Pristimerin, 5-fluorouracil, cisplatin and propidium iodide (PI) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Pristimerin (molecular structure shown in Fig. 1A) was prepared as a 20 mM stock solution in dimethyl sulfoxide. The CAL-27 and SCC-25 cells, which were initially isolated from the epidermal tongue tissue of patients with OSCC, were kindly donated by Professor Hongzhang Huang (Department Oral & Maxillofacial Surgery, Sun Yat Sen University, Guangzhou, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) F-12 (Biological Industries, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Biological Industries), at 37°C in a humidified atmosphere containing 5% CO₂.

Cell viability was measured by MTS assay (CellTiter 96 AQueous MTS Reagent; Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. Briefly, CAL-27 and SCC-25 cells (5×10³ cells/well) were seeded into a 96-well plate for 12 h to allow cell attachment, and were then treated with increasing concentrations of pristimerin (0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 µM), cisplatin (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 µM) or 5-fluorouracil (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160 µM) at 37°C for 68 h. Thereafter, 20 µl MTS/PMS (20:1 in volume) was added and incubated for an additional 4 h. Cell viability was finally determined using a microplate reader (Detie, Nanjing, China) at 490 nm. The half maximal inhibitory concentration (IC₅₀) of pristimerin was calculated.

CAL-27 and SCC-25 cells (2×10⁵/ml) were treated with increasing concentrations of pristimerin (0, 0.25, 0.5, 1 µM) at 37°C for 12 or 24 h. They were then collected, washed three times with PBS and seeded in a 12-well plate (10³/well) in DMEM F-12 medium containing 0.3% agar and 20% FBS. After a further 10–14 days of culture at 37°C, colonies containing >50 cells were counted under an inverted phase-contrast microscope.

CAL-27 and SCC-25 cells (2×10⁵/ml) were treated with various concentrations of pristimerin (0, 0.25, 0.5, 1 µM) at 37°C for 12 or 24 h. After collection, cells were washed with PBS and stained with Annexin V-fluorescein isothiocyanate (FITC)/PI (Annexin V-FITC Apoptosis Detection kit; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. The number of viable, necrotic and apoptotic cells were assessed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

CAL-27 and SCC-25 cells (2×10⁵/ml) were treated with various concentrations of pristimerin (0, 0.25, 0.5, 1 µM) at 37°C for 12 or 24 h. After collection, they were washed twice with cold PBS and fixed with cold 75% ethanol at 4°C overnight. Ethanol was eventually discarded, cells were resuspended in PBS containing PI (50 µg/ml) and were incubated in a water bath (37°C) for 1 h. The cell cycle distribution was finally examined by flow cytometry.

Following treatment with pristimerin (0, 0.25, 0.5, 1 µM for 24 h and 1 µM for 12 h), total cellular RNA was extracted from CAL-27 and SCC-25 cells with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. RNA was reverse transcribed into cDNA (MMLV reverse transcriptase; Promega Corporation), according to the manufacturer's protocol, and the mRNA expression levels were measured by GoTaq qPCR Master Mix (Promega, Corporation) using the ABI7000 cycler (Applied Biosystems; Thermo Fisher Scientific Inc.). The primers for RT-qPCR were designed as follows: p21, forward 5′-TCTTGTACCCTTGTGCCTCG-3′, reverse 5′-GAAGATCAGCCGGCGTTTG-3′; p27, forward 5′-GTCAAACGTAAACAGCTCGAAT-3′, reverse 5′- TGCATAATGCTACATCCAACG-3′; p53, forward 5′- GAGGTTGGCTCTGACTGTACC-3′, reverse 5′- TCCGTCCCAGTAGATTACCAC-3′; cyclin D1, forward 5′-GTGCTGCGAAGTGGAAACC-3′, reverse 5′-ATCCAGGTGGCGACGATCT-3′; cyclin E, forward 5′-GTTATAAGGGAGACGGGGAGC-3′, reverse 5′-TGCTCTGCTTCTTACCGCTC-3′; and GAPDH, forward 5′-CGACCACTTTGTCAAGCTCA-3′; and reverse 5′-AGGGGTCTACATGGCAACTG-3′. PCR was performed at 94°C for 5 min, followed by 40 cycles at 94°C for 30 sec and 56°C for 30 sec. Relative quantification of gene expression was performed using the threshold cycle difference 2^−ΔΔCq method (17), and the geometric mean of GAPDH levels was used as an internal control to normalize the variability in expression level.

Cells were washed with cold PBS and lysed with lysis buffer (1X PBS, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) supplemented with freshly added 1 mM phenylmethylsulfonyl fluoride, 1X Roche complete Mini protease inhibitor cocktail (Roche Diagnostics, Shanghai, China), 10 mM glycerophosphate, 10 mM NaF, and 1 mM sodium orthovanadate. DNA contained in the lysate was sheared by sonication with 10 1-sec bursts with a 3 sec interval at medium power. Following quantification using the bicinchoninic acid method, proteins (60 µg) were separated by 10–15% SDS-PAGE. Thereafter, proteins were transferred to polyvinylidene difluoride membrane and blocked with 5% non-fat milk in PBS-Tween (PBST) at 37°C for 1 h. Antibodies against poly (ADP-ribose) polymerase (PARP) (cat. no. 9532), caspase-3 (cat. no. 9665), p21 (cat. no. 2947), Erk1/2 (cat. no. 4695), phosphorylated (p)-Erk1/2 (cat. no. 4370), Akt (cat. no. 4691) and p-Akt (cat. no. 4060) were purchased from CST Biological Reagents Co., Ltd. (Shanghai, China). Horseradish peroxidase-conjugated goat antibodies against mouse and rabbit (cat. no. 31430, cat. no. 31460) were purchased from Thermo Fisher Scientific Inc. Antibodies was prepared in PBST with dilutions of 1:500 (p-Erk1/2 and p-Akt), 1:8,000 (β-actin) and the rest of the antibodies were 1:1,000. Primary antibody was added to the membrane and incubated at 4°C overnight. Following washing in triplicate with PBST, the secondary antibody was added (1:5,000) and incubated at room temperature for 1 h, subsequently washed 2 times with PBST and 1 time with PBS. Enhanced chemiluminescence reagent (Immobilon Western; cat. no. WBKLS0500, EMD Millipore, Billerica, MA, USA) was added and the x-ray film was exposed. Protein bands were quantified and normalized to β-actin by using Image-Pro Plus 6.0 system (Media Cybernetics, Inc., Rockville, MD, USA).

Each experiment was performed at least three times. GraphPad 5.0 Software (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. Data are expressed as the means ± standard deviation, and differences between groups were assessed by one-way analysis of variance with post-hoc intergroup comparisons using Turkey test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the possible effects of pristimerin on OSCC cells, CAL-27 and SCC-25 cells were treated with increasing concentrations of pristimerin for 68 h, prior to determining cell viability with the MTS assay. Results demonstrated that pristimerin effectively inhibited the growth of CAL-27 and SCC-25 cells in a dose-dependent manner with IC₅₀ values of 0.70 and 0.73 µM, respectively (Fig. 1B). The toxic effects of pristimerin on CAL-27 and SCC-25 viability were compared to the ones observed after 72 h treatment with the commonly used antitumor drugs cisplatin and 5-fluorouracil. The results demonstrated that the IC₅₀ values of cisplatin and 5- fluorouracil in CAL-27 cells were 7.69 and 11.98 µM, respectively. The IC₅₀ values of cisplatin and 5-fluorouracil in SCC-25 cells were 15.96 and 11.45 µM, respectively (Fig. 1C). Compared with cisplatin and 5-fluorouracil, the IC₅₀ values of pristimerin in oral cancer cells were lowest (~0.7 µM). Because clonogenicity is believed to better reflect the malignant behavior of tumor cells, colony formation was determined after CAL-27 and SCC-25 cells were treated either with increasing concentrations of pristimerin for 24 h, or with 1 µM pristimerin for various durations. The results demonstrated that pristimerin potently inhibited the colony formation o CAL-27 and SCC-25 cells in a dose- and time-dependent manner (Fig. 1D).

To explore the underlying mechanism of pristimerin-induced inhibition of cell proliferation, the effects of pristimerin on cell cycle distribution were examined by flow cytometry (Fig. 2A). The results revealed that the proportion of G₀/G₁ phase CAL-27 cells treated with 0, 0.25, 0.5 and 1 µM pristimerin for 24 h were 51.92, 55.53, 66.32 and 74.80%, respectively. Similarly, in SCC-25 cells, G₀/G₁ phase distributions were 60.63, 71.63, 85.73 and 89.63% following treatment with 0, 0.25, 0.5 and 1 µM pristimerin, respectively, for 24 h. In addition, the proportion of G₀/G₁ phase CAL-27 cells was 65.08% and of G₀/G₁ phase SCC-25 cells was 76.50% following treatment with 1 µM pristimerin for 12 h (Fig. 2A). In parallel, the S and G₂/M phase distributions were reduced. These data revealed that pristimerin significantly increased the proportion of G₀/G₁ phase CAL-27 and SCC-25 cells, thus suggesting that G₀/G₁ phase arrest may be induced following pristimerin treatment. Furthermore, the expression levels of molecules involved in the cell cycle process, including cyclin D1, cyclin E, cyclin-dependent kinase (CDK) inhibitors (CDKIs) p21 and p27, and p53, were assessed by RT-qPCR. Results demonstrated that p21, p27 and p53 expression levels were significantly increased following pristimerin treatment (Fig. 3A-C), whereas cyclin D1 and cyclin E expression levels were significantly decreased under the same conditions (Fig. 4A and B). These modulating effects of pristimerin were observed in a dose- and time-dependent manner in both cell lines. Moreover, western blot analysis exhibited increased levels of p21 protein in a dose- and time-dependent manner in both cell lines (Fig. 5A and B). These results were consistent with the ones obtained from RT-qPCR analysis of p21 expression (Fig. 3).

The effects of pristimerin on cancer cell apoptosis were assessed by flow cytometry. CAL-27 and SCC-25 cells were treated with various concentrations of pristimerin for various durations (Fig. 6), and were double-stained with Annexin V-FITC/PI. Results demonstrated that apoptotic cells were significantly increased by pristimerin in a dose- and time-dependent manner (Fig. 6A and B). The mean apoptotic cell rates from three independent experiments were 1.29, 18.81, 39.1 and 47.7% in CAL-27 cells, and were 2.1, 21.1, 30.05 and 50.23% in SCC-25 cells following treatment with pristimerin at 0, 0.25, 0.5 and 1 µM, respectively for 24 h. Following cell treatment with 1 µM pristimerin for 12 h, the mean apoptotic cell rates were 13.61% in CAL-27 cell and 16.67% in SCC-25 cells. Additionally, PARP and caspase-3, the hallmark proteins in apoptosis, were examined by western blot analysis. As presented in Fig. 7, caspase-3 expression levels were significantly reduced in the two cell lines in a dose- and time-dependent manner following pristimerin treatment. In addition, cleaved-PARP (85 kDa) levels were substantially increased in a dose- and time-dependent manner following pristimerin treatment in CAL-27 and SCC-25 cells (Fig. 7). The upregulated cleaved-PARP level and downregulated caspase-3 level further suggested that apoptosis was induced following pristimerin treatment in OSCC cells (Fig. 7).

To determine whether the MAPK/Erk1/2 and PI3K/Akt pathways were involved in the anticancer effects of pristimerin in OSCC cells, p-Akt, Akt, p-Erk1/2 and Erk1/2 levels were detected by western blotting (Fig. 8). Results demonstrated that p-Akt and p-Erk1/2 expression levels were decreased in a dose- and time-dependent manner; however, there were no significant differences in the expression levels of total Akt and Erk1/2.