Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells
- Authors:
- Published online on: April 24, 2018 https://doi.org/10.3892/or.2018.6398
- Pages: 414-424
Abstract
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 (5–7), 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 (12–16). 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 (27–29). 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.
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. |
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 (32–36). 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 |
References
|
Terasaki M, Hirose A, Narayan B, Baba Y, Kawagoe C, Yasui H, Saga N, Hosokawa M and Miyashita K: Evaluation of recoverable functional lipid components of several brown seaweeds (phaeophyta) from Japan with special reference to fucoxanthin and fucosterol contents. J Phycol. 45:974–980. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Terasaki M, Narayan B, Kamogawa H, Nomura M, Stephen NM, Kawagoe C, Hosokawa M and Miyashita K: Carotenoid profile of edible Japanese seaweeds: An improved HPLC method for separation of major carotenoids. J Aquat Food Prod Technol. 21:468–479. 2012. View Article : Google Scholar | |
|
Beppu F, Niwano Y, Tsukui T, Hosokawa M and Miyashita K: Single and repeated oral dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice. J Toxicol Sci. 34:501–510. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Iio K, Okada Y and Ishikura M: Single and 13-week oral toxicity study of fucoxanthin oil from microalgae in rats. Shokuhin Eiseigaku Zasshi. 52:183–189. 2011.(In Japanese). View Article : Google Scholar : PubMed/NCBI | |
|
Okuzumi J, Takahashi T, Yamane T, Kitao Y, Inagake M, Ohya K, Nishino H and Tanaka Y: Inhibitory effects of fucoxanthin, a natural carotenoid, on N-ethyl-N'-nitro-N-nitrosoguanidine-induced mouse duodenal carcinogenesis. Cancer Lett. 68:159–168. 1993. View Article : Google Scholar : PubMed/NCBI | |
|
Kim JM, Araki S, Kim DJ, Park CB, Takasuka N, Baba-Toriyama H, Ota T, Nir Z, Khachik F, Shimidzu N, et al: Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation. Carcinogenesis. 19:81–85. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Nishino H: Cancer prevention by carotenoids. Mutat Res. 402:159–163. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Shiratori K, Ohgami K, Ilieva I, Jin XH, Koyama Y, Miyashita K, Yoshida K, Kase S and Ohno S: Effects of fucoxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo. Exp Eye Res. 81:422–428. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Nishikawa S, Hosokawa M and Miyashita K: Fucoxanthin promotes translocation and induction of glucose transporter 4 in skeletal muscles of diabetic/obese KK-Ay mice. Phytomedicine. 19:389–394. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Maeda H, Hosokawa M, Sashima T, Funayama K and Miyashita K: Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochem Biophys Res Commun. 332:392–397. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Hitoe S and Shimoda H: Seaweed fucoxanthin supplementation improves obesity parameters in mildly obese Japanese subjects. Funct Food Health Dis. 7:246–262. 2017. | |
|
Hosokawa M, Kudo M, Maeda H, Kohno H, Tanaka T and Miyashita K: Fucoxanthin induces apoptosis and enhances the antiproliferative effect of the PPARgamma ligand, troglitazone, on colon cancer cells. Biochim Biophys Acta. 1675:113–119. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Das SK, Hashimoto T and Kanazawa K: Growth inhibition of human hepatic carcinoma HepG2 cells by fucoxanthin is associated with down-regulation of cyclin D. Biochim Biophys Acta. 1780:743–749. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Murakami C, Takemura M, Sugiyama Y, Kamisuki S, Asahara H, Kawasaki M, Ishidoh T, Linn S, Yoshida S, Sugawara F, et al: Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases. Biochim Biophys Acta. 1574:85–92. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Okuzumi J, Nishino H, Murakoshi M, Iwashima A, Tanaka Y, Yamane T, Fujita Y and Takahashi T: Inhibitory effects of fucoxanthin, a natural carotenoid, on N-myc expression and cell cycle progression in human malignant tumor cells. Cancer Lett. 55:75–81. 1990. View Article : Google Scholar : PubMed/NCBI | |
|
Kotake-Nara E, Terasaki M and Nagao A: Characterization of apoptosis induced by fucoxanthin in human promyelocytic leukemia cells. Biosci Biotechnol Biochem. 69:224–227. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Asai A, Sugawara T, Ono H and Nagao A: Biotransformation of fucoxanthinol into amarouciaxanthin A in mice and HepG2 cells: Formation and cytotoxicity of fucoxanthin metabolites. Drug Metab Dispos. 32:205–211. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Asai A, Yonekura L and Nagao A: Low bioavailability of dietary epoxyxanthophylls in humans. Br J Nutr. 100:273–277. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Hashimoto T, Ozaki Y, Mizuno M, Yoshida M, Nishitani Y, Azuma T, Komoto A, Maoka T, Tanino Y and Kanazawa K: Pharmacokinetics of fucoxanthinol in human plasma after the oral administration of kombu extract. Br J Nutr. 107:1566–1569. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Takahashi K, Hosokawa M, Kasajima H, Hatanaka K, Kudo K, Shimoyama N and Miyashita K: Anticancer effects of fucoxanthin and fucoxanthinol on colorectal cancer cell lines and colorectal cancer tissues. Oncol Lett. 10:1463–1467. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH, Simeone DM, et al: Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA. 104:10158–10163. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, Alea Perez M, Richel DJ, Stassi G and Medema JP: Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci USA. 105:13427–13432. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Findlay VJ, Wang C, Watson DK and Camp ER: Epithelial-to-mesenchymal transition and the cancer stem cell phenotype: Insights from cancer biology with therapeutic implications for colorectal cancer. Cancer Gene Ther. 21:181–187. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Kanwar SS, Yu Y, Nautiyal J, Patel BB and Majumdar AP: The Wnt/beta-catenin pathway regulates growth and maintenance of colonospheres. Mol Cancer. 9:212–225. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Terasaki M, Maeda H, Miyashita K, Tanaka T, Miyamoto S and Mutoh M: A marine bio-functional lipid, fucoxanthinol, attenuates human colorectal cancer stem-like cell tumorigenicity and sphere formation. J Clin Biochem Nutr. 61:25–32. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Yoshida M, Hatano N, Nishiumi S, Irino Y, Izumi Y, Takenawa T and Azuma T: Diagnosis of gastroenterological diseases by metabolome analysis using gas chromatography-mass spectrometry. J Gastroenterol. 47:9–20. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Dazard JE, Sandlers Y, Doerner SK, Berger NA and Brunengraber H: Metabolomics of ApcMin/+ mice genetically susceptible to intestinal cancer. BMC Syst Biol. 8:722014. View Article : Google Scholar : PubMed/NCBI | |
|
Yoshie T, Nishiumi S, Izumi Y, Sakai A, Inoue J, Azuma T and Yoshida M: Regulation of the metabolite profile by an APC gene mutation in colorectal cancer. Cancer Sci. 103:1010–1021. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Han XY, Wei B, Fang JF, Zhang S, Zhang FC, Zhang HB, Lan TY, Lu HQ and Wei HB: Epithelial-mesenchymal transition associates with maintenance of stemness in spheroid-derived stem-like colon cancer cells. PLoS One. 8:e733412013. View Article : Google Scholar : PubMed/NCBI | |
|
Warburg O: On the origin of cancer cells. Science. 123:309–314. 1956. View Article : Google Scholar : PubMed/NCBI | |
|
Sato M, Kawana K, Adachi K, Fujimoto A, Yoshida M, Nakamura H, Nishida H, Inoue T, Taguchi A, Takahashi J, et al: Spheroid cancer stem cells display reprogrammed metabolism and obtain energy by actively running the tricarboxylic acid (TCA) cycle. Oncotarget. 7:33297–33305. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Vermeersch KA, Wang L, Mezencev R, McDonald JF and Styczynski MP: OVCAR-3 spheroid-derived cells display distinct metabolic profiles. PLoS One. 10:e01182622015. View Article : Google Scholar : PubMed/NCBI | |
|
Penkert J, Ripperger T, Schieck M, Schlegelberger B, Steinemann D and Illig T: On metabolic reprogramming and tumor biology: A comprehensive survey of metabolism in breast cancer. Oncotarget. 7:67626–67649. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Lin SH, Liu T, Ming X, Tang Z, Fu L, Schmitt-Kopplin P, Kanawati B, Guan XY and Cai Z: Regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker CD133 under low glucose conditions. Sci Rep. 6:211842016. View Article : Google Scholar : PubMed/NCBI | |
|
Chen KY, Liu X, Bu P, Lin CS, Rakhilin N, Locasale JW and Shen X: A metabolic signature of colon cancer initiating cells. Conf Proc IEEE Eng Med Biol Soc. 2014:4759–4762. 2014.PubMed/NCBI | |
|
Küntzer J, Eggle D, Lenhof HP, Burtscher H and Klostermann S: The roche cancer genome database (RCGDB). Hum Mutat. 31:407–413. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Matsuda Y, Miura K, Yamane J, Shima H, Fujibuchi W, Ishida K, Fujishima F, Ohnuma S, Sasaki H, Nagao M, et al: SERPINI1 regulates epithelial-mesenchymal transition in an orthotopic implantation model of colorectal cancer. Cancer Sci. 107:619–628. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Russo P, Malacarne D, Falugi C, Trombino S and O'Connor PM: RPR-115135, a farnesyltransferase inhibitor, increases 5-FU-cytotoxicity in ten human colon cancer cell lines: Role of p53. Int J Cancer. 100:266–275. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Hara K, Hamada C, Wakabayashi K, Kanda R, Kaneko K, Horikoshi S, Tomino Y and Suzuki Y: Scavenging of reactive oxygen species by astaxanthin inhibits epithelial-mesenchymal transition in high glucose-stimulated mesothelial cells. PLoS One. 12:e01843322017. View Article : Google Scholar : PubMed/NCBI | |
|
Festuccia C, Mancini A, Gravina GL, Scarsella L, Llorens S, Alonso GL, Tatone C, Di Cesare E, Jannini EA, Lenzi A, et al: Antitumor effects of saffron-derived carotenoids in prostate cancer cell models. BioMed Res Int. 2014:1350482014. View Article : Google Scholar : PubMed/NCBI | |
|
Mondul AM, Sampson JN, Moore SC, Weinstein SJ, Evans AM, Karoly ED, Virtamo J and Albanes D: Metabolomic profile of response to supplementation with β-carotene in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Am J Clin Nutr. 98:488–493. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Qin XY, Tatsukawa H, Hitomi K, Shirakami Y, Ishibashi N, Shimizu M, Moriwaki H and Kojima S: Metabolome analyses uncovered a novel inhibitory effect of acyclic retinoid on aberrant lipogenesis in a mouse diethylnitrosamine-induced hepatic tumorigenesis model. Cancer Prev Res. 9:205–214. 2016. View Article : Google Scholar | |
|
Huang YT, Lin YW, Chiu HM and Chiang BH: Curcumin induces apoptosis of colorectal cancer stem cells by coupling with CD44 marker. J Agric Food Chem. 64:2247–2253. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Amelio I, Cutruzzolá F, Antonov A, Agostini M and Melino G: Serine and glycine metabolism in cancer. Trends Biochem Sci. 39:191–198. 2014. View Article : Google Scholar : PubMed/NCBI |
