Chemoprevention of Colonic Aberrant Crypt Foci by Novel Schiff Based Dichlorido(4-Methoxy-2-{[2-(Piperazin-4-Ium-1-Yl)Ethyl]Iminomethyl}Phenolate)Cd Complex in Azoxymethane-Induced Colorectal Cancer in Rats

Abstract

Schiff-based complexes as a source of cancer chemotherapeutic compounds have been subjected to the variety of anticancer studies. The in-vitro analysis confirmed the CdCl2(C14H21N3O2) complex possess cytotoxicity and apoptosis induction properties in colon cancer cells, so lead to investigate the inhibitory efficiency of the compound on colonic aberrant crypt foci (ACF). Five groups of adult male rats were used in this study: Vehicle, cancer control, positive control groups and the groups treated with 25 and 50 mg/kg of complex for 10 weeks. The rats in vehicle group were injected subcutaneously with 15 mg/kg of sterile normal saline once a week for 2 weeks and orally administered with 5% Tween-20 (5 ml/kg) for 10 weeks, other groups were injected subcutaneously with 15 mg/kg azoxymethane once a week for 2 weeks. The rats in positive groups were injected intra-peritoneally with 35 mg/kg 5-Flourouracil four times in a month. Administration of the complex suppressed total colonic ACF formation up to 73.4% (P < 0.05). The results also showed that treatment with the complex significantly reduced the level of malondialdehyde while increasing superoxide dismutase and catalase activities. Furthermore, the down-regulation of PCNA and Bcl2 and the up-regulation of Bax was confirmed by immunohistochemical staining.

Figure 1. Histological sections of liver (first row) and kidney (second row) in female rats.

(A,D) Control pre-treated with vehicle (10% Tween-20), (B,E) pre-treated with 250 mg/kg of CdCl₂(C₁₄H₂₁N₃O₂) complex in female group. No structural differences were observed between the compound treated group and the control group. (H,E) stain; 100 × magnification.

Figure 2. Histological sections of liver (first row) and kidney (second row) in male rats.

(A,D) Control pre-treated with vehicle (10% Tween-20), (B,E) pre-treated with 250 mg/kg of CdCl₂(C₁₄H₂₁N₃O₂) complex in male group. No structural differences were observed between the compound treated group and the control group. (H,E) stain; 100 × magnification.

Figure 3. Topographic views of colon mucosa.

(A) Vehicle colon mucosa, (B) cancer control group, (C) positive control group, (D) treatment group with 25 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex, (E) treatment group with 50 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex in methylene blue staining of rat colonic tissue. ACF were distinguished from the surrounding of normal crypts by their increased in size, increased distance from lamina to basal surfaces of cells and easily discernible pericryptalzone. Magnification, ×20.

Figure 4. Histological study of colon cancer in rats.

(A) Vehicle colon mucosa, (B) cancer control group, (C) positive control group, (D) treatment group with 25 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex, (E) treatment group with 50 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex. The section was cut parallel to the muscle layer. (H,E)stain; 100 × magnification).

Figure 5. Immunohistochemical analysis of expression of Bcl2 in the colon of different group of rats.

(A) Normal colon mucosa. (B) Colon mucosa of the group exposed to AOM. (C) Colon mucosa of the group treated with 5-fluorouracil., (D) Colon mucosa of the treatment group with 25 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex, (E) Colon mucosa of the treatment group with 50 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex. Immunohistochemistry staining showed upregulation of Bcl2 protein expression in rats treated with CdCl₂(C₁₄H₂₁N₃O₂) Schiff base compounds. Magnification, ×100.

Figure 6. Immunohistochemical analysis of expression of Bax in the colon of different group of rats.

(A) Normal colon mucosa. (B) Colon mucosa of the group exposed to AOM. (C) Colon mucosa of the group treated with 5-fluorouracil. (D) Colon mucosa of the treatment group with 25 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex, (E) Colon mucosa of the treatment group with 50 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex. Immunohistochemistry staining of Bax proteins showed downregulation of expression of Bax protein in rats treated with CdCl₂(C₁₄H₂₁N₃O₂) Schiff base compounds. Magnification, ×100.

Figure 7. Immunohistochemical analysis of expression of PCNA in the colon of different group of rats.

(A) Normal colon mucosa. (B) Colon mucosa of the group exposed to AOM. (C) Colon mucosa of the group treated with 5-fluorouracil. (D) Colon mucosa of the treatment group with 25 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex, (E) Colon mucosa of the treatment group with 50 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex. Immunohistochemistry staining of PCNA proteins showed downregulation of PCNA protein in rats treated with CdCl₂(C₁₄H₂₁N₃O₂) Schiff base compounds. Magnification, ×100. All values were expressed at ± standard error of mean. The mean differences were significant at p < 0.05 when compared to the cancer control group.

Figure 8. Effects of the compound on antioxidant enzyme activities in homogenised tissue.

(A) Vehicle colon group, (B) group exposed to AOM, (C) group treated with 5-fluorouracil, (D) treatment group with 25 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex, (E) treatment group with 50 mg/kg CdCl₂(C₁₄H₂₁N₃O₂) complex. All values were expressed at ± standard error of means. Mean differences were significant at p < 0.05 when compared with cancer control group.