Tocotrienol suppresses colitis-associated cancer progression through TLR4 signaling in a mouse model of colorectal cancer

Abstract

This study aimed to evaluate the preventive efficacy of tocotrienol in inhibiting the nuclear factor-kappa B (NF-κB) mediated inflammation pathways in colorectal cancer. We utilized the azoxymethane (AOM) and dextran sulfate sodium salt (DSS) to induce colitis-associated colorectal cancer (CAC) mice model. In generating a CAC model, mice were intraperitoneally injected with AOM at a concentration of 10 mg/kg body weight. Seven days after the AOM injection, mice drinking water containing 3 % DSS for 1 week, followed by a 2-week period of regular water. This cycle of DSS treatment (1-week 3 % DSS+2-week water) was repeated for two additional cycles. Mice were randomly divided into five groups (n = 20/group), including Blank group, Model group, three different dosages tocotrienol groups (Low dose group [50 mg/kg], Medium dose group [75 mg/kg], and High dose group [100 mg/kg]). The protective effects of tocotrienol were assessed using histological, flow cytometry, western blot and mouse Luminex assay. Compared with the blank group, expressions of toll-like receptor 4 (TLR4), myeloid differentiation protein 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF-6), NF-κB, Interleukin (IL)-6 and tumor necrosis factor (TNF) -α were increased in model group, while IL-4 and IL-10 were decreased in model group (P<0.05). Tocotrienol prevented carcinogenesis and decreased the IL-6, TNF-α, MyD88, TLR4, TRAF-6 and NF-κB expression levels, compared with the model group (P<0.05). Compared with the model group, the expression of IL-10 was increased in medium dose group and high dose group (P<0.05). The protective effects of tocotrienol may be related to the inhibition of TLR4 /MyD88 /NF-κB mediated inflammatory signaling pathways. Therefore, the use of tocotrienol can improve the abnormal expression of cytokines in a mouse model of colorectal cancer and inhibit the occurrence and development of colorectal cancer.

Keywords: Annatto-tocotrienol; Colorectal cancer; Cytokines; TLR4 signaling.

Graphical abstract

Fig. 1

Effects of annatto-tocotrienol on the body weight and survival in mice. A. Changes of body weight in the mice model for 20 weeks, Repeated measures ANOVA. The number of mice is as follows: Blank group, n = 20; Model group, n = 15; Low dose group, n = 16; Medium dose group, n = 17; High dose group, n = 18. B Survival curves in mice, Survival analysis. The data are presented as the mean ± SD. *P<0.05 compared with the model group.

Fig. 2

Effects of annatto-tocotrienol on the organ coefficients in mice. (A) The organ coefficients of spleen in each group. (B) The organ coefficients of colon in each group. The data are presented as mean ± SD. Group comparisons were analyzed using ANOVA. Pairwise comparisons were conducted using the Bonferroni method. *P<0.05 compared with the model group; **P<0.01 compared with the model group.

Fig. 3

Pathological changes of spleen in mice. A) Blank group (400 × ). B) Model group (400 × ). C) Low dose group (400 × ). D) Medium dose group (400 × ). E) High dose group (400 × ). The blank group had a smooth tunica serosa. The red and white pulp structures of the spleen were clear, with the red pulp spleenic sinuses filled with red blood cells, and there was no increase or decrease in the number of splenic nodules in the white pulp. In the model group: the number of splenic nodules decreased, the structure of the nodules and the marginal zone were less clear, and the lymphocytes in the red pulp were sparse. In the low dose group, medium dose group and high dose group, the number of splenic nodules, the structure of the splenic nodules, the structure of the marginal zone, and the content of lymphocytes in the red pulp tended to improve or recover close to the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Pathological changes of colon in mice. A) Blank group (100 × ). B) Model group (100 × ). C) Low dose group (100 × ). D) Medium dose group (100 × ). E) High dose group (100 × ). E) Blank group (400 × ). F) Model group (400 × ). Under the microscope, the blank group showed that the intestinal glandular epithelial cells were arranged in a single layer of columnar cells with normal size and morphology. In the model group, the mice displayed adenomatous structures with pseudostratification, loss of cell polarity, inconsistent cell size and morphology, deepened nuclear staining, and other obvious atypical features, involving cases from the mucosal layer to the submucosal layer. The tumor formation rate in the model group was 100 %, in the tocotrienol high-dose group was 84 %; in the tocotrienol medium-dose group was 88 %; and in the tocotrienol low-dose group was 84 %.

Fig. 5

Effects of annatto-tocotrienol on the IL-6, TNF-α, and IL-10 levels in mice. The IL-6 (A), TNF-α (B), and IL-10 levels in the serum were measured in mice by flow cytometry (FACS). Group comparisons were analyzed using ANOVA. Pairwise comparisons were conducted using the Bonferroni method. The data are presented as mean ± SD. *P<0.05 compared with the blank group; ^**P<0.01 compared with the blank group.

Fig. 6

Effects of annatto-tocotrienol on the IL-4 and MIP-1β levels in mice. The IL-4 (A) and MIP-1β (B) levels in the serum were measured in mice by Luminex assay. The data are presented as mean ± SD. Group comparisons were analyzed using ANOVA. Pairwise comparisons were conducted using the Bonferroni method. *P<0.05 compared with the blank group; ^**P<0.01 compared with the blank group.

Fig. 7

MyD88 expression in the colon of mice treated with AOM/DSS or annatto-tocotrienol. MyD88 expression in mice colon in each group was determined by immunohistochemistry. Immunohistochemistry showed that MyD88 staining was mainly in the cytoplasm. A–E MyD88 expression in the blank (A, 400 × ), model (B, 400 × ), low dose group (C, 400 × ), medium dose group (D, 400 × ) and high dose group (E, 400 × ), respectively.

Fig. 8

NF-κB expression in the colon of mice treated with AOM/DSS or annatto-tocotrienol. NF-κB expression in mice colon in each group was determined by immunohistochemistry. Immunohistochemistry showed that NF-κB staining was mainly in the nucleus. A–E NF-κB expression in the blank (A, 400 × ), model (B, 400 × ), low dose group (C, 400 × ), medium dose group (D, 400 × ) and high dose group (E, 400 × ), respectively.

Fig. 9

MyD88 and NF-κB levels in the mice colon in mice. The total protein was extracted from mouse colon each group. The MyD88 and NF-κB levels were measured by IHC. The data are presented as mean ± SD. Group comparisons were analyzed using ANOVA. Pairwise comparisons were conducted using the Bonferroni method. *P<0.05 compared with the Model group; ^**P<0.01 compared with the Model group.

Fig. 10

The protein expression of TLR4, MyD88, TRAF6, NF-κB and β-actin in the mice colon in mice. The total protein was extracted from mice colon each group. The tissue were examined by western blotting as described in the Materials and Methods section. The data are presented as mean ± SD. Representative images of at least three independent experiments are shown. Group comparisons were analyzed using ANOVA. Pairwise comparisons were conducted using the Bonferroni method. *P<0.05 compared with the model group; **P<0.01 compared with the model group.