Curcumin (Nagara Science Co., Ltd., Gifu, Japan) was purchased and dissolved in dimethyl sulfoxide (DMSO).

Human breast cancer MCF-7 (estrogen and progesterone receptor-positive, luminal type), MDA-MB-231 (triple-negative, basal type) and human prostate cancer PC-3 cells were obtained from the NCI-60 cancer cell line panel of the National Cancer Institute Developmental Therapeutics Program (NCI DTP). MCF-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 4 mM glutamine, 50 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂. The MDA-MB-231 and PC-3 cells were cultured in RPMI-1640 medium with 10% FBS, 2 mM glutamine, 50 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂. It has been identified that the activity of NF-κB in MDA-MB-231 cells is higher than that in MCF-7 cells (26).

The cell growth was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The CCK-8 solution was added to the medium of curcumin-treated or siRNA-transfected MCF-7 and MDA-MB-231 cells. The absorbance (450 nm) of the medium was determined using Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA) after 4 h of incubation.

The MCF-7 and MDA-MB-231 breast cancer cells treated with curcumin or transfected with each siRNA for RPS3 were harvested by trypsinization. The cells were washed with phosphate-buffered saline (PBS) and suspended in PBS containing 0.1% Triton X-100, 150 µg/ml RNase A and 50 µg/ml propidium iodide to stain the nuclei. The DNA content in the stained nuclei was analyzed by FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ, USA) and the hypodiploid population (sub-G1) was detected and quantified.

The MCF-7 and MDA-MB-231 breast cancer cells were treated with curcumin or transfected with each siRNA and lysed with RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM DTT and 0.5 mM PMSF] at 4°C for 30 min and centrifuged. The supernatants were separated by 12% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline containing 5% skim milk and incubated with primary antibodies at room temperature for 1 h. The primary antibodies were a mouse monoclonal anti-XIAP antibody (MAB822; R&D Systems, Minneapolis, MN, USA), a rabbit monoclonal anti-RPS3 antibody (#9538; Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal anti-β-actin antibody (A5441; Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Each protein was visualized on BioMax XAR film (Carestream Health, Inc., Rochester, NY, USA) using Chemi-Lumi One L (Nacalai Tesque, Kyoto, Japan) or Immobilon Western (Millipore).

The magnetic FG beads with epoxy linkers were purchased from Tamagawa Seiki Co., Ltd. (Nagano, Japan). The beads were mixed with curcumin in dimethylformamide (DMF) containing potassium carbonate (K₂CO₃) at 37°C overnight. The resulting beads were washed three times with deionized water and stored at 4°C.

The MCF-7 and PC-3 cells were lysed with binding buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1 mM DTT and 0.5 mM PMSF] for 30 min at 4°C and centrifuged. The collected supernatants were used as whole cell extracts. The extracts were incubated with curcumin-fixed or empty beads for 4 h at 4°C. After the beads were washed three times with the binding buffer, the proteins binding to these beads were eluted with Laemmli dye. The binding proteins were subjected to SDS-PAGE and stained by silver staining. Subsequently these proteins were subjected to in-gel digestion with Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) and the resulting peptide fragments were analyzed by Autoflex II (Bruker Daltonics, Billerica, MA, USA).

The total cellular RNA was extracted from the MCF-7 and MDA-MB-231 cells transfected with each siRNA using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan) and complementary DNA (cDNA) was synthesized from total RNA with High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR analysis was performed by an ABI 7300 Real-Time PCR system (Applied Biosystems) with the cDNA and TaqMan probes (Applied Biosystems) to XIAP (Hs00745222_s1) and GAPDH (Hs02758991_g1).

The MCF-7 and MDA-MB-231 cells were transfected with each siRNA using Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA). The following siRNAs (Life Technologies) were used: siRPS3 #1 (HSS184418), 5′-GAGCUGGCUGAAGAUGGCUACUCUG-3′; siRPS3 #2 (HSS184419), 5′-GCGGAUUCGGGAACUGACUGCUGUA-3′; siRPS3 #3 (HSS184420), 5′-UGAUCCACAGCGGAGACCCUGUUAA-3′; and negative control siRNAs (cat. no. 12935-112 and no. 12935-115). Only RNA sequences of sense strands are shown.

The nuclear extracts were prepared from the MCF-7 and MDA-MB-231 cells transfected with each siRNA using Nuclear Extract kit (Active Motif, Carlsbad, CA, USA). Subsequently, the activity of the p65 subunit of NF-κB in the nuclear extracts was determined by the NF-κB p65 Transcription Factor Assay kit (TransAM NFκB p65; Active Motif) and Multiskan FC (Thermo Fisher Scientific).

The data from triplicate samples are represented as the mean ± standard deviation (SD). Statistical analysis was performed using non-repeated measures ANOVA followed by Bonferroni post hoc test due to comparisons of more than two groups. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have identified that XIAP is strongly expressed in breast cancer tissues (16,17). In order to elucidate the mechanisms responsible for the overexpression of XIAP, we searched for compounds that downregulate XIAP in breast cancer cells. Curcumin, one of the most common polyphenols, inhibited the growth of the MCF-7 breast cancer cells (Fig. 1A) and induced apoptosis (Fig. 1B). We then found that curcumin downregulated the XIAP protein (Fig. 1C). These results indicate that curcumin reduces the protein levels of XIAP in the MCF-7 breast cancer cells.

In an attempt to clarify the mechanisms by which curcumin downregulates XIAP in MCF-7 cells, we examined the curcumin-binding proteins using magnetic FG beads. Curcumin was conjugated onto the beads with K₂CO₃ (Fig. 2A). We initially used PC-3 prostate cancer cells to identify the curcumin-binding proteins and identified RPS3 as one of the curcumin-binding proteins (data not shown). RPS3 was also purified from the whole cell extracts of the MCF-7 cells using curcumin-fixed beads (Fig. 2B). These results reveal that RPS3 is a target of curcumin.

Several extraribosomal functions of the ribosomal proteins have recently been identified (21,27–29). RPS3 is known to be a subunit in NF-κB complexes and was found to promote the transcription of anti-apoptotic molecules, including XIAP in Jurkat leukemia cells (21). Therefore, we examined whether RPS3 also regulates XIAP at the mRNA level in breast cancer cells. The knockdown of RPS3 inhibited cell growth (Fig. 3A) and induced apoptosis (Fig. 3B). Although the knockdown of RPS3 downregulated the XIAP protein (Fig. 3C), unexpectedly the mRNA level of XIAP was not reduced (Fig. 3D). These findings reveal that RPS3 regulates the expression of XIAP independently of the NF-κB pathway in the MCF-7 breast cancer cells.

Additionally, in order to confirm that RPS3 regulates the expression of XIAP independently of the NF-κB signaling, we used another breast cancer cell line, MDA-MB-231. This cell line is classified as triple-negative breast cancer, which is the most aggressive subtype (30,31) and the NF-κB pathway is activated in this cell line (26). Similar to the MCF-7 breast cancer cells, the knockdown of RPS3 inhibited the cell growth (Fig. 4A) and induced apoptosis (Fig. 4B). The RPS3 depletion downregulated the XIAP protein (Fig. 4C), but not the mRNA level of XIAP in the MDA-MB-231 cells (Fig. 4D). Collectively, these results clearly reveal that RPS3 regulates the expression of XIAP independently of the NF-κB pathway in breast cancer cells, which is inconsistent with a previous study (21).

In addition, we investigated the activation of NF-κB by quantifying the activity of the p65 subunit of the NF-κB. As shown in Fig. 5A, the activity of NF-κB in the MDA-MB-231 breast cancer cells was higher than that in the MCF-7 breast cancer cells as previously reported (26). The knockdown of RPS3 did not reduce the activity of NF-κB in the MCF-7 and MDA-MB-231 cells (Fig. 5B and C), whereas the RPS3 knockdown downregulated the XIAP protein (Figs. 3C and 4C). These findings further reveal that RPS3 regulates the expression of XIAP independently of NF-κB.