period of 10 days. Colonies were visualized by crystal violet staining. B. Left panel: Quantification of colony number in mock versus RAMB1 treated HeLa cells. Middle panel: Quantification of colony size in mock versus RAMB1 treated cervical cancer cells. Right panel: Representative experiment of HeLa cells (102) which were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentration over a period of 10 days. Colonies were visualized by crystal violet staining and manually counted using an inverted microscope. Inserts: Representative example of colony size in mock versus RAMB1-treated cells. Middle panel: Quantification of colony number. Right panel: Quantification of colony size, representative of three independent experiments. **, P,0.02. C. CaSki cells were treated with checkerboard dilution series of RAMB1 and Chloroquine over a period of 24 hours. Cell viability was measured by XTT assay and calculated as percent of control untreated cultures. Synergy is expressed as combination index (CI). doi:10.1371/journal.pone.0023888.g008 promising anticancer properties are members of the flavonoid and triterpene families. While the chemical common denominator within these two families capable of inducing preferential killing in cancer cells is yet to be identified, a number of studies support the idea that they share an ability to inhibit one or multiple catalytic activities of the proteasome, and that their anti-cancer activities stems from this inhibition [14,15,17,21]. While we have recently shown that in chalcone-based small molecule inhibitors of proteasome the nature of aminoacidic component determines the selectivity toward individual catalytic activities of the proteasome [14], a substantial body of evidence indicates that chalcones may have within their b-carbons to a,b- unsaturated carbonyls the molecular determinant for inhibition of ubiquitin-dependent protein degradation upstream of proteasomes [22,23,24,25]. Thus, they may exert anticancer activity by induction of UPS stress via a mechanism independent from direct inhibition of the catalytic activities of the 20S proteasome. Given these observations and based on our recent work [14], we have synthesized a library of chalcone-based small molecules (RAMBs) containing a,b-unsaturated carbonyl systems and lacking aminoacid substitutions and tested their antineoplastic potential via inhibition of ubiquitin-mediated protein degradation PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23888