The flavonoid quercetin suppresses cell proliferation and enhances apoptosis in vitro. In this study, we determined whether quercetin protects against colon cancer by regulating the protein level of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt or by suppressing the expression of proinflammatory mediators [cyclooxygenase (COX)-1, COX-2, inducible nitric oxide synthase (iNOS)] during the aberrant crypt (AC) stage. Forty male rats were randomly assigned to receive diets containing quercetin (0 or 4.5 g/kg) and injected subcutaneously with saline or azoxymethane (AOM; 2 times during wk 3 and 4). The colon was resected 4 wk after the last AOM injection and samples were used to determine high multiplicity AC foci (HMACF; foci with >4 AC) number, colonocyte proliferation and apoptosis by immunohistochemistry, expression of PI 3-kinase (p85 and p85alpha subunits) and Akt by immunoblotting, and COX-1, COX-2, and iNOS expression by real time RT-PCR. Quercetin-fed rats had fewer (P = 0.033) HMACF. Relative to the control diet, quercetin lowered the proliferative index (P = 0.035) regardless of treatment and diminished the AOM-induced elevation in crypt column cell number (P = 0.044) and expansion of the proliferative zone (P = 0.021). The proportion of apoptotic colonocytes in AOM-injected rats increased with quercetin treatment (P = 0.014). Levels of p85 and p85alpha subunits of PI 3-kinase and total Akt were unaffected by dietary quercetin. However, quercetin tended to suppress (P < 0.06) the expression of COX-1 and COX-2. Expression of iNOS was elevated by AOM injection (P = 0.0001). In conclusion, quercetin suppresses the formation of early preneoplastic lesions in colon carcinogenesis, which occurred in concert with reductions in proliferation and increases in apoptosis. It is possible the effects on proliferation and apoptosis resulted from the tendency for quercetin to suppress the expression of proinflammatory mediators.

We have demonstrated that 0.45% quercetin added to a diet containing corn oil (15% w/w), as the lipid source, and cellulose (6% w/w), as the fiber source, was able to suppress the formation of high multiplicity aberrant crypt foci (ACF > 4 AC/focus), to lower proliferation and enhance apoptosis in a rat model of colon cancer. This experiment determined whether quercetin was acting as an antiinflammatory molecule in an in vivo model of colon cancer. We used weanling (21 d old) Sprague Dawley rats (n = 40) in a 2×2 factorial experiment to determine the influence of quercetin on iNOS, COX-1 and COX-2 expressions, all of which are elevated in colon cancer. Half of the rats received a diet containing either 0 or 0.45% quercetin, and within each diet group, half of the rats were injected with saline or azoxymethane (AOM, 15 mg/kg BW, sc, 2× during wk 3 and 4). The colon was resected 4 wk after the last AOM injection, and the mucosa scraped and processed for RNA isolation. Data from this experiment were analyzed using a mixed model in SAS for main effects and their interaction. AOM injection stimulated (P < 0.0001) iNOS expression. However there was an interaction such that, relative to rats injected with saline, AOMinjected rats consuming diets without quercetin had significantly elevated iNOS expression (5.29-fold), but the expression in AOM-injected rats consuming the diet with quercetin was not significantly elevated (1.68-fold). COX-1 expression was 20.2% lower (P < 0.06) in rats consuming diets containing quercetin. COX-2 expression was 24.3% higher (P < 0.058) in rats consuming diets without quercetin. These data suggest inflammatory processes are elevated in this early stage of colon carcinogenesis, yet quercetin may protect against colon carcinogenesis by down-regulating the expressions of COX-1 and COX-2.

Quercetin (Q) may protect against colon cancer due to its antioxidant activity. In contrast, we have shown that a fish oil diet (FO; n‐3 fatty acids), compared to corn oil (CO; n‐6 fatty acids), protects against colon cancer by decreasing endogenous antioxidant activities leading to increased reactive oxygen species (ROS), an inducer of apoptosis. We hypothesized that adding an antioxidant to a pro‐oxidant diet could negate the protective effect of FO by counteracting FO effects on colonocyte redox status. To test this, we provided 40 rats with FO or CO (fiber = pectin) diets supplemented with 0 or 0.45% Q for 10 wk. All rats were injected with AOM on d 21 and 28. Aberrant crypts (AC) (marker for colon cancer) and apoptosis (TUNEL) were measured. Catalase (CAT) and Mn superoxide dismutase (SOD) activity were indicators of endogenous antioxidant capacity. AC numbers were lower (p=0.0001) in FO vs CO rats, but AC tended to increase (P<0.098) for FO diets containing Q. Surprisingly, dietary lipid did not affect apoptosis, yet apoptosis was elevated (P=0.0002) by Q for FO and CO diets. CAT activity was higher (p=0.0204) in FO vs CO rats, however, Q lowered (P=0.068) CAT activity in FO rats. SOD activity was not affected by diet. Despite increasing apoptosis, Q did not lower AC formation. The small increase in AC caused by Q in the FO diet may result from the lower CAT activity caused by the combination. The long‐term consequences of supplementing antioxidants to a diet thought to exert its anticancer effect through a pro‐oxidant mechanism are unknown and deserve further study. Funding: USDA‐CSREES 2005‐34402‐16401, NIEHS P30‐ES09106.

We have demonstrated changes in antioxidant enzyme activities and glutathione metabolism in colonocytes from rats consuming diets containing 0.45% quercetin combined with corn oil or fish oil. The goal of this study was to query whether these changes were regulated at the transcriptional level. Therefore, real‐time PCR was used to assess the relative expression of 15 genes involved in maintaining cellular antioxidant levels and xenobiotic metabolism. Fish oil alone enhanced the level of catalase, and did not affect the expression of any other genes tested. Quercetin decreased the expression of six genes (SOD2, GPx2, GPx4, GLR2, Ephx2, Cyp1a1) and elevated the expression of Ephx1. When the interaction between quercetin and dietary lipid was analyzed, it was noted that quercetin and fish oil tended to lower the expression levels of seven genes, when compared with the fish oil rats not consuming quercetin. However, when quercetin was combined with corn oil, there was a tendency for mixed responses with four genes enhanced and two suppressed. These data suggest that the ability of quercetin to suppress expression of genes involved in regulating cellular antioxidant status and xenobiotic activation/ detoxification is amplified by its combination with fish oil. Funding USDA/CSREES 2005‐34402‐16401, 2007‐118409, and NIEHS P30‐ES09106.