The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Abstract

Curcuma purpurascens BI. rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia that is traditionally used in assorted remedies. Dichloromethane extract of C. purpurascens BI. rhizome (DECPR) has previously been shown to have an apoptosis-inducing effect on colon cancer cells. In the present study, we examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM) (15 mg/kg) by determining the percentage inhibition in incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, three groups of rats were orally administered once a day for 2 months either 10% Tween 20 (5 mL/kg, cancer control), DECPR (250 mg/kg, low dose), or DECPR (500 mg/kg, high dose). Meanwhile, the control group was intraperitoneally injected with 5-fluorouracil (35 mg/kg) for 5 consecutive days. After euthanizing the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and proliferating cell nuclear antigen (PCNA) protein expressions were examined using immunohistochemical and Western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. The percentage inhibition of ACF was 56.04% and 68.68% in the low- and high-dose DECPR-treated groups, respectively. The ACF inhibition in the treatment control group was 74.17%. Results revealed that DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied by reduced expression of PCNA. Upregulation of Bax and downregulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Taken together, the present data clearly indicate that DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

Keywords: PCNA; Zingiberaceae; colon cancer.

Figure 1

Effect of DECPR on ACF formation in proximal and distal parts of the colons separated from the treated rats. Notes: The five groups of rats were A: normal control; B: cancer control; C: low dose of DECPR; D: high dose of DECPR; and E: 5-fluorouracil treatment control. Data are shown as mean ± standard error of the mean (n=6). Values are statistically significant at *P<0.05. Abbreviations: ACF, aberrant crypt foci; DECPR, dichloromethane extract of Curcuma purpurascens rhizome.

Figure 2

Effect of DECPR on gross appearances of aberrant crypt foci (arrows) in the colon mucosa separated from the treated rats and stained with methylene blue dye. Notes: The five groups of rats were (A) normal control; (B) cancer control; (C) low dose of DECPR; (D) high dose of DECPR; and (E) 5-fluorouracil treatment control. Scale bar: 500 µm. Abbreviation: DECPR, dichloromethane extract of Curcuma purpurascens rhizome.

Figure 3

Immunohistochemical expression of PCNA in colon tissues of control and experimental groups of rats. Notes: (A and B) (a) Group A. (b) Group B. (c) Group C. (d) Group D. (e) Group E. PCNA protein expression is illustrated as brown staining. (A) Scale bar: 10 µm. (B) Scale bar: 100 µm. (C) Quantitative data expressing the PCNA protein level illustrate that there was significant downregulation in groups C–E compared with the cancer control group. Data are shown as mean ± standard error of the mean (n=6). Values are statistically significant at *P<0.05. The five groups of rats were A: normal control; B: cancer control; C: low dose of DECPR; D: high dose of DECPR; and E: 5-fluorouracil treatment control. The yellow arrows show brown staining demonstrating a significant downregulation of PCNA in groups C–E compared with the cancer control group. Abbreviations: DECPR, dichloromethane extract of Curcuma purpurascens rhizome; PI, proliferation index; PCNA, proliferating cell nuclear antigen.

Figure 4

Immunohistochemical expression of Bax in colon tissues of control and experimental groups of rats. Notes: (A and B) The five groups of rats were (a) normal control; (b) cancer control; (c) low dose of DECPR; (d) high dose of DECPR; and (e) 5-fluorouracil treatment control. Bax protein expression is illustrated as brown staining. The yellow arrows depict the up regulation of Bax in groups C–E is shown as brown staining. (A) Scale bar: 10 µm. (B) Scale bar: 100 µm. Abbreviation: DECPR, dichloromethane extract of Curcuma purpurascens rhizome.

Figure 5

Immunohistochemical expression of Bcl-2 in colon tissues of control and experimental groups of rats. Notes: (A and B) The five groups of rats were (a) normal control; (b) cancer control; (c) low dose of DECPR; (d) high dose of DECPR; and (e) 5-fluorouracil treatment control. Bcl-2 protein expression is illustrated as brown staining. The yellow arrows depict the down regulation of Bcl-2 in groups C–E is shown as brown staining. (A) Scale bar: 10 µm. (B) Scale bar: 100 µm. Abbreviation: DECPR, dichloromethane extract of Curcuma purpurascens rhizome.

Figure 6

Western blot analysis of PCNA, Bax, and Bcl-2 proteins extracted from colon tissues of rats. Notes: The five groups of rats were A: normal control; B: 10% Tween 20 (cancer control); C: low dose of DECPR; D: high dose of DECPR; and E: 5-fluorouracil treatment control. β-actin Western blotting was used as the control band. Abbreviation: DECPR, dichloromethane extract of Curcuma purpurascens rhizome; PCNA, proliferating cell nuclear antigen.

Figure 7

Levels of enzymatic antioxidants and MDA in the colon tissues of control and experimental groups. Notes: (A) CAT. (B) GPx. (C) SOD. (D) MDA. Data are shown as mean ± standard error of the mean (n=6). Values are statistically significant at *P<0.05. The five groups of rats were A: normal control; B: cancer control; C: low dose of DECPR; D: high dose of DECPR; and E: 5-fluorouracil treatment control. Abbreviation: DECPR, dichloromethane extract of Curcuma purpurascens rhizome; CAT, catalase; GPx, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde.