Cyanidin chloride (cat. no. 80022), peonidin chloride (cat. no. 80085), cyanidin-3-glucoside (C3G; cat. no. 89616) and peonidin-3-glucoside (P3G; cat. no. 89754) were purchased from PhytoLab GmbH & Co. Sulforhodamine B (SRB; cat. no. S1402) and trichloroacetic acid (TCA; cat. no. T0699) were purchased from Sigma-Aldrich (Merck KGaA). TRIzol™ reagent (cat. no. 15596026) and the BCA Protein Assay Kit (cat. no. 23225) were obtained from Thermo Fisher Scientific, Inc. SensiFAST cDNA Synthesis Kit (cat. no. BIO-65053) was purchased from Bioline. LightCycler^® 480 SYBR Green I Master Mix (cat. no. 04707516001) was purchased from Roche Diagnostics. Cell culture reagents, including DMEM (cat. no. 12100-046), Eagle's minimum essential medium (MEM; cat. no. 61100-061), penicillin-streptomycin (cat. no. 15140-122) and FBS (cat. no. 10270-098), were purchased from Gibco (Thermo Fisher Scientific, Inc.). Primary antibodies against phosphorylated AKT (pAKT; cat. no. 4060S) and total AKT (cat. no. 4685S) were purchased from Cell Signaling Technology Inc. Anti-claudin-1 (cat. no. sc-81796), vimentin (cat. no. sc-6260), slugs (cat. no. sc-166476) and β-actin (cat. no. sc-47778) antibodies were purchased from Santa Cruz Biotechnology, Inc. HRP-conjugated anti-mouse (cat. no. NXA931V) and anti-rabbit (cat. no. NA934V) secondary antibodies, ECL prime blocking reagent (cat. no. RPN415V) and ECL prime western blot detection (cat. no. RPN2236) were obtained from Cytiva. HRP-conjugated anti-goat secondary antibodies (cat. no. A15999) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). A total of 16 biotinylated lectins and ABC-peroxidase solution (cat. no. PK-4000) were purchased from Vector Laboratories, Inc. (Maravai LifeSciences). The SignalStain^® DAB substrate kit (cat. no. 8059) was obtained from Cell Signaling Technology, Inc. Alexa fluor 488 conjugated streptavidin (cat. no. S11223; 1:400) was purchased from Thermo Fisher Scientific, Inc.

Black rice bran from black-pigmented rice (Oryza sativa L.) was collected from a rice mill in Krabueang Yai (Nakhon Ratchasima, Thailand). The black rice bran extract enriched in anthocyanins was prepared as previously described (7). Briefly, black rice bran was soaked in n-hexane followed by 0.1% HCl in MeOH at room temperature for 24 h. The MeOH extracts were filtered and evaporated to obtain powdered extracts. The powder extracts were subjected to silica column chromatography (CC) using a gradient solvent system of hexane [hexane-ethylacetate (EtOAc), EtOAc, EtOAc-MeOH and MeOH]. A total of eight fractions were obtained, and the anthocyanin contents were investigated via thin-layer chromatography (TLC) and compared with the cyanidin, peonidin, C3G and P3G standards. Fractions with high contents of C3G and P3G via TLC were further purified using Sephadex LH20 CC in MeOH:H₂O (80:20) to produce fractions. A total of four fractions, including BBR-M-10 from Sephadex LH20 CC were analyzed via TLC. TLC analysis demonstrated that the main anthocyanin components in BBR-M-10 were C3G and P3G when compared with the standard compounds. Anthocyanin content was further analyzed using high-performance liquid chromatography and compared with the cyanidin, peonidin, C3G and P3G standards. HPLC analyses were performed using the 1260 Infinity instrument (Agilent Technologies, Inc.) The separation of anthocyanins was analyzed using a ZORBAX SB-C18 StableBond Analytical 4.6x250 mm 3.5 µM column. The mobile phases used were: A, 0.1% TFA in deionized water and B, acetonitrile. The gradient conditions were as follows: B at 10% for 5 min, followed by a linear increase to 15% B over the following 15 min, a hold at 15% B for 5 min, then an increase in B from 15-18% over 5 min followed, by 18-35% B over 20 min, then B at 35-90% for 10 min. The detection wavelength was 520 nm. The chromatographic conditions were as follows: Flow rate, 1 ml/min; column temperature, 35˚C; 20 µl injection; stop time, 60 min; and post time, 10 min. The total anthocyanin content of BBR-M-10 was 108 mg CGE/100 g DW (100 mg/l). BBR-M-10 comprises C3G (94.5 mg/l) and P3G (2.47 mg/l).

Human CCA cell lines (KKU-055 and KKU-213A), a normal human lung fibroblast line (IMR-90) and an immortalized human cholangiocyte (MMNK-1) cell line were used. KKU-055 (cat. no. JCRB1551) and KKU-213A (cat. no. JCRB1557) were previously established and authenticated (21). Certificates of analysis were obtained from the Japanese Collection of Research Bioresources Cell Bank. The immortalized human cholangiocyte MMNK-1 cell line was provided by Dr. Sopit Wongkham (Khon Kaen University, Thailand). CCA cell lines and MMNK-1 were cultured in DMEM supplemented with inactivated 10% FBS and 100 U/ml penicillin-streptomycin. IMR-90 cells (cat. no. CCL-186) were obtained from the American Type Culture Collection and cultured in EMEM supplemented with inactivated 10% FBS and 100 U/ml penicillin-streptomycin. All the cell lines were incubated at 37˚C in a humidified incubator containing 5% CO₂. PCR assays were used to verify the lack of mycoplasma contamination in all cell lines.

KKU-055, KKU-213A, IMR-90 and MMNK-1 cells were seeded at 7x10³ cells/well into 96-well plates and incubated overnight. The cells were then treated with various concentrations of BBR-M-10 from 0-5,000 µg/ml and incubated for 24 h. Cell viability was assessed using the SRB assay. Briefly, treated cells were fixed with 10% TCA at 4˚C for overnight and stained with 0.4% (w/v) SRB in 1% (v/v) acetic acid at room temperature for 30 min. The unbound SRB was removed, washed three times with 1% (v/v) acetic acid and dried. Next, the stained cells were solubilized in 10 mM Tris-base (pH 10) and mixed in a shaker at room temperature for 15 min. Absorbance at 564 nm was measured using a TECAN Infinite 200 Pro microplate reader (Tecan Group, Ltd.). The IC₅₀ was calculated using GraphPad Prism (version 8; Dotmatics).

KKU-055 and KKU-213A cells were seeded at 3.5x10⁵ cells/well into 6-well plates. After 24 h, cells were treated with BBR-M-10 at 0 and 200 µg/ml for 24 h. Total RNA was extracted using TRIzol™ reagent according to the manufacturer's instructions. RNA quantity and quality were measured using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) and agarose gel electrophoresis, respectively. A SensiFAST cDNA Synthesis Kit was used to synthesize cDNA. Quantitative PCR was performed using the LightCycler^® 480 SYBR Green I Master Mix to investigate gene expression. The primer sequences for all genes were obtained from previous studies (Table I) (22,23). Gene amplification was performed by initial denaturation at 95˚C for 5 min, followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 50˚C for ST6GAL1, annealing at 60˚C for ST3GAL1, 3, 4, and 6 for 10 sec and a final extension at 72˚C for 10 sec. b-actin was used as the endogenous control. The relative mRNA levels of each gene were normalized to b-actin and calculated using the 2^-∆∆Cq method (24).

KKU-055 and KKU-213A cells were seeded at a density of 3x10⁵ cells/well in 24-well plates. After 24 h of growth to 90% cell confluence, a vertical wound was scratched through the cell monolayer using a sterile 200 µl plastic micropipette tip. Cell debris was removed and replaced with BBR-M-10 at 50, 100 and 200 µg/ml in serum-free DMEM. Cell migration during the wound healing process was observed and digitally photographed using a light microscope at 0, 12, 18 and 24 h (magnification, x100). The wound area was evaluated using Image J software (version 1.53a; National Institutes of Health) and calculated as follows: (Area of original wound-area of wound during healing)/area of the original wound.

Transwell inserts were coated with 100 µl of Matrigel 200 µg/ml and incubated at 37˚C for 2 h. KKU-055 and KKU-213A cells at a density of 1x10⁵ cells in 200 µl of FBS-free medium with or without BBR-M-10 at 50, 100 and 200 µg/ml were seeded into Transwell inserts for the migration assay Matrigel-coated Transwell inserts for the invasion assay. Next, 600 µl of 10% FBS-containing medium was loaded into the lower chamber to create a chemotactic gradient and incubated at 37˚C. After 12 h, the Transwell inserts were removed from the plate and a cotton-tipped applicator was used to remove the cells on the upper side of the membranes. The migrated cells on the bottom side of the membranes were fixed with 10% TCA at 4˚C overnight, stained with SRB at room temperature for 30 min, washed with 10% acetic acid and dried. Images of migrated cells were captured using a light microscope and counted. Migrated cells were counted in five fields of view. The presented values were the number of total migrated cells per fields of view at x100 magnification.

KKU-055 and KKU-213A cells were seeded at a density of 3x10⁴ cells/well in 24-well plates and incubated overnight. The cells were treated with 0 and 200 µg/ml BBR-M-10 for 24 h. The cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% Triton X-100 in PBST for 10 min at room temperature. The endogenous hydrogen peroxide-generating activity was blocked with 0.3% hydrogen peroxide for 30 min at room temperature. Nonspecific binding was blocked with 3% BSA (HIMedia Laboratories, LLC) for 30 min at room temperature. A 1:500 dilution of biotinylated lectins (Table II) was added and incubated at 4˚C overnight on a shaker. The ABC-Peroxidase Solution was then used as the secondary antibodies for 1 h at room temperature to determine the lectin signal. A SignalStain^® DAB substrate kit was used to visualize the signal under a light microscope at x100 magnification.

KKU-055 and KKU-213A cells were seeded at a density of 3x10⁵ cells/well in 6-well plates and incubated at 37˚C. After 24 h, the cells were treated with 0 and 200 µg/ml BBR-M-10 and incubated at 37˚C for 24 h. The cell pellet was harvested and washed with 2% FBS in PBS. The cell pellet was fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and nonspecific binding was blocked with 3% BSA for 30 min on ice. The cell pellet was then incubated for 1 h on ice with biotinylated SNA lectins (cat. no. PK-4000; 1:500). Alexa fluor 488 conjugated streptavidin (cat. no. S11223; 1:400) was then added for 1 h on ice to determine the lectin signal. Cell pellets were resuspended in 2% FBS in PBS and analyzed by flow cytometry using Attune NxT fluorescent detector and Attune Cytometric Software (version 5.3.2415.0; Thermo Fisher Scientific, Inc.). Then mean fluorescence intensity was analyzed using the Flowing software (version 2; Turku Bioscience).

KKU-055 and KKU-213A cells were seeded at a density of 2.5x10⁴ cells/well in 8-well cell culture slides and incubated at 37˚C and 5% CO₂ overnight. After 24 h of incubation, cells were treated with 0 and 200 µg/ml BBR-M-10 for 24 h. The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. The fixed cells were permeabilized with 0.1% Triton-X100 in PBS for 10 min on ice. A blocking solution (2% BSA in 0.1% PBST) was added to the cells for 30 min on ice, and then the cells were incubated at 4˚C overnight with phalloidin Alexa Fluor 647 antibodies (cat. no. #8940S; 1:50; Cell Signaling Technology, Inc.). After washing with PBS, nuclear counterstaining with Hoechst 33258 solution (1:1,000) was performed on ice for 10 min. Stained cells were visualized using a confocal microscope (Nikon Corporation).

KKU-055 and KKU-213A cell lysates were extracted using protein lysis buffer containing 10% TritonX, protease inhibitor cocktail (Roche diagnostics GmbH) and Tris-lysis buffer pH 7.5. The protein determination method was a BCA protein assay kit (Thermo Fisher Scientific Inc.). A total of 40 µg of protein extracts were separated using 10-15% SDS-PAGE and transferred to a nitrocellulose membrane (Cytiva). Non-specific binding was blocked with 5% ECL prime blocking reagent (Cytiva) containing 0.05% Tween 20 at room temperature for 1 h. Then, the membranes were probed with primary antibodies at dilutions of 1:1,000 for vimentin, claudin-1, pAKT and AKT and 1:500 for ST6GAL1 in 0.05% PBST at 4˚C overnight. The membranes were then washed three times with 0.1% PBST three times for 10 min each. Membranes were incubated with secondary antibodies at room temperature for 1 h. ECL™ Prime Western blotting detection reagents (Cytiva) were used to determine the target protein signals and visualized using the ImageQuant™ LAS 500 (Cytiva). The density of each target protein was determined using ImageJ software and normalized to β-actin.

The results were expressed as the mean ± SD (n=3). The one-way ANOWA followed by Bonferroni's multiple comparisons test was used to analyze data. GraphPad Prism was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of BBR-M-10 on CCA cell viability, KKU-055 and KKU-213A cells were treated with BBR-M-10 at various concentrations. A normal fibroblast cell line (IMR-90) was used as the normal control cell line because IMR-90 cells were derived from a human embryo with a normal karyotype and have a finite lifespan (25). The cell viability test demonstrated that BBR-M-10 was not toxic to KKU-055, KKU213A and IMR-90 cells. The respective IC₅₀ value for BBR-M-10 was 2.94 and 3.47 mg/ml, for the CCA cell lines KKU-055 and KKU-213A, respectively, and for IMR-90 cells it was 4.30 mg/ml (Fig. 1A). Additionally, the effect of BBR-M-10 in MMNK-1 cells was measured. The IC₅₀ value for BBR-M-10 was 0.75 mg/ml for MMNK-1 (Fig. S1). Taken together, the decreases in cell viability of MMNK-1 and CCA cell lines were observed at high doses of BBR-M-10 (1,250-5,000 µg/ml), whereas high doses of BBR-M-10 showed less toxicity to the IMR-90 cell line (Fig. S2). Our previous study demonstrated that the main anthocyanin components in BBR-M-10 are C3G and P3G (7). Thus, the effects of C3G and P3G on CCA cell viability were investigated in both CCA cell lines. These results showed that CCA cell viability was not significantly impacted by C3G or P3G treatment at 0-500 µM (Fig. 1B and C). Thus, low doses of BBR-M-10 (50, 100 and 200 µg/ml) were selected to assess the effect of BBR-M-10 on CCA cell migration and invasion.

The effects of anthocyanins on crucial cancer cell properties, including cell proliferation, migration and invasion, have been documented in various types of cancers such as squamous cell carcinoma, liver cancer, CCA and cervical cancer (10,19). To address whether BBR-M-10 affected cancer cell activity, the migration and invasion properties of CCA cell lines upon BBR-M-10 treatment were assessed using wound healing, Transwell migration and invasion assays. KKU-055 and KKU213A cells were treated with BBR-M-10 at 0, 50, 100 and 200 µg/ml. The wound healing assay results demonstrated a statistically significant dose-dependent decrease in the migration ability of both KKU-055 and KKU-213A cells (Fig. 2A and B). Additionally, BBR-M-10 treatment at 100 and 200 µg/ml significantly decreased the migration area in both KKU-055 and KKU-213A cells. Transwell migration assay results demonstrated that BBR-M-10 treatment at 50, 100 and 200 µg/ml significantly reduced the migration ability of KKU-055 and KKU-213A cells (Fig. 3A and B). Moreover, BBR-M-10 treatment at 200 µg/ml significantly decreased the cell invasion ability of KKU-055 and KKU-213A cells (Fig. 3C). These findings suggested that BBR-M-10 may inhibit the signaling pathway that promotes CCA cell migration and invasion.

Actin polymerization serves a crucial role in regulating cell structure during cancer cell migration and invasion (26). To address the effect of BBR-M-10 on lamellipodium formation, KKU-055 and KKU213A cells were treated with BBR-M-10 at 0, 100 and 200 µg/ml. The distribution of filamentous actin (F-actin) and the overall shape of cells were detected by phalloidin staining and visualized using confocal microscopy. BBR-M-10 reduced F-actin accumulation at the edges of both KKU055 and KKU-213 cells, suggesting a decrease in migration ability (Fig. 4A). Epithelial-to-mesenchymal transition (EMT) is a crucial initiating step in cancer invasion and metastasis and is modulated via multiple signaling pathways, including AKT signaling. Several EMT markers, including claudin-1, Slug and vimentin, are regulated by this pathway (27). The effect of BBR-M-10 on CCA cell migration and invasion via EMT was also investigated. Western blotting analysis demonstrated that BBR-M-10 treatment at 100 and 200 µg/ml markedly increased the protein expression levels of expression of claudin-1 in KKU-055 (Fig. 4B). BBR-M-10 treatment at 200 µg/ml significantly decreased vimentin protein expression levels compared with cells treated with 100 µg/ml BBR-M-10. Additionally, a marked reduction in the ratio of phosphorylated to non-phosphorylated AKT (pAKT/AKT) was observed in KKU-055 cells treated with BBR-M-10 at 100 and 200 µg/ml. The protein expression levels of claudin-1 were markedly increased, whilst the protein expression levels of vimentin were markedly decreased in KKU-213A cells treated with 100 and 200 µg/ml BBR-M-10 (Fig. 4B). A marked decrease in the pAKT/AKT ratio was observed in KKU-213A cells treated with BBR-M-10 at 200 µg/ml only.

Aberrant glycosylation is a hallmark of cancer and is associated with certain behaviors exhibited by cancer cells, including EMT (28). To investigate whether BBR-M-10 induced glycosylation changes in CCA cell lines, a panel of 16 lectins were used to identify differences in glycan expression between BBR-M-10-treated and control CCA cells (Table II). Lectin cytochemistry demonstrated that low expression levels of SNA binding α2,6-sialylated glycan was observed in BBR-M-10-treated KKU-213A cells compared with the control (Fig. 5A). Additionally, flow cytometry analysis confirmed that surface SNA binding α2,6-sialylated glycan showed a significant decrease in expression levels in BBR-M-10-treated KKU-213 cells (Fig. 5B). Next, it was further investigated whether BBR-M-10 altered the expression of sialyltransferase genes, including α2,3 sialyltransferase genes ST3GAL1, 2, 3, 4 and 6 and α2,6 sialyltransferase gene ST6GAL1. Gene expression level analysis demonstrated that the mRNA expression levels of ST3GAL4 and ST6GAL1 were significantly reduced after BBR-M-10 treatment in KKU-213 cells compared with the control cells (Fig. 5C).

Moreover, the protein expression level of ST6GAL1 was significantly decreased after BBR-M-10 treatment in KKU-213 cells (Fig. 5D). The altered expression of α2,3-sialylated glycans in both CCA cell lines was not detected via MAL II Lectin staining after BBR-M-10 treatment. Therefore, the protein expression levels of ST3GAL4 were not included in the present study. Taken together, these findings suggest that BBR-M-10 may affect CCA progression via the reduction of glycoprotein sialylation.