AEC samples were prepared as described previously (17) with slight modification. Firstly, dried samples of E. cava were purchased from Para Jeju Co. Ltd., and were deposited as voucher specimens (accession no. WPC-19-001) at the Functional Materials Bank of the Wellbeing RIS Center at Pusan National University (Pusan, Korea). After extraction using mixture of AEC samples and distilled dH₂O solvent (1:15 ratio), the lyophilized AEC pellets were harvested in an N-1100 series rotary evaporator (Tokyo Rikakikai Co., Ltd.).

PT was extracted according to the method previously described by Lee et al (18) with slight modification. The dried E. cava powder (30 g) was mixed with 70% ethanol (300 ml; v/v) and the extract was then collected by shaking for 12 h at 37°C. After repeating the extraction process three times, the total extracted solution was filtered using a membrane with 8 mm pore size and then evaporated at 40°C. These extracts were dissolved in dH₂O, and sequentially fractionated using n-hexane, chloroform and ethyl acetate. Finally, ethyl acetate fraction was evaporated at 40°C to remove the solvent, and used as the PT-rich sample, as reported previously (18,19).

Total phenolic content (TPC) concentration was assessed using the Folin-Denis method, as previously described (20). Briefly, after consistently mixing the AEC solution, Folin-Ciocalteu reagent and 10% Na₂CO₃ solution for 1 h at 37°C, the absorbance of each well was measured at 725 nm. The concentration of TPC in AEC was estimated using a standard calibration curve for caffeic acid (MilliporeSigma) because it is considered as one of the major phenolic acids. Total flavonoid content (TFC) was assessed using the Davis method as previously described (21). Briefly, after constantly mixing AEC solution, 1 N NaNO₂ and 90% C₄H₁₀O₃ for 1 h at 37°C, the absorbance of each well was measured at 420 nm. The concentration of TFC in AEC was estimated using a standard calibration curve for naringin (MiliporeSigma) because naringin and its aglycone naringenin belong to this series of flavonoids. Total condensed tannin content (TTC) was assessed using the Vanillin method as previously described (22). Briefly. the concentration of TTC in AEC was estimated based on a standard calibration curve for a purified (+)-catechin hydrate standard (MilliporeSigma) because catechin has all the characteristics of condensed tannin. Finally, the concentration of total PT content (TPTC) was determined using the Folin-Ciocalteu method as described previously (23). The concentration of TPTC in AEC was estimated using a standard calibration curve for phloroglucinol (MilliporeSigma) because PT is a complex polymer of phloroglucinol, which is a specific compound found in brown algae. TPC, TFC, TTC and TPTC in AEC were presented as caffeic acid, naringin, catechin hydrate and phloroglucinol equivalents (mg) of AEC, respectively.

The scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was analyzed as previously described by Go et al (23). Briefly, after incubation of AEC and 0.1 mM DPPH solution (MilliporeSigma) for 30 min at room temperature, the absorbance of each well was measured at 517 nm using a VersaMax plate reader (Molecular Devices, LLC). AEC scavenging activity for DPPH radicals activity was presented as the half-maximal inhibitory concentration (IC₅₀) based on the percentage decrease in absorbance relative to the control group.

Bioactive compounds in AEC were assessed using an Agilent 1290 Infinity high-performance liquid chromatography (HPLC) system (Agilent Technologies Deutschland GmbH), coupled with a hybrid quadrupole time-of-flight mass spectrometer (Agilent Technologies Deutschland GmbH). Detailed conditions and the column for LC-MS analysis as well as data analysis were performed as described previously (17). Briefly, LC-MS signals were detected on a mass spectrometer operating in the positive ionization mode. A HSS T3 Column (2.1×100 mm, 1.8 µm; Waters Corporation) was used for chromatographic separation under the following conditions: 0.2 ml/min in flow rate, 10 µl of injection volume, water as mobile phase A, and 100% acetonitrile as mobile phase B. For MS detection, the operating parameters were as follows: Gas temperature, 300°C; gas flow, 9 l/min; nebulizer pressure, 45 psi; sheath temperature, 350°C; sheath gas flow, 11 l/min; VCap, 4,000 V; fragmentor voltage, 175 V. All the acquisition and analysis of data were controlled by MassHunter software (version B. 0600, Agilent Technologies).

Murine colorectal carcinoma CT26 cells from a BALB/c mouse, were used to evaluate the anti-tumor effects of AEC because they successfully form solid tumors and metastasize into other organs in BALB/c or immunocompromised mice. A HCT116 cancer cell line from the colon tissue of an adult male with colon cancer, was used to evaluate whether anti-tumor effects of AEC in colon cancer cells of murine were similar in human colon cancer cells. Fibroblast Detroit 551 cells from the skin of a normal human embryo, were used to assess whether AEC was toxic to normal cells. These three cell lines were purchased from the American Type Culture Collection. The medium with 10% fetal bovine serum (Welgene, Inc.) were prepared appropriately for each cell line; a Roswell Park Memorial Institute 1640 Medium (Welgene, Inc.) for CT26 cells, McCoy's 5a Medium (Welgene, Inc.) for HCT116 cells and Minimum Essential Medium Eagle (Welgene, Inc.) for Detroit 551. The viability of the CT26, HCT116 and Detroit 551 cells was determined after treatment with AEC using the tetrazolium compound, MTT (MilliporeSigma) as described previously (21). The optimal concentration of AEC was determined based on the anti-tumor activity of E. cava extract in human breast cancer cells (21) and the dose-response curve of AEC in CT26, HCT116 and Detroit 551 cell lines (Fig. S1A, B and C). Cells at 70–80% confluence were treated with either the dH₂O (Vehicle treated group, Vehicle) or pretreated with 5 µg/ml Cisplatin (Cis treated group, Cis), 600 (Low dose AEC, LoAEC treated group), 800 (Medium dose AEC, MiAEC treated group) or 1,000 µg/ml (High dose AEC, HiAEC treated group) AEC for 24 h at 37°C. In the case of the PT treatment, the CT26 cells were treated with 100 (Low dose PT, LoPT treated group), 150 (Medium dose PT, MiPT treated group) or 200 µg/ml (High dose PT, HiPT treated group) PT for 24 h at 37°C. The optimal dosage of PT was determined based on previous results which reported IC₅₀ from 56.3 to 219 µg/ml in MKN-28, Caco-2 and HT-29 cells (24) and the dose-response curve of PT in CT26 cell lines (Fig. S1D). After incubation for 24 h, 200 µl of fresh medium and 50 µl of MTT solution (2 mg/ml in 1× PBS) were added to each well, with the supernatants discarded. After incubation at 37°C for 4 h, the formazan precipitate in each well was dissolved entirely in dimethyl sulfoxide (DMSO, Duchefa Biochemie, B.V.), and the absorbance was read at 570 nm using a Vmax plate reader (Molecular Devices LLC). Before the detection of the level of the formazan precipitate, the morphological changes of cells were observed using an optical microscope (Leica Microsystems GmbH) at a 400× magnification.

Furthermore, the cell viability assessed using the MTT assay was also evaluated based on the quantification of the ATP present, because ATP concentration indicates the presence of metabolically active cells, using CellTiter-Glo assay kit (Promega Corporation) according to the manufacturer's protocol. After mixing cell culture medium and CellTiter-Glo^® Reagent, the luminescent signal from each well was measured under an GloMax^® 20/20 Luminometer (Promega Corporation) and ATP concentration were determined using an ATP standard curve.

AEC treated CT26 cells were fixed using 4% formaldehyde for 1 h at room temperature and permeabilized using 1% Triton X-100, were incubated with 0.5% bovine serum albumin (BSA) for 1 h at room temperature and then Ki67/MKI67 primary antibodies (1:20; cat. no. NB500-170s; Novus Biologicals, LLC) for 12 h at 4°C. The cells were subsequently incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:100; cat. no. A11008; Thermo Fisher Scientific, Inc.) secondary antibodies for 1 h at room temperature. Ki67 positive cells in CT26 cells were detected based on the green fluorescence intensity using an EVOS™ M5000 Imaging System (Thermo Fisher Scientific, Inc.). Their number was counted in the total area of the field of view (67,500 µm²) for each well of CT26 cells using the Imaging System.

The number of apoptotic cells was analyzed using a Muse™ Annexin V and Dead Cell Kit (MilliporeSigma). Briefly, the wells containing the CT26 cells were divided into six groups (No, Vehicle, Cis, LoAEC, MiAEC, and HiAEC) for the AEC experiment. After being treated with aforementioned concentrations of AEC and Cis for 24 h at 37°C, the cells were treated with annexin V and 7-aminoactinomycin D (7-AAD) for 20 min at room temperature. The distribution of the stained cells were analyzed using a Muse™ Cell Analyzer (MilliporeSigma). After gating analyzed data based on the cell size, cells were classified into four groups: non-apoptotic cells, early apoptotic cells, late apoptotic cells and primarily nuclear debris as described previously (21).

Total protein was collected from the CT26 cells and CT26 tumors of BALB/cKorl syngeneic mice using the Pro-Prep Protein Extraction Solution (Intron Biotechnology, Inc.). The tissue homogenates were prepared from two to three solid tumors per group based on the results of the measurement of weight and hematoxylin and eosin (H&E) staining analysis. After homogenizing the tumor, total proteins were harvested, and their concentration was determined using a SMART™ Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific, Inc.). Total proteins (30 µg/lane) were separated on 4–20% gradient SDS-PAGE for 2 h at 100 V, and they were subsequently transferred to nitrocellulose blotting membranes with a 0.45 µm pore size for 2 h at 40 V. Proteins bounded on nitrocellulose membranes were incubated with primary antibodies (Table SI) overnight at 4°C. After removing the non-specifically bound antibodies using washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 0.05% Tween 20), the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:1,000; cat. no. HS101; Transgene Biotech Co., Ltd.). The intensity of each protein was analyzed using a Chemiluminescence Reagent (Pfizer, Inc.) using the FluorChem^® FC2 imaging system (ProteinSimple). Finally, the density of each protein was quantified using AlphaView (Version 3.2.2, Cell Biosciences, Inc.).

The protocol for experimental animal study was reviewed and approved by the Pusan National University Institutional Animal Care and Use Committee (approval no. PNU-2020-0108). To ensure the reliability of the data from the animal study, the total number of animals was determined as 35 using G-POWER 3.1.9.7 (Heinrich-Heine-Universität Düsseldorf, Germany) with the α probability of 0.05, effect size of 0.8 and a power of 0.80. Seven week old male BALB/cKorl mice (n=35) were supplied by the National Institute of Food and Drug Safety Evaluation (Cheongju, Korea) of the Korea Food and Drug Administration (KFDA). The BALB/cKorl syngeneic mice were bred at the Pusan National University-Laboratory Animal Resources Center, which is accredited by the KFDA (accredited unit 000231) and The Association for Assessment and Accreditation of Laboratory Animal Care International (accredited unit 001525). Mice were provided, ad libitum, with filtered tap water and a standard irradiated chow diet (Samtako Co., Ltd.). All mice were maintained in a specific pathogen-free state, with a strictly regulated 12 h light/dark cycle, and constant temperature (22±2°C) and relative humidity (50±10%).

The anti-tumor activity of AEC was evaluated in the BALB/cKorl syngeneic mice for a 37 day period using a slight modification of methods reported previously (25,26). Briefly, 4×10⁵ CT26 cells were injected subcutaneously into the back region of BALB/cKorl mice on day 1. CT26 tumor-bearing BALB/cKorl syngeneic mice (n=35) were divided randomly into one of five groups (n=7/group). The first group (Vehicle treated group, n=7) was orally administrated the same volume (200 µl) of 1× PBS every day for five weeks. The second group (Cis treated group, n=7) was injected intraperitoneally with Cis (4 mg/kg) every two days from day 26 to day 37, because long-term administration of Cis causes serious toxicity and can lead to death (27). The other three groups were administered a low (250 mg/kg; LoAEC treated group, n=7), medium (500 mg/kg; MiAEC treated group, n=7) or high concentration of AEC (1,000 mg/kg; HiAEC treated group, n=7) orally every day for 37 days. The health and behavior of all mice were monitored every day before treatment was administrated. No animals died during the experimental period. Bedding was abundantly provided as special housing condition to help animals maintain their body temperature. Furthermore, humane endpoints were set when the tumor exceeded 4,400 mm³ in volume (28) and when body weight of mice decreased >20% within 1–2 weeks, to prevent pain or distress of mice. At 24 h after the final treatment, all BALB/cKorl syngeneic mice were euthanized by a trained researchers using an appropriate chamber with gas regulator and CO₂ gas with a minimum purity of 99.0% based on the AVMA Guidelines for the Euthanasia of Animals. A cage containing animals was placed in the chamber and CO₂ gas of 99.0% was introduced into chamber without pre-charging, with a fill rate of ~50% of the chamber volume per minute. The death of mice were confirmed by ascertaining cardiac and respiratory arrest, or dilated pupils and fixed body. The solid tumors were then collected from all euthanized BALB/cKorl syngeneic mice (Fig. S2).

The size of CT26 tumors was measured daily during oral administration of AEC (from day 1 to 36). The tumor volume was determined based on the measurement results for length and width of the tumors using the following formula: Tumor volume (mm³)=(length × width²)/2. Images of each tumor formed in the mice were taken after the last administration using a micro computed tomography (microCT) scanner (Nano Focus Ray Co., Ltd.). The tumor volume in the microCT image was quantified using ImageJ 1.52a (National Institutes of Health). The weight of each tumor was assessed, once excised, using an electrical balance in duplicate.

The CT26 solid tumor collected from the BALB/cKorl syngeneic mice model was fixed in a 10% formalin solution for 48 h at room temperature, and then the middle region of solid tumor was embedded into paraffin block. Following sectioning the tumor block into 4 mm thick slices, these section were stained with a H&E solution (Sigma-Aldrich; Merck KGaA) for 4 min 15 sec at room temperature. After optical microscopic examination of the tumor sections at 400× magnification, the tumor type and pathological features were characterized by Professor Beum Seok Han at Hoseo University (Asana, Korea). The necrotic areas on the H&E stained tumor sections were measured and quantified, as described previously (29).

The aforementioned tissue sections of CT26 solid tumor (two to three samples per group) were deparaffinized with xylene, rehydrated using a decreasing EtOH series, and then washed three times with dH₂O for 5 min at room temperature. These sections were blocked using 10% goat serum (Vector Laboratories, Inc.; Maravai LifeSciences) in 1× PBS solution for 30 min at room temperature. The tissue slides were incubated with anti-Ki67/MKI67 (1:100; cat. no. NB500-170s; Novus Biologicals) primary antibodies for 12 h at 4°C, and then with goat fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG secondary antibodies (1:100; cat. no. 65-6111; Thermo Fisher Scientific, Inc.) for 45 min at room temperature. Finally, Ki67 positive cells in tumor sections were detected based on the green fluorescence intensity using an EVOS™ M5000 Imaging System (Thermo Fisher Scientific, Inc.). Their number was counted in the total area of the field of view (67,500 mm²) for each CT26 tumor section using the Imaging System.

After anesthesia with an intraperitoneal injection of anesthetic mixture (4:1 ratio) containing of 40 mg/kg Alfaxan (Jurox Pty Limited) and 10 mg/kg Rompun (Bayer-Korea, Ltd.) based on previous report (30), the abdominal region of BALB/cKorl syngeneic mice were opened by sterile scissors. Before opening, adequate anesthesia of mice was ensured by testing the pedal withdrawal reflex (clasping one's toes with tweezers) and lacking the eye blink reflex. The whole blood sample was collected from the abdominal veins using 1 ml syringe (26 SWG), and subsequentially the mice were terminated by cervical dislocation. The levels of 12 key factors were analyzed by an automated cell counter (Beckman-Coulter, Inc.) using the Vetscan HM5 Reagents Pack (cat. no. 89126-098; Abaxis, Inc.; Zoetis Services LLC) according to the manufacturer's protocols. The levels of white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, corpuscular hemoglobin concentration mean, corpuscular hemoglobin content, hemoglobin concentration distribution width, platelets and mean platelet volume were measured in duplicate for each sample.

Serum samples were obtained from whole blood sample after centrifugation at 1,500 × g for 10 min at 4°C. The concentrations of 5 serum biochemical components were analyzed using a Hitachi 747 automatic serum analyzer (Hitachi, Ltd.). All analysis were performed in duplicate using fresh samples.

One-way ANOVA (SPSS for Windows, Release 10.10, IBM Corp.) was used to assess statistical significance between the treatment groups, and followed by Tukey's post-hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference, and all values were presented as the mean ± standard deviation (SD).

To evaluate the potential antioxidative activity of AEC, the main phytochemical classes and DPPH radical scavenging activity of AEC were analyzed. Among the three phytochemicals, TPC was detected at the highest concentrations (108.05 mg/g), whereas the TTC and TFC concentrations were relatively low (29.19 and 6.88 mg/g, respectively). Furthermore, the concentration of TPTC was 2.60 mg/g (Fig. 1A). In addition, the scavenging activity of AEC against the DPPH radical increased markedly in a dose-dependent manner, with an IC₅₀ value of 21.96 µg/ml (Fig. 1B). These concentrations of phytochemicals and DPPH radical scavenging activity were similar to those reported elsewhere (31). Furthermore, eleven active components, including 9,12-octadecadienoic acid (32), aspartic acid (33), eckol (34), phlorofucofuroeckol (35) and dieckol (36), were identified and characterized using LC-MS analyses (Figs. 1C and S3). Among them, six compounds, including phloroglucinol, eckol, 6,6′-bieckol, phlorofucofuroeckol, fucodiphloroethol G and dieckol, belonged to PT, which contains numerous separate compounds, and is classified into four subclasses: the fuhalols and phlorethols group, the fucols group, the fucophlorethols group and the eckols group (5,35,36). Therefore, these results indicated that AEC had potential antioxidant activity.

The viability of CT26 cells was determined using an MTT assay, cell morphological analysis and ATP concentration assay after exposure to three different AEC concentrations for 24 h to determine if exposure to AEC induced the cytotoxicity of CT26, HCT116 and Detroit 551 cells. The viability of CT26 cells in MTT assay was decreased significantly in a dose-dependent manner, and the highest cytotoxicity was demonstrated in the HiAEC treated group (Fig. 2A). The results detected using the MTT assay were reflected in the morphological changes seen in CT26 cells (Fig. 2A) and indicated that AEC exerted significant cytotoxicity on CT26 cells at concentrations <1,000 µg/ml. Furthermore, the number of cells stained with Ki67 proteins, a marker for cell proliferation, was significantly decreased after the AEC treatment compared with the No or Vehicle treatment groups (Fig. 2B). Furthermore, the effects of AEC on the cell viability assessed using the MTT assay were also demonstrated in the cell viability assessed based on the concentration of ATP although there are differences in dose-dependent responses (Fig. 2C). Moreover, the cytotoxicity of AEC was similarly demonstrated in HCT116 cells which are colorectal carcinoma cells derived from an adult male (Fig. 2D). However, AEC did not cause any toxicity in Detroit 551 normal fibroblast cell line derived from a skin tissues of human fetus (Fig. 2E). These results indicated that AEC exerts significant cytotoxicity in CT26 and HCT116 colon cancer cells at concentrations <1,000 µg/ml without any significant toxicity to normal skin fibroblast cells.

Experiments in CT26 cells were performed to evaluate if the cytotoxicity effects of AEC were related to changes in the apoptosis-associated response. The number of live and apoptotic CT26 cells were assessed using Annexin V/PI staining analysis, and the protein expression levels of the mitogen-activated protein kinases (MAPK) signaling pathway members and apoptosis regulatory proteins were assessed using specific antibodies. After treatment with only HiAEC, the number of apoptotic cells was significantly increased, with a concomitant decrease in the number of live cells, compared with the Vehicle group (Fig. 3A). The increase in the number of apoptotic cells was reflected by the protein expression levels of the proteins related to the Bax/Bcl-2 and MAPK signaling pathways. The AEC-treated group demonstrated significantly enhanced p-c-Jun N-terminal kinase (p-JNK), p-extracellular signal-regulated kinase (p-ERK), and p-p38 protein expression levels compared with the Vehicle group, even though the increase rate of phosphorylation of each protein was varied (Fig. 3B). A similar response was demonstrated in the relative protein expression levels of Cleaved Cas-3/Cas-3 and Bax/Bcl-2, which were increased significantly in the LoAEC, MiAEC and HiAEC treated groups compared with the Vehicle group (Fig. 3B). These results indicated that the cytotoxic effects of AEC may be tightly linked to the promotion of the apoptosis-related response and activation of the MAPK and Bax/Bcl-2 signaling pathways.

The present study evaluated whether the anti-tumor activity of AEC in CT26 colon cancer cells could be reproduced entirely in the BALB/cKorl syngeneic mice with CT26 tumors. The changes in the volume and histopathological structure of the tumor were analyzed in CT26 tumors of BALB/cKorl syngeneic mice treated with three different doses of AEC for five weeks. The volume of the tumor markedly decreased in the AEC treated groups compared with the Vehicle group, and a significant reduction in the weight of them was demonstrated in the MiAEC treated group compared with the Vehicle group, but not HiAEC treated group (Fig. 4A and B). A similar effect on the tumor volume was also demonstrated in actual and microCT imaging (Fig. 4C). Furthermore, few spots containing cell necrosis were detected in the H&E stained sections of CT26 tumors in the Vehicle treated group. The necrotic region of these spots was markedly expanded after the AEC treatment compared with the Vehicle treated group. Certain pathological alterations, including hemorrhage, cyst formation and angiogenesis, were observed in the AEC treated tumors of BALB/cKorl syngeneic mice (Fig. 5A and B). By contrast, no significant differences in the toxicity parameters, including body, kidney and liver weight, cell composition of the blood, metabolites of the serum, and the histopathological structure of the liver and kidney were demonstrated between the Vehicle and AEC treated groups (Fig. S4, Fig. S5, Fig. S6, Fig. S7). These results indicated that AEC treatment could inhibit the growth of CT26 tumors in BALB/cKorl syngeneic mice without significant toxicity.

The tissue distribution of Ki67 and proliferating cell nuclear antigen (PCNA) proteins were evaluated using specific antibodies in AEC treated tumors of BALB/cKorl syngeneic mice to evaluate if the inhibitory activity of AEC on CT26 tumor growth was accompanied by a change in cell proliferation ability. The fluorescent intensity for the Ki67 proteins significantly decreased with increasing AEC dose in CT26 tumors of BALB/cKorl syngeneic mice (Fig. 6A). A similar pattern was observed with a significant decrease of the protein expression level of PCNA (Fig. 6B). These results indicated that the inhibitory activity of AEC on CT26 tumor growth could be linked to the suppression of tumor cell proliferation in BALB/cKorl syngeneic mice.

Alterations to the protein expression levels of the major proteins within the MAPK and Bax/Bcl-2 signaling pathways were assessed in the AEC treated tumors of BALB/cKorl syngeneic mice to evaluate if the inhibitory effects of AEC on CT26 tumor growth were accompanied by changes in the apoptosis-associated response. In the MAPK signaling pathway, the phosphorylation of ERK, JNK and p38 increased significantly in all AEC treated groups compared with the Vehicle treated group (Fig. 7A). Furthermore, a similar pattern of increase was demonstrated in the Bax/Bcl-2 signaling pathway, where the protein expression levels of Bax/Bcl-2 and Cleaved Cas-3 proteins were increased significantly in the AEC treated group compared with the Vehicle group (Fig. 7B). These results indicated that the inhibitory effects of AEC on CT26 tumor growth may be associated with the enhanced phosphorylation of several members of the MAPK signaling pathway and the critical proteins in the Bax/Bcl-2 signaling pathway.

The alteration of the protein expression levels of key proteins in the myosin light chain (MLC)/focal adhesion kinase (FAK)/Akt signaling pathway were assessed in the AEC treated tumors of BALB/cKorl syngeneic mice to evaluate if the inhibitory activity of AEC on the CT26 tumor growth were accompanied by suppression of the migration ability. Protein expression levels of the vascular endothelial growth factor A (VEGFA), matrix metallopeptidase (MMP)2, and MMP9 proteins were significantly lower in the AEC treated group compared with the Vehicle treated group, even though their rate was varied (Fig. 8A). Furthermore, a similar decreasing pattern was demonstrated in the regulation of the MLC/FAK/Akt signaling pathway proteins. In the MLC/FAK signaling pathway, the phosphorylation levels of FAK and MLC decreased markedly in the AEC treated groups compared with the Vehicle treated group (Fig. 8B). In the phosphoinositide 3-kinases (PI3K)/Akt signaling pathway, the phosphorylation levels of PI3K and Akt proteins were significantly decreased in the AEC treated group compared with the Vehicle group (Fig. 8C). Therefore, the inhibitory effects of AEC on CT26 tumor growth may be associated with suppression of the migration ability of tumor cells through alternative control of the MLC/FAK/Akt signaling pathway.

Whether the inhibitory activity of AEC on CT26 tumor growth was accompanied by changes in the protein expression level of tumor suppression-related proteins and the NF-κB signaling pathway was evaluated. Changes in the protein expression levels of the tumor suppression-related and the NF-κB signaling pathway proteins were evaluated in the AEC treated tumors of BALB/cKorl syngeneic mice. The protein expression levels of p53, mouse double minute 2 (MDM2) and p27 increased markedly in a dose-dependent manner in the MiAEC and HiAEC treated groups compared with the Vehicle group (Fig. 9A). However, protein expression levels in the NF-κB signaling pathway were significantly inhibited under the same conditions, where the protein expression levels of p-NF-κB and p-IκB-α were significantly lower in the AEC treated group compared with the Vehicle group (Fig. 9B). Therefore, the inhibitory effects of AEC on CT26 tumor growth could be linked to the upregulation of tumor suppression-related proteins through the inhibition of the NF-κB signaling pathway in CT26 tumors in BALB/cKorl syngeneic mice.

Finally, the anti-tumor effects of PT as the bioactive compound candidates in AEC were confirmed in CT26 cells. Changes in the cytotoxicity and apoptosis activation were assessed in CT26 cells after PT treatment. The viability of CT26 cells was significantly decreased in the PT treated groups compared with the No or Vehicle group (Fig. 10A). The number of cells stained with Ki67 protein were significantly decreased in the PT treated groups compared with the No or Vehicle groups (Fig. 10B). The PT treated groups in CT26 cells demonstrated an increase in the phosphorylation levels of ERK, JNK and p38 proteins compared with Vehicle group although the increased rate of JNK was the lowest among them (Fig. 10C). Also, similar enhancements in the protein expression levels of Bax/Bcl-2 and Cleaved Cas-3/Cas-3 were observed in the CT26 cells treated with PT and HiAEC compared with the No or Vehicle group (Fig. 10D). Therefore, PT in AEC can be considered one of the main bioactive compounds which exert an anti-tumor effect of AEC.