The CAL 27 parental human oral cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), which is a cell line identified as tongue squamous cell carcinoma. The cisplatin-resistant oral cancer CAR cells were established by gradient induction of increasing concentrations of cisplatin up to 80 µM in CAL 27 cells, as previously described (1,8,27). In brief, the CAL 27 cells were initially incubated with 10 µM cisplatin for 24 h, and the culture media was replaced by cisplatin-free fresh culture medium until the CAL 27 cells reached a confluence of 80–90%. The procedure was repeated with increasing concentrations of cisplatin, and the CAL 27 cells were cultured with each concentration (10–80 µM) of cisplatin for five cycles to obtain the cisplatin-resistant CAR cells. The CAR cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Normal human primary gingival fibroblast (HGF) was obtained from CLS Cell Lines Service GmbH (Eppelheim, Germany) and cultivated in DMEM/F12 1:1 medium (HyClone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% FBS, 100 µg/ml streptomycin, 100 U/ml penicillin and 2 mM L-glutamine. All cells were cultured in a 37°C humidified incubator with 5% CO₂.

DMEM, DMEM/F12 1:1 medium, FBS, L-glutamine, and penicillin/streptomycin were purchased from HyClone Laboratories; GE Healthcare Life Sciences. Ursolic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other chemicals were of analytical grade from Sigma-Aldrich; EMD Millipore (Billerica, MA, USA) unless otherwise specified. The pan-caspase inhibitor z-VAD-fmk was purchased from EMD Millipore. Caspase-3 and Caspase-9 Colorimetric Assay kits were from R&D Systems, Inc. (Minneapolis, MN, USA). The anti-caspase-3 (cat. no. GTX110543), anti-caspase-9 (cat. no. GTX112888), anti-phosphorylated (p)-AKT^Ser473 (cat. no. GTX28932), anti-AKT (cat. no. GTX121937), anti-p-BAD^Ser136 (cat. no. GTX50136), anti-BAD (cat. no. GTX130108), anti-Bax (cat. no. GTX109683), anti-Bcl-2 (cat. no. GTX100064), anti-Bcl-xL (cat. no. GTX84834), and anti-β-actin (cat. no. GTX109639) antibodies, and the anti-rabbit (cat. no. GTX213110-01) and anti-mouse (cat. no. GTX213111-01) IgG horseradish peroxidase (HRP)-linked antibodies were all purchased from GeneTex, Inc. (Hsinchu, Taiwan). The reactive oxygen species (ROS) indicator H₂DCFDA and the mitochondrial membrane potential (ΔΨm) detector DiOC₆(3) were obtained from Molecular Probes; Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Cell viability was evaluated with an MTT method, as previously described (23,28). Briefly, the CAR or HGF cells were seeded in a 96-well plate at a density of 1×10⁴ cells/well and treated with different concentrations (50, 100, 150 or 200 µM) of ursolic acid prior to pre-incubation with or without 10 µM z-VAD-fmk (pan-caspase inhibitor) for 1.5 h at 37°C. After 24 h, the medium was removed, and the cells were cultured with 0.5 mg/ml MTT solution for an additional 2 h at 37°C. Subsequently, 100 µl DMSO was used to dissolve the blue formazan product, and cell viability was spectrophotometrically measured at the absorbance of 570 nm, as previously described (29,30).

The cell confluence experiment was monitored using the IncuCyte ZOOM system (Essen BioScience, Ann Arbor, MI, USA), as previously described (1,31). In brief, the CAR cells at a density of 1×10⁴ cells per well were seeded in a 96-well plate and then exposed to 0, 50, 100 and 200 µM of ursolic acid for 48 h. Data collection was performed every 2 h until 48 h, and images of the morphological changes were captured and collected every 12 h using the IncuCyte ZOOM system (Essen BioScience).

The activities of caspase-3 and caspase-9 were detected using Caspase-3 and Caspase-9 Colorimetric Assay kits (R&D Systems Inc.) with synthetic tetrapeptides [Asp-Glu-Val-Asp (DEAD) for caspase-3; Leu-Glu-His-Asp (LEHD) for caspase-9] labeled with p-nitroaniline (pNA) to link to the caspase-specific substrate. The CAR cells (5×10⁶ cells per 75T flask) were treated with or without 50, 100, 150 and 200 µM of ursolic acid for 24 h. The cell lysates were then harvested, and the supernatants were incubated with the supplied reaction buffer with dithiothreitol and DEAD-pNA or LEHD-pNA as substrates at 37°C for 2 h in the dark according to the manufacturer's protocols.

The CAR cells (5×10⁶ cells per 75T flask) were treated with or without 100, 150 and 200 µM of ursolic acid for 12 h. The cells were then harvested and lysed with Trident RIPA lysis buffer (GeneTex, Inc.). The Pierce BCA protein assay kit was used to detect the protein concentration, following which an equal quantity of the protein sample (40 µg) was subjected to electrophoresis on a 10–12% sodium dodecyl sulfate-polyacrylamide gel, as previously described (32,33). The separated protein was transferred onto the Immobilon-P Transfer membrane (Merck Millipore, Darmstadt, Germany) via use of electroblotting. Thereafter, the membranes were soaked in 5% skim milk and individually incubated overnight with primary antibodies, including caspase-3, caspase-9, p-AKT^Ser473, AKT, p-BAD^Ser136, BAD, Bax, Bcl-2, Bcl-xL (all 1:1,000 dilution) and β-actin (1:5,000 dilution) at 4°C, followed by incubation with the appropriate HRP-conjugated secondary antibodies (1:10,000 dilution) for 1 h at room temperature to hybridize targeted protein using Immobilon Western Chemiluminescent HRP substrate (Merck Millipore), as previously described (34,35). All bands of immunoblots were normalized to β-actin, and their densitometric quantification was performed using NIH ImageJ 1.47 software (National Institutes of Health, Bethesda, MD, USA).

The CAR cells (2×10⁵ cells/ml) in 12-well plates were exposed to 0, 50, 100, 150 and 200 µM of ursolic acid for 12 h. The cells were then collected and incubated with 500 µl of H₂DCF-DA (ROS detector dye, 10 µM) and 50 nM of the cell-permeant ΔΨm probe, DiOC₆(3), at 37°C for 30 min using flow cytometry, as previously described (33,36).

The values are expressed as the mean ± standard deviation from at least three separate experiments. Data analysis was performed using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). The differences were analyzed using one-way analysis of variance followed by Dunnett's test. P<0.001 was considered indicate a statistically significant difference.

The cytotoxicity of ursolic acid towards CAR cells was first investigated. The cells were cultured with various concentrations of ursolic acid (50, 100, 150 and 200 µM) for 24 h. Cell viability was evaluated using the MTT assay. The results demonstrated that ursolic acid at 100, 150 and 200 µM significantly reduced the viability of CAR cells in a concentration-dependent manner (Fig. 1A). By contrast, ursolic acid exerted no toxicity towards the normal HGF cells (Fig. 1B). Similarly, the cell confluence of the cultured CAR cells following exposure to the different concentrations (0, 50, 100 and 200 µM) of ursolic acid was monitored using the IncuCyte ZOOM system instrument at the 2-h period. The data showed that the inhibition of CAR cell confluence appeared following incubation with 200 µM ursolic acid, compared with the control, when incubated for up to 48 h (Fig. 2). In addition, images of the cultured CAR cells captured at 12-h intervals demonstrated that ursolic acid at 200 µM induced cell morphology changes and a decrease of cell confluence, and triggered CAR cell death (Fig. 3). Therefore, these finding suggested that ursolic acid at 200 µM produced a marked reduction in the viability of CAR cells.

To further examine whether the observed suppression of cell viability involved apoptotic machinery, the cells were pretreated with 10 µM z-VAD-fmk and then exposed to 200 µM ursolic acid for 24 h. The results showed that, without prior incubation of the CAR cells with 10 µM z-VAD-fmk, the inhibition of cell viability was significantly inhibited by ursolic acid at 200 µM (Fig. 4). Therefore, ursolic acid inhibited CAR cell viability via the caspase pathway.

To investigate whether the ursolic acid-induced apoptosis in CAR cells is associated with the intrinsic pathway (caspase-3 and caspase-9) following treatment with various concentrations (0, 50, 100, 150 and 200 µM) of ursolic acid, the activities and protein levels of caspase-3 and −9 were individually assayed using a colorimetric assay and western blot analysis. The results indicated that ursolic acid significantly promoted the activities of caspase-3 at 100, 150 and 200 µM concentrations in a concentration-dependent manner (Fig. 5A). Similarly, the activity of caspase-9 was markedly enhanced in the CAR cells exposed to ursolic acid (150 and 200 µM; Fig. 5B). Ursolic acid markedly increased the protein level of the active form of caspase-3 (Fig. 5C). In addition, it promoted an increase in the protein level of cleaved caspase-9 (Fig. 5C). On the basis of these results, it was suggested that the apoptotic mechanism of ursolic acid in CAR cells was mediated via caspase-3/-9-dependent signaling.

As ursolic acid affected the activation of caspase-9, it was hypothesized that ursolic acid-induced apoptosis may be regulated via the mitochondria-dependent pathway. The CAR cells were treated with ursolic acid at various concentrations for 12 h. The levels of ROS production and ΔΨm were measured by flow cytometric assays. The results indicated that ursolic acid promoted the production of ROS (Fig. 6A), but decreased the level of ΔΨm (Fig. 6B) in the CAR cells, and these effects were concentration-dependent. Based on these findings, mitochondrial dysfunction may be required for the ursolic acid-induced apoptosis of CAR cells.

To further understand the mechanism of apoptosis in CAR cells, the protein signals of AKT, BAD, Bax, Bcl-2 and Bcl-xL were determined in the ursolic acid-treated cells. Ursolic acid at 100, 150 and 200 µM for 12 h decreased the phosphorylation of AKT on Ser473 (p-AKT) and BAD on Ser136 (p-BAD), decreased the protein levels of Bcl-2 and Bcl-xL, and increased the expression of BAD and Bax (Fig. 7A and B). These findings showed that ursolic acid induced apoptotic CAR cell death through a mitochondria-dependent pathway.