HyClone RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Thermo Scientific (Logan, UT). The following antibodies were used: 4′-6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), anti-Sp1 (1C6), anti-poly (ADP-ribose) polymerase (PARP) (BD Biosciences, San Diego, CA), anti-cyclin D1 (M-20), anti-myeloid cell leukemia (Mcl)-1, anti-survivin, anti-Bid, anti-Bax, anti-Bcl-xL (Cell Signaling Technology, Inc., Danvers, MA), anti-caspase-3 (H-277) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-β-actin (AC-74) (Sigma-Aldrich, Inc. St. Louis, MO). RNase A was supplied by Sigma-Aldrich.

MSTO-211H cells were obtained from the American Tissue Culture Collection (Manassas, VA). Cells were maintained in RPMI-1640 medium supplemented with 5% FBS and 100 U/ml each of penicillin and streptomycin at 37°C in a 5% CO₂ incubator.

The effects of Qu on cell viability were determined using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (Promega, Madison, WI). MSTO-211H cells were seeded in a 96-well plate for 24 h and treated with Qu (20–80 μM) for 24 or 48 h. MTS solution was then added for 2 h at 37°C in 5% CO₂. The absorbance at 490 nm was recorded using a GloMax-Multi Microplate Multimode Reader (Promega).

The level of nuclear condensation and fragmentation was observed by nucleic acid staining with DAPI. MSTO-211H cells were treated with Qu, harvested by trypsinization, and fixed in 100% methanol at room temperature for 20 min. The cells were spread on slides, stained with DAPI solution (2 μg/ml), and analyzed under a FluoView confocal laser microscope (Fluoview FV10i; Olympus Corporation, Tokyo, Japan).

Following treatment with Qu (20–80 μM) for 72 h, the detached MSTO-211H cells (floaters) were collected by centrifugation and combined with adherent cells. The cells were fixed with 70% ice-cold ethanol overnight at −20°C, and subsequently treated with 150 mg/ml RNase A and 20 mg/ml PI. DNA contents were analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

MSTO-211H cells treated with Qu (20–80 μM) for 48 h were washed with phosphate-buffered saline (PBS), and were then homogenized with PRO-PREP™ Protein Extraction Solution (Intron Biotechnology, Korea). Extracted proteins were measured using DC protein assay reagent (Bio-Rad Laboratories Inc., Hercules, CA). The equal amounts of protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred onto membranes, which were blocked for 2 h at room temperature with 5% non-fat dried milk in PBS containing 0.05% Tween-20, and then incubated overnight at 4°C with specific antibodies. The protein bands were observed after treating them with horseradish peroxidase-conjugated secondary antibody using a Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL).

This method has been described previously (19,20). Briefly, MSTO-211H cell lysates were reacted with sepharose 4B beads or Qu-sepharose 4B beads in reaction buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, 2 μg/ml bovine serum albumin, 0.02 mM phenylmethylsulfonyl fluoride and 1X proteinase inhibitor), and washed 5 times with washing buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, 0.02 mM phenylmethylsulfonyl fluoride). Proteins bound to the beads were analyzed by western blot analysis using anti-Sp1 antibody.

Total RNA was extracted from the cells using TRIzol^® Reagent (Life Technologies, Carlsbad, CA), and 2 μg of RNA were used to synthesize cDNA using the HelixCript™ first-strand cDNA synthesis kit (NanoHelix, Korea). cDNA was obtained by PCR amplification using β-actin-specific and Sp1-specific primers (as described below) using the following PCR conditions: 25 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C. The β-actin primers used were: forward, 5′-GTG-GGG-CGC-CCC-AGG-CAC-CA-3′ and reverse, 5′-CTC-CTT-AAT-GTC-ACG-CAC-GAT-TTC-3′; and the Sp1 primers were: forward, ATG CCT AAT ATT CAG TAT CAA GTA and reverse, CCC TGA GGT GAC AGG CTG TGA. PCR products were analyzed by 2% agarose gel electrophoresis.

MSTO-211H cells (6x10⁴) were added to each well of a 24-well plate and incubated at 37°C in a humidified atmosphere of 5% CO₂ for 24 h. Transient transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). The survivin-269 promoter construct was kindly provided by Dr Sung-Dae Cho (Chonbuk National University, Jeonju, Korea). The Mcl-1 promoter was obtained from Addgene (Cambridge, MA). Cells were transfected with 250 ng of cyclin D1 (-1745-luc), Mcl-1 (-325-luc), or survivin (-269-luc) and 20 ng of β-gal using Lipofectamine 2000 reagent (Invitrogen) for 24 h (21). After cafestol and kahweol treatment for 48 h, the cells were disrupted with 100 ml of lysis buffer [0.1 M potassium phosphate (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol (DTT), 2 mM EDTA], and firefly luciferase activity and galactosidase were determined with a Promega luciferase assay kit according to the manufacturer’s instructions. Relative luciferase units were calculated by normalizing to the galactosidase activity.

The results are presented as the means ± SD of at least 3 independent experiments performed in triplicate. Data were analyzed for statistical significance using one-way analysis of variance. A P-value <0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of Qu (Fig. 1A) on the viability of malignant mesothelioma cells we used a MTS assay. It was found that Qu suppressed cell viability with an IC₅₀ of 58 μM in MSTO-211H cells for 48 h (Fig. 1B). Qu treatment also resulted in a significant concentration-dependent inhibition of cell growth with IC₅₀ values of approximately 73 μM in HT28 cells (another malignant mesothelioma cell line) for 48 h (data not shown). To investigate the morphological changes, MSTO-211H cells were treated with various concentrations (20–80 μM) of Qu for 48 h. The results obtained showed that the cells had decreased in size and that they had became rounded (Fig. 1C).

The effect of Qu treatment on the initiation of apoptosis in MSTO-211H cells was determined by nuclear morphology using DAPI staining. The Qu treatment of mesothelioma cells increased nuclear condensation and fragmentation when compared to the control group (Fig. 2A). In order to evaluate whether the increase in the sub-G₁ cell population induced by Qu was related to apoptosis, the Qu-treated cells were used for PI staining. The number of MSTO-211H cells in the sub-G₁ phase increased from 25 to 50% in the presence of 20–80 μM Qu (Fig. 2B and C).

Sp1 contributes to cell progression and apoptotic cell death via the regulation of the expression of a number of genes, such as cyclin D1, Mcl-1, survivin, Bcl-xL, Bax and caspase-3 in various cancers (7,13,22). The interaction between Sp1 and Qu was examined by conducting a Qu-sepharose 4B affinity experiment by immunoblotting with anti-Sp1. The results obtained indicated that Qu bound with Sp1 in cell lysates from human MSTO-211H cells (Fig. 3A). Furthermore, we found that Qu at 20, 40 and 80 μM downregulated Sp1 protein (Fig. 3D) and mRNA levels (Fig. 3B), and Sp1 protein expression level monitoring showed that Qu time-dependently reduced protein levels over 0 to 48 h (Fig. 3C). Qu clearly attenuated the cyclin D1, Mcl-1 and survivin promoter activities (Fig. 4A–C). In addition, significant decreases in the protein levels of cyclin D1, Mcl-1 and survivin were observed following Qu treatment (Fig. 4D).

The treatment of cells with Qu regulated the expression levels of several apoptosis-related proteins. MSTO-211H cells were treated with various concentrations (20–80 μM) of Qu for 48 h and harvested. The protein expression levels of Bid, Bcl-xL, caspase-3, PARP and Bax were analyzed by western blot analysis. The results showed that Qu induced the activation of Bid, caspase-3 and PARP, decreased Bcl-xL, and increased Bax levels in the MSTO-211H cells (Fig. 5).