following addition of the Suc-LLVY-GloTM substrate (Promega, Madison, WI) specific for the chymotrypsin-like activity of the proteasome according to the manufacturer’s recommendations. Assay of 4XUbiquitin-Luciferase degron The 4Xubiquitin–luciferase fusion construct designated Ub-FL and the control plasmid designed CMV-FL were kindly provided by Dr. David Piwnica-Worms (Washington University, St. Louis, MO) [26]. Sub-confluent cultures of HeLa cells were transfected with plasmids DNA by using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA). FL and Ub-FL transfected HeLa cells were seeded at 50,000 cells/well in 24-well plates or 200,000 cells/well in 6-wells plate 24 hours post transfection and incubated with compounds or vehicle (DMSO) at the doses and times indicated. Luciferase activity in cell lysate was determined with a luciferase assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. Images were acquired for 1 min with a Xenogen IVIS 200 (Caliper, Hopkinton, MA). Equally sized areas were analyzed using Living Image 2.20 software. Clonogenic assay Exponentially growing SiHa, CaSki and HeLa cervical cancer cells were seeded at either 1,000 or 100 cells/well in 6 well plates. One day post seeding, the cells were treated with either vehicle alone (mock) or RAMB1 at the indicated doses, and incubated at 37 C in 5% CO2 for 10 days. At the end of the treatment the medium was removed and the cells were rinsed with PBS prior fixation and staining of the colonies using a mixture of 6.0% glutaraldehyde and 0.5% crystal violet as previously described [27]. Colonies were imaged and counted using a Nikon Eclipse TE 2000E inverted microscope and images captured with Spot 3.5.8 acquisition software (Diagnostic Instruments, Sterling Heights, MI). All the experiments were conducted in triplicate. Statistical analysis Results are reported as mean 6 Standard Deviation (SD). Statistical significance of differences was assessed by two-tailed Student’s t using Prism (V.5 Graphpad, San Diego, CA) and Excel. The level of significance was set at p#0.05. The combination index (CI) of RAMB1 and Chloroquine was calculated by the median-effect analysis according to the method of Chou and Talaly [28]. CI,1 indicates synergism, CI = 1 indicates additivity, and CI.1 indicates antagonism. Further regression analyses were performed to stabilize estimates. Results RAMB compounds selectively reduce the viability of cervical cancer cells independently of HPV genotype via blockade of proteasomal degradation Flavonoids and triterpenoids family members including celastrol [19], resveratrol [29,30], curcumin [17], epigallocatechin-3-gallate [20,31,32,33,34] and chalcones [35,36] exhibit anti-cancer properties associated with their activity as proteasome inhibitors [14,15,16,21]. We recently reported that in chalcone-based proteasome inhibitors the nature of the aminoacid portion of the molecule confers specificity toward catalytic activities of the 20S proteasome [14]. Based upon several prior studies [22,23,37], we hypothesized that the a-b ketone system of chalcones may represent the minimum molecular determinant for inhibition of ubiquitin-mediated protein degradation upstream of proteasomes. To test this hypothesis we initially screened a library of chalcone-based derivatives carrying various substituents on the Causing UPS Stress for Cervical Cancer Treatment aromatic rings adjacent to the a-b ketone system and lacking aminoacidic portions, for their cell growth inhibitory capacity in exponentially growing HPV18-positive HeLa cervical cancer cell line in a range of concentration from 100 to 0.01 mM (not shown). Four chalcone derivatives, hereafter termed RAMBs, capable of reducing the cell viability of exponentially growing HeLa cervical cancer cells in a dose-dependent fashion with IC50 values ,5 mM, were chosen for further evaluation (Figure 1). To determine the feasibility of using RAMBs compounds for treatment of cervical cancer, we tested whether RAMB1–4 treatment would specifically hinder the cell viability of cervical cancer cells over normal keratinocytes and whether the reduction in cell viability in cervical cancer cells is dependent upon the HPV-genotype. As shown in Figure 2, RAMB1 or RAMB4 treatment produced a dose- dependent reduction in the viability of HPV16-positive SiHa and Caski cells and HPV-39-positive ME180 cervical cancer cell lines respectively with minimal effects on the viability of primary human keratinocytes and with IC50 similar to the obtained with HeLa. Similar results with slightly higher IC50 were obtained using RAMB2 and RAMB3 (not shown). To test whether the reduction in cell viability observed in cervical cancer cells following exposure to the RAMB1–4 compounds was due to their capacity of interfere with ubiquitin-mediated protein degradation, we monitored the levels of accumulation of poly-ubiquitinated proteins following treatment. Specifically, HeLa cells were treated with 5 mM of RAMB1, RAMB2, RAMB3, or RAMB4 or 10 nM of Bortezomib, the latter being used as UPS-stressor positive control, over a period of 6 hours. As shown in Figure 3 (left panel), immunoblot analysis of ubiquitinated protein expression levels in HeLa cells revealed a clear pattern of accumulation of poly-ubiquitinated proteins in RAMBs -treated HeLa cultures. A semi-quantitative analysis of the polyubiquitinated protein levels shows that the GAPDH- normalized levels of polyubiquitinated proteins are consistently higher (up to 3-fold) in RAMB-treated versus mock-treated cells (Figure 3, right panel). These findings suggest that the decrease in cell viability observed in the cervical cancer cell panel but not in normal cells following RAMBs treatment is associated with the perturbation of ubiquitin-mediated protein degradation and occurs regardless of the oncogenic HPV type. RAMB treatment triggers a Ubiquitin-Proteasome-System (UPS)-stress response without affecting 20S proteasome catalytic activities To test whether the rapid (six hours or less, unpublished data) accumulation of poly-ubiquitinated proteins following RAMBs exposure occurs concomitantly with direct inhibition of the Figure 1. Summary of the structure of compounds RAMB1–4 and their IC50 values in HeLa cervical cancer cell line. IC50 values were determined by XXT assay. The IC50 value reported are average of three independent determinations. doi:10.1371/journal.pone.0023888.g001 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23888