Combination of selenium and green tea improves the efficacy of chemoprevention in a rat colorectal cancer model by modulating genetic and epigenetic biomarkers

Dietary supplementation of selenium and green tea holds promise in cancer prevention. In this study, we evaluated the efficacies of selenium and green tea administered individually and in combination against colorectal cancer in an azoxymethane (AOM)-induced rat colonic carcinogenesis model and determined the underlying mechanisms of the protection. Four-week old Sprague-Dawley male rats were fed with diets containing 0.5% green tea extract, 1ppm selenium as selenium-enriched milk protein, or combination of 1ppm selenium and 0.5% green tea extract. Animals received 2 AOM (15 mg/kg) treatments to induce colonic oncogenesis. Rats were killed 8 or 30 wk later after the last AOM to examine the effect of dietary intervention on aberrant crypt foci (ACF) formation or tumor development. On sacrifice, colons were examined for ACF and tumors, the mRNA levels of SFRP5 and Cyclin D1, and the proteins levels of ß-catenin, COX-2, Ki-67, DNMT1 and acetyl histone H3. The combination of selenium and green tea resulted in a significant additive inhibition of large ACF formation, this effect was greater than either selenium or green tea alone, P,0.01; the combination also had a significant additive inhibition effect on all tumor endpoints, the effect of the combination diet on tumor incidence, multiplicity and size was greater than selenium or green tea alone, P,0.01. Rats fed the combination diet showed marked reduction of DNMT1 expression and induction of histone H3 acetylation, which were accompanied by restoration of SFRP5 mRNA in normal-appearing colonic crypts. The combination diet also significantly reduced ß-catenin nuclear translocation, Cyclin D1 expression and cell proliferation. These data show, for the first time, that combination of selenium and green tea is more effective in suppressing colorectal oncogenesis than either agent alone. The preventive effect is associated with regulation of genetic and epigenetic biomarkers implicated in colonic carcinogenesis

Repair and removal of azoxymethane-induced O6-methylguanine in rat colon by O6-methylguanine DNA methyltransferase and apoptosis

Azoxymethane (AOM) is an alkylating agent that generates mutagenic and carcinogenic O6-methylguanine (O6meG) adducts in DNA. O6meG has been detected in human colonic DNA; hence, understanding the innate cellular events occurring in response to the formation of O6meG is important in developing preventive strategies for colorectal cancer. We explored the time-course, dose–response, and kinetics of O6meG formation and its removal by the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), and apoptosis. In rats given AOM (10 mg/kg), the formation of O6meG occurs within 2 h of exposure, accompanied by rapid depletion of MGMT activity and followed by the induction of an acute apoptotic response that peaks at 6–8 h. MGMT repair and apoptosis are dependent on AOM dose and O6meG load. Apoptosis is initiated only when a high O6meG load is present and MGMT activity is fully depleted. AOM, 10 mg/kg, overwhelms MGMT repair for about 96 h and renewed MGMT activity is only observed once O6meG is no longer detectable. A threshold for apoptosis is observed at 6 h after 6 mg/kg AOM, when a high O6meG persists and MGMT activity is very low. These data suggest that apoptosis is probably triggered by O6meG, but only once the capacity of MGMT to repair O6meG is exhausted. In the colonic epithelium, apoptosis may be complementary to MGMT, in terms of minimising potentially mutagenic events and maintaining a healthy genome.

Experimental design for evaluation of Se, green tea and the combination diet for chemopreventive effects against colonic carcinogenesis in an AOM-induced rat CRC model.

<p>ACF study (A): groups of rats (n = 15) were fed control, Se, green tea or diet containing Se and green at age of 5 wk. 2 wk later, rats were given 15 (mg/Kg/body weight) AOM, once a week for 2 wk. 8 wk after last AOM treatment, rats were euthanized and colons were evaluated for ACF; normal-appearing crypts were also examined for β-catenin, COX-2 and Ki-67 expression. Tumor study (B) : groups of rats (n = 25) were fed control, Se, green tea or diet containing Se and green at age of 5 wk. Rats were given two weekly AOM treatments similar to ACF study. 30 wk after AOM treatment, rats were euthanized and colons were historically evaluated for tumor outcomes; normal-appearing crypts were also examined for β-catenin, DNMT1, Ac-H3 as well as <i>SFRP5</i> and <i>Cyclin D1</i> expression.</p

Effect of Se alone, green tea alone, and their combination on the formation of AOM-induced ACF in rats.

<p>AOM, azoxymethane; Se, selenium; ACF, aberrant crypt foci; SEM, standard errors of mean.</p><p>ACF study was undertaken by feeding rats with 4 experimental diets (15 rats per group), containing either control diet, green tea diet, Se diet and the combination diet. Animals received two weekly AOM (15 mg/kg) injections to induce ACF and were killed 8 wk later.</p>#<p>Total number of ACF was calculated as the sum of the small and large ACF.</p>†<p>Small ACF (Crypt multiplicity) was classified by the number of crypts per focus (1–3).</p>‡<p>Large ACF (Crypt multiplicity) was classified by the number of crypts per focus (≥4).</p><p>Values with different superscripts (a, b, c) in each column were statistically different (<i>P</i><0.05).</p

Effects of dietary Se, green tea and the combination of Se and green tea on β-catenin, DNMT1 and Ac-H3 expression (tumor study).

<p>A representative section of immunohistochemical staining β-catenin, DNMT1 and Ac-H3 in AOM-untreated normal crypts (A), AOM-treated normal-appearing crypts from the control, green tea, Se and the combination diet (B) and tumors; labelling index for β-catenin (membrane and nuclear positive cells) (n = 12) (C) and labelling index for DNMT1 and Ac-H3 (n = 12) (D). Western blot analysis of β-catenin, DNMT1 and Ac-H3 for colon samples (n  = 6) (E), expression of β-catenin and DNMT1 was normalized to that of β-actin and expression of Ac-H3 was normalized to that of H3, data was presented as precent of control (F). Increased strong β-catenin nuclear staining and DNMT1 overexpression were displayed in AOM-induced tumors, whereas weaker Ac-H3 expression was noted in colon tumors. β-catenin nuclear staining was significantly decreased by the combination diet and Se alone, but not green tea; DNMT1 was significantly decreased by the combination diet and green tea alone, but not Se; Ac-H3 nuclear staining was significantly increased by the combination diet and Se alone, but not green tea. Statistical significance of dietary intervention between the groups was analysed by ANOVA, values with different superscripts in each column were statistically different (<i>P</i><0.05), Bars: mean ± SEM.</p

Effect of Se and green tea alone, and their combination on tumor incidence, multiplicity and size in AOM-treated rats.

<p>AOM, azoxymethane; Se, selenium; SEM, standard errors of mean.</p><p>Tumor study was undertaken by feeding animals with 4 different diets (25 rats per group), containing either control diet, green tea diet, Se diet and the combination diet. Animals received two weekly AOM (15 mg/kg) injections to induce tumor formation and killed 30 wk later.</p>#<p>No statistical analysis undertaken for adenocarcinomas due to the small numbers.</p><p>Values with different superscripts (a, b, c) in each column were statistically different (<i>P</i><0.05).</p

Effects of dietary Se, green tea and the combination of Se and green tea on β-catenin, COX-2 and Ki-67 expression (ACF study).

<p>A representative section of immunohistochemical staining of β-catenin, Ki-67and COX-2 in AOM-untreated normal crypts (A), AOM-treated normal-appearing crypts from control, green tea, Se and the combination diet (B); labelling index for β-catenin (membrane and nuclear positive cells, calculated as the number of positive cells per crypt column divided by the total number of cells and multiplied by 100 (n = 12) (C); labelling index for COX-2 and Ki-67 (n = 12) (D). β-catenin and Ki-67 nuclear staining was significantly decreased by the combination diet and Se alone, but not green tea. COX-2 cytoplasmic staining was significantly decreased by green tea, Se and the combination diet. Statistical significance of dietary intervention between the groups was analysed by ANOVA, values with different superscripts in each column were statistically different (<i>P</i><0.05), Bars: mean ± SEM.</p

Effects of dietary Se, green tea and the combination of Se and green tea on <i>SFRP5</i> and <i>Cyclin D1</i> mRNA expression (tumor study).

<p>Quantitative RT-PCR analysis of expression of <i>SFRP5</i> (A) and <i>Cyclin D1</i> (C) mRNA in AOM-untreated normal colons and AOM-induced tumors (n = 6); expression of <i>SFRP5</i> (B) and <i>Cyclin D1</i> (D) mRNA from the control, green tea, Se and the combination diet (n = 6) in AOM-treated normal-appearing crypts. Significant repression of <i>SFRP5</i> and marked increase <i>Cyclin D1</i> mRNA in AOM-induced colon tumors were noted compared with normal colons. <i>SFRP5</i> was significantly increased by the combination diet, Se alone and green tea alone. <i>Cyclin D1</i> was also significantly inhibited by the combination diet and Se alone, but not green tea. Statistical significance of dietary intervention between the groups was analysed by ANOVA, values with different superscripts in each column were statistically different (<i>P</i><0.05), Bars: mean ± SEM.</p

Composition of experimental diets (g/100 g diet).

<p>The experimental diets consisted of a modified AIN-76A diet achieved by adding 19% sunflower oil and 20% protein.</p>#<p>Milk protein was used as source of protein for control diet and green tea diet</p>†<p>Se-enriched milk protein was used as source of protein and Se for Se diet and the combination diet.</p>‡<p>No Se was included in mineral mix in the diets.</p