containing single aminoacid substitutions in their structure act as proteasome inhibitors and that the nature of the aminoacidic portion determines their selectivity toward the different catalytic activities of the 20S proteasome [14]. However other findings suggest that chalcone molecules might contain within their a- unsaturated carbonyl system the molecular determinant for inhibition of ubiquitin-mediated protein degradation upstream of the 20S proteasome [22,23,24,25]. We report for the first time that a series of chalcone-derivatives lacking aminoacidic components, here termed RAMBs, are ubiquitin-proteasome system (UPS)-stressors via inhibition of ubiquitin-mediated protein degradation upstream of the 20S proteasomal catalytic activites. Specifically, our RAMBs com- pounds are capable of selective killing of cervical cancer cells via accumulation of poly-ubiquitinated protein followed by triggering of unfolded protein responses including aggresome formation and Hsp90 stabilization. Further, this accumulation of poly-ubiquiti- nated proteins is accompanied by a compensatory activation of lysosome-dependent protein degradation, stabilization of p53, the destabilization of cyclin D1 and the onset of apoptosis. Our findings suggest that treatment RAMB compound, possibly combined with the lysosome inhibitor Chloroquine, has promise as new avenue for the treatment of cervical cancer. Materials and Methods Cell culture Cervical cancer cell lines HeLa, SiHa, CaSki and ME180, were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin at 5% CO2. Keratinocytes were obtained from Invitrogen (Carlsbad, CA) and cultured in defined Keratinocyte-SFM. Cell viability assay Cell viability was determined by 2,3-bis[2-methoxy-4-nitro- 5- sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Diagnostics GmbH, Mannheim, Germany). Cells seeded at the concentration of 1,000 per well in 100 mL medium in 96-well plate were treated with chalcone-based derivatives at specified concentrations. After the indicated periods, the cells were incubated according to the manufacturer’s protocol with the XTT labeling mixture for 4 hours. Formazan dye was quantified using a spectrophotometric plate reader to measure the absorbance at 450 nm (ELISA reader 190; Molecular Devices, Sunnyvale, CA). All experiments were done in triplicate. Determination of apoptotic cells by flow cytometry Induction of apoptosis was determined by Annexin-V/7-AAD staining. Annexin-V/7-AAD staining was done using Annexin V- PE Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA) according to manufacturer’s protocol. Briefly, 16105 cells were re- suspended in Binding Buffer, 5 ml of Annexin V-PE and 5 ml of 7- AAD were then added into the cells which were then incubated at room temperature for 15 minutes, and analyzed by flow cytometry on a Becton Dickinson FACSCalibur. Data analysis was done with CellQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA). Antibodies and Western Blot Analysis Total cellular protein (10–20 mg) from each sample was separated by SDS-PAGE, transferred to PVDF membranes and subjected to Western blot analysis. Antibodies for Western Blot analysis were obtained by the following commercial sources: anti-ubiquitin (Santa Causing UPS Stress for Cervical Cancer Treatment Cruz Biotechnology, Santa Cruz, CA), anti-p53 clone DO-1 (Calbiochem, Gibbstown, NJ) anti-PARP (BD Pharmingen, San Diego, California), anti-GAPDH (Sigma, St. Louis, MO), peroxi- dase-linked anti-mouse Immunoglobulin G (Amersham, Piscat- away, NJ) and utilized at the concentration recommended by the manufacturer. Anti-ubiquitin and anti-vimentin antibodies for immunofluorescence analysis were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Texas red-labeled goat anti-mouse Immunoglobulin G and Fluorescein-labeled horse anti-rabbit Immunoglobulin G were obtained from Molecular Probes (Carls- bad, CA), and Vector Laboratories (Burlingame, CA) respectively and used at the concentration recommended by the manufacturer. Cellular morphology and immuno-fluorescence analyses A Nikon Eclipse TE 2000E inverted microscope was used for the imaging of cellular morphology with phase contrast and immuno- fluorescence. For analysis of ubiquitin and vimentin sub-cellular localization, cultures of HeLa cells were grown as described in Lab- Tek II chambered coverglass (Nalge Nunc International, Rochester, NY). At the indicated times, cells were fixed and permeabilized with methanol and incubated with the indicated primary antibodies. Fluorescent secondary antibodies were used to visualize protein localization and nuclear DNA visualized by 4,6-diamidino-2- phenylindole (DAPI) staining. Mounted samples were viewed under a Nikon Eclipse TE 2000E inverted microscope and images captured with Spot 3.5.8 acquisition software (Diagnostic Instru- ments, Sterling Heights, MI). Measurement of Proteasomal activity in 20S Purified Proteasomes Cells (56108) were washed in cold PBS and resuspended in buffer containing 50 mM TRIS-HCl (pH 7.5), 5 mM MgCl2, 1 mM DTT (Sigma), 2 mM ATP and 250 mM sucrose. Glass beads equivalent to the volume of the cell suspension were added, and the mixture was vortexed for 1 min at 4uC. Beads and cell debris were removed by 5 min centrifugation at 1,000 g, followed by 20 min centrifugation at 10,000 g [27]. Lysates were cleared by ultracentrifugation for 1 hr at 100,000 g, and supernatants were further ultracentrifuged for 5 hr at 100,000 g. Proteasome- containing pellets were resuspended in 0.5 ml of homogenization buffer [50 mM TRIS-HCl (pH 7.5), 100 mM KCl, 15% glycerol]. Protein concentration was determined using the BCA protocol (Pierce, Rockford, IL). Fluorogenic substrates Suc-LLVY-AMC, Boc-LRR-AMC and Ac-YVAD-AMC were used to measure chymotryptic-like, tryptic-like and caspase-like activities, respec- tively. Semipurified proteasomes (10 ml), pretreated or not with inhibitors for 30 min at 37uC, were assayed at 37uC for 45 min using the different peptide substrates in a buffer containing 50 mM TRIS-HCl (pH 7.5), 5 mM MgCl2 and 1 mM DTT (final volume 100 ml). The reaction was quenched with 1 ml 1% SDS and fluorescence determined by fluorimeter (Perkin-Elmer, Beacons- field, UK) with excitation at 380 nm and emission at 440 nm [18]. Data are expressed as the percent inhibition relative to untreated proteasomal preparations. Measurement of Proteasomal activity in 26S Proteasomes in living cells Exponentially growing cells (16106) were plated in 60 mm dishes and either mock treated or treated with different concentration RAMB1 over a period of 4 hours. Proteasomal activity in cell lysates (NP-40 lysis buffer: 0.1% NP-40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol and 1 mM DTT) was determined by measuring residual luminescence activity PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23888