Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin were purchased from HyClone; GE Healthcare Life Sciences (Logan, UT, USA). Acridine orange (AO), Hoechst 33342, LysoTracker Red DND-99, trypsin-EDTA, the High Capacity cDNA Reverse Transcription kit, Pierce bicinchoninic acid (BCA) protein assay kit and SYBR-Green PCR Master mix were sourced from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Immobilon-P polyvinylidene difluoride transfer membrane and Immobilon western chemiluminescent horseradish peroxidase (HRP) substrate were purchased from EMD Millipore (Billerica, MA, USA). Caspase-3 (cat. no. K106-100), -8 (cat. no. K113-100), and -9 (cat. no. K119-100) colorimetric assay kits were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). All primary antibodies, as well as anti-mouse and anti-rabbit immunoglobulin (Ig)G HRP-linked secondary antibodies were all procured from GeneTex International Corporation (Hsinchu, Taiwan). Pterostilbene, 3-methyladenine (3-MA), chloroquine (CQ), carbobenzoxyvalyl-alanyl-aspartyl fluoromethyl ketone (Z-VAD-FMK), monodansylcadaverin (MDC) and all other chemicals and reagents were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), unless otherwise specified.

Cisplatin-resistant human oral cancer CAR cells were established from the human oral cancer cell line CAL 27 (American Type Culture Collection, Manassas, VA, USA) following a previously reported method (43,44). CAR cells were then cultured in DMEM containing 10% FBS, 1% penicillin/streptomycin (100 μg/ml streptomycin and 100 U/ml penicillin), 2 mM L-glutamine and 80 μM cisplatin. Cells were maintained at 37°C in a 5% CO₂/95% air humidified incubator. Prior to administration, pterostilbene was dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO was <0.5%. Control cells were exposed to 0.5% DMSO alone.

CAR cells were seeded onto 96-well culture plates at a density of 1×10⁴ cells/well (100 μl/well) and subsequently incubated with 5, 10, 25, 50, 75 and 100 μM pterostilbene for 24, 48 and 72 h. The effect of pterostilbene on cell viability was then determined using an MTT assay, as previously described (45,46). For the inhibition assays, cells were pretreated with autophagy inhibitors (10 mM 3-MA and 20 μM CQ) and 15 μM Z-VAD-FMK (a pan-caspase inhibitor) for 1 h prior to exposure to 50 or 75 μM pterostilbene for 24 or 48 h. Following the exposure of cells to pterostilbene, MTT (5 mg/ml) was dissolved in phosphate-buffered saline, and 10 μl MTT solution was added to each well at a final concentration of 500 μg/ml for 2 h. The blue formazan crystals were then dissolved in DMSO (100 μl/well) by constant shaking for 10 min. The absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader at a test wavelength of 570 nm, with a reference wavelength of 620 nm. Half maximal inhibitory concentrations (IC₅₀) were calculated using the improved Karber method, using the following formula: (lgIC₅₀ = Xm-I[P-(3-Pm-Pn)/4], where Xm, lg(maximum dose); I, lg(maximum dose/adjacent dose); P, sum of the positive reaction rates; Pm, maximum positive reaction rate; and Pn, minimum positive reaction rate (47). For the examination of cell morphology, cells were observed without any treatments (control), observed following treatment with pterostilbene alone, treatment with the autophagy inhibitors (10 mM 3-MA or 20 μM CQ) alone, and following treatment with pterostilbene and each of the autophagy inhibitors. Cells were incubated and subsequently observed and photographed using a phase-contrast microscope at a magnification of ×400.

CAR cells (1×10⁴ cells/well) were seeded onto a 96-well plate and treated with 0, 25, 50, 75 or 100 μM pterostilbene. The cell confluence experiment was conducted over 48 h with data collection every 2 h and was monitored using the IncuCyte ZOOM System instrument (Essen BioScience, Ann Arbor, MI, USA), as previously described (48,49).

CAR cells were plated onto sterile chamber slides onto 10-cm tissue culture dishes at a density of 1×10⁶ cells/plate. Cells were treated with 0, 25, 50 or 75 μM pterostilbene for 24 h following pretreatment with or without 10 mM 3-MA or 20 μM CQ for 1 h. Cells were then fixed with 4% paraformaldehyde on ice for 15 min and were individually probed with either 1 μg/ml AO, 100 μM MDC, 1 μg/ml LysoTracker Red or 1 μg/ml Hoechst 33342 for 15 min at room temperature, as previously described (50,51). Lysosomal activity in the pterostilbene-treated cells were determined using the Magic Red Cathepsin B assay kit (ImmunoChemistry Technologies, LLC, Bloomington, MN, USA) following the manufacturer's protocol. Following staining, each slide was mounted with coverslips, and photomicrographs of acidic vesicular organelles (AVOs), autophagic vacuoles, lysosomal biogenesis, and nuclei were taken using a fluorescence microscope.

CAR cells (5×10⁶) in T75 flasks were treated with 0, 50 and 75 μM pterostilbene for 24 h. Total RNA was then isolated using QIAGEN RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's instructions and cDNA synthesis was completed using the High Capacity cDNA Reverse Transcription kit. qPCR was performed under the following conditions: 10 min at 95°C, 40 cycles of 15 sec at 95°C and 1 min at 60°C. qPCR was performed using 2X SYBR-Green PCR Master mix and 200 nM forward and reverse primers for light chain 3 (LC3)-II, autophagy-related gene (Atg)12 and MDR1. GAPDH was used as a reference gene. The sequences of the primers used were as follows: LC3-II, forward, 5′-CCGACCGCTGTAAGGAGGTA-3′ and reverse, 5′-AGGACGGGCAGCTGCTT-3′; Atg12, forward, 5′-TGTGGCCTCAGAACAGTTGTTTA-3′ and reverse, 5′-CGCCTGAGACTTGCAGTAATGT-3′; MDR1, forward, 5′-GTGTGGTGAGTCAGGAACCTGTAT-3′ and reverse, 5′-TCTCAATCTCATCCATGGTGACA-3′; GAPDH, forward, 5′-ACACCCACTCCTCCACCTTT-3′ and reverse, 5′-TAGCCAAATTCGTTGTCATACC-3′. Each assay was run on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) in triplicate, and the fold-changes of gene expression were derived using the comparative 2^−ΔΔCq method, as previously described (50,52).

CAR cells at a density of 5×10⁶ cells per 75T flask were incubated with 0, 50 and 75 μM pterostilbene for 24 or 48 h. Cell samples were lysed in the Trident RIPA Lysis Buffer (cat. no. GTX400005; GeneTex) and collected as previously described (53,54). Protein concentration was determined using the Pierce BCA protein assay kit. Equal amounts of the protein sample (40 μg) were prepared and subsequently 10–12% SDS-PAGE was performed. Proteins were transferred to the Immobilon-P polyvinylidene difluoride transfer membrane prior to blocking with Trident Universal Protein Blocking Reagent (cat. no. GTX30963; GeneTex) for 1 h at room temperature. The membrane was subsequently incubated with primary antibodies against Atg5 (cat. no. GTX113309), Atg7 (cat. no. GTX113613), Atg12 (cat. no. GTX629815), Beclin-1 (cat. no. GTX631396), LC3 (cat. no. GTX39752; LC3-I, 17 kDa; LC3-II, 14 kDa), Bcl-2 (cat. no. GTX100064), Bax (cat. no. GTX109683), cytochrome c (cat. no. GTX108585), caspase-9 (cat. no. GTX112888; active form, 19 kDa), caspase-3 (cat. no. GTX110543; active form, 19 kDa), caspase-7 (cat. no. GTX22301; active form, 20 kDa), poly (ADP-ribose) polymerase (PARP; cat. no. GTX100573; p85, 85 kDa), MDR1 (cat. no. GTX108370), phosphorylated (p)-AKT (Ser473) (cat. no. GTX28932), AKT (cat. no. GTX121937) and β-actin (cat. no. GTX109639) at 4°C overnight. All antibodies were purchased from GeneTex and used at a dilution of at 1:1,000. Membranes were then incubated with appropriate anti-mouse (cat. no. GTX213111-01) and anti-rabbit (cat. no. GTX213110-01) IgG HRP-linked secondary antibodies at a dilution of 1:10,000 for 1 h at room temperature. Blot visualization was performed using the Immobilon Western Chemiluminescent HRP Substrate and all bands of immunoblots were normalized to the densitometric value of β-actin. The bands were quantified by densitometry using ImageJ software version 1.41 (National Institutes of Health, Bethesda, MA, USA).

CAR cells (1×10⁵ cells/ml) on 12-well plates were harvested following treatment with 0, 25, 50, 75 and 100 μM pterostilbene for 48 h and re-suspended with the In Situ Cell Death Detection kit, Fluorescein (Sigma-Aldrich; Merck KGaA) to perform terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL), following the manufacturer's protocol. TUNEL-positive cells were then assessed using a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and the data were quantified using the BD CellQuest Pro Software version 5.1 (BD Biosciences), as previously described (55).

CAR cells (5×10⁶ cells/75T flask) were exposed to 0, 25, 50, 75 and 100 μM pterostilbene for 48 h. Cell lysates were harvested and the activities of caspases-3 and -9 were then measured using the caspase-3 and -9 colorimetric assay kits, following the manufacturer's protocol.

All results are expressed as the mean ± standard deviation of triplicate samples. Statistical analysis was performed using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). Differences among groups were determined using one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

Following treatment with different concentrations (5, 10, 25, 50, 75 and 100 μM) of pterostilbene for 24, 48 and 72 h, cells underwent apoptotic changes, including cell shrinkage, membrane blebbing and rounding and acquire autophagic characteristics (Fig. 1A). Furthermore, pterostilbene treatment decreased the number of CAR cells compared with the untreated control, as recorded by a phase-contrast microscope. These effects occurred in a time- and concertation-dependent manner (Fig. 1A). Furthermore, incubation with pterostilbene for 24, 48 and 72 h significantly decreased cell viability in a time- and concentration-dependent manner (Fig. 1B). The IC₅₀ values of pterostilbene in CAR cells following 24, 48 and 72 h incubation were 78.26±4.33, 48.04±3.68 and 20.65±4.88 μM, respectively. Interestingly, administration of 0, 25, 50, 75 and 100 μM pterostilbene suppressed cell confluence over a 48-h period in a time- and concentration-dependent manner (Fig. 2 and supplementary data: https://youtu.be/P7MCMTJnO00). Thus, pterostilbene may induce CAR cell death via autophagy and apoptosis.

To determine whether autophagic cell death is caused by pterostilbene, AVOs, autophagosome vesicles or lysosome activity were detected using different molecular probes, including AO, MDC, LysoTracker Red and cathepsin B. AO and MDC staining indicated that pterostilbene markedly increased the number of AVOs within the cytoplasm compared with the untreated control (Fig. 3A and B). LysoTracker Red and cathepsin B staining also indicated that treatment with pterostilbene caused the accumulation of autophagic vacuole marker and suppressed lysosome activity (Fig. 3C and D). In addition, increased DNA condensation occurred in cells treated with 25, 50 and 75 μM pterostilbene for 24 h, as indicated by Hoechst 33342 staining (Fig. 3E). These results demonstrate that pterostilbene-elicited CAR cell death is mediated by autophagic and apoptotic responses.

To further investigate the effect of pterostilbene on autophagy, the gene and protein levels of key autophagic regulators, including Atg5, Atg7, Atg12, Beclin-1 and LC3 were assessed using RT-qPCR analysis and western blotting. Following treatment of cells with 50 and 75 μM pterostilbene for 24 h, there was a significant increase in the mRNA expression of LC3-II (Fig. 4A) and Atg12 (Fig. 4B). Treatment with 50 and 75 μM pterostilbene also markedly increased the protein expression of Atg5, Atg7, Atg12, Beclin-1 and LC3-II in CAR cells. These results imply that pterostilbene provokes the autophagy of CAR cells by increasing the expression of Atg/Beclin-1/LC3-associated molecules.

Cells were pretreated with 10 mM 3-MA or 20 μM CQ and subsequently exposed to 75 μM pterostilbene for 24 h. Cell viability, autophagic characteristics and AVOs were individually monitored using an MTT assay, microscopic examination of cell morphology and staining with AO, followed by fluorescence microscopy. The results demonstrated that 3-MA and CQ significantly increased the viability of CAR cells following pterostilbene treatment, compared with cells treated with pterostilbene alone (Fig. 5A). Similarly, pretreatment of CAR cells with 3-MA and CQ suppressed the development of autophagic characteristics (Fig. 5B) and AVOs (Fig. 5C) following exposure to pterostilbene. These results suggest that pterostilbene-induced autophagy in CAR cells may be mediated by phosphatidylinositol-4,5-bisphosphate 3-kinase class III signaling.

The effects of pterostilbene on cell apoptosis and its underlying mechanism of action was subsequently assessed. Following treatment with 25, 50, 75 and 100 μM pterostilbene for 48 h, cells were detected for DNA breaks and direct apoptotic responses. Pterostilbene significantly increased the number of TUNEL-positive cells in a concentration-dependent manner (Fig. 6A), indicating that it induces apoptosis. To determine if the caspase cascade contributes to this pterostilbene-induced apoptosis, the pan-caspase inhibitor Z-VAD-FMK was used to pretreat CAR cells prior to incubation with 50 μM pterostilbene. Z-VAD-FMK significantly reversed the viability of cells treated with pterostilbene alone, by 32.6% (Fig. 6B). These results indicate that pterostilbene induces the apoptosis of CAR cells via caspase-dependent signaling.

Following the determination of the apoptosis-inducing effect of pterostilbene on molecular signals, its underlying mechanism of action was further investigated. Treatment with 50, 75 and 100 μM pterostilbene for 48 h significantly increased caspase-3 (Fig. 7A) and caspase-9 (Fig. 7B) activity in a concentration-dependent manner, compared with untreated control cells. Furthermore, there was no significant increase in caspase-8 activity (data not shown) following pterostilbene treatment. To determine changes in the expression of mitochondria-modulated pro- and anti-apoptotic proteins, levels of these proteins in pterostilbene-treated cells were measured. The results demonstrated that 50 and 75 μM pterostilbene upregulated the expression of Bax, cytochrome c, the active forms of caspase-9, caspase-3, caspase-7 and PARP, but it downregulated the expression of Bcl-2 (Fig. 7C). These results indicate that pterostilbene induces CAR cell death by activating the intrinsic (caspase-9-and caspase-3-dependent) apoptotic cascade.

Following treatment with 50 and 75 μM pterostilbene for 24 h, the mRNA and protein expression of MDR1 was monitored. Pterostilbene treatment significantly decreased the expression of MDR1 mRNA (Fig. 8A) and protein (Fig. 8B). Furthermore, treatment with 50 and 75 μM pterostilbene decreased the phosphorylation of AKT on the Ser473 site but had no effect on total AKT protein expression (Fig. 8B). Collectively, these results indicate that pterostilbene triggers the autophagy and apoptosis of CAR cells by suppressing MDR1 expression and AKT signaling. The proposed schematic representation of the plausible molecular signaling induced by pterostilbene in CAR cells is summarized in Fig. 9.