Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells

  • Authors:
    • Masaru Terasaki
    • Masatoshi Mima
    • Satoki Kudoh
    • Tetsuya Endo
    • Hayato Maeda
    • Junichi Hamada
    • Kazumi Osada
    • Kazuo Miyashita
    • Michihiro Mutoh
  • View Affiliations

  • Published online on: April 24, 2018     https://doi.org/10.3892/or.2018.6398
  • Pages: 414-424
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Abstract

Fucoxanthinol (FxOH) is a strong anticancer metabolite of fucoxanthin that accumulates in abundance in edible brown algae and promises human health benefits. FxOH has been shown to suppress tumorigenicity and sphere formation in human colorectal cancer stem cell (CCSC)-like spheroids (colonospheres, Csps). In the present study, we aimed to clarify the inhibitory activity of FxOH on epithelial-mesenchymal transition (EMT), which is essential for cancer recurrence and distant metastasis, and to identify intracellular low-molecular-weight metabolites that may be useful for evaluating the cellular effects of FxOH on CCSCs. FxOH significantly suppressed sphere-forming activity, migration and invasion in a dose-dependent manner. In addition, treatment with 50 µmol/l FxOH suppressed N-cadherin and vimentin expression and the activation of integrin signaling linked to EMT suppression by western blot analysis. MAPK signaling and STAT signaling related to cell growth and apoptosis in Csps derived from human CRC HT-29 and HCT116 cells were also altered. According to our metabolite profiling by GC-MS analysis, reduced glycine and succinic acid levels were correlated with EMT suppression and apoptosis induction in Csps. Our data indicate that simple amino acids such as glycine and succinic acid may be good prognostic indicators of physiological changes to CCSCs induced by FxOH treatment.

Introduction

Fucoxanthin (Fx) is a non-provitamin A of high polar xanthophyll that has an unusual allenic bond, an epoxide group and a conjugated carbonyl group in a polyene chain. Fx occurs dominantly in marine brown algae and diatoms and is responsible for photosynthesis and photoprotection. Undaria pinnatifida (wakame), Hizikia fusiforme (hiziki) and Sargassum horneri (akamoku) are particularly excellent sources of Fx among Japanese algal foods (1,2). It has been demonstrated that Fx is extremely safe in terms of toxicity, showing no adverse effects in animal experiments (3,4). Several researchers have conclusively reported the anticancer (57), anti-inflammatory (8), anti-diabetic (9) and anti-obesity effects of Fx in animals and humans (10,11). Moreover, Fx possesses strong potential for cell growth inhibition and the induction of apoptosis in human neuroblastoma, gastric cancer, hepatoma, colorectal cancer (CRC) and promyelocytic leukemia cells (1216). Fx is metabolically converted to fucoxanthinol (FxOH) (Fig. 1) and amarouciaxanthin A (Amx A) in the mouse intestine and liver (17). After the oral administration of wakame or Fx concentrate in humans, higher levels of FxOH and lower levels of cis-FxOH have been detected in human plasma (18,19). Thus, FxOH is an important indicator of Fx function and may be a promising candidate for human cancer chemoprevention. FxOH significantly attenuated the proliferation of cancer cells derived and cultured from human CRC tissue (20). However, despite the strong anticancer effects of FxOH, its underlying mechanisms are not well known.

CRC is the third most common cause of cancer-related death worldwide, and therefore it is urgent to reduce CRC prevalence (21). Although non- or less polar carotenoids such as β-carotene and lutein exhibit cancer preventive effects, as shown in many epidemiological studies, their utility has ‘insufficient evidence’. In the case of Fx or brown algae, prospective clinical trials, cohorts or follow-back studies for cancer prevention have not been attempted.

CRC stem cells (CCSCs) occupy only a small subset of CRC tissue, but they are thought to play a central role in cancer development. Self-renewal, differentiation, sphere formation, and tumorigenicity in immunodeficient animals have been characterized for CCSCs (22,23). CCSCs often acquire an epithelial-mesenchymal transition (EMT) phenotype accompanied by the activation of related proteins, and EMT not only promotes their migration and invasion, but is also believed to be a leading cause of CRC recurrence and distant metastasis (24). Therefore, attenuation of the EMT phenotype of CCSCs may represent a promising approach for cancer prevention, cancer recurrence/metastasis prevention and survival rate elongation. It is well known that spheroids formed from CRC cells, called colonospheres (Csps), are considered a representative CCSC model phenotype since they contain a high abundance of CCSCs and possess sphere reconstruction and tumorigenic capacities (22,23,25). We recently revealed that FxOH strongly induces the apoptosis of HT-29 Csps and attenuates tumorigenicity in a xenograft mouse model (26). However, little information regarding the EMT-suppressing effects of FxOH on CCSCs or Csps is available to date. Moreover, there are no studies reports regarding the anti-metastatic effects of FxOH in in vivo setting. Thus, we aimed to clarify the inhibitory activity of FxOH on EMT in the present study.

Intracellular low-molecular-weight metabolites that are essential for regulating energy systems such as glycolysis and the TCA cycle are suggested to be prognostic indicators representing cellular genomic and proteomic alterations after encounters with various endogenous and exogenous stimuli. Comprehensive metabolite analyses have revealed many marker metabolite candidates in serum, plasma, urine and cancer tissue in CRC patients and an animal model (2729). Therefore, intracellular low-molecular-weight metabolites likely represent a convenient approach to promptly diagnose the cellular conditions of CCSCs with complicated genetic and protein backgrounds. However, the marker metabolites representing the EMT phenotype in CCSCs remain elusive.

In the present study, we investigated the EMT-suppressive effects of FxOH on Csps formed from human CRC HT-29 and HCT116 cells. The molecules through which FxOH exerted EMT suppression were investigated. In addition, we examined the alterations exerted by FxOH on the metabolite profiles of Csps and identified marker metabolites with EMT potential in Csps.

Materials and methods

Chemicals and cell culture

All-trans-FxOH (purity, ≥98%) was provided by Dr Hayato Maeda (Hirosaki University, Japan) (Fig. 1). EGF, bFGF and DMEM/F12 medium were purchased from Wako Pure Chemicals (Osaka, Japan). B27 was obtained from Miltenyi Biotec, Inc. (Auburn, CA, USA). HT-29 and HCT116 human CRC cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 mM L-glutamine, 40,000 U/l penicillin and 40 mg/l streptomycin. All other chemicals and solvents were of analytical grade.

Colonosphere formation

HT-29 and HCT116 parental cells (PCs) were trypsinized from culture plates, washed twice with PBS, suspended in stem cell medium (SCM) composed of DMEM/F12 medium, 20 ng/ml EGF, 10 ng/ml bFGF, 0.2% B27 and an antibiotic-anti-mycotic agent, plated at a density of 3×104 cells/ml SCM in 10-cm dishes or 24-well ultra-low attachment plates (Corning Inc., Corning, NY, USA) and incubated for 2 days at 37°C in a humidified atmosphere containing 5% CO2. All experiments utilizing colonospheres (Csps) described below were performed using Csps grown for 2 days.

Analysis of the suppression of colonosphere formation

Csps derived from HT-29 and HCT116 PCs were formed in a 24-well ultra-low attachment plate for 2 days. After the Csps formed, a total of 2–10 mM FxOH reconstituted in dimethyl sulfoxide (DMSO) was applied to the cell medium at a final concentration of 10–50 µM (0.5 v/v%), or vehicle alone (DMSO) was applied. The cells were harvested and trypsinized after incubation for 24 h. Viable cells in Csps were counted using a trypan blue exclusion method. The Csps treated with FxOH were treated with ribonuclease A, stained with propidium iodide (PI) and subjected to flow cytometry. The percentage of apoptotic cells (sub-G1, hypodiploid cells) was estimated by a FACSaria-III flow cytometer (BD Biosciences, San Jose, CA, USA).

Western blot analysis

pAkt (Ser473) (cat. no. 4060), β-catenin (cat. no. 9582), pβ-catenin (Ser31/37/Thr42) (cat. no. 9561), cyclin D1 (cat. no. 2922) and PPARγ (cat. no. 2435) antibodies and phospho-ERK1/2 pathway (cat. no. 9911) and phospho-Stat antibody sampler (cat. no. 9914) kits were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). β-actin (cat. no. GTX109639), E-cadherin (cat. no. GTX100443), N-cadherin (cat. no. GTX127345), caspase-3 (cat. no. GTX110543), pFAK (Tyr397) (cat. no. GTX24803), LGR5 (cat. no. GTX129862) and vimentin (cat. no. GTX132610) antibodies were obtained from GeneTex (Irvine, CA, USA). The paxillin (Tyr31) antibody (MAB61641) was obtained from R&D Systems (Minneapolis, MN, USA). CD44 (cat. no. MS-668-P0), MMP-9 (cat. no. MS-817-P0) and p53 (cat. no. MS-105-P0) antibodies were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). EpCAM (cat. no. 11-581-C025) and integrin β1 (cat. no. 11-219-C100) antibodies were obtained from Exbio (Prague, Czech Republic). Epithelial type cells obtained immediately before preparing Csps were used as the parental cells (PCs) of the Csps. Csps derived from HT-29 and HCT116 PCs formed in 10-cm ultra-low attachment plates for 2 days and were then treated with 50 µM FxOH and vehicle (DMSO) for 4–24 h. The cells were harvested, washed twice with phosphate-buffered saline (PBS) and then lysed in a lysis buffer to obtain whole-cell lysates. The protein concentrations were photometrically measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Fifty micrograms of whole-cell proteins were separated on SDS-polyacrylamide minigels. The gels were then electroblotted onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated in 1% BSA blocking buffer at room temperature and probed with each of the primary antibodies (1:1,000 dilution) in blocking buffer overnight at 4°C following the manufacturer's instructions. The membranes were washed and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. The membranes were again washed and subsequently subjected to chemiluminescence reagents. The stripping process to avoid the detection of previous bands may induce unclear blot when we use a new antibody on the same membrane. Thus, we repeated western blotting, loading the same amount of sample, to obtain the new membrane for several times.

Migration and invasion analyses

Migration and invasion assays were performed using a 24-well Transwell chamber with an 8-µm pore size and a 24-well Matrigel invasion chamber (pore size, 8 µm) (Corning Costar, Cambridge, MA, USA). Csps derived from HT-29 and HCT116 PCs formed in 10-cm ultra-low attachment plates for the assays, were trypsinized and were washed twice with PBS. Then, 3×104 suspended cells were seeded in 500 µl of SCM in a 15-ml centrifuge tube and treated with FxOH (20 and 50 µM) or vehicle (DMSO). One hundred microliters of the cell suspension containing FxOH was applied to the upper compartment of each chamber. DMEM containing 10% FBS was loaded into the lower compartment in both chambers. The migration and invasion chambers were then incubated for 6 and 24 h, respectively, at 37°C. Cells that migrated or invaded to the underside of the upper compartment were fixed with formalin and stained with Giemsa solution/(1:00 dilution). Migrated or invaded cells were visualized at ×40 magnification and counted as the numbers of migrated or invaded cells/field in three random fields.

GC-MS analysis

2-Isopropylmalic acid (2-IPMA), methoxyamine hydrochloride and N-methyl-N(trimethylsilyl)-trifluoroacetate (MSTFA) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), MP Biomedicals (Solon, OH, USA) and GL Sciences (Tokyo, Japan), respectively. Csps treated with FxOH were harvested from culture plates, trypsinized, and washed twice with PBS. The pelleted cells were re-suspended in 50 µl of cold PBS, to which 2 µl of 0.1 mg/ml 2-IPMA was added as an internal standard, and then the suspensions were disrupted by sonication for 5 sec on ice. Total protein contents were determined using the Bradford method with 1 µl of each suspension. Metabolites were extracted with 250 µl of CH3OH/CHCl3/DW (2.5:1:1, v/v/v), centrifuged at 16,000 × g for 5 min and the upper phase was washed with 200 µl of DW. The extracts obtained were evaporated to dryness. The residues were oxymated with 30 µl of 20 mg/ml methoxyamine hydrochloride in dry pyridine at 30°C for 90 min, followed by the application of 15 µl of MSTFA at 37°C for 30 min. All GC-MS analyses were carried out using a GCMS-QP5000 system (Shimadzu, Kyoto, Japan) equipped with a non-polar capillary column [Rxi-5ms, 30 m × 0.25 mm i.d., film thickness, 0.25 µm; Restek, Co. Ltd., GmbH (Bad Homburg, Germany)] and online analysis software (CLASS 5000). The carrier gas (He) flow rate was at 0.5 ml/min (15.7 kPa), and injections were 1 µl in split-ratio mode (split ratio, 33%). The column temperature was initially 80°C for 2 min, increased to 330°C at 4°C/min and then maintained at 330°C for 8 min. The interface and source temperatures were 250 and 230°C, respectively. Identification was confirmed by comparing the spectra of single components with those stored in the acquisition system library. All metabolite contents were expressed as pmol metabolite/µg of total protein content.

Statistical analysis

All experiments were performed at least twice and are presented as representative data. Significant differences for multiple comparisons were determined by one-way ANOVA followed by Tukey-Kramer post hoc test. Differences were considered statistically significant at P<0.05 as indicated with the relevant symbols in the figures.

Results

Stemness and metabolite characteristics of colonospheres

Among the three CCSC surface markers (CD44, EpCAM and LGR5), CD44 variant forms (CD44v) and EpCAM were overexpressed in both HT-29 and HCT116 Csps compared with the PCs (Fig. 2). CD44 standard form (CD44s) and LGR5 were strongly increased in HT-29 Csps, while LGR5 was weakly increased in HCT116 Csps. Among the three key proteins representing the EMT phenotype (E-cadherin, N-cadherin and vimentin), the expression of E-cadherin and vimentin was elevated in both Csps compared with the expression in the PCs. N-cadherin expression did not discriminate between Csps and PCs for both cell types.

In the next experiment, metabolite profiles were constructed using GC-MS (Fig. 3). The quantitative data obtained for the Csps and PCs derived from both cell types for all 20 metabolites analyzed are presented in Table I. Four amino acids, specifically glycine, serine, threonine and glutamic acid, as well as succinic acid, a TCA cycle metabolite, were significantly increased in the HCT116 Csps compared with the PCs. Although no significant changes were observed between HT-29 Csps and PCs, there was a tendency toward a changing pattern for these five metabolites similar to that of HCT116 cells. Overall, metabolites were weakly increased in Csps compared with the PCs, but no significant change was observed for other metabolites from Csps or PCs derived from both cell lines.

Table I.

Metabolite profiles of the colonospheres from HT-29 and HCT116 parental cells.

Table I.

Metabolite profiles of the colonospheres from HT-29 and HCT116 parental cells.

pmol metabolite/µg total protein content

HT-29 cellsHCT116 cells


Peak no.aGroup - compoundCspsPCsCspsPCs
Amino acid
3   Valine2.9±2.10.6±0.60.1±0.12.7±2.7
5   Leucine1.4±0.81.6±1.61.6±0.7ND
6   Proline6.6±2.3NDNDND
7   Glycine19.3±3.96.9±3.1 16.6±2.2b1.0±0.5
11   AlanineNDND0.6±0.6ND
12   Serine2.2±0.50.6±0.6   2.6±0.2bND
13   Threonine6.2±0.72.4±1.6 10.6±1.1b0.5±0.5
15   Aspartic acid3.8±0.81.8±1.13.7±3.7ND
18   Glutamic acid9.0±2.32.6±1.1 13.2±1.4b0.9±0.5
19   Phenylalanine0.5±0.40.7±0.40.1±0.1ND
Dicarboxylic acid (TCA cycle)
8   Succinic acid2.8±1.80.5±0.5   0.9±0.3bND
9  Fumaric acid0.5±0.5ND0.1±0.1ND
14  Malic acid3.2±1.25.6±1.11.5±1.50.5±0.5
Carboxylic acid
1  Pyruvic acid1.4±0.81.1±1.12.0±0.31.7±0.4
2   Propionic acid74.2±10.097.1±23.954.5±6.259.4±19.4
4  Butyric acid34.3±5.141.5±6.963.7±10.351.6±4.6
10   Pelargonic acid3.8±0.83.1±0.54.9±0.33.2±0.8
16   γ-aminobutyric acid5.6±2.09.1±1.16.7±1.72.3±0.6
17   2,3,4-trihydroxybutyric acidNDND0.9±0.90.8±0.4
20  Lauric acid10.1±4.512.9±11.48.8±4.08.6±3.4

{ label (or @symbol) needed for fn[@id='tfn1-or-40-01-0414'] } All data are expressed as mean ± SE (n=3).

a The number of peak shown in Fig. 3

b P<0.05 (one-way ANOVA followed by post hoc Tuker-Kramer test vs. parental cells of each cell type). ND, a metabolite was not detected over signal/noise ratio (3:1) by GC-MS. Csps, colonospheres; PCs, parental cells.

Antiproliferative effects of FxOH in colonospheres

Treatment with 10, 20 and 50 µM FxOH inhibited the growth of Csps from the HT-29 and HCT116 cells in a dose-dependent manner (Fig. 4A and B). Sphere formation was as follows for Csps from HT-20 and HCT116 cells: 10 µM FxOH, 86.7±7.1 and 116.3±10.4%, respectively; 20 µM, 77.0±7.0 and 95.0±9.4%, respectively; and 50 µM, 57.7±11.0 and 52.1±5.4%, respectively. Vehicle (DMSO) alone exerted no effects on cell proliferation. Flow-cytometric analyses exhibited that the percentage of sub-G1 phase cells (apoptosis-induced cells) in the FxOH-treated Csps (35.4±0.5%) was higher than that noted in the control Csps (5.6±1.0%) (Fig. 4C). In addition, FxOH abrogated p53 expression and increased the p17 and p19 active subunits of caspase-3, suggesting that this growth inhibition is linked to apoptosis (Fig. 7).

Suppressive effects of FxOH on the migration and invasion of colonospheres

Treatment with 20 and 50 µM FxOH inhibited the migration and invasion of both HT-29 and HCT116 Csps in a dose-dependent manner (Fig. 5). Migration activities were as follows for Csps from HT-29 and HCT116 cells: 20 µM FxOH, 82.1±9.3 and 75.7±13.2%, respectively; and 50 µM, 15.0±7.4 and 16.7±4.2%, respectively. Invasion activities were as follows for HT-29 and HCT116 Csps: 20 µM FxOH, 52.8±3.3 and 47.4±2.7%, respectively; and 50 µM, 13.4±2.7 and 7.4±1.3%, respectively. Vehicle (DMSO) alone exerted no effects on both migration and invasion capacities.

Changes in the metabolite profiles of colonospheres with and without FxOH

Csps derived from both cell types were treated with 50 µM FxOH, and a total of 20 metabolites were analyzed using GC-MS. The quantitative data are presented in Table II and Fig. 6. Among them, three amino acids, specifically glycine, threonine and glutamic acid, were significantly decreased in both or either Csps compared with control Csps. Succinic acid, a carboxylic acid involved in the TCA cycle, was significantly decreased in both HT-29 and HCT116 Csps compared with control Csps. Although no significant change was observed in other metabolites, the majority of metabolites were weakly decreased in both HT-29 and HCT116 Csps treated with FxOH compared with control Csps.

Table II.

Metabolite profiles of colonospheres from the HT-29 and HCT116 cells following FxOH treatment at 24 h.

Table II.

Metabolite profiles of colonospheres from the HT-29 and HCT116 cells following FxOH treatment at 24 h.

pmol metabolite/µg total protein content

HT-29 cellsHCT116 cells


Peak no.aGroup - compoundCspsPCsCspsPCs
Amino acid
3   Valine2.5±1.51.0±1.01.8±1.81.5±0.7
5   Leucine3.7±2.10.9±0.91.6±0.50.7±0.7
6   Proline8.6±4.421.0±11.04.3±4.3ND
7   Glycine35.9±0.7 14.1±6.4b29.6±5.7 6.5±2.5b
11   Aranine2.7±2.70.3±0.34.5±4.51.9±1.0
12   Serine3.8±2.12.4±1.55.0±1.80.6±0.6
13   Threonine15.3±5.16.7±3.822.7±3.9 3.3±0.8b
15   Aspartic acid10.1±4.82.2±1.15.9±3.40.8±0.8
18   Glutamic acid29.1±8.211.3±4.931.0±2.1 7.8±3.0b
19   Phenylalanine2.6±0.30.7±0.70.8±0.80.9±0.9
Dicarboxylic acid (TCA cycle)
8   Succinic acid6.7±1.4   0.6±0.6b1.6±0.3NDb
9  Fumaric acid1.5±1.20.6±0.60.9±0.90.3±0.3
14  Malic acid13.9±4.04.2±2.16.2±2.32.1±1.4
Carboxylic acid
1  Pyruvic acid1.0±1.01.1±1.12.7±0.52.4±0.3
2   Propionic acid188.5±42.8110.2±31.5121.2±32.091.0±27.9
4  Butyric acid44.9±11.631.2±3.942.6±14.521.4±13.2
10   Pelargonic acid3.0±1.73.1±0.23.6±1.21.6±1.0
16   γ-aminobutyric acid16.0±2.75.4±2.830.7±18.18.2±6.9
17   2,3,4-Trihydroxybutyric acid2.7±0.81.7±1.0ND0.3±0.3
20  Lauric acid9.7±2.710.1±3.015.3±1.614.7±4.3

{ label (or @symbol) needed for fn[@id='tfn4-or-40-01-0414'] } All data are expressed as the mean ± SE (n=3).

a The number of peaks shown in Fig. 6.

b P<0.05 (one-way ANOVA followed by post hoc Tuker-Kramer test vs. parental cells of each cell type). ND, a metabolite was not detected over signal/noise ratio (3:1) by GC-MS.

Expression of molecules related to FxOH treatment

To clarify the proteins involved in the inhibition of EMT, invasion and migration, we performed a western blot assay. FxOH treatment increased E-cadherin at 24 h and decreased N-cadherin at 24 h and vimentin at 8 h in Csps in both cell lines (Fig. 7). FxOH also decreased the activation of integrin, MAPK and Stat signaling by inhibiting phosphorylation of their key proteins in Csps in both cell lines. Regarding integrin signaling, protein levels of Integrin β1 and phosphorylation levels of paxillin and Akt are clearly reduced at 24 h. The phosphorylation levels of pFAK and protein levels of PPARγ were reduced in a time-dependent manner. Regarding MAPK signaling, the phosphorylation levels of C-Raf decreased in a time-dependent manner. The phosphorylation levels of MEK were reduced at 24 h. The phosphorylation levels of ERK were downregulated only in HT-29 Csps. However, phosphorylation levels of p90RSK did not changed during the experiment. Regarding Stat signaling, phosphorylation levels of Stat3, Stat5 and Stat6 were reduced clearly at 24 h. We also observed reduced pro-caspase-3 at 24 h and cleaved p17/p19 (active forms of caspase-3) at 24 h and reduced p53 levels. As shown in the figure, phosphorylated MSK1, Stat1 and Stat2 were not detected in this experiment.

Time-dependent metabolite levels in colonospheres after FxOH treatment

To investigate FxOH-induced metabolite alterations in Csps, levels of the two metabolites were evaluated in HT-29 and HCT116 Csps treated with 50 µM FxOH for 4, 8 and 24 h. FxOH significantly increased glycine levels from 0 until 8 h and decreased glycine levels from 8 until 24 h in HT-29 Csps. Succinic acid levels in both Csps treated with FxOH were drastically decreased at 8 or 24 h compared with levels in each Csps at 0 h (Fig. 8).

Discussion

The results of the present study suggest that fucoxanthinol (FxOH) suppressed EMT. Conversely, glycine and succinic acid were found to be prognostic indicators of physiological changes in Csps treated with FxOH. This is the first study demonstrating EMT inhibition by FxOH treatment accompanied by detectable metabolite alterations. In addition, FxOH induced apoptosis by inhibiting integrin, MAPK and Stat signaling activations. Moreover, the decrease in p53 expression and activation of caspase-3 were suggested to be involved in FxOH-induced apoptosis in HT-29 and HCT116 Csps.

We first confirmed the existence of CCSC surface protein markers and EMT phenotype protein markers in HT-29 and HCT116 Csps (Fig. 2). The enhancement of CD44v, EpCAM, LGR5 and vimentin, an EMT marker, suggested that both Csps possessed CCSC and EMT properties. In general, the suppression of E-cadherin expression in cancer cells indicated their transformation into the EMT phenotype. Thus, the upregulation of E-cadherin and vimentin observed in this study represented a lack of consistency. Indeed, other researchers observed decreased E-cadherin and increased vimentin in Csps formed from HT-29 and HCT116 PCs (30). Although we have no idea why E-cadherin was increased in the Csps compared with PCs in this experiment, we used these Csps as a near-Csp model. Regarding N-cadherin, it is generally acknowledged that this marker as well as vimentin may be elevated upon transformation into the EMT phenotype.

We also found that incubation with SCM for both HT-29 and HCT116 Csps resulted in metabolic reprogramming during the PC to Csp steps, in which glycine, serine, threonine, glutamic acid and succinic acid levels were significantly elevated. In addition, these metabolites showed a similar increase in Csps formed from HT-29 PCs, although without significance (Fig. 3 and Table I). It is speculated that the activation of cytosol glycolysis and serine metabolism and the promotion of the mitochondrial GSH/GSSG redox system and TCA cycle are essential metabolic pathways in Csps derived from HT-29 and HCT116 PCs. Positive aerobic glycolysis and subsequent metabolic reprogramming in cancer cells are collectively known as the Warburg effect (31). Various researchers have demonstrated the impact of the Warburg effect on the metabolite profiles of CSCs or CSC-like spheroids reconstructed from original breast, colorectal, hepatic and ovarian cancer PCs (3236). Among the amino acids, aspartate, serine, glutamic acid and glutamine are assumed to be particularly good targets for cancer therapeutics in CSCs. Furthermore, several pathways, such as amino acid metabolism, the redox system, the TCA cycle and fatty acid biosynthesis, are also targets of CSCs. However, the precise mechanisms and physiological significance underlying the metabolite contents of CSCs remain unclear.

Properties such as gene expression, morphology and chemoresistance differ between HT-29 and HCT116 cells. For example, HT-29 is p53-mutant and HCT116 is p53-wild-type (37). Although HCT116 cells possess a phenotype that more closely resembles EMT compared with that of HT-29 cells, both cell types demonstrated similar capacity in terms of invasion, sphere formation and tumorigenicity (30,38). HCT116 cells are more sensitive to 5-fluorouracil treatment than HT-29 cells (39). In the present study, FxOH inhibited sphere formation, migration and invasion to the same degree in both Csp types and induced apoptosis through the same molecular regulations with similar temporal expression patterns (Figs. 4, 5 and 7). We previously reported that FxOH induced apoptosis along with the downregulation of pAkt (Ser473), peroxisome proliferator-activated receptor (PPAR)β/δ and PPARγ in HT-29 Csps (26). In the present study, we further showed that FxOH treatment began to suppress PPARγ and inhibit pC-Raf (Ser338) starting at 4 h, and decreased amounts of vimentin or increased E-cadherin levels were observed at 8 h, followed by caspase-3 activation and p53 depression at 24 h in both Csps. A highly polar xanthophyll, astaxanthin, as well as FxOH both inhibit EMT accompanied by the attenuation of reactive oxygen species production, inflammatory cytokine production and NF-κB activation in rat peritoneal mesothelial cells (40). The apocarotenoids crocetin and crocin promote EMT attenuation by inhibiting N-cadherin and β-catenin expression and increasing E-cadherin expression in aggressive prostate cancer PC3 and 22rvl cells (41).

In addition to regulating proteins, the metabolite changes in Csps treated with FxOH were very similar between each cell type (Figs. 6 and 8, and Table II). FxOH treatment markedly decreased glycine and succinic acid in both Csps at 8 or 24 h. Therefore, FxOH may attenuate the mitochondrial GSH/GSSG redox system and TCA cycle in Csps. Previously, Fx was shown to rapidly elicit the mitochondrial membrane potential in human promyelocytic leukemia HL-60 and HP100-1 cells (16). Our findings indicate that FxOH treatment may accompany the mitochondrial disruption of spheroids, regardless of the different phenotypes of cancer cells. Little is known about changes to anti-metabolism capacity by carotenoids in CSCs or CSC-like spheroids. A randomized, double-blind, placebo-controlled study, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, demonstrated that β-carotene significantly increased 17 metabolites in the sera of male smokers (42). In a diethylnitrosamine (DEN)-induced mouse hepatic tumor model, acyclic retinoid (0.06%) administration resulted in significant changes in 88 metabolites in liver tumor tissue compared with mice that did not receive DEN treatment (43). In contrast, a colorimetric lipid, curcumin, induced apoptosis accompanied by glutamine reduction in CD44-positive CSC-like cells derived from HT-29 cells (44). Some anticancer drugs such as 5-fluorouracil and gemcitabine alter cellular metabolism pathways (45). In the present study, we revealed that FxOH exerts anti-metabolism activity in cancer cells similar to that observed for other carotenoid or carotenoid-derived compounds and anticancer drugs.

In summary, FxOH attenuated EMT, inhibited the activation of integrin, MAPK, and Stat signaling and altered metabolite profiles in CSC-like cells of Csps derived from human CRC HT-29 and HCT116 cells. Glycine and succinic acid were suggested to be metabolite markers of EMT suppression induction in Csps. Further studies may reveal that these two metabolites are helpful in understanding the cellular conditions of CCSCs in human or animal colorectal mucosal tissue following Fx or FxOH administration.

Acknowledgements

Not applicable.

Funding

The present study was supported in part by a grant from Japan Society for the Promotion of Science KAKENHI (no. 16K07880).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

MT and MM conceived and designed the study. MT, MM and SK performed the experiments. MT and MM wrote the paper. TE, HM, JH, KO and KM performed interepretation of data, reviewed and edited the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors state that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

CCSCs

colorectal cancer stem cells

CD44s

CD44 standard form

CD44v

CD44 variant forms

Csps

colonospheres

DEN

diethylnitrosamine

DMEM

Dulbecco's modified Eagle's medium

DMSO

dimethyl sulfoxide

DW

distilled water

EMT

epithelial-mesenchymal transition

FBS

fetal bovine serum

FxOH

fucoxanthinol

Fx

fucoxanthin

2-IPMA

2-isopropylmalic acid

MSTFA

N-methyl-N(trimethylsilyl)-trifluoroacetate

PCs

parental cells

PPAR

peroxisome proliferator-activated receptor

SCM

stem cell medium

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July 2018
Volume 40 Issue 1

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APA
Terasaki, M., Mima, M., Kudoh, S., Endo, T., Maeda, H., Hamada, J. ... Mutoh, M. (2018). Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells. Oncology Reports, 40, 414-424. https://doi.org/10.3892/or.2018.6398
MLA
Terasaki, M., Mima, M., Kudoh, S., Endo, T., Maeda, H., Hamada, J., Osada, K., Miyashita, K., Mutoh, M."Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells". Oncology Reports 40.1 (2018): 414-424.
Chicago
Terasaki, M., Mima, M., Kudoh, S., Endo, T., Maeda, H., Hamada, J., Osada, K., Miyashita, K., Mutoh, M."Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells". Oncology Reports 40, no. 1 (2018): 414-424. https://doi.org/10.3892/or.2018.6398