Bakuchiol (Fig. 1), MTT reagent and dimethyl sulfoxide (DMSO) were ordered from Sigma Aldrich; Merck KGaA (Darmstadt, Germany). Cell culturing RPMI-1640 medium, fetal bovine serum (FBS) and penicillin/streptomycin antibiotics were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-rabbit monoclonal B cell lymphoma-2 associated X, apoptosis regulator (Bax; cat. no. ab32503; 1:1,000), anti-rabbit monoclonal B cell lymphoma-extra large (Bcl-xL; cat. no. ab32370; 1:1,000), anti-rabbit monoclonal procaspase-3 (cat. no. ab32150; 1:500), anti-rabbit monoclonal procaspase-6 (cat. no. ab3263, 1:800), anti-mouse monoclonal procaspase-8 (cat. no. ab38271; 1:1,000) and anti-rabbit polyclonal procaspase-9 (cat. no. ab135544; 1:800), anti-rabbit monoclonal poly (ADP-ribose) polymerase (PARP; cat. no. ab191217; 1;1,000), anti-rabbit monoclonal cleaved PARP (cat. no. ab32064; 1:1,200), anti-rabbit monoclonal protein kinase B (AKT; cat. no. ab183758; 1:1,000), anti-rabbit monoclonal phosphorylated (p-AKT; cat. no. ab81283; 1;800), anti-rabbit monoclonal, p-c-Jun N-terminal kinase (JNK; cat. no. ab179461; 1:1,200), anti-rabbit monoclonal extracellular signal-regulated kinase 1/2 (ERK1/2; cat. no. ab184699; 1:1,000), anti-rabbit polyclonal cleaved caspase-3 (cat. no. ab2302; 1:1,200), anti-rabbit monoclonal β-actin (cat. no. ab32575; 1;1,500) and anti-mouse monoclonal β-actin (cat. no. ab8226; 1:1,500) antibodies were purchased from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; cat. no. ab97023; 1:10,000) and anti-rabbit IgG (cat. no. ab6721; 1:10,000) were also obtained from Abcam (Cambridge, UK). The Muse Annexin V and Dead Cell Assay kit (cat. no. MCH100105) and the Muse Cell Cycle Assay kit (cat. no. MCH100106) were ordered from EMD Millipore (Billerica, MA, USA). DAPI was obtained from Vector Laboratories, Inc. (Burlingame, CA, USA). All other chemicals and materials utilized for electrophoresis were acquired from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

NUGC3 human gastric cancer cell lines were purchased from the Japanese Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in RPMI-1640 medium and supplemented with heat-inactivated 10% FBS (v/v) and 1% penicillin and streptomycin in humidified conditions of 5% CO₂ at 37°C.

To test the effect of bakuchiol on human gastric cancer cell viability, NUGC3 cells were seeded in a 96 well plate at a density of 6×10⁴ cells/ml, then treated with 0, 20, 40, 60, 80, 100 or 120 µg/ml bakuchiol and incubated for 24 h at 37°C. The negative control cells were treated with DMSO. Following incubation, 0.5 g/ml MTT was added to each well and the plates were further incubated for 3 h at 37°C. The medium was then removed and DMSO was added to dissolve the formazan crystals formed. The absorbance was recorded at a 540 nm wavelength using an iMark (model 550) ELISA microplate absorbance reader (Bio-Rad Laboratories, Inc.).

NUGC3 cells were treated with 0, 50 and 100 µg/ml bakuchiol and incubated for 24 h at 37°C. The cell lines were collected and cleaned using cold PBS and centrifuged at 1,000 × g for 10 min at 37°C. Cold ethanol 70% (v/v) was used to fix the pellet at −20°C for 3 h, and the cells were then washed using PBS and 200 µl was transferred to a fresh tube. Muse Cell Cycle Assay kit solution (100 µl) was added to each well and incubated for 30 min in the dark at room temperature. The pellet was resuspended in 1 ml RPMI-1640 medium and 100 µl was transferred to a fresh tube for the analysis of apoptosis. Again, 100 µl of Muse Annexin V and Dead Cell Assay kit solution was added and incubated at room temperature for 30 min in the dark at 37°C and analyzed using the Muse cell analyzer (0500–3115) using Muse count and viable software (version 1.05.0) from EMD Millipore (Billerica, MA, USA).

NUGC3 cells were treated with 0, 50 and 100 µg/ml bakuchiol and incubated for 24 h at 37°C. A total of 1×10⁴ cells were then washed with cold PBS at room temperature. Then the cells were fixed with 3.7% paraformaldehyde (1 ml) and 95% ethanol at room temperature for 10 min and followed by washing with PBS. The fixed cells were stained using DAPI (DAPI staining kit solution). The morphological nuclear changes were examined using fluorescence microscopy at ×400 magnification (Model-DMLS) from Leica Microsystems (GmbH, Wetzlar, Germany).

NUGC3 cells were treated with 0, 50 and 100 µg/ml bakuchiol at 37°C for 72 h and then lysed with NP-40 (1% w/w) ice cold radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), sodium deoxycholate (1% w/v), SDS (0.1% w/v), NaCl (0.15 M), EDTA (2 mM) sodium phosphate buffer (0.01 M, pH 7.2) and sodium fluoride (50 mM). The resultant cell lysates were centrifuged at 3,000 × g for 10 min at 37°C and total cellular protein concentration was determined by Bio-Rad Bradford protein assay method (Bio-Rad Laboratories, Inc.) (9). Equal quantities of protein (50 µg/lane) was separated by SDS PAGE on 12% gel and then transferred onto a polyvinyldene fluoride membrane. The membrane was blocked with Tris-buffered saline containing Tween-20 and 5% skimmed milk for 4 h at 37°C and probed with the primary antibodies overnight at 37°C, followed by incubation with secondary antibodies conjugated to horseradish peroxidase for 2 h at 37°C. The blots were examined using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences, Chalfont, UK) and quantification was performed using Image J software from the National Institutes of Health (version 2.8; Bethesda, MD, USA). All assays were performed in triplicate.

The statistical analyses were performed using SPSS software (version 20.0; SPSS, Inc., Chicago, IL, USA). The results were expressed as the mean ± standard deviation of three independent experiments. Differences between the experimental groups were determined using one-way analysis of variance followed by Dunnett's multiple comparison post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the concentration of bakuchiol that inhibits cell viability, NUGC3 cells were treated with 0, 20, 40, 60, 80, 100 or 120 µg/ml bakuchiol for ~24 h, and cell viability was then measured by MTT assay. Following bakuchiol treatment and 24 h incubation, cell viability was inhibited in a concentration-dependent manner when compared with the control, without bakuchiol treatment (P<0.05; Fig. 2A). The concentration that produced 50% inhibition (IC₅₀ value) was revealed to be ~120 µg/ml. Therefore, bakuchiol concentrations of 0, 50 and 100 µg/ml were used for further examination. Microscopic study indicated changes in the cell structure, including visible shrinkage of cells and reduced cell counts, in bakuchiol treated cells compared with controls (Fig. 2B).

Cell cycle distribution and the apoptotic cell population in bakuchiol-treated NUGC3 cells was determined using flow cytometry analysis. Bakuchiol treatment elevated the percentage of sub-G1 cells from 13% to 28 and 39% at 50 (P<0.05) and 100 µg/ml (P<0.01), respectively (Fig. 3). Meanwhile, bakuchiol treatment significantly reduced the cell population of G₀/G1, S and G2/M compared with the control (P<0.05; Fig. 3). In addition, the effect of bakuchiol on apoptosis induction in NUGC3 cells was examined using Annexin V-fluorescein isothiocyanate/propidium iodide double staining and flow cytometry. The mild elevation of sub-G1 cells in the untreated group was attributed to the effect of DMSO on NUGC3 cells.

Treatment with bakuchiol (50 µg/ml; P<0.05 and 100 µg/ml; P<0.01) significantly increased the percentage of early apoptosis, late apoptosis and total apoptosis in NUGC3 cells in a concentration dependent manner compared with the control (Fig. 4A and B). Nuclear fragmentation and apoptotic organelles were observed following 50 and 100 µg/ml bakuchiol treatment, using DAPI staining (Fig. 4C). These data suggested that bakuchiol treatment induced cell death in NUGC3 cells.

To confirm whether bakuchiol-induced apoptosis was caspase dependent, western blotting analysis was performed (Fig. 5A). Furthermore, the levels of apoptosis-associated proteins, Bax and Bcl-xL, were measured in bakuchiol-treated NUGC3 cells. Levels of procaspase-3,6,8 and 9 were significantly decreased following treatment with 50 and 100 µg/ml bakuchiol treatment compared with the control (P<0.05; Fig. 5B-E, respectively), while cleaved caspase-3 and cleaved PARP levels were significantly elevated following 50 and 100 µg/ml bakuchiol treatment compared with the control (P<0.01; Fig. 5F and G, respectively). The ratio of Bax/Bcl-xL in NUGC3 cells was also significantly upregulated following bakuchiol (50 µg/ml; P<0.05 and 100 µg/ml; P<0.01) treatment compared with the control (Fig. 5H). These data suggested that bakuchiol treatment induced caspase-dependent apoptosis in NUGC3 cells.

Cell proliferation and apoptosis are regulated by the PI3K/AKT and MAPK signaling pathways. As AKT activity is maintained by phosphorylation, the phosphorylation of PI3K/AKT and MAPK was analyzed using western blotting during bakuchiol-induced apoptosis in NUGC3 cells (Fig. 6A). Bakuchiol treatment (50 and 100 µg/ml) resulted in significant decrease (50 µg/ml; P<0.01 and 100 µg/ml; P<0.05) in the levels of phosphorylated AKT (p-Akt) compared with the control (Fig. 6B). Furthermore, phosphorylated ERK1/2, JNK and p38 levels were significantly upregulated upon treatment with bakuchiol compared with the control (P<0.01; Fig. 6C-E, respectively). These experimental findings suggested that bakuchiol induced apoptosis in NUGC3 cells by regulation of the PI3K/AKT and MAPK signaling pathways.