In this study, we demonstrated that a selective RXR agonist bexarotene inhibited intestinal tumorigenesis in ApcMin/+
mice, a model for human familial adenomatous polyposis. Bexarotene treatment caused a decrease in serum TGs and enhanced uptake of oleate, and caused tumor growth inhibition, suggesting that the RXR pathway is an important target for colon cancer prevention. The suppressive effect of bexarotene on the development of colonic tumors also was closely correlated with a decrease in proinflammatory cytokines. Although the value of the ApcMin/+
mouse as model for predicting efficacy of chemopreventive agents in humans is a subject of debate, results obtained in this model [18,19
] did forecast the efficacy of COX inhibitors, such as sulindac and celecoxib, to retard adenoma recurrence in humans [20,21
The expression of proliferation marker PCNA is significantly reduced in the treated polyp, with no effect on normal crypt (). This could be due to tumor-specific suppression of proliferation by bexarotene, and it is well known that tumor cells that proliferate much faster contain very high levels of PCNA compared with normal crypt cells. Additional experiments would be needed to determine whether and to what extent bexarotene influences the normal crypt cell PCNA levels. The chemopreventive effect of bexarotene in the ApcMin/+
mouse was, to a certain extent, dose related. Bexarotene at 200 ppm, which reduced SI polyp number by approximately 75%, could not extend mouse survival because of significant toxicity; after 12 weeks of feeding, only 50% of the mice survived. However, lower subtoxic doses of bexarotene (30 and 60 ppm) significantly inhibited SI tumorigenesis by 38.2% and 59.9% (P
< .015 to P
< .0001) or 8.5% and 36.9% (P
< .007), for male and female mice, respectively. It is intriguing that bexarotene showed better efficacy against SI tumors in males compared to female mice and, in contrast, better efficacy against colon tumors in female than in male mice. The reasons for these differences need further investigation. However, it is possible that hormonal differences or differences in bioavailability of bexarotene in male versus
female mice might have played a role. The differences between male and female mice in the dose-response relationship suggest that the bexarotene dose must be optimized separately for males and females for optimal colon cancer chemoprevention. The recommended dosage of bexarotene is 300 mg/m2
per day clinically (cancer patients). Data reported by others suggest that dosages of retinoids up to 10 and 50 mg/kg per day used in mice (female MMTV-erbB2 transgenic mice) can achieve peak plasma levels that are similar to those from human trials using 200 to 400 mg/m2
per day [22–24
], which is equivalent to approximately 6 to 10 mg/kg body weight per day. Thus, 60 ppm (~10 mg/kg body weight per day) of bexarotene used in the present study should be very close to the clinical dosage (by kilogram body weight); however, it is much lower than the dosage used by others in previous studies in mice.
The regulation of growth and differentiation by rexinoids results from direct and indirect effects on gene expression mediated by the RAR and RXR nuclear receptors. RXRs form heterodimers with LXR receptors and regulate the levels of fatty acids [25
]. Hence, in the presence of bexarotene, the increased expression of RXR and its interaction with LXR might have caused the increase in oleate in colonic mucosa. Kim et al. [26
] showed that the expression of several rexinoid-regulated biomarkers is modulated in vivo
in mammary glands from mice treated with bexarotene. Microarray analysis showed up-regulation of stearoyl-CoA desaturase (regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate) and down-regulation of COX-2. Bexarotene binding to RXR may induce the absorption of oleate, which affects the expression of COX-2, an important gene in colon tumor development. COX-2 is induced by a variety of mitogenic and inflammatory stimuli. It is well known that COX-2 expression is markedly elevated in colorectal cancer in both humans and rodents, and COX-2 plays an important role in cancer cell proliferation [27
The COX-2 molecule could be a second messenger for transcriptional action of the fatty acid oleate as reported by Duplus et al. [28
]. In the current study, COX-2 levels in colon polyps from ApcMin/+
mice were much greater than those in wild-type mice. Treatment with bexarotene increased oleate levels in colon mucosa, and this increase somewhat correlated with reduced colon tumors. Oleate is reported to lower cholesterol levels, thereby reducing atherosclerosis and the risk of cardiovascular disease. It reduces insulin resistance, thereby improving glucose maintenance, improves immune function, and provides protection against certain types of cancer [29
]. Our findings also suggest that RXR may regulate COX-2 and, thus, the production of prostaglandins, which are known to play a key role in colon tumor development.
Epidemiologically, a positive association between hypertriglyceridemia and colorectal cancer development has been reported [30
]. Also, an experimental rodent study showed a positive effect of serum TG on the development of aberrant crypt foci [31
]. Niho et al. [32–34
] and our laboratory [16
] recently showed that a hyperlipidemic state is associated with intestinal polyp formation in Apc
-deficient mice, and this was attributed mainly to down-regulation of lipoprotein lipase in Apc mutant mice. They observed that peroxisome proliferator-activated receptor ligands and lipoprotein lipase activator reduce serum TG levels and suppress intestinal polyp formation in Apc
-deficient mice [32–34
]. Other laboratory investigations also have shown that RXR-selective agonists increase the transcription of apolipoprotein C-III, possibly interfering with plasma lipid clearing [35
]. Lipid metabolism and the intracellular transport of bioactive molecules are critical in the modulation of proliferation through interactions with nuclear receptors. Our finding that administration of bexarotene improved hyperlipidemia and suppressed intestinal polyp formation in the ApcMin/+
mice is consistent with these other findings. ApcMin/+
mice showed approximately six-fold higher serum TG levels compared to wild-type control mice, but both male and female ApcMin/+
mice fed bexarotene showed a dose-dependent suppression of serum TG levels. Importantly, decreased serum TG levels were highly correlated with decreased intestinal polyp number and tumor size induced by bexarotene.
The ability of bexarotene to interfere with tumorigenesis also could be due to the inhibition of inflammatory cytokines. We observed inhibition of IL-2, IL-12, IFN-γ, and other proinflammatory cytokines. Recent studies show that retinoids inhibit the production of several inflammatory cytokines by activated macrophages through functional interactions between their receptors (RXR and RAR) and nuclear factor-κB, a crucial transcription factor for IL-12 gene expression [36
]. Furthermore, IL-1β has been shown to enhance the production of vascular endothelial growth factor through IL-2, which was shown to induce angiogenesis in colon cancer cells [37
]. TNF-α and IL-1β are key cytokines involved in inflammation, immunity, and cellular organization [38
]. In this study, the expression of TNFα and IL-1β proteins was substantially upregulated in the serum of control ApcMin/+
mice. Bexarotene treatment reduced the expression of TNFα and IL-1β protein in the serum of ApcMin/+
mice. Because the induction of COX-2 is regulated by TNFα and IL-1β, these cytokine effects may play important roles in tumor promotion. Thus, the suppression of TNFα and IL-1β expression by bexarotene may contribute to the low frequency of large tumors (size ~1.5-2 mm) as observed in this study.
In conclusion, the dietary administration of bexarotene could effectively suppress intestinal tumor development in the ApcMin/+ mice by affecting multiple factors, including up-regulation of RXR-α, and by decreasing COX-2, inflammatory cytokines, and hyperlipidemia, all of which are involved in colon carcinogenesis. Collectively, these data suggest a role of bexarotene in chemoprevention of intestinal cancers.