Although epidermal growth factor receptor (EGFR) mutations serve an important therapeutic role in patients with NSCLC, EGFR mutations are rarely (0–14.6%) identified in lung SqCC (12). Therefore, the present study used EGFR-negative SqCC cell lines (13,14). NCI-H520 and NCI-H226 cells, which are human lung SqCC cell lines, which were purchased from the Food Industry Research and Development Institute (15,16). These cells were maintained in RPMI-1640 supplemented with 10% FBS and an antibiotic-antimycotic agent containing amphotericin B, penicillin and streptomycin. All cell culture reagents were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The cells were cultured at 37°C in a humidified incubator with 5% CO₂. The stock dose of pterostilbene (Sigma-Aldrich; Merck KGaA) was 50 mM and was dissolved in a DMSO solution (Sigma-Aldrich; Merck KGaA).

H520 cells (3×10⁴) were seeded into a 24-well plate (Corning, Inc,), and allowed to adhere overnight. The cells were then treated with 1.56, 3.13, 6.25, 12.5 25 and 50 µM pterostilbene for 24 and 48 h. Following 24 and 48 h treatment, cells were incubated with 200 µl 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA) for 4 h. Cells treated with 0.1% DMSO were used as the control. The formazan was then dissolved in DMSO, and the optical density (OD) value at 570 nm was measured using an ELISA reader (Tecan Group, Ltd.). The IC50 was calculated by polynomial regression analysis using Microsoft Excel software version 2016 (Microsoft Corporation), and the mean (OD) ± SD for each group of triplicates was calculated.

H520 cells (3×10⁵) were seeded into a 6-well plate (Corning, Inc.), and allowed to adhere overnight in the aforementioned culture conditions. The next day, the cells were treated with 12.5, 25 and 50 µM pterostilbene for 48 h. Following 48 h treatment, cells were collected into a flow tube containing trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and centrifuged at 1,000 × g for 5 min at 4°C. Then, the cells were fixed for 16 h in 75% ethanol at −20°C. The cells were washed using PBS and incubated with 500 µl PBS with 0.1% (v/v) Triton X-100, 100 µg/ml RNase A and 50 µg/ml PI (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. The sub-G1 population containing apoptotic cells was detected using an Accuri™ C5 flow cytometer (BD Biosciences). Data were further analyzed with the C6 Accuri system software 1.0.264.21 (BD Biosciences).

H520 cells (3×10⁵) were seeded into a 6-well plate, and allowed to adhere for 24 h in the aforementioned culture conditions. The cells were then treated with 12.5–50 µM pterostilbene for 48 h. At these time points, the cells were collected into a flow tube containing trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and centrifuged at 1,000 × g for 5 min at 4°C. Then, the cells were stained according to the protocol of an Annexin V-FITC apoptosis detection kit (cat. no. 556547; BD Biosciences) for 15 min at room temperature. Finally, the green and red fluorescence of Annexin V/PI were detected with an Accuri™ C5 cytometer in the FL-1 and FL-2 graphs. Data were further analyzed with the C6 Accuri system software 1.0.264.21.

H520 cells (3×10⁵) were seeded into a 6-well plate. The next day, the cells were treated with 12.5–50 M of pterostilbene for 48 h. Following 48 h treatment, the cells were collected into a flow tube containing trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and centrifuged at 1,000 × g for 5 min at 4°C. The cells were then stained with 2 µM JC-1 (10 µg/ml; Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. The red and green fluorescence were detected with an Accuri™ C5 cytometer in the FL-1 and FL-2 graphs. Data were further analyzed with the C6 Accuri system software 1.0.264.21.

H520 cells (5×10⁵) were seeded onto a 6-cm plate. The next day, the cells were treated with 12.5–50 µM pterostilbene for 48 h. Following 48 h treatment, the cells were collected into a flow tube containing trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and centrifuged at 1,000 × g for 5 min at 4°C. The cells were lysed in RIPA buffer containing 1% protease inhibitor cocktail and 2% PMSF (all from Sigma-Aldrich; Merck KGaA) on ice for 30 min, and the protein concentration in the cell lysates was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). Cytosolic fractions were isolated using the Mitochondria/Cytosol Fraction kit (BioVision, Inc.) according to the manufacturer's instructions and the protein concentration in the cytosolic fraction was quantified using the BCA protein assay kit. The samples were separated using 12% SDS-PAGE and transferred onto a PVDF membrane (EMD Millipore). The membrane was blocked with a blocking buffer containing 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: Anti-cyclin A (1:500; cat. no. sc-239, clone BF683; Santa Cruz Biotechnology, Inc.), anti-cyclin E (1:500; cat. no. sc-247, clone HE12; Santa Cruz Biotechnology, Inc.), anti-CDK2 (1:500; cat. no. sc-6248, clone D12; Santa Cruz Biotechnology, Inc.), anti-p21 (1:1,000; cat. no. 2947, clone 12D1; Cell Signaling Technology, Inc.), anti-p27 (1:1,000; cat. no. 3686, clone D69C12; Cell Signaling Technology, Inc.), anti-cytochrome c (1:500; cat. no. 4280, clone 136F3; Cell Signaling Technology, Inc.), anti-Bax (1:1,000; cat. no. 5023, clone D2E11; Cell Signaling Technology, Inc.), anti-Bcl-2 (1:1,000 dilution; cat. no. 4223, clone D55G8; Cell Signaling Technology, Inc.) and anti-GAPDH (1:2,000 dilution; cat. no. sc-32233, clone 6C5; Santa Cruz Biotechnologies, Inc.). The next day, membranes were incubated with horseradish peroxidase AffiniPure goat anti-rabbit immunoglobulin G (H+L) (1:2,000; cat. no. 111-035-144; Jackson ImmunoResearch Laboratories, Inc.) or horseradish peroxidase AffiniPure Goat Anti-mouse IgG (H+L) (1:2,000; cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.) at 4°C overnight. Finally, a Western Lightning^® Plus-ECL kit reagent (PerkinElmer, Inc.) was added to the membranes so the immunofluorescence signals could be detected using Hansor Luminescence Image system (Hansor). All bands in the blots were normalized to the level of GAPDH for each lane. The intensity of the bands was quantified using ImageJ 1.47 software for Windows (National Institutes of Health).

H520 cells (3×10⁵) were seeded into a 6-well plate. The next day, the cells were treated with 12.5–50 M pterostilbene for 48 h. Following 48 h treatment, the cells were collected into a flow tube containing trypsin-EDTA and centrifuged at 1,000 × g for 5 min at 4°C to remove the supernatant. The cells were stained according to the manufacturer's protocol for the CaspGLOW™ fluorescein active caspase-3/-8/-9 staining kit (BioVision, Inc.).

Female BALB/c athymic nude mice (18–22 g, 6-week-old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). A total of 12 animals were categorized into two groups, each comprising 6 mice (n=6). During the whole period of experimental study, animals were housed under standard environmental conditions of temperature and humidity (22±2°C and 60±10%, respectively) and with 12-h light/dark cycle. All animals had free access to regular rat chow and water ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of National Chung Hsing University.

H520 cells (1×10⁷ in 100 µl) was mixed with 0.2 ml of extracellular matrix gel (BD Biosciences) that was inoculated under the skin of the nude mice. When the tumor volume had reached ~10 mm³, the nude mice (n=6 per cage) were intraperitoneally administered 50 mg/kg pterostilbene or the vehicle control (10% DMSO + 90% glyceryl trioctanoate; Sigma-Aldrich; Merck KGaA) every other day continuously until day 37. On day 38, the nude mice were sacrificed using 30 psi CO₂ for 15–20 sec until cardiac arrest. Subsequently, the tumors were excised from the nude mice, images of the tumors were obtained, the tumors were weighed and the tumor volumes were measured three times a week using an electronic caliper (Mitutoyo Inc.). Tumor volume was calculated according to the following formula: (a × b² ×0.5), where a and b indicate the tumor's long and short diameters, respectively. The dosage and route of pterostilbene injection were based on previous research (17). To evaluate the toxicity of the pterostilbene treatment on the mice, the weights of the body, heart, lung, liver, kidney and spleen were recorded.

For comparison between two groups, an unpaired two-tailed t-test (Student's t-test) was conducted. One-way or two-way ANOVA followed by Tukey's HSD post hoc test was used to compare multiple groups. Data are presented as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effect of pterostilbene on the viability of SqCC cancer cell lines was determined using an MTT assay. In the present study, H520 and H226 human SqCC cancer cell lines were treated with different concentrations of pterostilbene for 24 and 48 h. As illustrated in Fig. 1A, pterostilbene induced cytotoxicity in human SqCC cancer cell lines in a dose-dependent manner. Notably, these two SqCC cell lines exhibited different sensitivities to pterostilbene; the IC50 values of pterostilbene for H520 cells were 47.7±5.3 and 31.4±4.6 µM at 24 and 48 h, respectively, while the IC50 values for H226 cells were >50 and 44.3±3.7 µM at 24 and 48 h, respectively. In addition, the cell morphology and shape were assessed using an inverted microscope; this assessment indicated apoptotic morphological changes, including cell shrinkage and cytoplasmic blebbing, in the treated cells (Fig. 1B). The results demonstrated that the H520 cell line was highly sensitive to pterostilbene treatment; therefore H520 was selected for the subsequent analysis and evaluation of the cytotoxic potency of pterostilbene.

The present study subsequently investigated the impact of pterostilbene on different cell cycle phases in H520 cells. The H520 cells were exposed to different concentrations of pterostilbene for 48 h and were subjected to flow cytometric analysis following staining with PI. As Fig. 2A illustrates, the percentage of cells in S phase was markedly increased following exposure to 12.5–50 µM pterostilbene for 48 h. Additionally, the sub-G1 cell population, which is indicative of cell mortality, was increased in the presence of 50 µM pterostilbene. To confirm whether apoptotic mechanisms may have been involved in the cell death associated with treatment with pterostilbene, H520 cells were treated with pterostilbene for 48 h and subjected to flow cytometric analysis following Annexin V-FITC and PI staining. A quantitative flow cytometric analysis revealed that the percentages of early apoptotic H520 cells (Annexin V⁺/PI⁻, lower right quadrant) were increased by pterostilbene in a dose-dependent manner (Fig. 2B). Taken together, these results indicated that pterostilbene induced S phase arrest and early apoptosis in H520 SqCC cells.

To investigate the molecular mechanisms of pterostilbene in the induction of S phase arrest, the present study examined the effect of pterostilbene on the expression of key cell cycle regulators of S phase progression through western blot analysis. The Cip/Kip family members, p21 (Cip1) and p27 (Kip1), can inhibit the activity of cyclin E-CDK2 and cyclin A-CDK2 complexes. The results indicated that treatment with pterostilbene reduced the protein expression of cyclin A and cyclin E, but did not affect the expression of CDK2 (Fig. 3), as indicated by the fold change in the band intensity compared with that of the vehicle-treated cells. In addition, p21 and p27 protein expression was increased by treatment with pterostilbene (Fig. 3). These findings suggested that pterostilbene induced S phase arrest by regulating the expression of S phase cell cycle regulatory proteins in the H520 SqCC cell lines.

In order to determine whether pterostilbene induced apoptotic cell death in H520 cells through the activation of caspase-3, −8 and −9, H520 cells were treated with pterostilbene for 48 h, harvested and examined using flow cytometry. Pterostilbene increased the activity of caspase-3, −8 and −9 (Fig. 4) in a dose-dependent manner and induced cell death through the activation of caspase-3, −8 and −9 in H520 cells.

The loss of mitochondrial membrane has a major role in the induction of apoptosis. Therefore, the membrane-permeable JC-1 dye was used to examine the mitochondrial membrane in H520 cells that has been treated with pterostilbene. As presented in sFig. 5A, following treatment with pterostilbene, a dose-dependent increase in the green fluorescence was observed in H520 cells, suggesting that pterostilbene produced significant mitochondrial depolarization. Cytochrome c release from the mitochondria to the cytosol is also a key initial step in apoptosis. Therefore, in order to determine whether cytochrome c was released during pterostilbene-induced apoptosis, western blot analysis was performed to analyze the cytochrome c level in the cytosolic fractions of H520 cells that had been exposed to pterostilbene. As presented in Fig. 5B, an increase in cytochrome c at 48 h was observed in the cytosolic fraction of the pterostilbene-treated cells, compared with the cytochrome c level in the control cells. In terms of the expression of Bcl-2 family members, such as the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax, have been identified during critical events in the mitochondrial apoptotic pathway; therefore, the present study further investigated the effects of pterostilbene on the expression of Bax and Bcl-2 in H520 cells by western blot analysis, and there was an increase in Bax protein expression and a decrease in Bcl-2 expression in H520 cells (Fig. 5C). These data suggested that pterostilbene induced the caspase-dependent mitochondrial apoptotic pathways in H520 cells.

The present study then investigated whether pterostilbene had an effect against H520 ×enografts in nude mice. As illustrated in Fig. 6A-C, compared with the effect of the vehicle control, pterostilbene (50 mg/kg) was able to reduce the tumor volume (Fig. 6A and B) and weight (Fig. 6C) (day 38), indicating that pterostilbene also inhibited the growth of SqCC cells in vivo. The toxic effect of treatment with pterostilbene was investigated in H520 ×enograft models. As illustrated in Fig. 6D and E, there was no significant difference in body weight (Fig. 6D), or heart, lung, liver, kidney, and spleen weights (Fig. 6E) between the pterostilbene-treated group and the vehicle-treated group, which suggested that treatment with pterostilbene did not obviously exhibit adverse side effects.