The test sample, which consisted of multi-lamellar vesicles, was prepared by hydrating dietary soy phosphatidylcholine with an aqueous solution containing bLF. Briefly, 10.2% (w/w) soy phosphatidylcholine solubilised in glycerine and 19.8% (w/w) bLF were mixed at a ratio of 1.00:1.54, and emulsified (R&D Division, Sunstar Inc., Osaka, Japan). The emulsified solution was then liposomalised using a high-pressure homogenizer. The diameter of the liposomes was determined using a particle size analyser, and the mean diameter was ~70 nm. The control solution (glycerine) was prepared in a similar manner.

A total of 36 male 5-week-old F344 rats (weighing 70–90 g) were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). The animals were cared for in compliance with the principles and guidelines of the Ethical Committee for Animal Care of the Prefectural University of Hiroshima (Hiroshima, Japan) and the Prefectural University of Hiroshima Animal Ethics Committee in accordance with the Japanese National Law on Animal Care and Use. The Ethical Committee for Animal Care of the Prefectural University of Hiroshima (Hiroshima, Japan) approved the experiments undertaken. The rats were housed in an air-conditioned room at the Laboratory Animal Research Centre of the Prefectural University of Hiroshima, Japan. The room provided a 12-h light/dark cycle, a controlled ambient temperature of 23±2°C and a humidity of 50±10%. The rats had free access to drinking water and were fed a moderate fat basal diet (Oriental Yeast Co., Ltd., Tokyo, Japan).

DMH (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in 0.9% NaCl solution, and the pH was adjusted to 6.5 using NaHCO₃. As indicated in Fig. 1, the drinking water of all 36 rats was supplemented with 1% DSS for one week (week-1), starting at 5 weeks of age. Upon reaching 6 weeks of age (week 0), the rats were randomly allocated into three groups of 12 rats each. Each group received water (control), 500 or 1,000 mg/kg/day LbLF from week 0–25. All rats were injected with DMH (20 mg/kg body weight) once per week for 8 consecutive weeks (weeks 0–8). The body weights and LbLF dilution intake of the rats were recorded every week to determine the correct dose of LbLF. All rats were sacrificed by anaesthesia (45 mg/kg body weight of sodium pentobarbital). 25 weeks following the commencement of DMH administration to allow for tissue examination.

After the rats were sacrificed, the colons were quickly removed, flushed with saline solution and opened longitudinally from the cecum to the anus. The colons were placed on a paper towel and fixed in 10% buffered formalin for 24 h at room temperature. They were stained with 0.5% methylene blue for 15–30 min at room temperature and then placed on a glass slide, with the luminal side facing up. The stained colons were observed under a light microscope at a magnification of ×20–30. The number of ACFs was recorded.

After the ACFs had been recorded, the 10% buffered formalin-fixed colons were embedded in paraffin, sectioned at a thickness of 4 µm, stained with haematoxylin and eosin (H&E) and examined under a light microscope (magnification, ×100; Olympus Corporation, Tokyo, Japan). The counted tumours were classified into two types: Adenomas (including mild, moderate or severe dysplasia categorisations) and adenocarcinomas (including well, moderately or poorly differentiated tubular adenocarcinoma, signet ring cell or mucinous carcinoma categorisations).

RKO and RCN-9 human CRC cells, provided by the Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan), were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂. For the growth assay, 5×10³ cells were plated onto 24-well plates (Falcon; Corning Incorporated, Corning, NY, USA) and cultured in DMEM with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂. Subsequently, trypsinized cells were counted at 0, 1, 2, and 3 days using a Cell Counter (Coulter Z1, Coulter Co., Hialeah, FL, USA).

RKO and RCN-9 cells were seeded into 60-mm culture dishes (5×10⁵ cells/well) and cultured in DMEM supplemented with 10% FBS as aforementioned. The cells were then cultured in fresh DMEM supplemented with 10% FBS with or without lipopolysaccharide (A.a-LPS; 100 ng/ml), LPS from Aggregatibactor Actinomycetemcomitance (ATCC29522 strain; A.a.-LPS) was provided by Professor Tatsuji Nishihara of the Kyusyu Dental College (Kyusyu, Japan). The cultured cells were harvested 0, 2, 4 and 6 h after LPS stimulation. The expression level of TNFα mRNA was determined. Furthermore, following a 4-h treatment with LbLF (1, 10 or 100 µg/ml) or a control treatment (no LbLF) at 37°C in a humidified atmosphere of 5% CO₂, the culture plates were briefly washed twice with PBS and the cells were incubated with A.a.-LPS (100 ng/ml) with or without a 2-h pre-treatment. The cultured cells were collected and expression level of TNFα mRNA was evaluated.

The total RNA from the harvested fully confluent cells was isolated using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). An ultramicro spectrophotometer (ND-2000: NanoDrop 2000; Thermo Scientific, Inc.,) was used to detect the concentration of RNA. The cDNA was synthesized from 1 µg of total RNA was produced using a transcriptase PCR kit (ReverTra Dash; Toyobo Biochemicals, Osaka, Japan) according to the manufacturer's protocol. The following primers were used: Human tumour necrosis factor α (TNFα): 5′-GCCCATGTTGTAGCAAACC-3′, forward and 5′-CCAAAGTAGACCTGCCCAGA-3′, reverse (product size, 239 bp); human GAPDH: 5′-TCCACCACCCTGTTGCTGTA-3′, forward and 5′-ACCACAGTCCATGCCATCAC-3′, reverse. Aliquots of total cDNA (0.05 µg) were amplified with 1.25 U rTaq-DNA Polymerase (Qiagen GmbH) in a thermal cycler (MyCyler; Bio-Rad Laboratories, Inc., Hercules, CA). The PCR protocol for all primers consisted of 30 cycles of the following: An initial 30 sec of denaturation at 94°C, annealing for 30 sec at 60°C and extension for 1 min at 72°C. The amplification reaction products were resolved on 1.2% agarose/Tris-acetate-EDTA gels (Nacalai Tesque, Inc., Kyoto, Japan). The final PCR products were separated by electrophoresis on 1.2% agarose gels at 100 mV for 20–40 min and visualised using ethidium bromide.

The Statcel software package (KaleidaGraph version 4.1, Reading, PA, USA), was used for statistical analysis. The data in the current study are presented as means ± standard error. The significance differences between control group and LbLF group in the in vivo and in vitro experiments were evaluated using unpaired Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.

Fig. 2 presents the body weight of rats from the three groups measured during the experiment. Upon reaching 6 weeks of age (defined as week 0), the rats were randomly allocated into 3 groups of 12 rats. Each group received water (control), 500 or 1,000 mg/kg/day LbLF from week 0–25. The body weights of the rats were recorded every week. Groups of rats were compared for body weight from week 0–25. Body weight was not observed to significantly differ between any of the groups of rats at any point of the experiment.

The inhibitory influence of LbLF on the growth and development of DMH-induced total number of colonic ACF in rats is presented in Fig. 3 and Table I. ACF expression in the colons of rats treated with DMH-DSS was analysed using 0.5% methylene blue stain (Fig. 3A). Rats treated with DMH exhibited a 100% incidence of ACF. Furthermore, the mean number of ACF was significantly lower in the 500 and 1,000 mg/kg/day LbLF groups, as compared with in the control group (Fig. 3B; Table I; P<0.01). Arrows indicate the ACFs in the colon (Fig. 3A).

The colon tissue sections obtained from the rats were subjected to histopathological investigation, and the colon epithelial lesions were classified as adenomas or adenocarcinomas (Fig. 4). Table II indicates that rats from the 500 and 1,000 mg/kg/day LbLF groups harboured significantly fewer colon adenomas than rats from the control group (500 mg/kg/day, P<0.05; 1,000 mg/kg/day, P<0.01). The mean ± standard deviation (SD) of adenomas identified in the control, 500 and 1,000 mg/kg/day LbLF groups were 33.70±12.91, 23.60±7.86 and 17.10±5.81, respectively. Table II also demonstrates that rats from the 500 and 1,000 mg/kg/day LbLF groups harboured significantly fewer colon adenomas with moderate atypia than rats from the control group (500 mg/kg/day, P<0.05; 1,000 mg/kg/day, P<0.01). Furthermore, rats from the 1,000 mg/kg/day LbLF group harboured significantly fewer colon adenomas with mild or severe atypia than rats from the control group (mild, P<0.01; severe, P<0.05; Table II).

Table III indicates that the 500 and 1,000 mg/kg/day LbLF groups harboured significantly fewer colon adenocarcinomas than rats from the control group (P<0.05 and P<0.01, respectively). In addition, the mean ± SD for adenocarcinomas identified in the control, 500 and 1,000 mg/kg/day LbLF groups were 1.25±0.62, 0.67±0.65 and 0.50±0.67, respectively. Table III also reveals that rats from the 1,000 mg/kg/day LbLF group harboured significantly fewer differentiated carcinomas (well and moderate adenocarcinoma; P<0.05), but not undifferentiated carcinomas (poorly, mucinous, signet ring cell carcinoma), compared with rats from the control group. Thus, LbLF for low malignant grade carcinoma inhibition effect is marked.

The cell growth of LbLF-treated RKO and RCN-9 cells was examined. Compared with the control (no treatment with LbLF), it was observed that treatment with ≥10 µg/ml LbLF significantly inhibited the growth of RKO and RCN-9 cells (Fig. 5; P<0.01).

The RKO cells were harvested 0, 2, 4 and 6 h following LPS stimulation. The expression levels of TNFα mRNA were determined. LPS was observed to upregulate the expression of TNF-α mRNA in RKO cells after 2 h (Fig. 6A). Subsequently, treatment with LbLF (1, 10 or 100 µg/ml) was demonstrated to inhibit TNF-α mRNA expression in CRC cells (RKO and RCN-9 cells). Pre-treatment with a high concentration LbLF (10 or 100 µg/ml) blocked the LPS-induced upregulation of TNF-α mRNA in RKO and RCN-9 cells (Fig. 6B).