Thymoquinone subdues tumor growth and potentiates the chemopreventive effect of 5-fluorouracil on the early stages of colorectal carcinogenesis in rats

Abstract

Colorectal cancer (CRC) is one of the most prevalent cancers and has a high mortality rate. Insensitivity and the limited therapeutic efficacy of its standard chemotherapeutic drug, 5-fluorouracil (5-FU), represents an important challenge in CRC treatment. The robust antitumor properties of thymoquinone (TQ), the main bioactive constituent of Nigella sativa, have recently been demonstrated on different cancers. We investigated whether TQ could potentiate the chemopreventive effect of 5-FU to eradicate the early stages of CRC and elucidated its underlying mechanisms. An intermediate-term (15 weeks) model of colorectal tumorigenesis was induced in male Wistar rats by azoxymethane (AOM), and the animals were randomly and equally divided into five groups: control, AOM, AOM/5-FU, AOM/TQ, and AOM/5-FU/TQ. TQ (35 mg/kg/d; 3 d/wk) was given during the seventh and 15th weeks post-AOM injection, while 5-FU was given during the ninth and tenth weeks (12 mg/kg/d for 4 days; then 6 mg/kg every other day for another four doses). At week 15, the resected colons were subjected to macroscopic, histopathological, molecular, and immunohistochemical examinations. Interestingly, 5-FU/TQ combination therapy resulted in a more significant reduction on AOM-induced colorectal tumors and large aberrant crypts foci than treatment with the individual drugs. Mechanistically, 5-FU and TQ remarkably cooperated to repress the expression of procancerous Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, and TBRAS and upregulate the expression of anti-tumorigenesis DKK-1, CDNK-1A, TGF-β1, TGF-βRII, Smad4, and GPx. Overall, our findings present the first report describing the in vivo enhancement effect of combined TQ and 5-FU against early stages of CRC; however, further studies are required to determine the value of this combination therapy in an advanced long-term model of CRC and also to realize its clinical potential.

Keywords: 5-fluorouracil; colorectal tumors; combination therapy; rats; thymoquinone.

Figure 1

Representative photos of macroscopic and microscopic appearance of colorectal mucosa. Notes: Shown are the pictures of normal control group (A), AOM group (B), AOM/5-FU group (C), AOM/TQ group (D), and AOM/5-FU/TQ group (E). The colorectal mucosa of the different groups was examined by naked eyes after formaline fixation (i); dissecting microscopy at magnifications ×100 following staining with 0.2% methylene blue (ii), and light microscopy at magnifications ×100 and ×200 following staining with H&E (iii). Black arrowheads: gross tumors observed by naked eye; yellow arrow: microtumor under dissecting microscopy; red arrows: large aberrant crypts foci (ACF) (containing four or more aberrant crypts). Scale bars =8 μm. Abbreviations: AOM, azoxymethane; 5-FU, 5-fluorouracil; TQ, thymoquinone; H&E, hematoxylin and eosin.

Figure 2

Findings of quantitative real-time polymerase chain reaction. Notes: The figures show the modulatory effects of thymoquinone (TQ), 5-fluorouracil (5-FU), and their combination therapy on the relative mRNA expression of (A) Wnt, (B) β-catenin, (C) NF-κB, (D) COX-2, (E) DKK-1, (F) CDKN-1A, (G) TGF-β1, and (H) Smad4 genes in azoxymethane (AOM)-induced rat colorectal tumors. ^aP<0.01 vs normal controls; ^bP<0.05 vs normal controls; ^cP<0.05 vs AOM group; ^dP<0.05 vs AOM/5-FU group; ^eP<0.05 vs AOM/TQ group; and ^fP<0.01 vs AOM group.

Figure 3

Enzyme-linked immunosorbent assay findings. Notes: Shown are the modulatory effects of thymoquinone (TQ), 5-fluorouracil (5-FU), and their combination therapy on the protein concentrations of (A) β-catenin, (B) TGF-β1, (C) COX-2, (D) VEGF, (E) GPx, and (F) TBARS in the colorectal tissues of azoxymethane (AOM)-induced colorectal tumors in rats. Data are represented as mean ± SD. ^aP<0.01 vs normal controls; ^bP<0.05 vs normal controls; ^cP<0.05 vs AOM group; ^dP<0.05 vs AOM/5-FU group; ^eP<0.05 vs AOM/TQ group; ^fP<0.01 vs AOM group.

Figure 4

Representative photos of immunohistochemical findings. Notes: The figures show the modulatory effects of thymoquinone (TQ), 5-fluorouracil (5-FU), and their combination therapy on the protein concentrations of tissue growth factor-β1 (TGF-β1; left column), TGF-β receptor II (TGF-βRII; middle column), and Smad4 (right column) in the colorectal tissues of normal control (A, F, and K), azoxymethane (AOM; B, G, and L), AOM treated with 5-fluorouracil (AOM + 5-FU; C, H, and M), AOM treated with thymoquinone (AOM + TQ; D, I, and N), and AOM treated with 5-FU/TQ combination therapy (E, J, and O) groups (×200 magnification and the scale bars are 8 μm).

Figure 5

Representative photos of immunohistochemical findings. Notes: The pictures show the regulatory effects of thymoquinone (TQ), 5-fluorouracil (5-FU), and their combination therapy on the protein concentrations of β-catenin and inducible nitric oxide synthase (iNOS) in the colorectal tissues of normal control (A and F), azoxymethane (AOM; B and G), AOM treated with 5-fluorouracil (AOM + 5-FU; C and H), AOM treated with thymoquinone (AOM + TQ; D and I), and AOM treated with 5-FU/TQ combination therapy (E and J) groups (×200 magnification and the scale bar is 8 μm). Abbreviation: iNOS, inducible nitric oxide synthase.