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

Figures & data

Table 1 Primer sequences used in the real-time quantitative PCR for detection of the transcription activities of Wnt, β-catenin, NF-κB, COX-2, DKK-1, CDNK-1A, TGF-β1, Smad4, and β-actin genes including the corresponding gene’s accession numbers

Gene	Forward	Reverse
Wnt (NCBI: NM_001105714.1)	5′ AGC TGG GTT TCT GCT ACG TT 3′	5′ AAT CTG TCA GCA GGT TCG TG 3′
β-catenin (NCBI: AF397179.1)	5′ TTC CTG AGC TGA CCA AAC TG 3′	5′ GCA CTA TGG CAG ACA CCA TC 3′
NF-κB (NCBI: NM_001008349.1)	5′ CAG AGC TGG CAG AGA GAC TG 3′	5′ TAC GAA GGA GAC TGC CAC TG 3′
COX-2 (NCBI: AF233596.1)	5′ AAT CGC TGT ACA AGC AGT GG 3′	5′ GCA GCC ATT TCT TTC TCT CC 3′
DKK-1 (NCBI: NM_001106350.1)	5′ ATT CCA GCG CTG TTA CTG TG 3′	5′ GAA TTG CTG GTT TGA TGG TG 3′
CDNK-1A (NCBI: NM_080782.3)	5′ AGA AGG GAA CGG GTA CAC AG 3′	5′ ACC CAT AAG AAG GGC AGT TG 3′
TGF-β1 (NCBI: NM_021578.2)	5′ GGTGGACCGCAACAACGCAATCTA 3′	5′ GGGTGGCCATGAGGAGCAGGAA 3′
Smad4 (NCBI: NM_019275.3)	5′ CCACCAACTTCCCCAACATT 3′	5′ TGCAGTCCTACTTCCAGTCCAG 3′
β-actin (NCBI: NM_031144.3)	5′ CGG TCA GGT CAT CAC TAT CG 3′	5′ TTC CAT ACC CAG GAA GGA AG 3′

Abbreviation: PCR, polymerase chain reaction.

Table 2 Effects of thymoquinone (TQ) and/or 5-fluorouracil (5-FU) therapy

Parameter	Normal group	AOM group	AOM/5-FU group	AOM/TQ group	AOM/5-FU/TQ group
Body weight (g)	231.5±20	221.9±23	238.4±13.6	237.8±24	231.8±21
Colon surface area (cm^2)	19.1±2.2	18.89±3.43	19.81±1.84	20.7±2.72	20.3±3.7
Tumor count
Gross	N/A	12.5±3.21	8.7±2.9a	9.1±2.5a	7.3±1.3b–d
Dissecting microscope	N/A	17.36±4.6	8.8±1.75a	7.9±2.8a	6.1±1.4b–d
Total	N/A	29.16±2.92	16.66±4.41a	17.5±2.1a	13.8±2.8b–d
Large ACF count	N/A	45.7±5.4	23.2±3.7a	25.0±4.0a	16.4±3.1b–d
AST (U/L)	92.4±24.2	105.8±26.7	109±21.6	100±23.2	104.8±13.1
ALT (U/L)	67.6±2.4	71.2±6.7	68.7±4.1	66.7±16.9	65.2±15.7
ALP (IU/L)	122.6±11.2	125.7±9.7	122.8±12.4	124.3±11.9	121.6±11.1
Creatinine (mg/dL)	0.22±0.03	0.23±0.06	0.23±0.05	0.20±0.02	0.21±0.04
Urea (mg/dL)	48.6±5.1	50.3±4.3	50.6±9.5	48.8±6.2	49.3±7.4
BUN (mg/dL)	22.2±2.4	24.4±1.9	24.3±4.4	21.2±3.8	22.5±3.4

Notes: The table shows the effect on the body weight, colon surface area (length × width in cm²), numbers of grown tumors and large aberrant crypts foci (ACF), liver enzymes (AST, ALT, and ALP), and renal function parameters (creatinine, urea, and BUN) in azoxymethane (AOM)-induced colorectal tumors in rats. The gross colorectal tumors were counted by the naked eye, while both microtumors and large ACF (containing four or more aberrant crypts) were counted under a dissecting microscope after methylene blue staining. Data are represented as mean ± SD.

a P<0.05 vs AOM group.

b P<0.05 vs AOM/5-FU group.

c P<0.05 vs AOM/TQ group.

d P<0.01 vs AOM group.

Abbreviations: N/A, not applicable; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen.

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.

Table 3 Immunohistochemistry scores for the effects of thymoquinone (TQ) and/or 5-fluorouracil (5-FU)

Parameter	Normal group	AOM group	AOM/5-FU group	AOM/TQ group	AOM/5-FU/TQ group
TGF-β1	315.5±31.2	136.4±23.4a	301.48±44.3b	307.3±37.2b,c	371.7±35.3a–c
TGF-βRII	333.7±28.7	121.4±19.2a	135.7±17.6a	185.6±26.4a–c	305.4±31.8a–d
Smad4	301.1±26	147.2±20.3a	216.1±23.9a,b	248.4±27.7a–c	322.7±31.5a–d
β-catenin	61±16.7	370.6±28.7a	267.6±35.3a,b	251.6±35.1a,b	193.4±29.9a–d
iNOS	39.4±9.5	373.1±26.8a	261.8±47.2a,b	117.5±41.1a–c	79.5±22.3a–d

Notes: The table shows the colorectal expression levels of tissue growth factor-β1 (TGF-β1), TGF-β receptor II (TGF-βRII), Smad4, β-catenin, and inducible nitric oxide synthase (iNOS) in azoxymethane (AOM)-induced colorectal tumors in rats. Data are represented as mean ± SD.

a P<0.05 compared with normal control group.

b P<0.05 compared with AOM group.

c P<0.05 compared with 5-FU group.

d P<0.05 compared with TQ 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.

Figure S1 Corresponding histopathological features of colorectal tissues stained with Alcian blue.

Notes: The figure shows the pre-neoplastic mucin-depleted foci (MDF) that had lost mucin expression (blue color) and was abundantly present in the colorectal tissues of rats injected with azoxymethane and left without treatment (AOM group) but not in rats injected with AOM and then treated with TQ and 5-FU combination therapy. Light microscopy at magnifications ×2,000.
Abbreviations: AOM, azoxymethane; TQ, thymoquinone; 5-FU, 5-fluorouracil.