Cancer is a genetic disorder characterized by an altered balance between proliferation and mechanisms of cell death. Colorectal cancer (CRC) worldwide ranks third in terms of incidence, but second in terms of mortality. Most CRC risk factors are related to dietary and lifestyle factors, which are increased after high meat consumption due to the stimulation of insulin secretion, and increased fat intake. Carcinogenesis caused by insulin resistance leads to increased cell proliferation and reduced apoptosis [1
Recently, the evidence highlights the insulin/IGF system as an important molecular target for the pathogenesis, progression and treatment of CRC. Among the important components in CRC, the IGF-1 factor and its receptor (IGF-1R) promote both the growth and malignant transformation of adenomatous polyps. IGF-1 factor is found to be more highly expressed in adenocarcinomas compared to adenomas and normal colon samples from patients, while IGFBP3 is lower in patients than in healthy individuals [2
]. This strongly suggests a positive and a negative association of these two proteins with CRC risk, respectively. Moreover, IGF-1R is frequently overexpressed in cancer cells, which inhibits apoptosis and enhances cell cycle progression [4
]. Overall, the levels of these IGF system components might play a central role in cell-cycle progression, differentiation, and proliferation in CRC. However, the underlying pathophysiological links are still barely understood, and the molecular mechanisms still remain unsolved.
The dietary bioactive compound lycopene (LYC), an acyclic isomer of β-carotene found mainly in tomatoes, exerts excellent antioxidant, singlet oxygen quencher and anti-inflammatory properties, and it has been found to be protective against different types of cancer in animal models and epidemiological interventions, with minimal to no toxic effects along with its pleiotropic effects. LYC has promising chemopreventive effects on CRC by modulating IGF-1 system components through direct and indirect actions on pathways such as the Ras/MAPK and PI3K/AKT/Wnt signaling pathways [2
]. Increasing evidence from epidemiological studies and in vivo models suggests that probiotics, prebiotics and their combination with bioactive compounds might modulate host resistance against intestinal infections. Prebiotics are non-digestible food carbohydrates that selectively stimulate the growth of probiotics in the colon and potentiate the beneficial effects of these microorganisms providing protective effects against colon cancer development [5
]. In this regard, the consumption of probiotics such as Bifidobacterium longum
(BF) has been suggested.
The anticarcinogenic mechanisms associated with the administration of B. longum
include: control of cell growth and differentiation, fermentation of non-digestible carbohydrates to produce short-chain fatty acids, modulation of the gut microbiome, reduction of pH caused by the excessive presence of bile acids in feces, inhibition of colon carcinoma cell proliferation as well as induction of apoptosis in colon carcinoma cells [9
]. A daily intake of at least 1 × 107
viable cells has been suggested as the minimum intake to provide a protective effect. However, several factors have been claimed to affect the viability of probiotics, including heat processing and gastrointestinal conditions [11
]. Due to this, microencapsulation has been developed to protect the cells in order to ensure their effectiveness and ability to induce beneficial effects in the host [13
]. The spray drying encapsulation technique is suitable for heat-sensitive materials, and it gives satisfactory results with minimal loss of viability [14
]. Although there are previous studies on the microencapsulation of lactic acid bacteria [15
], there is no information from in vivo studies regarding the use of mixtures of anionic polysaccharides as encapsulating agents, used independently without the need to be incorporated into food systems, in addition to the evaluation of the beneficial effects of combined strategies using chemopreventive agents and lactic acid bacteria on carcinogenesis. Therefore, the aim of this work was to evaluate the chemopreventive efficacy of a thermomechanically suitable microencapsulation system for BF using spray drying and its evaluation along the gastrointestinal tract (GIT) co-administrated with LYC, through the expression and modulation of IGF-1/IGF-1R/IGFPs system components and its relationship with intestinal parameters related to the specific colon carcinogenesis azoxymethane (AOM)-dextran sulfate sodium (DSS) model.
Bifidobacteria are extensively used as probiotics. Alternatively, lycopene (LYC) is a carotenoid that selectively stimulates the growth of probiotics in the GIT and potentiates the beneficial effects of these microorganisms [5
]. Using an AOM + DSS model, we investigated a combined probiotic BF + LYC supplementation strategy in the development of CRC by providing thermomechanically enhanced, microencapsulated BF during a 16-week experimental period.
Several factors have been claimed to affect the viability of probiotics, such as gastrointestinal conditions [11
]. Therefore, microencapsulated BF detectable viability in fecal samples during the experimental period was evaluated by using BSM as selective enumeration of Bifidobacteria. However, the levels of BF in all BF- and LYC-treated groups were significantly reduced at the 5th week after the administration of AOM and DSS probably due to the association between the disturbance of gut ecology and the induction of inflammation by DSS-treatment did not promote colonization of the murine gastrointestinal tract (GIT) by BF from the 5th week. The above may be associated to disruption of the colonic epithelial barrier, ulcerations and heavy infiltration of inflammatory cells into the mucosa with changes in the bile acid enterohepatic circulation and alterations (adaptation or imbalance) in the bacterial flora caused by DSS-treatment [23
]. Despite its minor colonization of the mouse GIT in the first 4 weeks, BF displayed a protective effect on colorectal cancer when administered daily, maintaining a stable count of BF from the 5th to 16th weeks without statistical difference in each group, according to the analysis by Tukey test at <0.05 (Table S1
), confirming that a constant dosage of microencapsulated BF (8.992 × 1010
) might exert protection in a CRC model. Conversely, Singh and collaborators [25
] reported a significant decrease in a Bifidobacterium
strain of two Log cell counts/g feces in only 24 h after the intragastric administration at day 14 under healthy conditions.
The adhesion to intestinal epithelial cells and mucus as a feature that supports the colonization and persistence of Bifidobacteria in the GIT; cell surface components that promote colonization and adhesion to the intestinal epithelium include sortase-dependent proteins, exopolysaccharides and lipoproteins, as well as the pili of Bifidobacteria, wich were shown to modulate immune responses. Another aspect contributing to the high or low ability of BF to stably colonize mice might be a better adaptation to its original habitat, i.e., the human infant gut, due to the fact that host adaptation of bifidobacteria has been shown on the level of carbohydrate utilization. BF has the capacity to utilize plant oligo- and polysaccharides that are derived from the diet of the host [24
]. As expected, the preferential colonization site of Bifidobacterium strains was in the caecum and colon, with a higher bacterial concentration in the luminal zone [26
]. In this regard, our results showed a negative correlation between the β-GA, pH and the bacterial colonization; therefore, the overactivity of the β-GA enzyme and basicity of pH contribute to a lesser bacterial colonization in the luminal zone (p
< 0.05), while in the tissue-adherent zone, the bacterial colonization was only negatively correlated with the overactivity of β-GA in the caecum, colon, and feces. Our results agree with the anticarcinogenic mechanisms associated to the administration of BF [9
]. However, this is the first report to demonstrate that LYC- and Metformin-treatments decrease colonic fecal pH and β-GA values in mouse colon carcinogenesis, thus confirming their application as therapeutic intestinal targets in the AOM-DSS model.
In this study, tumor (macroscopic quantitative evaluation) and adenocarcinoma incidence (histopathological classification) induced by AOM + DSS were significantly correlated (p
< 0.05), thus confirming that these circumscribed masses of cells that project above the surface (polyps) might develop to colorectal carcinomas [23
]. Although colitis was practically absent from AOM + DSS-treated mice after 16 weeks of treatment, we still observed higher inflammation grade in colonic samples from all BF- and LYC-treated groups (Table 3
). Therefore, we pursued for evaluating whether combined probiotic BF + LYC supplementation strategy could modulate the presence of resident inflammatory cells and others inflammatory scores, and found an improvement in BF- and Metformin-treated groups. This is of major relevance as Tanaka and collaborators [27
] have showed that the number of inflammatory cells infiltrated in the colonic neoplasms and lamina propia mucosa, as well as, crypt abscess formation suggest that, these analyses are statistical correlated with the development from premalignancy to malignancy in the colon with inflammation. Similarly, Doulberis and collaborators [28
] observed that mice did not show clinical signs of colitis after 3.5 months of experimental period in BALB/cJ mice treated with AOM and DSS (1%, three cycles), but an increased number of inflammatory cells persist in the colon of mice even at 3.5 months after the episodes of colitis induced by DSS. This finding is consistent with our observation that at the end of the 16-weeks of experimental period, mice in BF- and LYC-treated groups had still had resident inflammatory cells. Despite this observation, our results suggest that BF and metformin administration were effective in reducing number of tumors, the composition of resident inflammatory cells and other inflammatory scores, and the incidence of adenocarcinomas in the colon of AOM + DSS treated mice.
The protective role of LYC in the prevention of chronic diseases including cancer has been previously reported [6
], and its co-administration with B. lactis
+ oligofructose/inulin showed a synergistic effect on the initiation phase of 1,2-Dimethylhydrazine (DMH)-induced colon carcinogenesis (total number of Aberrant crypt foci (ACF)) by significantly increasing apoptosis as compared to LYC-, oligofructose + B. lactis
- and DMH-treated groups [5
]. However, it has been previously concluded that dietary LYC is absorbed and then distributed to various tissues, including liver, lung and adipose tissue, thus, deviating its protective role against the development of early and protuberant lesions of the colon [8
]. This finding is consistent with our observation that at the end of the 16-weeks of experimental period, LYC co-administration did not further improve the protective effect of microencapsulated BF. On the contrary, metformin administration was effective in reducing number of tumors, the composition of resident inflammatory cells and other inflammatory scores, and the incidence of adenocarcinomas in the colon of AOM + DSS treated mice after 16-weeks. Similarly, the chemopreventive effect of metformin has been previously demonstrated [22
] and the effects of IGFBP3 knockout and metformin were observed in a murine model of ulcerative colitis, showing significantly reduced colitis [30
New evidence has demonstrated that the overexpressions of IGF-1 and IGF-1R contribute to the development and progression of colon cancer in patients [4
] and in an AOM-DSS-induced colorectal carcinogenesis model [31
]. We also found that IGF-1 and IGF-1R expressions correlated with tumor and adenocarcinoma incidence, as well as caecal and fecal pH and β-GA values (p
< 0.05). However, a negative correlation between IGF-1 and IGF-1R expressions and caecal/colonic-adherent and fecal viable bacteria was observed (p
< 0.05) (Table S2
); thus, the significant protection against adenocarcinoma development observed in BF-treated groups is confirmed. The lower positive rates of IGF-1 in the BF + LYC-treated (up to 50%) and Metformin-treated groups (by 67%) might be of major relevance in our study, since in a previous study, the incidence of tumor growth on the caecum was significantly lower in liver-specific IGF-1-deficient mice orthotopically transplanted with colon 38 adenocarcinoma tissue fragments [32
], in which serum IGF-1 levels were 25% of those in control mice.
Because it is currently well established that IGF-1 is mostly bound to IGFBP3, the finding that the positive rate of IGFBP3 was lower in the AOM + DSS control group as compared to the Normal group, while IGF-1 expression was higher in colon samples from the AOM + DSS control group than in animals of the Normal group, confirms the positive and negative correlations (p
< 0.05) (Table S2
), respectively, of these two proteins. Our findings agree with those previously observed in patients with colon cancer [4
]. Conversely, IGF2BP1 and IGFBP2 expressions significantly correlated with IGF-1 and IGF-2 expressions and adenocarcinoma incidence (p
< 0.05) (Table S2
Similar to the higher IGF-2 and IGFBP2 expressions in the AOM + DSS control group, mean IGFBP2 SD scores and expressions were significantly higher in sera and tumors from patients with colonic neoplasia compared to healthy individuals, whereas mean IGF-2 SDs were elevated in Dukes A and Dukes B cases compared with controls, but not in advanced disease [34
As an effective strategy for colon cancer chemoprevention, is worth mentioning that the BF + LYC 20 treatment group showed the best similarity to the Normal group (p
> 0.05) and a relevant fecal BF viability during the 16 weeks of treatment. Additionally, the preferential colonization site of BF was in the caecum and colon, with a higher bacterial concentration in the luminal zone. Moreover, this group was shown to exert beneficial changes in colonic physiology by decreasing caecal, colonic, and fecal pH, along with β-GA activity, and showing lower inflammation grade, local lymphocyte infiltration, and the suppression of adenocarcinoma incidence compared to those of the AOM + DSS control group. Therefore, it has become clear that the modulation of gut microbiome is likely to be the key element of the health-promoting activity of BF in the colon. Our results, with a low concentration of lycopene, are in agreement with Amir and collaborators [36
], who showed a synergistic suppression of HL-60 (promyelocytic leukemia cells) cell growth with a combination of low doses of lycopene and retinoic acid; their results are directly associated with the additive inhibition of cell cycle progression through the G1 phase. Several investigations [37
] have reported that the absorption of lycopene appears to be more efficient at lower doses due to a complex process involving release from the food matrix, dissolution into mixed chylomicrons, uptake by the liver, distribution to the tissues and secretion into very low-density lipoproteins. In addition, lycopene tends to lose its ability to reduce oxidative damage and exert anticancer effects at higher concentrations. Also, high concentrations of one carotenoid may interfere with the bioavailability of others, leading to imbalance, as occurs between β-carotene and lycopene [39
]. Although the mechanism of such a response is unclear, lycopene is presumed to be toxic at higher doses.
However, having mentioned that the underlying pathophysiological links with the IGF-1/IGF-1R/IGFPs system are still barely understood and the molecular mechanisms still remain unsolved [4
], the BF + LYC 20 supplementation showed strongly decreased expressions of IGF-1, IGFBP2, and IGF-1R; while the IGFBP3 protein expression increased in the colon after AOM + DSS treatment (Figure 4
), which has not been previously reported with a new form of as BF microencapsulated using the spray drying technique. In the absence of IGFBP3, the enhanced bioactivity of IGF-1/2 and binding to IGFBP2 might lead to an increase in epithelial proliferation and repair of the mucosal barrier, thereby decreasing DSS-induced inflammation [3
]. Due to this, restored IGFBP3 expression levels in BF + LYC-treated groups might lead to binding with high affinity to IGF-1 and then subsequent activation of the signaling pathways by IGF-2 bound to IGFBP2, but at a minor rate. These restored expression levels operating as positive regulators of IGF activity, such as stimulation of the cell cycle and induced apoptosis, thereby inhibiting colon carcinogenesis through the modulation of the PI3K/AKT and MAPK pathways [3
In all studied groups, LYC supplementation did not impede the beneficial actions of microencapsulated BF alone. Although inflammatory cells still persist in the colon of mice even at 16 weeks after the DSS-induced promotion of AOM-initiated carcinogenesis, BF and metformin administration were effective in reducing number of tumors, the composition of resident inflammatory cells, other inflammatory scores, and the incidence of adenocarcinomas in the colon of AOM + DSS treated mice. In our study, we could suggest that the DSS-induced inflammation does not favor the evolution to adenocarcinomas in BF- and Metformin-treated groups and the modulation of the expression of IGF-1/IGF-1R/IGFBP3 protein might be involved in the suppressive mechanisms underlying the evolution to adenocarcinomas in the AOM + DSS model of colon carcinogenesis.