Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/dbmice

Obesity, a growing health concern worldwide, is a result of excess energy intake and insufficient exercise. Obesity is associated with increased risk of diseases with high mortality, such as ischemic heart disease, stroke, and cancer [1]. In particular, the risk of colorectal cancer (CRC), which is the third most common malignancy in men and the second in women, globally [2], is especially higher when combined with obesity [3]-[5]. The five-year survival for early stage CRC is 80–90% but decreases to 65% for all stages [6]. Therefore, in addition to early detection and treatment, chemoprevention with effective agents is considered extremely important for the comprehensive management of CRC [7],[8].

Several pathophysiological mechanisms linking obesity and the development of CRC have been elucidated, including the emergence of insulin resistance, imbalance of adipokines, induction of oxidative stress, and a state of chronic inflammation [3]-[5]. Obese and diabetic mice are susceptible to chemically induced colon tumorigenesis [9]. Diet-induced obesity significantly promotes colon tumor development in mice [10]. On the other hand, recent studies have demonstrated that certain types of phytochemicals such as curcumin and (−)-epigallocatechin gallate inhibit the development of obesity-related colorectal carcinogenesis in mice by attenuating chronic inflammation [11],[12]. Administration of angiotensin-converting enzyme inhibitor also suppresses the early phase of colorectal carcinogenesis in diabetic and hypertensive rats by attenuating inflammation and oxidative stress [13]. These reports suggest that targeting obesity-related metabolic abnormalities including chronic inflammation and oxidative stress using phytochemicals and specific agents is an effective strategy for preventing CRC development in obese individuals [3].

Astaxanthin, a conventional red-colored xanthophyll, is an oxygenated carotenoid derivative occurring naturally in a wide variety of living organisms including microalgae, fungi, salmon, trout, shrimp, and some birds [14],[15]. Astaxanthin has been shown to exert numerous pharmacological effects due to its antioxidant, anti-inflammatory, antidiabetic, and antineoplastic properties [8],[14],[15]. Supplementation with astaxanthin actually decreased oxidative stress and inflammation in a clinical trial [16]. In preclinical animal studies, dietary astaxanthin was found to significantly inhibit chemically induced colorectal [17],[18], urinary bladder [19], and oral carcinogenesis [20]. In particular, anti-inflammatory activity is one of the key mechanisms by which astaxanthin prevents colitis-related CRC development [17],[18].

C57BL/KsJ-db/db (db/db) mice, which lack the long form of the leptin receptor, develop hyperphagic obesity and diabetes [21]. Interestingly, the development of colonic premalignant lesions induced by azoxymethane (AOM), a colonic carcinogen widely used to produce preneoplastic and neoplastic colonic lesions that mimic those observed human colon, is significantly enhanced in db/db mice [22]. A preclinical animal model using AOM and db/db mice [22] has proved useful in investigating specific agents for their ability to prevent inflammation-related colorectal carcinogenesis caused by obesity [11],[12],[23]-[25]. In the present study, we investigated the effects of astaxanthin on the development of colonic premalignant lesions, i.e., aberrant crypt foci (ACF) and β-catenin accumulated crypts (BCAC) [26]-[28], using this obesity-related colorectal carcinogenesis model, with special focus on the reduction of oxidative stress and the attenuation of inflammation.

Obesity, which is one of the most serious healthcare problems worldwide, is a significant risk factor for the development of CRC [4],[5]. Oxidative stress and chronic inflammation are key mechanisms linking obesity and colorectal carcinogenesis [37],[38]. In particular, increased adipose tissue creates an oxidative environment that can upregulate the expression of various pro-inflammatory cytokines including TNF-α and IL-6, and this is critically associated with CRC development because these cytokines stimulate tumor growth and progression [31],[39]. The results of the present study show that astaxanthin exerts preventive effects on the development of the AOM-induced colonic premalignant lesions ACF [26],[27] and BCAC [28] in db/db obese mice, mainly through the reduction of oxidative stress and attenuation of inflammation.

Saturated fatty acids from obesity-induced lipolysis are capable of activating macrophages and thereby activating NF-κB signaling, which in turn leads to transcriptional activation of genes encoding pro-inflammatory factors including IL-1β and IL-6 [39]. IL-1β plays a key role in obesity-induced inflammation [39] and inflammation-related carcinogenesis by modulating the gene expression involved in proliferation, survival, and angiogenesis [40]. The expression of CCL2, which is associated with infiltration and migration of tumor-related macrophages, has been demonstrated in several tumor tissues including CRC [32],[33]. In addition, CXCL2, an inflammatory cytokine, has been shown to participate in the early phase of colorectal carcinogenesis [34]. In the present study, the expression levels of IL-1β, IL-6, F4/80, CCL2, and CXCL2 mRNA were decreased by astaxanthin in the experimental mice. These findings indicate that overexpression of these inflammatory mediators, which connect obesity and carcinogenesis, may be one of the critical targets of astaxanthin in preventing the development of obesity-related CRC. These findings are also consistent with those from a previous study showing that feeding with astaxanthin significantly suppressed colitis and colitis-related colorectal carcinogenesis by inhibiting NF-κB activation, decreasing the expression of IL-1β and IL-6, and suppressing cell proliferation in mice [18]. In particular, NF-κB signaling pathway is regarded as one of the key targets of astaxanthin to exert chemopreventive effects [41].

Obesity and chronic inflammation are often accompanied with increased generation of reactive oxygen species (ROS), which are derivatives of molecular oxygen such as superoxide and hydrogen peroxide. These derivatives can induce mutagenic changes and may damage DNA repair proteins, resulting in cancer development [40],[42]. Cancer cells are also known to cause oxidative stress by generating ROS and modulating antioxidant enzymes [43]. In the present study, oxidative stress markers, including urinary levels of 8-OHdG and serum levels of d-ROMs, were significantly suppressed, while the expression levels of GPx1, SOD1, and CAT mRNA, which encode antioxidant enzymes, in the colonic mucosa were increased by astaxanthin intake in AOM-injected db/db mice. These results strongly indicate that attenuation of oxidative stress and recuperation from high oxidation state via antioxidative effects are critical mechanisms by which astaxanthin suppressed the occurrence of premalignant lesions, ACF and BCAC, in obese mice. It should be mentioned that astaxanthin has even been called a super-antioxidant because it is a superior antioxidant and scavenger of free radicals as compared with other carotenoids such as β-carotene [44].

Epidemiologically, it is still unclear whether the intake of carotenoids such as astaxanthin is associated with a reduced risk of CRC development. The results of randomized trials using β-carotene supplementation provided no evidence to support an effect of carotenoids on CRC chemoprevention [45],[46]. Rather, intervention trials using high-dose β-carotene supplements showed an increase in the incidence of lung cancer in high-risk patients, like smokers and/or workers exposed to asbestos [47],[48]. On the other hand, astaxanthin has been demonstrated to be safe in several human clinical trials [16],[49],[50]. Moreover, astaxanthin supplementation has positive effects on lipid profiles and oxidative stress in overweight and obese subjects, at least in part by activating the antioxidant defense system [49],[50]. Taken together, these observations suggest that obese individuals, who are at high-risk of developing CRC and colorectal adenomas [5], may be appropriate subjects for interventional trials using astaxanthin for the prevention of colorectal tumorigenesis.

Previous reports using metabolic syndrome animal models have shown that astaxanthin reduces insulin resistance, recovers insulin sensitivity, and increases serum levels of adiponectin [51],[52]. A double-blind randomized controlled trial also reported that astaxanthin consumption significantly increases blood adiponectin levels [53]. Furthermore, targeting insulin resistance and adipokine imbalance are suggested to be effective methods of preventing obesity-related colorectal tumorigenesis [3]. Therefore, we initially expected that astaxanthin would inhibit the development of ACF and BCAC in the AOM-treated db/db mice by ameliorating insulin resistance and improving adipokine imbalance. However, abnormalities in serum levels of glucose, insulin, adiponectin, and leptin were not improved by astaxanthin administration in this study. We suggest that this was likely due to the duration of the experiment (8 weeks) and the particular animal model studied, because previous studies demonstrating effects of astaxanthin on insulin sensitivity were long-term studies (22 weeks) [51] and the animals were not genetically obese [51],[52]. Our results were consistent with another study investigating the effect of astaxanthin on the apoptosis of retinal ganglion cells using db/db mice, in which insulin resistance was not improved by astaxanthin, but apoptosis of retinal ganglion cells was attenuated via the suppression of oxidative stress [54]. Future long-term studies should be conducted to confirm that astaxanthin inhibits the early phase of obesity-related colon tumorigenesis by improving insulin resistance and the imbalance of adipokines in several animal models. In our experimental model, astaxanthin suppressed the development of obesity-related colorectal tumorigenesis by targeting oxidative stress, inflammation, and cell proliferation.

In summary, the results from this study showed that reduction of oxidative stress and attenuation of inflammation in the colonic mucosa are crucial mechanisms by which astaxanthin acts to prevent the early phase of obesity-related colorectal carcinogenesis. Since the risk of CRC increases with obesity and obesity-related metabolic abnormalities, which are impending health crises worldwide, targeting obesity-related metabolic abnormalities, including chronic inflammation and induction of oxidative stress, may be an attractive and effective strategy for preventing the development of CRC in obese individuals. Astaxanthin seems to be a potentially effective and viable candidate for this purpose because this agent attenuates chronic inflammation while reducing oxidative stress without causing side effects.