Fucoxanthin Inhibits Development of Sigmoid Colorectal Cancer in a PDX Model With Alterations of Growth, Adhesion, and Cell Cycle Signals

Materials and Methods

Chemicals. A high-purity Fx powder (≥99%) was kindly donated by PATH Co., Ltd. (Tokyo, Japan), ALNUR Co., Ltd. (Tokyo, Japan), and Kampoikagakukenkyujyo Co., Ltd. (Osaka, Japan). All-trans-FxOH (purity, ≥98%), a metabolite of Fx, was enzymatically prepared from marine brown algal extracts by Dr. Hayato Maeda (Hirosaki University, Japan). RPMI-1640 cell culture medium (cat.no. 183-02165), Hank’s balanced salt solution (HBSS) (cat.no. 082-08961), dimethylsulfoxide (DMSO) (cat.no. 041-07217), isoflurane (cat.no. 099-06571), poly(oxyethylene 20 sorbitan) monolaurate (Tween 20) (cat.no. 166-21213), 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) (cat.no. 013-16385), sodium dodecyl sulfate (SDS) (cat.no. 194-13985), and neutral buffered formaldehydes (37%) (cat.no. A16163) and (10%) (cat.no. 060-01667) were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). Fetal bovine serum (FBS) (cat.no.10437), RNAlater (cat.no. AM7021), GlutaMAX (cat.no. 35050061), Lipofectamine RNAiMAX (cat.no. 13-778-075), Opti-MEM I reduced serum medium (cat.no. 31985062), and penicillin/streptomycin (cat.no. 15-140-122) were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Bovine serum albumin (BSA) (cat.no. 01281-97) and premix water-soluble tetrazolium salt cell proliferation assay system (WST-1) (cat.no. MK400) were obtained from Nacalai Tesque (Kyoto, Japan) and Takara Bio (Shiga, Japan), respectively. DynaMarker RNA High for Easy Electrophoresis (cat.no. DM170) was purchased from BioDynamics Laboratory (Tokyo, Japan). Supporting information (source, cat.no., and brand name) for each primary antibody was indicated to Table I. Goat anti-rabbit (cat.no. #7074) and anti-mouse (cat.no. SA00001-1) secondary antibodies conjugated with horseradish peroxidase (HRP) were obtained from Cell Signaling Technology (Danvers, MA, USA) and Proteintech (Rosemont, IL, USA), respectively. Recombinant human decorin (DCN or DCN-A, about 40 kDa) (cat.no. 143-DE) was obtained from R&D Systems/BioTechne (Minneapolis, MN, USA). Human colorectal cancer HT-29 (ATCC No. HTB-38) and HCT116 (ATCC No. CCL-247) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). All other chemicals and solvents were of high-grade quality.

Human cancer tissue. An experimental scheme is shown in Figure 2. A Japanese PDX (J-PDX) library was prepared at the National Cancer Center Hospital in Japan using a part of cancer tissue resected from a patient with CRC, who provided written informed consent and agreed to give the sample collection and the information transmission, as described in a previous study (37). The protocol was conducted according to the principles outlined in the Declaration of Helsinki and approved by the Institutional Review Board of National Cancer Center (identification code, 2015-123 and 2021-163; authorization, 2015 and 2021). The surgical specimen of the patient’s cancer tissue was conveyed to a suitable facility for pathological and multi-omics analyses, and to establish PDX-derived tissue. The primary tissue represented as F₀ was characterized by race, age, sex, location of sample collection, clinical characteristics of the disease, pathological type, TNM status, clinical stage, and prior treatment history (chemotherapy, radiotherapy and immunotherapy).

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Figure 2.

Experimental scheme. In the first step, an establishment of Japanese patient-derived xenograft (J-PDX) tumor was conducted with analyses of pathological finding in primary tumor and gene alteration of a Trans generation 3 (TG₃) PDX tumor. In the second step, a preclinical trial of fucoxanthin (Fx) administration was performed using a PDX murine model transplanted a colorectal cancer J-PDX tumor (CRC-PDX). The anticancer effects of Fx were assessed by body weight and tumor size changes, pathological characterization, proteome profiles using LC-MS/MS, and protein expressions and activations using western blot. In last step, the molecular mechanisms suggesting the anticancer effects of Fx in the mice were confirmed using human colorectal cancers cells treated with fucoxanthinol that was a main metabolite for dietary Fx. The effects were evaluated by cell viability, protein treatment and gene knockdown assays, and protein expressions and activations using western blot.

Establishment of PDX tumor from human colorectal cancer tissue. The Trans Generation 3 (TG₃) PDX tumor tissue was prepared using the F₀ primary CRC tissue from the patient described in the Section Human cancer tissue in the Materials and Methods. Six-week-old female NOD.Cg-Prkdc^scid Il2rg^tm1Sug/ShiJic (NOG) mice were obtained from In-Vivo Science (Kanagawa, Japan) and acclimated for 1 week under controlled temperature, humidity and light/dark cycle (12 h light/dark). Standard solid chow and tap water were given ad libitum. The F₀ CRC tissue was cut into 2 mm³ pieces and implanted subcutaneously in the right flank of the mice (7-week-old) (designated hereafter as CRC-PDX mice) using a 13G transplantation needle (Natsume Seisakusyo, Tokyo, Japan). Estimated tumor volumes in the mice were measured weekly. The estimated tumor volume (mm³) was expressed as the formula: a (mm) × b² (mm)/2 (a, long range; b, short range). After the tumor volume was reached the range of 200 to 2,000 mm³, it was excised, cut into 2 mm³ pieces, and transferred to another NOG mouse using the same protocol (TG₁). TG₃ CRC-PDX tumors were prepared using the same protocol and stored in a J-PDX library in Japan (37). To analyze genetic variations in the tissue, the DNA and RNA from the TG₃ tissue were extracted and purified using the KingFisher Cell and Tissue DNA kit (cat.no. 97030196) (Thermo Fisher Scientific, Waltham, MA, USA) and MagMAX mirVana Total RNA Isolation kit (cat.no. A27828) (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Genetic profiles of the tissue for 161 cancer-related genes were analyzed using a next generation sequencing panel with Oncomine Comprehensive Assay v3 and the Ion Torrent Genexus System (software version 6.6) (cat.no. A46296) (Thermo Fisher Scientific) was performed in the tissue. Animal experiments were performed according to the guidelines of the Institute for Laboratory Animal Research, National Cancer Center Research Institute (identification codes, T17-020, T17-073 and T19-008; authorization, 2017 and 2021).

Animal experiments of Fx administration. The experimental design is shown in Figure 2. The TG₃ CRC-PDX tumor tissue was provided by the National Cancer Center Research Institute (Tokyo, Japan). A 4-weeks-old female CB17.Cg-Prkdcscid Lystbg-J/CrlCrlj (SCID-Beige) mouse was purchased from Oriental Yeast (Tokyo, Japan) and acclimated for one week under controlled temperature, humidity, and 12 h light/dark cycle. Standard solid food (Grade: MF, Oriental Yeast Co. Ltd.) and tap water were given ad libitum. Two pieces of the tumors (size, 2 mm³) were implanted subcutaneously into the right flank of the mouse (designated hereafter as TG₄-CRC-PDX) using a 13G transplantation needle (Natsume Seisakusyo, Tokyo, Japan). The mouse was observed twice a week for clinical signs and estimated tumor volume. After three months, the mouse was sacrificed under 2% isoflurane anesthesia (air, 2.0 l/min), and the tumor (TG₄ CRC-PDX) was resected and cut into many pieces of 2 mm³ size tumors under HBSS (4°C) to generate TG₅ CRC-PDX mice.

Female SCID-Beige mice (4-weeks-old) were purchased from Oriental Yeast, randomly assigned to three groups (3-4 mice/cage, 6-12 mice per group), and maintained in similar temperature, humidity, and 12 h light/dark cycle conditions. Standard solid MF and tap water were given ad libitum. After a week of acclimation, two pieces of the TG₄ CRC-PDX tumors (2 mm³) were implanted subcutaneously in the right flank of the mice (5-week-old) (designated as TG₅ CRC-PDX mice) using a 13G transplantation needle on day 0. Fx powder was mixed with standard MF powder (Grade: MF, Oriental Yeast Co. Ltd.) at a concentration of 0.3% Fx (3.0 mg Fx/g MF powder, Fx-high) and 0.1% Fx (1.0 mg Fx/g MF powder, Fx-low). The control diet contained only MF powder (control diet). Groups 1 and 2 mice were administered Fx-high (n=11) and Fx-low (n=6) diets, respectively, ad libitum for 20 days from the day 0, and then implanted with TG₄ PDX tumors until sacrificed. Mice in Group 3 mice were given control diet (n=12) ad libitum during the same period. The animals were inspected routinely for clinical signs, mortality, dietary intake amount, body weight, and estimated tumor volume. The estimated tumor volume (mm³) was expressed as the formula: a (mm) × b² (mm)/2 (a, long range; b, short range). Mice were sacrificed under 2% isoflurane anesthesia (air, 2.0 l/min). The entire tumor from each mouse was excised, washed with cold phosphate-buffered saline (PBS), and cut into several pieces for pathological, proteome, and western blot analyses. For histopathological examination, one piece of the tumor was fixed in 10% formalin for 2 days and prepared the hematoxylin and eosin (HE)-stained sections. Histopathological findings were examined to determine the degree of the tumor lesions in the HE sections prepared by Morphotechnology (Hokkaido, Japan). Histopathological findings of the tumor lesions were evaluated by an expert pathologist. Other tumor pieces were immediately permeated with RNAlater (500 μl) overnight at 4°C, added to equal volume of cold-PBS and supernatants were then removed by centrifugation. Next, the tumor tissues were frozen at −80°C until proteome and western blot analyses. The experiments were performed in compliances with the Institutional Ethics Review Boards of the National Cancer Center Research Institute (identification code, 2021-163; authorization, 2021) and of the Health Sciences University of Hokkaido (identification code, 21P003; authorization, 2021), according to the Institutional Guidelines for Animal Care and Use in the Health Sciences University of Hokkaido (identification code, 22-003; authorization, 2021).

Proteome analysis. Whole tumor tissues from Groups 1 and 3 were taken, sonicated in SDS lysis buffer (50 mM Tris-HCl, 1%SDS, pH 6.8), and denatured. Protein concentrations in tumor or cell samples were determined photometrically using the Bradford assay (Bio-Rad, Hercules, CA, USA) and the proteins were then denatured at 95°C for 5 min. Total proteins from Group 1 and 3 were prepared as a mixed sample (total 75 μg) with equivalent amount of proteins taken from five mice. The samples were digested with trypsin and subjected to LC-MS/MS-based proteomics analysis using a quantitative LC-MS/MS system (UltiMate 3000 RSLCnano LC System) equipped with a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Carlsbad, CA, USA) at the Kazusa DNA Research Institute (Chiba, Japan) (Analysis Registration number: KK1657). Human tissue proteins were identified using the Human UniProtKB/Swiss-Prot database (downloaded, 26, Nov, 2021) and the Prosit spectral library (https://www.proteomicsdb.org/prosit/). The proteins were filtered by the cutoffs related to peptide false discovery rate (FDR) (≤1.0%) and protein FDR (≤1.0%). The protein expressions in the mice of group 1 compared with those of group 3 was analyzed using Scaffold DIA software (Promega Software, Portland, OR, USA). Among the identified up-regulated and down-regulated proteins, the proteins suggested to contribute to the anticancer effects of Fx was selected based on a comprehensive literature review regarding cancer development and suppression on each molecule.

Cell viability assay by FxOH treatment. HT-29 and HCT116 cells were seeded at a density of 5×10⁴ cells/ml into a 24-well plate in 10%FBS/RPMI (2.5×10⁴ cells/well) and allowed to adhere to the wells for 1 day. The cells were exposed to 10%FBS/RPMI with FxOH (final concentrations, 5 and 20 μmol/l) and further incubated for 1 day. Control cells were treated with the vehicle (DMSO) alone. Cell viability was measured using the WST-1 assay. Absorbance at 450 nm was determined using an ELISA plate reader (TECAN Japan, Tokyo, Japan). To measure the expression and activation of these cells, they were seeded at a density of 5×10⁴ cells/ml into a 10-cm dish in 10%FBS/RPMI (50×10⁴ cells/well) and allowed to adhere for 1 day. The cells were exposed to 10%FBS/RPMI with FxOH (final concentrations, 5 and 20 μmol/l) and incubated for 1 day. Control cells were treated with the vehicle (DMSO) alone. Protein expression and activation of the cells were measured using western blot analysis as described in Section Western blot analysis of the Materials and Methods.

Western blot analysis. Whole tumor tissues sampled from mice in Groups 1 and 3 were collected and sonicated in SDS lysis buffer. The HT-29 and HCT116 cells with gene knockdown or FxOH treatment were harvested, washed twice with PBS, and sonicated in lysis buffer. Protein concentrations in the tumors or cells were determined photometrically using the Bradford assay, and the proteins then denatured at 95°C for 5 min. The tumor or cell proteins (10 μg each) were separated on a 10% gel using SDS electrophoresis. The gels were immersed in a transfer buffer, and transferred onto Hybond PVDF membranes (cat.no. 10600058) (Amersham Bioscience, Little Chalfont, UK). The PVDF membranes were washed with Tris-buffered saline containing 0.1% Tween 20 (TBS-T), incubated with a blocking buffer of TBS-T containing 1 w/v% BSA (1% BSA/TBS-T) at room temperature for 1 h, and probed with each primary antibody (diluted 1:1,000) in 1% BSA/TBS-T overnight at 4 °C. The membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies in TBS-T at room temperature for 1 h. Protein bands were visualized using Immobilon Western chemiluminescence HRP reagent (cat.no. WBKLS0500) (Millipore, Billerica, MA, USA). The image of each protein band was normalized to that of the loading control (β-actin) and quantified by comparing with that of control group using FIJI Image J2 software (version 2.9.0/1.53t) (NIH, http://rsb.info.nih.gov/ij/).

Cell viability assay in DCN-treated cells. HCT116 cells were seeded at a density of 5×10⁴ cells/ml into a 96-well plate in 10%FBS/RPMI (5×10³ cells/well) and allowed to adhere for 1 day. The cells were incubated in 10%FBS/RPMI with recombinant human DCN (final concentrations, 10 and 50 μg/ml) and incubated for 1 day. Control cells were treated with 10%FBS/RPMI only. Cell viability was measured using the WST-1 assay, as described in Section Cell viability assay by FxOH treatment of the Materials and Methods.

Gene knockdown. Twenty-seven mer of dicer-substrate short interfering RNAs (dsiRNAs) targeting the coding sequences of DSN1 mRNA in Homo sapiens were prepared using Integrated DNA Technologies (Coralville, IA, USA). The designed DSN1 dsiRNAs were as follows: DSN1 dsRNA duplex-1, 5′-GUA AAG GAA UGA UAC UAA UUU UCT A-3′ and duplex-2, 5′-GCA GGA GGC UAA AGA GAU AUU GUC C-3′; and negative control (NC), 5′-GUG UUC UAC ACC AUU ACU CAA UUC UUA-3′. DsiRNA/Lipofectamine RNAiMAX/Opti-MEM I complex solution was prepared according to the manufacturer’s instructions. HCT116 cells were routinely passaged in RPMI-1640 medium containing 10% heat-inactivated FBS and 1% GlutaMAX™ without antibiotics [10%FBS/RPMI (−)]. The cells were seeded into 6-well plates containing 10%FBS/RPMI (−) at a density of 10×10⁴ cells/ml (20×10⁴ cells/well). The cells were allowed to adhere for 1 day. The dsiRNA or NC/Lipofectamine RNAiMAX complex solution was added to the culture medium (final concentration of dsiRNA or NC: 10 nmol/l) for 1 day and boosted with the new complex solution for 1 day. Some of the DSN1 knockdown HCT116 cells were seeded into a 24-well plate at a density of 5×10⁴ cells/mL (2.5×10⁴ cells/well), and the plate was incubated for 1 day. Cell viability was measured using the WST-1 assay as described in Section Cell viability assay by FxOH treatment. The remaining DSN1 knockdown HCT116 cells were subjected to western blotting to analyze protein expression, as described in Section Western blot analysis.

Statistical analysis. Results are expressed as the mean±standard error (SE). Statistical differences in tumor incidence between Groups 1 and 3 were determined using Fisher’s exact probability test. The Shapiro-Wilk test was used to evaluate the normality of the distribution of the results. Differences between two groups with and without normal distribution were assessed using the post hoc Student’s t-test (parametric) or the Mann-Whitney U-test (nonparametric), respectively. In addition, differences among Groups 1-3 with and without normal distribution were analyzed using one-way ANOVA with post hoc Tukey test (parametric) or Kruskal-Wallis test with post hoc Dunn-Bonferroni test (nonparametric), respectively. Statistical analyses were performed using the SPSS Statistics version 25 (IBM Co. Ltd., Armonk, NY, USA). Differences were represented as statistically significant at *p<0.05, **p<0.01, and exact p-values.