Quantitative and qualitative analyses were performed twice, and the change in expression was considered significant if the intensity of the corresponding spot was reproducibly different and increased or decreased in relative volume between the cells treated with 50 μM nobiletin and DMSO (negative control) for 24 h. The treatment time was chosen based on the finding that 24 h-treated SNU-16 cells had 65.30% ± 9.76% viability (data not shown) compared to 41.65% viability of the cell treated cells for 48 h [4]. Sixty-two different spots between nobiletin treatment and the negative control were identified by repeated 2-DGE analysis. We excluded spots that were too weak and selected 20 spots that were dense on both gels. Seventeen of 20 selected protein spots were successfully identified (Figure 1), including nine upregulated and eight downregulated proteins (Table 1). mRNA levels of the six proteins related to cell survival and death—including the Rho GDP dissociation inhibitor 1 (RhoGDI), glucose-regulated protein 78 kDa (GRP78), thioredoxin domain-containing protein 5 (TXNDC5), COMM domain-containing protein 9 (COMD9), eukaryotic translation initiation factor 4E (EIF4E), and peroxiredoxin 3 (PRDX3)—were analyzed by RT-PCR. Among these, the GRP78 mRNA level was most significantly induced in nobiletin-treated SNU-16 cells (Figure 2). Next, the changes in GRP78 levels in SNU-16 cells were confirmed by western blotting.

GRP78 regulates a variety of biological functions, including protein folding, the activation of transmembrane ER stress sensors, and cell survival [12]. Importantly, GRP78 signaling is crucial for cell survival/apoptosis via various signaling pathways [13,14]. In addition, previous reports found that nobiletin associated with ER stress by upregulated expression of DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP) and TRIB3 (tribbles homolog 3 protein, also known as TRB3) genes and proteins, which are well known to contribute to apoptosis caused by ER stress, in SK-N-SH human neuroblastoma, HuH-7 human hepatoma, and 3Y1 rat fibroblast cell lines [15,16,17]. Therefore, we investigated whether ER stress was involved in response to nobiletin treatment in SNU-16 cells. XBP-1 mRNA is constitutively expressed at a low level as an intron-containing precursor mRNA, unspliced XBP-1 (XBP-1u), which is subject to an inositol-requiring-1 α (IRE1-α)-mediated splicing reaction upon ER stress to produce the active form of XBP-1 mRNA, a spliced form of XBP-1 (XBP-1s) [18].

Excision of a 26-nucleotide-long intron creates XBP-1s, resulting in expression of an active and stable transcription factor that regulates transcription of target genes involved in protein folding and ER-associated degradation [19]. As shown in Figure 3A,B, the level of the spliced form of XBP-1 (XBP-1s) was significantly increased by nobiletin treatment, suggesting that ER stress is involved in the response to nobiletin. Next, we determined whether ER stress-mediated apoptosis is induced in response to nobiletin treatment. As shown in Figure 3C,D, the levels of the ER-stress markers inositol-requiring-1 α (IRE1-α), activating transcription factor 4 (ATF-4), and C/EBP homology protein (CHOP) were increased, and the level of inactive procaspase-4 was decreased. The cleaved form of caspase-4 was not detected even though we have tried western blot of caspase-4 many times. We speculate that this may be due to the low level of caspase-4 in the cell since Tatsuta et al. [20] also reported the degradation of procaspase-4 as a sign of namely activation of caspase-4. As proteolytic cleavage of caspase-4 and upregulation of CHOP are reportedly involved in ER stress-mediated apoptosis [6], these results suggest that nobiletin induces ER stress-mediated apoptosis in SNU-16 cells.

Recent studies show that autophagy plays key roles in cancer treatment and is associated with apoptosis [21]. Furthermore, numerous chemotherapeutic drugs have been found to induce cellular autophagy [22,23]. To test whether nobiletin-induced apoptosis can induce autophagy, we examined the levels of Akt/mTOR signaling proteins, either in phosphorylated (activated) or unphosphorylated forms, by western blotting. PI3K/Akt and the downstream mTOR play important roles in regulating cell proliferation, cell cycle, and are key regulators of autophagy initiation [24]. Nobiletin treatment caused a significant decrease in phosphorylated Akt and mTOR, and it increased the ratio of LC3B II/LC3B I and decreased the level of p62, indicating that p62 is degraded by autophagy through a direct interaction with LC3 (Figure 4A,B).

Autophagy may have a protective effect on tumor cells and therapy-induced cell death can be potentiated through autophagy inhibition [25]; thus, we determined whether the autophagy signal induced by nobiletin was pro-survival or pro-death. Cells were treated with chloroquine (CQ), which inhibits the fusion of autophagosomes and lysosomes, for 2 h before nobiletin (NT) treatment. As shown in Figure 4C, the proliferation of NT-treated SNU-16 cells was significantly reduced when cells were pre-treated with CQ, while CQ treatment alone did not affect cell viability. Western blotting revealed that NT increased cleaved PARP in the presence of CQ (Figure 4D,E). We also examined the sub-G1 population in SNU-16 cells pretreated with CQ followed by nobiletin treatment. When cells were treated with nobiletin alone for 24 h, 17.2% ± 2.9% of the cells were in sub-G1 phase (Table 2). In cells pretreated with CQ and then treated with nobiletin, the sub-G1 population increased to 23.0% ± 3.1%. These findings indicate that nobiletin-induced autophagy plays a protective role against apoptosis and that the inhibition of nobiletin-induced autophagy could enhance apoptosis in SNU-16 cells.

Nobiletin, propidium iodide (PI), and anti-β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, trypsin/EDTA, fetal bovine serum (FBS), penicillin, streptomycin, goat anti-rabbit IgG, and goat anti-mouse IgG secondary antibodies were obtained from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO) and MTT were purchased from Amresco (Solon, OH, USA). Antibodies against various proteins for Western blotting such as cleaved PARP, caspase-4, IRE1-α, ATF-4, CHOP, p-Akt, Akt, p-mTOR, mTOR, and LC3B were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-GRP78 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

SNU-16 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ in RPMI-1640 medium containing 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Exponentially growing cells were treated with various concentrations of the solvent fractions as indicated.

Urea, thiourea, 3-[(3-cholamidopropy) dimethyammonio]-1-propanesulfonate (CHAPS), dithio-threitol (DTT), benzamidine, Bradford solution, acrylamide, iodoacetamide, bisacrylamide, SDS, acetonitrile, trifluoroacetic acid, and α-cyano-4-hydroxycinnamic acid were purchased from Sigma-Aldrich (electrophoresis grade, ACS reagents, Ultrapure). Pharmalyte (pH 3.5–10) was obtained from Amersham Biosciences (Piscataway, NJ, USA) and IPG DryStrips (pH 4–10 NL, 24 cm) were obtained from Genomine (Gyeongsangbuk-do, Korea). Modified porcine trypsin (sequencing grade) was purchased from Promega (Madison, WI, USA).

IPG strips were equilibrated for 12–16 h with 7 M urea, 2 M thiourea containing 2% CHAPS, 1% DTT, and 1% Pharmalyte, and loaded with 200 μg of sample. Isoelectric focusing (IEF) was performed at 20 °C using a Multiphor II electrophoresis unit and EPS 3500 XL power supply (Amersham Biosciences) following the manufacturer’s instructions. For IEF, the voltage was linearly increased from 150 to 3500 V over 3 h for sample entry followed by a constant voltage (3500 V) with focusing complete after 96 kVh. Prior to the second dimension, the strips were incubated for 10 min in equilibration buffer (50 mM Tris-HCl, pH 6.8, containing 6 M urea, 2% SDS, and 30% glycerol) first with 1% DTT and then with 2.5% iodoacetamide. Equilibrated strips were inserted onto SDS-polyacrylamide gels (20 × 24 cm, 10%–16%). SDS-PAGE was performed using the Hoefer DALT 2D system (Amersham Biosciences) following the manufacturer’s instructions. The two-dimensional gels were run at 20 °C for 1700 Vh then silver-stained as described, except that the fixation and sensitization steps with glutaraldehyde were omitted [50].

A quantitative analysis of digitized images was performed using PDQuest (version 7.0; Bio-Rad, Hercules, CA, USA) software according to the manufacturer’s protocols. The quantity of each spot was normalized by the total valid spot intensity. Protein spots were selected based on significant expression variation deviating more than twofold in expression level compared with control or normal samples.

For protein identification using PMF, protein spots were excised, digested with trypsin (Promega, Madison, WI, USA), mixed with α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% TFA, and subjected to matrix-assisted laser desorption/ionization- time-of-flight (MALDI-TOF) analysis (Ettan MALDI-TOF Pro; Amersham Biosciences) as described previously [51]. Spectra were collected from 350 shots per spectrum over the 600–3000 m/z range and calibrated by two-point internal calibration using trypsin autodigestion peaks (m/z 842.5099 and 2211.1046). The peak list was generated using the Ettan MALDI-TOF Pro Evaluation Module (version 2.0.16; GE Healthcare, Little Chalfont, UK). The thresholds used for peak-picking were 5000 for minimum resolution of monoisotopic masses and 2.5 for S/N. The search program MASCOT, developed by Matrix Science (Boston, MA, USA), was used for protein identification. The following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of ± 0.1 Da. Probability scoring was used as PMF acceptance criteria.

Total RNA was extracted using TRIzol reagent (Invitrogen, Frederick, MO, USA) and precipitated in ethanol. cDNA synthesis was performed with 1 μg of total RNA by reverse transcription (Promega). The primers used were: Rho GDP dissociation inhibitor 1 (RhoGDI) [5′-GAGCCTGCGAAAGTACAAGG-3′ (sense), and 5′-TCCTTCAGCACAAACGACTG-3′ (antisense)]; glucose-regulated protein 78 kDa (GRP78) [5′-CTCCTGAAGGGGAACGTCTG-3′ (sense), and 5′-AACACTTTCTGGACGGGCTT-3′ (antisense)]; thioredoxin domain-containing protein 5 (TXNDC5) [5′-GCACAAGGCGACCACTTTAT-3′ (sense), and 5′-ATCCCGCTTTCCCTTGTACT-3′ (antisense)]; COMM domain-containing protein 9 (COMD9) [5′-CCACCAAAACCTCAAAAACC-3′ (sense), and 5′-AGGCTGGGATCTTCTTGGAT-3′ (antisense)]; eukaryotic translation initiation factor 4E (EIF4E) [5′-CAGATGGGCACTCTGGTTTT-3′ (sense), and 5’-CTCCCCGTTTGTTTTTCTCA-3′ (antisense)]; peroxiredoxin 3 (PRDX3) [5′-GTTGTCGCAGTCTCAGTGGA-3′ (sense), and 5′-GACGCT CAAATGCTTGATGA-3′ (antisense)]; XBP-1 [5′-TTACGAGAGAAAACTCATGGCC-3′ (sense), and 5′-GGGTCCAAGTTGTCCAGAATGC-3′ (antisense)]; and GAPDH [5′-GAGAAGGCTGGGGCTC ATTT-3′ (sense), and 5′-AGTGATGGCATGGACTGTGG-3′ (antisense)]. The RT-PCR conditions were 95 °C pre-denaturation for 5 min, followed by 95 °C denaturation for 40 s, 55–60 °C annealing for 30 s, and 72 °C extension for 40 s, for a total of 35 cycles.

Harvested cells were washed with PBS and then lysed with RIPA lysis buffer containing protease inhibitor cocktail (BioVision, Milpitas, CA, USA) and PMSF. Protein concentrations were determined using BCATM Protein Assay Reagent (Pierce, IL, USA). Equal amounts of protein were separated by 7.5%–15% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) using glycine transfer buffer (192 mM glycine, 25 mM Tris-HCl (pH 8.8), and 20% (v/v) methanol). After blocking with 5% skim milk, the membrane was incubated for 2 h with primary antibodies and then for 30 min with secondary antibodies in milk containing Tris-buffered saline and 0.1% Tween 20. The membrane was then exposed to X-ray film (AGFA, Mortsel, Belgium) and the protein bands were detected using the WEST-ZOL^® plus Western Blot Detection System (iNtRON, Gyeonggi-do, Korea).

Cell growth inhibition was examined using an MTT assay. SNU-16 was seeded in 96-well culture plates (5 × 10⁴ cells/mL). After incubation overnight, the cells were treated with 40 µM chloroquine (CQ) and 25 µM nobiletin (NT). Next, 0.1 mg of MTT was added to each well and the cells were incubated at 37 °C for 4 h. The medium was removed and 150 µL of DMSO was then added to each well to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader (Tecan, Salzburg, Austria).

For cell cycle distribution analysis, cells (5 × 10⁴ mL) were plated and left untreated or treated with samples for 24 h. The cells were then collected, fixed in 70% ethanol, washed in 2 mM EDTA in phosphate-buffered saline (PBS), resuspended in 1 mL of PBS containing 1 mg/mL RNase and 50 mg/mL PI, and incubated at 37 °C in the dark for 30 min. The DNA content was analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The population of cells in each cell cycle phase was determined using CellQuest software (BD Dickson, Franklin Lakes, NJ, USA). The sub-G1 population showed apoptosis-associated chromatin degradation.

All experiments were repeated at least three times. The data are expressed as means ± standard deviation (SD). A statistical analysis was performed using one-way analysis of variance (ANOVA); values of * p < 0.01 were considered to indicate statistical significance.