Cell culture and chemicals

Human KB cancer cells from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Carlsbad, CA, USA) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO) at 37°C in a humidified atmosphere with 5% CO 2 . Capsaicin (Sigma, St. Louis, MO, USA) was dissolved in pure dimethyl sulfoxide (DMSO) (Sigma). All chemicals were of the highest grade available.

Cell viability and cytotoxicity assays

KB cells (8×103 cells/well) were seeded in 96-well plates and cultured overnight. The cells were treated with various concentrations of capsaicin (1, 50, 100, 150, 200 and 250 μM) or DMSO (control group). After 24, 48 and 72 h, cell proliferation and viability was determined by a sulforhodamine B (SRB) colorimetric assay [33]. Briefly, the cells were fixed in 10% w/v trichloroacetic acid (Sigma) and stained with 0.4% SRB (Sigma). The cells were then washed with tap water and 1% acetic acid (Merck, Darmstadt, Germany). Protein-bound precipitates were dissolved in 10 mM Tris buffer (pH 10.5) (Merck), and the plate was read at a wavelength of 492 nm (Multiskan Spectrum Microplate Reader; Thermo Labsystems, Waltham, MA, USA) to determine the cell viability. Trypan blue exclusion was used to examine the numbers of viable and dead cells in each treatment. KB cells (1×105 cells/well) cultured in 6-well plates were treated with various concentrations of capsaicin (50, 100, 150, 200 and 250 μM) or DMSO for 24, 48 and 72 h. After harvesting, cell suspensions were thoroughly mixed with equal volumes of a 0.4% trypan blue solution (GIBCO). Viable and dead cells were counted using a hemacytometer under an inverted phase contrast microscope (Nikon Eclipse Ti-U; Nikon Instruments, Kanagawa, Japan).

Detection of apoptotic cells

Cells cultured in 24-well plates were treated with capsaicin (150 and 250 μM) or DMSO for 24 and 48 h. Following fixation in freshly prepared ice-cold 4% paraformaldehyde (Sigma) and washing with PBS, 1 μg/ml Hoechst 33342 (Cell Signaling Biotechnology Inc., Beverly, MA, USA) was used to stain the cells. Images were acquired under an inverted fluorescence microscope equipped with a digital camera (Nikon Eclipse Ti-U; Nikon Instruments). The percentage of apoptotic cells were scored by counting at least 200 cells for each treatment.

Cell cycle analysis

To detect the proportion of KB cells in different phases of the cell cycle, the DNA content in KB cells was detected by propidium iodide staining (Sigma) and flow cytometry. KB cells seeded in 6-well plates were incubated with capsaicin (150 and 250 μM) or DMSO for 24, 48 and 72 h, and then collected, washed with PBS, and fixed with 70% ethanol at −20°C overnight. After centrifugation, the cells were resuspended in PBS containing 4 μg/ml propidium iodide, 0.1 mg/ml RNase A (Amresco Inc., Solon, OH, USA) and 0.1% Triton X-100 (Sigma), and then incubated at room temperature for 1 h in the dark. Finally, the cell cycle distribution of cells from each treatment was determined by flow cytometry (Beckman FC500; Beckman Instruments, Fullerton, CA, USA) with CXP software. The percentage of cells in different phases of the cell cycle was analyzed by MultiCycle DNA Content and Cell Cycle Analysis Software.

Measurement of mitochondrial membrane potential (ΔΨm)

To elucidate the role mitochondria play in KB cell death after treatment with capsaicin, changes of the mitochondrial membrane potential were detected by flow cytometry using the fluorescent dye 3,3’-dihexyloxacarbocyanine iodide (DiOC 6 ) (Sigma). KB cells (3×104 cells/well) were seeded in 12-well plates. After incubation with various concentrations of capsaicin (100, 200 and 250 μM) or DMSO for 24 and 48 h, the cells were washed with PBS and resuspended in 10 nM DiOC 6 . After incubation at 37°C for 30 min, the cells were immediately analyzed by flow cytometry.

Immunoblotting

After treatment of KB cells with capsaicin (50, 100, 150 and 250 μM) or DMSO for 24 h, the cells were collected by trypsinization, washed twice with cold PBS, and then lysed with ice-cold lysis buffer consisting of 50 mM Tris–HCl (pH 7.4), 0.1% SDS, 150 mM NaCl, 1 mM EDTA (Merck), 1% Triton X-100 and a protease inhibitor cocktail (BioVision Research Products, Mountain View, CA, USA) of 0.001% (w/v) aprotinin, 0.001% (w/v) leupeptin, 0.00035% (w/v) pepstain A, and 0.085% (w/v) PMSF. Cell lysates were kept on ice for 30 min after gentle vortexing, and then centrifuged at 11,752 g for 10 min at 4°C. The supernatants were immediately analyzed by western blotting or stored at −80°C until use. The protein concentration was measured by a Bio-Rad assay according to the manufacturer’s instructions. For western blot analysis, equal amounts of proteins (20~50 μg) from each treatment were loaded on 12% SDS-PAGE gels. SDS-PAGE and electrophoretic transfer of proteins onto PVDF membranes (Millipore, Billerica, MA, USA) were performed with Hoefer and Bio-Rad apparati, respectively. PVDF membranes were soaked for 1 h in blocking buffer consisting of 5% non-fat milk powder or 5% bovine serum albumin (Sigma) in 1× PBS containing 0.5% Tween-20. Then, PVDF membranes were incubated with antibodies against caspase 8 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), caspase 9, caspase 3, PARP (Cell Signaling), and β-actin (Sigma) for 1 h or overnight. Membranes were then incubated with the appropriate secondary antibody conjugated to horseradish peroxidase for 1 h. After the membranes were exposed to reagents for ECL immunodetection (Millipore), the labeled proteins were detected by autoradiographic film (Eastman Kodak Co., Rochester, NY, USA).

Statistical analysis

The Student’s t-test was performed to determine the statistical significance of the difference between capsaicin- and DMSO-treated groups. Results were represented as the mean ± SD and considered significant at P < 0.05, unless stated otherwise.