Ammonium sulfate was purchased from Fujifilm Wako Pure Chemical Corporation. Maple Farms Japan, Inc (Osaka, Japan). generously gifted grade A maple syrup made from sugar maple (A. saccharum) by Bascom Maple Farms, Inc. (Acworth, New Hampshire, USA).

DLD-1 colon adenocarcinoma cells were purchased from the American Type Culture Collection and cultured in RPMI-1640 medium (Fujifilm Wako Pure Chemical Corporation) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator supplied with 5% CO₂ maintained at 37°C. T4056-C human primary normal colon epithelial cells (passage 2) were cultured in Prigrow X Medium for T4056-C (both from Applied Biological Materials Inc.) according to the manufacturer's protocol.

Maple syrup (100 ml) was diluted in 200 ml ultrapure water and ultrafiltered through an Amicon Ultra-15, Ultracel-10K centrifugal filter unit (MilliporeSigma) according to the manufacturer's instructions. Filtrates and concentrates were fractionated and collected as a low molecular weight (MW) fraction of maple syrup (MW<10,000) and a high MW fraction (MW≥10,000).

To obtain MSpf, ammonium sulfate was added to the high MW fraction to a final concentration of 80% saturation. The sample was centrifuged at 3,000 × g at 4°C for 15 min following incubation at 4°C overnight and the supernatant was removed. These steps were repeated once. The precipitate was dissolved in 5 ml PBS (MilliporeSigma). Any ammonium sulfate that remained was removed by ultrafiltration using an Amicon Ultra-15, Ultracel-10K. The total protein concentration in solution was measured using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions.

DLD-1 cells were cultured in a 6-well plate at a density of 1×10⁵ cells/well and grown in culture medium as aforementioned. Following 24 h incubation at 37°C, the medium was replaced and cells were grown in maple syrup, high or low MW fraction, MSpf (2.5, 5.0 or 10 µg/ml) or PBS (untreated control) in culture medium. DLD-1 cells were treated with maple syrup as described in our previous study (13), and DLD-1 cells were also treated by high or low MW fraction to be the same as treated with an equal volume of maple syrup. The number of cells which were treated with maple syrup or high or low MW fraction was counted after 72 h using a Countess Automated Cell Counter (Thermo Fisher Scientific, Inc.). The number of cells which were treated with MSpf or PBS was counted after 24, 48, 72 and 96 h using a Countess Automated Cell Counter (Thermo Fisher Scientific, Inc.).

DLD-1 cells were also plated at a density of 5×10³ cells/well in a 96-well plate and grown in culture medium as aforementioned. The following day, the medium was replaced and cells were grown in MSpf (10 µg/ml) without ammonium sulfate or in PBS control in culture medium, as aforementioned. After 96 h, the cells were incubated with WST-8 cell counting reagent (Fujifilm Wako Pure Chemical Corporation) at 2 h and the optical density of the culture solution in the plate was measured at 450 nm using an ELISA plate reader.

To examine the effect of the protein component of maple syrup on the viability of normal colon cells, T4056-C cells (passage no. 3) were plated at a density of 5×10³ cells/well in a 96-well plate and grown in culture medium as aforementioned. The 96-well plate was coated with Applied Cell Extracellular Matrix (Applied Biological Materials Inc.) according to the manufacturer's instructions. Following 24 h incubation at 37°C, the medium was replaced and cells were grown in MSpf (10 µg/ml)- or PBS (control)-containing culture medium, as aforementioned. After 96 h, WST-8 assay was performed, as aforementioned.

To examine the effect of the protein component of maple syrup on cell autophagy, cells were plated at a density of 5×10³ cells/well in a 96-well plate as aforementioned. The following day, the medium was replaced and cells were grown in either MSpf (10 µg/ml) or PBS (control) in the presence or absence of bafilomycin A1 (10 nM; MilliporeSigma), as previously described (24), in culture medium. After 72 h, the WST-8 assay was performed, as aforementioned.

To determine the molecular weight distribution of MSpf, SDS-PAGE was performed on a gradient SuperSep Ace (5–20% gradient gel; Fujifilm Wako Pure Chemical Corporation) loaded 10 µg MSpf/lane. Protein bands were stained with Silver Stain II kit (Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions.

In vitro migration assays were performed using a Boyden chamber (BD Biosciences). DLD-1 cells were plated on the inner surface of the inserts for 24-well plate with 8.0 µm pore size at a density of 1×10⁵ cells/insert, followed by incubation at 37°C for 48 h in a humidified incubator. Culture medium containing 10% FBS with MSpf (10 µg/ml) or PBS (control) was added to the lower chamber as a chemoattractant. DLD-1 cells on the outer surface of the inserts were counted after 48 h, as described previously (25), and the cells that moved to the outer surface of the inserts were fixed, stained with a Diff-Quik staining kit (Sysmex, Corp.) according to the manufacturer's instructions. All assays were performed in triplicate and five fields of view (magnification, ×200) were counted on each membrane in a blinded manner using a light microscope, EVOS FLoid Cell Imaging Station (Thermo Fisher Scientific, Inc.). The cell invasion assay was performed in the same way, except a Matrigel Invasion Chamber 24-Well Plate 8.0 µm was used as the insert (cat. no. 354480; Corning, Inc.).

The MSpf (10 µg/lane) was separated by SDS-PAGE using 5–20% gradient gel (Fujifilm Wako Pure Chemical Corporation). Protein bands were stained with Silver Stain MS kit (Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions. The protein bands were excised manually and then digested using In-gel Tryptic Digestion kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, the digests were purified using PepClean C-18 Spin Columns (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Peptide samples (~2 µg) were injected into a peptide L-trap column (Chemicals Evaluation and Research Institute) with an HTC PAL autosampler (CTC Analytics). They were further separated through a Paradigm MS4 (AMR Inc.) with a reverse-phase C18-column (L-column, 3-µm-diameter gel particles, 120 Å pore size, 0.2×50 mm; Chemicals Evaluation and Research Institute). The column flow rate was 1 µl/min, and the mobile phase consisted of 0.1% formic acid in water (solution A) and acetonitrile (solution B), with a concentration gradient of 5% solution B to 40% solution B over 45 min. Gradient-eluted peptides were introduced into the mass spectrometer through the nanoelectrospray ionization (NSI) interface that had a separation column outlet directly connected with a NSI needle. We analyzed the peptides with an LTQ ion-trap mass spectrometer (Thermo Fisher Scientific, Inc.), using no sheath or auxiliary gas. The MS scan sequence was full-scan MS in the normal/centroid mode and sequential MS/MS in the normal/centroid mode. The positive ion mass spectra were acquired in a data-dependent manner, with MS/MS fragmentation performed on the two most intense peaks of every full MS scan with an isolation width of m/z 1.0 and a collisional activation amplitude of 35% in the m/z range of 300 to 2,000. All MS/MS spectral data were searched against the SwissProt Acer L (Maple tree) database using Mascot version 2.4.01 (Matrix Science). The search criteria were enzyme as trypsin, with the following allowances: ≤2 missed cleavage peptides; mass tolerance, ±2.0 Da; MS/MS tolerance, ±0.8 Da; cysteine carbamidomethylation for fixed modification; and methionine oxidation modifications for variable modification.

Advanced glycation end products (AGEs) in MSpf were quantified by ELISA using the OxiSelect Advanced Glycation End Product Competitive ELISA kit (cat. no. STA-817-T; Cell Biolabs, Inc.) according to the manufacturer's instructions.

DLD-1 cells were plated at a density of 1×10⁵ cells/dish in a 30-mm dish and grown in culture medium as aforementioned. Following 24 h incubation at 37°C, the medium was replaced and cells were cultured with MSpf (10 µg/ml) or PBS (control) in culture medium as aforementioned. The cells were imaged using an EVOS FLoid Cell Imaging Station (Thermo Fisher Scientific, Inc.) after 72 h incubation.

To detect autophagic cells, DLD-1 cells were plated at a density of 2×10⁵ cells/slide in a Nunc Lab-Tek Chamber Slide System (Thermo Fisher Scientific, Inc.) in the aforementioned culture medium. The following day, the medium was replaced and cells were grown in 0.15 µM DAPGreen (Fujifilm Wako Pure Chemical Corporation) in culture medium for 30 min, according to the manufacturer's protocol. Then, cells were grown in MSpf (10 µg/ml) or PBS (control) in the presence or absence of bafilomycin A1 (10 nM) in culture medium after washing with culture medium. After 72 h incubation at 37°C, the culture medium from all slides was removed and the cells were washed three times with 1X PBS. Next, cells were fixed with 4% paraformaldehyde at room temperature for 10 min and washed again with 1X PBS. Images were captured using an EVOS FLoid Cell Imaging Station.

DLD-1 cells were plated at a density of 5×10⁵ cells in a 100-mm dish and grown in culture medium as aforementioned. The following day, the medium was replaced and cells were grown in culture medium containing MSpf (10 µg/ml) or PBS (control) with or without bafilomycin A1 (10 nM) as aforementioned. After 72 h, cells were solubilized in urea lysis buffer (7 M urea, 2 M thiourea, 5% CHAPS and 1% Triton X-100). The protein concentration was measured using a Bradford assay.

Total protein (10 µg/lane) was mixed with loading buffer and boiled at 95°C for 10 min. The proteins were separated by SDS-PAGE [8% gel E-cadherin and N-cadherin; 10% gel for MMP-2, MMP-9, RAGE, phosphorylated (p)STAT3, pp44/42 MAPK, p-stress-activated protein kinase 1c (SAPK)/JNK, pp38 MAPK and pAKT; 15% gel for cleaved caspase-3, caspase-3 and Snail; 5–20% gradient gel for LC3A] and transferred to PVDF membranes (MilliporeSigma) for 30 min at 15 V. Following blocking with TBS +0.1% Tween-20 buffer containing 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following antibodies at 4°C overnight: Anti-Caspase-3 [1:1,000; cat. no. 14220; Cell Signaling Technology, Inc. (CST)], anti-cleaved Caspase-3 (1:1,000; cat. no. 9661; CST), anti-LC3A (1:1,000; cat. no. 4599; CST), anti-E-cadherin (1:1,000; cat. no. 3195; CST), anti-N-cadherin (1:1,000; cat. no. 13116; CST), anti-Snail (1:1,000; cat. no. 3879; CST), anti-MMP-2 (1:1,000; cat. no. 87809; CST), anti-MMP-9 (1:1,000; cat. no. 13667; CST), anti-RAGE (1:500; cat. no. ab216329; Abcam, Inc.), anti-pSTAT3 (1:1,000; cat. no. 9134; CST), anti-pp44/42 MAPK (Erk1/2; 1:1,000; cat. no. 4370; CST), anti-pSAPK/JNK (1:1,000; cat. no. 4668; CST), anti-pp38 MAPK (1:1,000; cat. no. 4511; CST) or anti-pAKT (1:1,000; cat. no. 4051; CST). The membranes were washed with 0.1% TBS-T) and incubated with HRP-conjugated anti-rabbit or anti-mouse immunoglobulin G antibody (both 1:4,000; cat. no. A102PU and A106PU; both American Qualex) at room temperature for 1 h. The blots were washed with TBS-T and signals were visualized using SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc.), and analyzed using myECL Imager system (version 2.0; Thermo Fisher Scientific, Inc.). The membranes were stripped using Restore Western Blot Stripping buffer (Thermo Fisher Scientific, Inc.) and re-probed with anti-β-actin (1:5,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.), anti-STAT3 (1:1,000; cat. no. 9139; CST), anti-AKT (1:1,000; cat. no. 2920; CST), anti-Erk1/2 (1:1,000; cat. no. 9102; CST), anti-SAPK/JNK (1:1,000; cat. no. 9252; CST), or anti-p38 MAPK (1:1,000; cat. no. 8690; CST) antibodies at 4°C overnight, which served as the protein loading or total protein controls. All western blot analyses were performed as three independent experiments.

Data are presented as the mean ± SEM of at least three independent experiments. Statistical analyses were performed using GraphPad Prism version 8.1.2 (GraphPad Software, Inc.; Dotmatics). Differences between groups were evaluated using unpaired Student's t test or one-way ANOVA followed by Tukey-Kramer's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the active ingredients in maple syrup that possessed putative antitumor effects against CRC cells, the maple syrup was separated into high MW and low MW fractions with a 10 kDa cut-off, and the fraction that contained the active ingredients was determined. Cell viability assays demonstrated that treatment with the high MW fraction significantly inhibited cell viability in DLD-1 cells, similar to treatment with the maple syrup and our previous reports (Fig. 1) (13,15). On the other hand, there was no significant difference between high and low MW fraction treatment. Proteins were considered to be the majority constituents in the high MW component of the maple syrup. Therefore, the protein components of maple syrup were suggested to be the active ingredients in maple syrup.

An overview of the preparation of MSpf is shown in Fig. 2A. MSpf was dissolved in PBS and used in subsequent experiments (Fig. 2B). Several bands between 20 and 100 kDa were observed following SDS-PAGE of MSpf (Fig. 2C). Based on the protein concentration of MSpf (0.50±0.05 mg/ml), obtained 5 ml MSpf solution contain 2.5 mg protein. Therefore, 100 ml maple syrup contained ~2.5 mg protein.

Compared with untreated control cells, 5 and 10 µg/ml MSpf significantly inhibited the viability of DLD-1 cells in a dose-dependent manner (Fig. 3A); 10 µg/ml MSpf significantly inhibited proliferation of DLD-1 cells compared with control cells at 48, 72 and 96 h (Fig. 3B). A WST-8 assay was used to confirm the inhibitory effect of MSpf on viability of DLD-1 cells; 10 µg/ml MSpf significantly inhibited the viability of DLD-1 cells compared with the control cells at 96 h (Fig. 3C). MSpf was dissolved in PBS following ammonium sulfate precipitation. To investigate whether the ammonium sulfate remaining in the MSpf solution exhibited a cytotoxic effect, solution was prepared to remove any residual ammonium sulfate by ultrafiltration and a WST-8 assay was performed using this solution. The results showed that 10 µg/ml MSpf without ammonium sulfate significantly reduced viability of DLD-1 cells compared with control (Fig. 3D). A WST-8 assay was also performed to determine whether MSpf treatment affected the viability of T4056-C normal colon cells; 10 µg/ml MSpf treatment did not affect proliferation of human primary colon cells (Fig. 3E).

Furthermore, the migration and invasion of DLD-1 cells treated with MSpf were assessed, and the data show that the number of migrating and invading cells was significantly decreased by MSpf treatment compared with the untreated control group (Fig. 3F and G, respectively).

Since proliferation of DLD-1 cells was inhibited by MSpf treatment, it was hypothesized that this suppression may be involved in the induction of cell death. To clarify whether MSpf treatment affected induction of apoptosis or autophagy of DLD-1 cells, the expression of cleaved caspase-3 (a marker of apoptosis) (26) and LC3A (a marker of autophagy; 27,28) in MSpf-treated DLD-1 cells was measured (Fig. 4A). LC3AI and LC3AII expression was notably increased in MSpf-treated DLD-1 cells compared with control cells, whereas cleaved caspase-3 expression was not detected.

In addition, it was determined whether MSpf treatment was associated with cell morphology. MSpf-treated cells exhibited a more uniform and adherent monolayer compared to the controls (Fig. 4B). Therefore, it was hypothesized that MSpf treatment may inhibit cell migration and invasion by decreasing epithelial-mesenchymal transition (EMT). To investigate this, the expression of EMT markers E-cadherin, N-cadherin and Snail in MSpf-treated DLD-1 cells was determined. E-cadherin expression was increased in MSpf-treated DLD-1 cells compared with control cells, whereas N-cadherin and Snail expression were not affected by MSpf treatment (Fig. 4C). The expression levels of MMP-2 and MMP-9 in MSpf-treated DLD-1 cells were examined to investigate the potential association with the inhibition of cell invasion following MSpf treatment. MMP-9 expression decreased in MSpf-treated DLD-1 cells compared with control cells, whereas MMP-2 expression was not affected by MSpf treatment (Fig. 4C).

To clarify the molecular mechanism of the effects of MSpf, proteins in MSpf were analyzed using proteomics (data not shown). However, identification was not possible due to insufficient registration of the proteins in the SwissProt database against Maple tree) taxonomy. It has been reported that the free amino groups of side chains of amino acid in proteins react with sugars due to the Maillard reaction during the heat treatment, and this led to formation of brown colored AGEs (29,30). As maple syrup is made by boiling sap, the obtained protein fraction has a dark brown color (Fig. 2B); therefore, MSpf might contain AGEs. In addition, maple syrup contains various unique sugars rarely found in natural products (31). Therefore, it was hypothesized that the AGEs in MSpf generated by such unique carbohydrate linkages had unique effect such an antitumor effect. The quantity of AGEs in MSpf was examined by ELISA; the mean concentration of AGEs in MSpf was 0.64±0.39 mg/ml. As protein concentration in MSpf was (0.50±0.05 mg/ml), almost all protein components in MSpf were predicted to be AGEs (127.07±45.33%).

AGEs bind to their receptor RAGE and activate intracellular transcription factors (32). Previous reports have shown that autophagy is induced by regulation of the RAGE/STAT3 pathway (33–36). To determine whether MSpf was associated with the regulation of this pathway, the expression of RAGE and pSTAT3 were determined. RAGE and pSTAT3 expression were decreased in MSpf-treated DLD-1 cells compared with control cells (Fig. 5A).

To investigate whether autophagy was induced by MSpf, the effect of MSpf treatment on autophagosome formation was assessed using DAPGreen staining. MSpf treatment increased the DAPGreen-positive cells compared with control cells, which suggested induction of autophagy by MSpf (Fig. 5B). To confirm the effect of MSpf treatment on induction of autophagy, bafilomycin A1 (a specific autophagy inhibitor of autophagosome-lysosome fusion; 24) was used. Autophagy was successfully inhibited, as shown by an increase in autophagosome formation following bafilomycin A1 treatment (Fig. 5C; control and bafilomycin A1). However, autophagosome formation induced by MSpf treatment was not enhanced by bafilomycin A1 treatment (Fig. 5C; MSpf and MSpf+ bafilomycin A1). This was confirmed by protein expression analysis of autophagic marker LC3A (Fig. 5D). The expression of LC3AI and II in MSpf-treated DLD-1 cells was significantly induced compared with control, bafilomycin A1-treated and MSpf+ bafilomycin A1-treated cells while there were no significant differences among other treated cells. Conversely, the inhibitory effect of MSpf treatment on cell viability was enhanced by inhibition of autophagy when cells were treated with bafilomycin A1 (Fig. 5E).

Additionally, MSpf treatment activated Akt, whereas no effect on ERK, JNK and p38 phosphorylation was observed (Fig. S1).