The mouse colon cancer CT26 cell line was provided by Professor Isaiah J. Fidler (MD Anderson Cancer Center, Houston, TX, USA). The mouse rectal carcinoma CMT93 cell line and the mouse lung cancer LL2 cell line were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). Cells were cultured in Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in a humidified condition with 5% CO2 atmosphere.

Cells in 6 dishes (1×10⁵ in 3.5 cm-diameter dishes) were treated with LA (50 µg/ml) or EA (20 µg/ml) and concurrently with 5-FU (1 µg/ml) at 37°C for 24 h. For sequential treatment, cells were seeded in 6 dishes (1×10⁵ in 3.5 cm-diameter dishes) and treated with LA (50 µg/ml) or EA (20 µg/ml) at 37°C for 24 h prior to 5-FU treatment (1 µg/ml at 37°C for 24 h).

The cell viabilities were assessed using a 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) Assay kit (Promega Corporation, Madison, WI, USA). Following treatment with LA or EA and/or 5-FU, MTS solution was added to each well for 2 h at 37°C in 5% CO₂. The absorbance at 490 nm was recorded using a microplate reader.

Male BALB/c mice (4-weeks-old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were maintained in the pathogen-free animal facility with a 12/12 h light/dark cycle in a temperature (22°C) and humidity-controlled environment, according to institutional guidelines approved by the Committee for Animal Experimentation of Nara Medical University (Kashihara, Japan; approval no. 9559), in accordance with the current regulations and standards of the Japanese Ministry of Health, Labor and Welfare.

To establish a subcutaneous tumor model, CT26 cancer cells (1×10⁷) were inoculated into the scapular subcutaneous tissue of BALB/c mice. Mice were euthanized and observed using immunohistochemistry 4 weeks following inoculation. 5-FU (10 mg/kg; Wako Pure Chemical Industries, Ltd.) was injected intraperitoneally twice a week for 2 weeks following inoculation. LA (Sigma-Aldrich; Merck KGaA) or EA (Wako Pure Chemical Industries, Ltd.) were administrated at 1.0 and 0.1% w/w, respectively, through the daily supplementation of a standard CE-2 diet (CLEA Japan, Inc., Tokyo, Japan). The mean intake of LA and EA was 4.2±0.78 and 0.31±0.07 mg/day, respectively. A total of five mice were used for per treatment group. The mean weight of the mice was 21.5±1.4 g at the start of treatment.

Total RNA (1 µg) was extracted using an RNeasy extraction kit (Qiagen, Inc., Valencia, CA, USA). RNA was extracted from 1×10⁷ CT26 cells and eluted in 50 µl RNase-free water. cDNA was synthesized using ReverTra Ace RT-qPCR kit (Toyobo, Tokyo, Japan). Quantification of PCR products was performed with QuantiTect Primer Assays using QuantiFast, QuantiTect, Rotor-Gene, and FastLane kit for SYBR-Green-based detection (Qiagen, Inc.), according to the manufacturer's protocol. mRNA quantification was performed according to the 2^−ΔΔcq method (19). The number of replicates was 30 cycles. The primer sets used for amplification were as follows: Mouse CD133 (prominin 1; accession no., BC028286.1) forward, 5′-GAAAAGTTGCTCTGCGAACC-3′ and reverse, 5′-TCTCAAGCTGAAAAGCAGCA-3′; mouse nucleostemin (NS; accession no. AY181025.1) forward, 5′-CAGGATGCTGACGATCAAGA-3′ and reverse, 5′-TTGATTGCTCAGGTGACAGC-3′; and mouse β-actin (accession no. NM_007393.4) forward, 5′-AGCCATGTACGTAGCCATCC-3′ and reverse 5′-CTCTCAGCTGTGGTGGTGAA-3′. The primers were synthesized by Sigma Genosys (Sigma-Aldrich; Merck KGaA). The PCR conditions were set according to the manufacturer's protocol. The number of replicates was 30 cycles. PCR products were electrophoresed on a 2% agarose gel and visualized using ethidium bromide.

Consecutive 4 µm tissue sections were immunohistochemically stained using the immunoperoxidase technique, as described previously (8). Mouse CD133 antibody (AG13328; ProteinTech Group, Inc., Chicago, IL, USA) and mouse nucleostemin antibody (sc-166430; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used at a concentration of 0.5 µg/ml at 37°C for 2 h. Color development was performed using 3–3′-diaminobenzidine (Dako; Agilent Technologies, Inc.), and the specimens were counterstained with Mayer's hematoxylin (Sigma-Aldrich; Merck KGaA) to visualize the nuclei. Following immunostaining, all slides were assessed to measure the number of positively stained nuclei using an all-in-one microscope (BZ-X700; Keyence, Osaka, Japan).

ALDH activity was measured using the ALDEFLUOR kit (Veritas Technologies LLC, Tokyo, Japan) according to the manufacturer's protocol. The activity was normalized to the negative control, diethylaminobenzaldehyde.

Statistical significance was calculated using the two-tailed Fisher's exact test, χ² test and unpaired Student's t-test, with the assumption of Gaussian distributions, by Kolmogorov and Smirnov tests Statistical analysis was performed using InStat software (GraphPad Software, Inc., La Jolla, CA, USA). The data are presented as the mean ± standard deviation. P<0.05 (two-sided) was considered to indicate a statistically significant difference.

Three cancer cell lines, CT26 (colon cancer), LL2 (lung cancer) and CMT93 (rectal cancer), were concurrently treated with either LA or EA and 5-FU, or with LA, EA or 5-FU alone. Treatment with LA alone decreased cell viability in all 3 cell lines (Fig. 1). LA also augmented the tumor cell inhibition observed following 5-FU treatment in all 3 cell lines. In contrast, EA alone did not significantly suppress tumor viability in any of the 3 cell lines (Fig. 1). In addition, concurrent treatment of EA with 5-FU did not recover or enhance 5-FU-induced growth inhibition in the three cell lines.

Sequential treatment with LA or EA followed by 5-FU was then examined (Fig. 1). Concurrent treatment of 5-FU and LA revealed a more pronounced decrease of cell viability in the 3 cell lines. In contrast, temporal treatment of LA to 5-FU demonstrated no additional decrease of viability compared with that of 5-FU treatment alone. (Fig. 1). In addition, EA pretreatment reversed the effect of 5-FU, increasing the viability of LL2 and CMT93 cells (Fig. 1).

Previous studies have demonstrated that the resistance of cancer cells to anticancer drugs is associated with CSCs (20,21). Therefore, alterations of stemness in LA- or EA-treated cancer cells were examined (Fig. 2). Expression of CD133 and NS was examined in cells treated with 5-FU alone, or concurrently with LA or EA (Fig. 2A-D). The expression of CD133 and NS was increased in cells treated with LA or EA when compared with untreated cells. Although expression of NS appears to be higher in cells treated with 5-FU and LA concurrently compared with cells treated with LA alone in CT26 and CMT93 cells, the expression of CD133 and NS was lower in cells treated concurrently with 5-FU and either LA or EA compared with cells treated with LA or EA alone. However expression was increased in cells treated concurrently with 5-FU and either LA or EA compared with cells treated with 5-FU alone. Activity of ALDH, a stem cell-associated enzyme, was increased in cells treated with LA or EA compared with in untreated cells (Fig. 2E). ALDH activity was higher in cells treated concurrently with 5-FU and either LA or EA compared with cells treated with 5-FU alone (Fig. 2E).

Finally, the effect of oral intake of LA or EA on the antitumor effects of 5-FU was examined in BALB/c mice inoculated with CT26 cells (Fig. 3). Treatment with 5-FU inhibited the growth of CT26-derived tumors, and this was abrogated by concurrent treatment with either LA or EA (Fig. 3A and B). The expression of NS in CT26-derived tumors was examined by immunohistochemistry (Fig. 3C and D). NS-positive CSCs were more abundant in tumors treated with 5-FU and either LA or EA compared with tumors treated with 5-FU alone.