Cafestol, Cycloheximide (CHX), and Mithramycin A (Mith A) were purchased from Sigma-Aldrich (St. Louis, Missouri). Kahweol was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The chemicals were dissolved in dimethyl sulfoxide (DMSO).
The following antibodies were purchased: anti-Cyclin D1 (M-20), anti-Sp1 (1C6), anti-Caspase-3 (H-277), horseradish-peroxidase-conjugated anti-mouse IgG, and horseradish-peroxidase-conjugated anti-rabbit IgG (all from Santa Cruz Biotechnology, Inc.), anti-Poly ADP-ribose polymerase (PARP) (BD Biosciences, San Diego, California), anti-Mcl-1, anti-Survivin, anti-Bid, anti-Bax, anti-Bclxl (Cell Signaling, Danvers, Massachusetts), and anti-β-Actin (AC-74) (Sigma-Aldrich, Inc. St. Louis, Missouri).
MSTO-211H and H28 cells, a type of human mesothelioma cells, were obtained from the American Tissue Culture Collection (Manassas, Virginia). The MSTO-211H cells and H28 were maintained in Hyclone RPMI-1640 containing 5% and 10% fetal bovine serum (FBS), respectively, and 100 U/ml each of penicillin and streptomycin (Thermo scientific, Logan, Utah) at 37°C in a humidified chamber with 5% CO2 and 95% air. The medium was changed every two days.
The cell viability of MSTO-211H and H28 cells was accessed using the MTS (3-(4,5-dimethylthiazol–2-yl)-5-(3–carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H–tetraz-olium) Assay Kit (Promega, Madison, Wisconsin), according to the manufacturer’s instruction. The MSTO-211H cells (3 × 103 cells/100ul/well) or H28 (1.5 × 103 cells/200 μl) were grown on a 96-well microtiter plate for 24 hours. Cafestol and kahweol were added directly to the culture media with no more than 0.1% as final concentration of DMSO. Cells were treated with various concentration of cafestol and kahweol for 24 hours and 48 hours. MTS cell proliferation assay reagent was added, and samples were incubated at 37°C in 5% CO2 for two hours. Absorbance was measured at 490 nm using GloMax-Multi Microplate Multimode Reader (Promega, Madison, Wisconsin), and the difference between the test and reference wavelength was calculated. The cell viability was calculated using an equation: (optical density ratio of cafestol or kahweol-treated sample/non-treated sample) × 100 (%).
Apoptosis of cafestol or kahweol-treated cells was observed using 4’-6-diamidino-2-phenylindole (DAPI) staining. Nuclear condensation and fragmentation were observed using nucleic acid staining with DAPI (Sigma-Aldrich, Inc. St. Louis, Missouri). The MSTO-211H cells treated for 48 hours with 0–90 μM cafestol and 0–60 μM kahweol were harvested by trypsinization, washed with cold Phosphate Buffered Saline (PBS), and fixed in 100% methanol at room temperature for 20 minutes. The cells were spread on slide, stained with DAPI solution (2 μg/ml), and observed through a FluoView confocal laser microscope (Fluoview FV10i, Olympus Corporation, Tokyo, Japan).
Propidium iodide staining
After 48 hours of cafestol or kahweol treatment, the detached cells (floaters) were collected by centrifugation and combined with adherent cells. The cells were washed with cold PBS, fixed in 70% ice-cold ethanol overnight at −20°C, and treated with 150 μg/ml RNase A and 20 μg/ml propidium iodide (PI; Sigma-Aldrich, Inc. St. Louis, Missouri). The stained cells were analyzed, and distribution of the cells in different phases of cell cycle was calculated using flow cytometry with a MACSQuant Analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
Reverse transcription-polymerase chain reaction
Total RNA was extracted from cells using TRIzol® Reagent (Life Technologies, Carlsbad, California), and first strand cDNA was synthesized from 2 μg of RNA using reverse transcriptase with Oligo-(dT) primer according to instructions for HelixCriptTM 1st-strand cDNA synthesis kit (NanoHelix, Korea). cDNA was obtained by PCR using β-actin-specific and Sp1-specific primers as described below under following PCR conditions (25 cycles: 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C). β-actin primers were: 5'-GTG-GGG-CGC-CCC-AGG-CAC-CA-3' (forward) and 5'-CTC-CTT-AAT-GTC-ACG-CAC-GAT-TTC-3' (reverse). Forward Sp1 primers were: ATG CCT AAT ATT CAG TAT CAA GTA; reverse primers were: CCC TGA GGT GAC AGG CTG TGA. The PCR products were analyzed using 2% agarose gel electrophoresis.
MSTO-211H cells (6 × 104) were seeded into 24 well plates in triplicate for 24 hrs. Transient transfection was performed using a LipofectAMINE2000 reagent (Invitrogen, Carlsbad, CA, USA). The Survivin-269 promoter constructs was kindly provided by Dr Sung-Dae Cho (Chonbuk National University, Jeon-ju, Korea). The mcl-1 promoter was obtained from Addgene (Cambridge, MA, USA). Cells were transfected with 250 ng of cyclin D1 (−1745-luc), mcl-1 (−325), or Survivin (−269), and 20 ng of β-gal using LipofectAMINE2000 reagent (Invitrogen, Carlsbad, CA, USA) for 24 h . 48 hrs after cafestol and kahweol treatment, whole cell lysates were harvested, and firefly luciferase and galactosidase activity were determined with a Promega luciferase assay kit (Madison, WI) according to the manufacturer’s manual. After PBS wash, passive lysis buffer (200 μl) was added, the cells were then incubated for 1 hr with gentle shaking. Cell lysates (100 μl each) were mixed with 100 μl luciferase assay reagent, and firefly luciferase light emission was measured by GloMax™ Multi + MicroplateMulti Reader (Promega). Subsequently, 50 μl of β-galactosidase substrate was added in order to normalize the firefly luciferase data.
The cafestol or kahweol treated cells were cultured for 48 hours and washed twice with cold PBS, then the cells were lysed with PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Korea) containing 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mM PMSF. The extracted protein was prepared in 200 μl of the extraction solution, containing about 1 × 107 cells. The protein concentration was measured using DC Protein Assay Reagent (BIO-RAD Laboratories Inc., Hercules, California). Fifty micrograms of total cellular protein per lane were separated with 10 and 15% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membrane was blocked for two hours at room temperature with 5% non-fat dried milk in PBS containing 0.1% tween-20, and incubated with specific antibodies overnight at 4°C. The secondary antibodies to IgG conjugated with horseradish peroxidase were used, and the membrane was developed with the Pierce ECL Western Blotting Substrate (Thermo scientific, Rockford, Illinois) according to the manufacturer’s instructions.
Data were presented as the mean ± SD from three independent experiments. Data analysis for statistical significance was done using Student’s t-test. Compared to the vehicle control, p value was less than 0.05, indicating statistical significance of the data.