Colorectal cancer (CRC) is a major worldwide health problem and is the third most frequent cause of cancer-related death in developed countries [1
]. The growing incidence of CRC is probably associated with a modern lifestyle typified by limited physical activity, alcohol consumption and dietary changes [2
]. CRC development is a multistep process, from normal epithelial cells via inflammation to aberrant crypt foci (ACF) and progressive adenoma stages, to carcinomas and then metastatic disease [3
]. In order to reduce the incidence of CRC, effective prevention and treatment strategies need to be identified. Due to the long precancerous stage of this disease, dietary intervention may exert a favorable effect on polyp formation and/or inhibition of the transformation of adenomas to CRC. Recent findings indicate that long-term consumption of a diet rich in vegetables may prevent the development of CRC [5
]. Vegetables contain a wide variety of bioactive secondary metabolites such as glucosinolates, polyphenols and/or polyacetylenic oxylipins, some of which have shown bioactivities that may contribute to their CRC protective effects [6
Strong evidence suggests the existence of an association between the development of CRC and the expression of cyclooxygenase (COX) enzyme complex that catalyzes the conversion of arachidonic acid released from membrane phospholipids to prostaglandins. Prostaglandins sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response [9
]. COX-1 is a constitutively expressed enzyme in most mammalian tissues and it is involved in normal physiological functions [9
]. In contrast, COX-2 levels are normally low but are rapidly induced as an early response to growth factors, cytokines and tumor promoters associated with inflammation, cell survival, abnormal proliferation, angiogenesis, invasion, and metastasis [9
]. In particular, the existence of an association between CRC and COX-2 overexpression has been established [9
]. Chronic inflammation is associated with a greater likelihood of carcinogenesis, and in the case of CRC, a microenvironment conducive for the development of early neoplastic lesions is created with relative high concentrations of prostaglandins [11
]. COX-2 expression but not COX-1 has been detected in 50% of colorectal adenomas and in up to 85% of colorectal carcinomas, and it is correlated with poor prognosis [12
]. Therefore, inhibition of the expression and/or activity of COX-2 by dietary bioactive secondary metabolites may be an important target for CRC chemoprevention. This information might have prognostic importance and may help in identifying new treatment strategies. Nonsteroidal anti-inflammatory drugs (NSAIDs) are known COX inhibitors, and they have been widely used for cancer prophylaxis; also, epidemiological studies have found them to have chemopreventive potential [13
]. However, several adverse effects, including gastrointestinal bleeding, asthma, hepatic, renal and cardiovascular toxicity have been associated with their long-term usage, and hence their clinical application has been limited [14
]. In this context, dietary bioactive natural products have attracted a great deal of research interest because they inherently show low toxicity and a promising safety profile with no known severe side effects [15
For the prevention of CRC by dietary measures, apiaceous vegetables such as carrots, celery, celeriac, fennel, parsley, and parsnip are highly interesting due to their content of the bioactive polyacetylenic oxylipins falcarinol (FaOH) and falcarindiol (FaDOH) (Figure 1
]. FaOH and FaDOH have shown many interesting bioactivities, including anti-inflammatory, anti-platelet-aggregatory, anti-diabetic, and cytotoxic activity as well as an anti-neoplastic effect [17
]. From in vitro studies, it is clear that FaOH is more cytotoxic than FaDOH and that their cytotoxicity depends on the cell lines [17
]. In addition, it has been demonstrated that FaOH inhibits the growth of the human epithelial colorectal adenocarcinoma cell line Caco-2 in vitro, and that this effect is enhanced synergistically when combined with FaDOH in ratios of 1:1, 1:5 or 1:10 [27
]. Furthermore, it has been shown that FaOH and FaDOH can lead to cell cycle arrest and apoptosis of cancer cells [25
]. The exact mechanisms of action for the anti-proliferative effect of FaOH and FaDOH in adenoma/cancer cells are unknown, but may be due to their alkylating properties, which can lead to the inhibition of pro-inflammatory markers, enzymes, and inflammatory transcription factors via covalent alkylation [30
]. The synergistic anti-proliferative effect of FaOH and FaDOH also seem to exist in vivo as recently demonstrated in a cancer primed rat model for CRC where a diet containing 7 μg FaOH and 7 μg FaDOH g−1
feed was shown to significantly inhibit neoplastic transformations in the colon epithelium [32
]. The amounts of FaOH and FaDOH used in this rat study correspond to a daily human intake of 250–300 g carrots and indicates that apiaceous vegetables could have a preventive effect against CRC.
FaDOH is an effective inhibitor of COX-1 and COX-2 in vitro, whereas the COX inhibitory activity of FaOH is less pronounced [21
]. In addition, FaOH and FaDOH have been shown to inhibit the formation of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNFα), and hence, the activation of their upstream nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway [19
]. This signaling pathway is crucial for neoplastic transformation and promotion [36
]. FaOH and FaDOH are also strong inhibitors of lipoxygenases that are involved in tumor-progression and activation of NF-κB [20
]. Thus, we hypothesized that a likely mechanism of action for the preventive effect of FaOH and FaDOH on colorectal precancerous lesions is mainly due to their anti-inflammatory activity and that this effect is dose-dependent.
In this study, we show that FaOH and FaDOH have an inhibitory effect on certain inflammatory markers in neoplastic lesions and that a possible mechanism of action in relation to CRC prophylactics could be as selective COX-2 inhibitors. Furthermore, we demonstrate that FaOH and FaDOH prevent the development of early neoplastic ACFs and neoplastic polyp lesions in the colorectal intestine in a dose-response relationship.
AOM is a potent carcinogen that has been shown to be an efficient inducer of ACF and other precancerous lesions as well as CRC in rats [32
]. Only a small number of early neoplastic ACF lesions will develop to neoplastic polyp lesions and only a small number of these lesions will have the potential to develop into adenomas and cancers [42
]. In the AOM-induced rat model, the AOM enters the circulation and is metabolized in the liver, and then it is mixed with the feed in the duodenum and transported to the large intestine where the metabolized carcinogen induces neoplastic lesions in the colon of the rats [32
]. The cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase 1 metabolism of which cytochrome P450 2E1 (CYP2E1) metabolizes many xenobiotics and procarcinogens, and thus they play an important role in the activation of AOM in vivo [45
]. Compounds that inhibit or suppress CYP2E1 activity affect AOM metabolism and hence may prevent hypermethylation and carcinogenesis of intestinal cells, and thereby early neoplastic ACF formation as well as other neoplasms [32
]. Since administration of FaOH and FaDOH in the present study started two weeks before the first carcinogen dose and continued during the carcinogen administration phase, one mechanism of action for the chemopreventive effect of FaOH and FaDOH could be an inhibition or suppression of AOM metabolism. However, the protective role of these dietary polyacetylenes against xenobiotics and procarcinogens are not due to inhibition or suppression of CYP2E1 activity and other phase 1 enzymes as demonstrated in carbon tetrachloride-induced hepatotoxicity in rodents [46
]. In fact, FaOH and FaDOH seem to exert their chemopreventive protection against carcinogens and other toxic compounds through the activation of the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor [erythroid-derived 2]-like 2 (Nrf2)/antioxidant response element (ARE) pathway due to their S-alkylating properties of cysteine, thereby inducing the formation of antioxidant and other cytoprotective phase 2 enzymes [47
]. The alkylating properties of these dietary polyacetylenes may not only explain their ability to protect against the formation of carcinogens through the Keap1/Nrf2/ARE pathway but also their role in the inhibition of inflammatory markers of the NF-κB signaling pathway. The latter primarily explain the effect of FaOH and FaDOH on neoplastic lesions and their effect on the development of CRC as discussed below. Furthermore, as described previously, FaOH and FaDOH exhibit a preventive effect on cancer cells grown in vitro. Still, it could be interesting in future investigations to determine more precisely the importance of FaOH and FaDOH in the detoxification of chemical carcinogens via the Keap1/Nrf2/ARE pathway and their overall cancer preventive role.
A reduction in the growth rate of early neoplastic ACF’s and neoplastic polyp lesions is effected after the administration of AOM, when it is no longer present in the rats. Hence, the cancer primed rat model used in this study makes it possible not only to evaluate the chemopreventive effect of FaOH and FaDOH during the carcinogenesis but also their potential as inhibitors of tumor growth. The doses of FaOH and FaDOH used in the rat feed in the present dose-response study corresponds to a realistic daily human intake of carrots, except maybe for the highest concentration of 35 µg FaOH and 35 µg FaDOH g−1
]. The results of this study may therefore be used to design clinical experiments with, for example, food products to determine a possible preventive effect of a dietary intake of FaOH and FaDOH.
The literature on the clinical significance of ACF as precursors of colorectal adenomas and CRC is modest and because ACF are the earliest precursor lesions in colorectal carcinogenesis, it is not likely that all early neoplastic ACF can be used as surrogate markers in CRC chemoprevention trials in humans [40
]. However, in carcinogen-induced rodent carcinogenesis models the frequency of dysplastic ACF is much higher compared to humans, and if dysplastic ACF are precursors of colorectal adenomas or CRC, then the ACF number may be a useful tool to identify promising chemopreventive agents in cancer primed rodent models [43
]. In the present investigation, a correlation appeared to exist between the number of early neoplastic ACFs and the number of neoplastic polyp lesions. The average number of ACF decreased with an increasing dose of FaOH and FaDOH in the diet, and a significant dose-response relation was observed for both ACF < 7 crypts and ACF > 7 crypts, although a further inhibitory effect on the formation of ACFs at the highest doses of FaOH and FaDOH was minimal. On the other hand, the largest effect on the total number of macroscopic polyp neoplasms was observed at the highest doses of FaOH and FaDOH in the feed, which indicates that the anti-neoplastic effect is most significant at these doses. Thus, it appears that an optimal anti-neoplastic effect is achieved with doses between 7–35 µg of FaOH and FaDOH g−1
feed in the cancer primed rat model used in this study. Interestingly, adenomas were in general smaller in the FaOH and FaDOH treated rats, especially at the highest doses, i.e., > 1.4 µg of FaOH and FaDOH g−1
feed, compared to the control group. This is also in accordance with our previous findings [32
]. These results not only demonstrate a clear dose-dependent chemopreventive effect of FaOH and FaDOH on the formation of colorectal early neoplastic ACF and neoplastic polyp lesions, but also indicate that these dietary polyacetylenic oxylipins may to some extent exert a growth inhibition of neoplastic lesions, as they not only reduce the number of macroscopic polyp neoplasms but also their size.
Chronic inflammation is believed to play an important role in the early stage of malignant transformation, including CRC [52
]. The use of NSAIDs, such as aspirin, reduces the overall number and size of adenomas in patients by inhibition of COX-1 and COX-2 [53
]. Furthermore, healthy individuals using NSAIDs regularly reduce their risk of developing colorectal cancers by 40% to 50% [12
]. Indeed, the accumulating clinical and experimental evidence now supports a potent anti-tumorigenic efficacy of inhibiting COX-2 with NSAIDs as a chemotherapeutic strategy for CRC [10
]. From studies in cell cultures, it is known that FaOH and FaDOH are able to inhibit pro-inflammatory cytokines and enzymes such as COX, as described in the introduction. These downstream inflammatory markers therefore seem to constitute a likely explanation for the anti-neoplastic effects of FaOH and FaDOH. However, it cannot be excluded that FaOH and FaDOH have an inhibitory effect on other inflammatory markers not included in the present investigation. Gene expression studies and immunohistochemical analysis showed, as expected, an upregulation of COX-2 in the neoplastic tissue [10
] whereas COX-1 was not significantly affected. However, a significant downregulation of COX-2 in adenomas from rats receiving SRD supplemented with FaOH and FaDOH compared to adenomas of the control group, implicate the involvement of COX-2 in CRC development and as an important target for the chemopreventive effects of these polyacetylenes. Based on the results of this study, it appears that FaOH and FaDOH in combination act as selective COX-2 inhibitors.
The NF-κB signaling pathway regulates the immune response and inflammation, which has also been implicated in carcinogenesis and is crucial for neoplastic transformation and promotion as it is the main regulator of the pro-inflammatory cascade, including the expression and formation of COX-2 [54
]. As expected, the gene expression of NF-κB was significantly downregulated in tumor tissue in rats receiving SRD supplemented with FaOH and FaDOH compared to the control group that only received the SRD. This further confirms that inflammation plays a key role in early neoplastic formation and that the chemopreventive effect of FaOH and FaDOH in the colon is linked to their anti-inflammatory activity. Further evidence for the anti-inflammatory action of FaOH and FaDOH was confirmed by the gene expression of TNFα and IL-6, which are members of the pro-inflammatory cytokine cascade and have been implicated in carcinogenesis [55
]. Increased TNFα levels are linked to increased leukocyte infiltration and tumor formation and upregulation of TNFα levels are usually detected in colorectal neoplasms, and in animal models of CRC [55
]. However, gene expression of TNFα was significantly downregulated in rats receiving SRD supplemented with FaOH and FaDOH compared to the control group, which indicates that these polyacetylenes exert an effect on the regulation of this pro-inflammatory biomarker. IL-6 is highly upregulated in many cancers and is considered as one of the most important pro-inflammatory cytokines during tumorigenesis and metastasis [57
]. IL-6 was significantly downregulated in rats receiving FaOH and FaDOH in the diet compared to the control group, which again confirmed the anti-inflammatory effect of FaOH and FaDOH in the NF-κB signaling pathway, and thus the downregulation of COX-2 formation.
Epithelial IL-1β is known to be regulated by NF-κB but may also be induced by DNA damage via a NF-κB-independent mechanism leading to mucosal inflammation in the gut [58
]. In the present study, FaOH and FaDOH showed no effect on the gene expression of IL-1β in neither healthy nor tumor tissue, which indicates that cytokines such as TNFα and IL-6, rather than IL-1β, play a role in the regulation of COX-2 in neoplastic tissue in this rat model.
PPARγ is an essential nuclear receptor controlling the expression of a large number of regulatory genes in lipid metabolism, insulin sensitization, inflammation, and in cell proliferation. The isoform PPARγ2 is mostly found in adipose tissue and the intestine and is highly expressed in these tissues [59
]. In the colon, PPARγ play a key role in the control of intestinal inflammation such as ulcerative colitis [59
]. In addition, PPARγ has been found to have an anti-neoplastic property as it can induce apoptosis and differentiation of colon cancer cells both in vivo and in vitro [60
]. Thus, PPARγ could play a significant role in the development of CRC. The regulation of PPARγ expression in the colon is unresolved but PPARγ expression may be upregulated by intestinal-microbial interactions involving lipopolysaccharides of Gram-negative bacteria and/or ligands of PPARγ [60
]. In the present study, PPARγ expression was significantly downregulated in rats receiving FaOH and FaDOH in the diet compared to the control group, which indicates that PPARγ is not an important target for the anti-neoplastic effect of these polyacetylenic oxylipins. FaOH and FaDOH have previously been shown to act as partial PPARγ agonists, which seems to explain their anti-diabetic properties [22
]; thus, an increased expression of PPARγ in epithelial cells would have been expected in rats receiving FaOH and FaDOH in the diet. However, recent research has also shown that FaOH and FaDOH alter the gut microbiota composition in the AOM-induced rat model [61
], which could be the main reason for the downregulated expression of PPARγ observed in rats receiving FaOH and FaDOH in the diet.