**3.3. Inhibition on colon cancer by probiotics and the possible action mechanisms of these microorganisms**

The evidence pointing to the beneficial effects of probiotics on colon cancer comes from *in vitro* tests, experiments with animals and clinical trials. Additionally, these has been much discussed on which step in the process of carcinogenesis might the effect by probiotics. It is likely that different probiotic strains act on different stages of carcinogenesis [20].

In general, the probiotics do not colonize the human gut, but some strains are can permanently colonize the indigenous microbiota [61].

The mechanisms by which probiotics may inhibit colon cancer are not yet fully characterized. However, several explanations have been suggested including: alteration of the metabolic activities of the intestinal microbiota; quantitative and qualitative changes in the intestinal microbial compositin; alteration of physicochemical conditions in the colon; binding and/or degradation of potential carcinogens; SCFA production; production of antitumorigenic or anti-mutagenic compounds; modulation of hosts's immune response, and/or physiology [3,62, 63].

Probiotics may modulate the metabolic activities of the intestinal microbiota by three possible mechanisms: competing with and displacing other components of the microbiota; producing antibacterial substances, including bateriocins, to control the growth of other members of the microbiota; producing lactic and other organic acids, which might lower the luminal pH and thus modulate enzyme activity [20,64].

Several investigations have shown that probiotics can influence bacterial enzymes activity related to the production of carcinogenic compounds, such as beta-glucuronidase, nitroreductase and azoreductase [65, 66, 67].

Bacterial glucuronidase appears to have an important role in the initiation of colon cancer, due to its ability to hydrolyze several glucuronides and carcinogenic aglycones in the intestinal lumen [65,68]. The nitroreductase and azoreductase take past in to the formation of aromatic amines harmful to the body [69].

224 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

nutritional studies [58, 59].

**of these microorganisms** 

physiology [3,62, 63].

reproduced in the TIM-1 small intestine model include bile secretion, motility, pH and absorption capacity of the upper intestine. Proximal colon simulator models such as TIM-2 include other host functions such as peristaltic mixing and water and metabolite absorption. The combination of TIM-1 and TIM-2 models led to the creation of an artificial digestive system which has been used to investigate pharmaceutical drug delivery and advanced

The use of a multidisciplinary biological systems approach, in combination with '-omics' platforms as outlined will facilitate the most advanced system for unraveling the complex

In vitro fermentation models are an innovative technological platform where the greatest advantages are exhibited by the virtually limitless experimental capacity as experimentation is not restricted by ethical concerns. Host intestinal function is only partially simulated in some model designs (e.g. TIM-1 and TIM-2) and together with microbial population

**3.3. Inhibition on colon cancer by probiotics and the possible action mechanisms** 

The evidence pointing to the beneficial effects of probiotics on colon cancer comes from *in vitro* tests, experiments with animals and clinical trials. Additionally, these has been much discussed on which step in the process of carcinogenesis might the effect by probiotics. It is

In general, the probiotics do not colonize the human gut, but some strains are can

The mechanisms by which probiotics may inhibit colon cancer are not yet fully characterized. However, several explanations have been suggested including: alteration of the metabolic activities of the intestinal microbiota; quantitative and qualitative changes in the intestinal microbial compositin; alteration of physicochemical conditions in the colon; binding and/or degradation of potential carcinogens; SCFA production; production of antitumorigenic or anti-mutagenic compounds; modulation of hosts's immune response, and/or

Probiotics may modulate the metabolic activities of the intestinal microbiota by three possible mechanisms: competing with and displacing other components of the microbiota; producing antibacterial substances, including bateriocins, to control the growth of other members of the microbiota; producing lactic and other organic acids, which might lower the

Several investigations have shown that probiotics can influence bacterial enzymes activity related to the production of carcinogenic compounds, such as beta-glucuronidase,

likely that different probiotic strains act on different stages of carcinogenesis [20].

permanently colonize the indigenous microbiota [61].

luminal pH and thus modulate enzyme activity [20,64].

nitroreductase and azoreductase [65, 66, 67].

microbial and host factors governing human gut microbiota functionality [60].

balancing remains a major challenge of in vitro gut fermentation modeling.

Both harmful and beneficial bacteria are commonly found in the intestines and differ in their enzymatic activity [70]. In general, bacteria from the genera *Bifidobacterium* and *Lactobacillus* produced a very little activity of enzymes that convert pro-carcinogens into carcinogens, compared with bacteria from the genera *Bacteroides* and *Clostridium* [71]. Therefore, the activities of these enzymes in the lumen might be correlated with the number of lactic acid bacteria (LAB) in the intestine [72]. This suggests that increasing the proportion of LAB in the gut could diminish the levels of xenobiotic metabolizing enzymes [71]. Thus, the effect of probiotic microorganisms on fecal enzyme activities might be explained by this mechanism.

In a preliminary study, on feces of small animal, the animal supplementation of a high cholesterol diet with a mixture of probiotic strains of *L. johnsonii* and *L. reuteri* for 5 weeks significantly decreased the activity of fecal-glucuronidase and azoreductase [67].

Gorbach and Goldin [65] studied, in humans, the effect of ingestion of *L. acidophilus* NCFM strains about the activity of-glucuronidase, nitroreductase and azoreductase. Both strains had a similar effect and caused a significant decline in the activity of these three enzymes. A reverse effect was found 10 to 30 days after the end of the intake of these bacteria, suggesting that continuous consumption of *L. acidophilus* is necessary for maintaining.

Benno and Mitsuoka [73] and Spanhaak et al. [66] also found in humans, a significant reduction in the activity glucuronidase after intake of *Bifidobacterium longum* and *L casei* Shirota, respectively. On the other hand, Marteau et al. [74] verified in healthy volunteers that the regular consumption of a fermented dairy product (100 g three times per day) containing *L. acidophilus*, *B. bifidum*, *Streptococcus thermophilus* and *S. cremoris* for 3 weeks decreased the feces nitroreductase activity from baseline but not that of β-glucuronidase or azoreductase.

Feces metabolites are also indicators of bacterial activity. Changes in enzyme activities and the concentration of ammonia, phenol and cresol have been detected in volunteers who consumed Lactobacilli [65]. Other metabolites with possible adverse effects are N-nitroso compounds, diacylglycerol and secondary bile acids [49].

A wide variety of microrganisms can produce ammonia, for example, enterobacteria, bacteroides and clostridia. Ammonia is considered a potential promoter of tumor in the colon and it can increase the rate of neoplastic transformation in the intestine. According to Benno and Mitsuoka [73], reducing the proportion of clostridia and bacteroides could explain the decrease in the concentration of ammonia in individuals who consumed fecal *B. longum.*

Epidemiological studies indicate an association between the risk of developing colon cancer and the consumption of high fat diets [7,75, 76]. For the digestion of fats, bile acids

conjugated to glycine or taurine molecules are released into the small intestine and reabsorbed in the same location. It is believed that the deoxycholic acids may be cytotoxic to the epithelial cells, which could lead to the development of colon cancer [71]. Probiotic modulation of the intestinal microbiota may affect the activity of one of the enzymes (7adehydroxylase) forming these toxic products, but probiotics may also reduce the toxicity of bile salts that bind to them [77]. Lidbeck et al. [68] found that administering *L. acidophilus* to colon cancer patients for 6 weeks resulted in reduction in the concentration of soluble bile acids in the stool.

Probiotics and Intestinal Microbiota: Implications in Colon Cancer Prevention 227

proliferation. These mechanisms may play an important role in the reduction and/or

Studies show that the LAB may be involved in the detoxification of various carcinogens such as polycyclic aromatic hydrocarbons and heterocyclic aromatic amines [86]. The mechanisms of action of these bacteria are poorly known, but it is possible that the LAB bind directly to the carcinogen and catalyze detoxification reactions [62]. It is worth noting that the protective effects conferred by LAB only appear when these are at a high density

Evidence is accumulating that heterocyclic aromatic amines (HCAs), which are derived from amino acids in meat during cooking, might be involved in the etiology of human cancer [88]. Zsivkovits et al. [89] showed that *L. bulgaricus* 291, *S. thermophilus* F4, *S. thermophilus* V3 and *B. longum* BB536 are highly protective against the genotoxic effects of HCAs in rats. Additionally, the inhibition of HCAs induced DNA damage was dose dependent and significant when 1 x 107 cells/animal were administered. Other authors showed that *L. casei* DN 114001 may metabolize or adsorb HCAs and reduce their

*In vivo* evidence that probiotics bond the carcinogens are still not conclusive. Hayatsu Hayatsu (1993) demonstrated the marked suppressive effect of orally administered *L. casei Shirota* (LcS) on the urinary mutagenicity arising from ingestion of fried ground beef by humans. In another clinical trial, the consumption of *L. acidophilus* decreased the urinary and fecal excretion of mutagens [68]. In view of the *in vitro* results, it is possible that the LAB supplements are inuencing excretion of mutagens by simply binding them in the intestine [62]. Even though the binding of carcinogens is a possible mechanism for the inhibition of genotoxicity and mutagenicity by LAB in vitro, some researchers have reported that it does not appear to have any influence *in vivo* [90]. Additionally, the extent of the binding

Several studies have also reported the effect of probiotics on the promotion phase of carcinogenesis. Rowland et al. [91] found that administration of *B. longum* (6 x 109 CFU/day) inhibited the formation of aberrant crypt foci (ACF) in rats that received an induced of carcinogenesis (azomethane). As the probiotic treatment began 1 week after exposure to the carcinogen, these results indicate an effect on the early promotional phase of carcinogenesis

Goldin et al. [92] observed a lower incidence of colonic tumors in rats who consumed *Lactobacillu*s GG before, during and after chemical induction with dimethylhydrazine (DMH) than in animals that were fed the probiotic after receiving carcinogen. The researchers concluded that probiotics acted by inhibiting the initiation stage of

Kumar et al. [93] tested the efficacy of *L. plantarum* AS1 in the suppression of colorectal cancer induced by DMH in rats and formed that AS1 was capable of diminishing colon

inhibition of promotion and progression of cancer [1, 85].

depends on the mutagen and bacterial strain used [71].

and when there is a regular intake [87].

genotoxicity *in vitro* [89].

[71].

carcinogenesis.

The consumption of fermented milk containing *L. acidophilus* may reduce the population of harmful bacteria, such as coliforms, and increased levels of lactobacilli in the intestine [78], suggesting that supplementation with this microorganism can have a beneficial effect since it inhibits the growth of bacteria that harmful are possibly involved in the production of tumor promoters and pro-carcinogens. Savard et al. [79] assessed the impact of four week's consumption of commercial yoghurt with *Bifidobacterium animalis* subsp. lactis (BB-12) and *Lactobacillus acidophilus* (LA-5) on fecal bacterial counts in healthy adults. The yoghurt had a positive effect on the bacterial population in that a the increase in beneficial bacteria and the reduction of potentially pathogenic bacteria was observed.

Not all studies show a correlation between the administration of probiotics and the activity of intestinal microbiota. Bartram et al. [80] argued that the fecal microbiota is relatively stable and generally unaffected by the administration of probiotics. In an intervention study, 12 individuals consumed yogurt (500 mL) enriched with *B. longum*. No significant difference was found in fecal weight, pH, concentration of fecal short chain fatty acids, bile acids and neutral sterols after 3 weeks of intervention. Despite the rise in the fecal concentration of *B. longum*, the results suggested litlle or no modulation of resident microbiota.

Some researchers have suggested that a high intestinal pH may be related to increased risk of colon cancer, whereas acidification of the colon could prevent the formation of carcinogens. Benno and Mitsuoka [73] found a significant reduction of faecal pH in health men who ingested *B. longum* for 5 weeks.

Evidence indicates that a high concentration of short chain fatty acids (acetate, propionate and butyrate) can assist in maintaining an appropriate pH in the lumen of the colon for the expression of many bacterial enzymes that probably metabolize carcinogens in the gut [81]. The activity of some dietary carcinogens, such as nitrosamines (resulting from commensal bacterial metabolic activity in individuals who consume a diet rich in proteins) can be neutralized by butyric acid produced by some probiotics [82]. Furthermore, production of ammonia, nitrosamines and secondary bile acids in the intestinal environment can be reduced by lowering the pH [83].

Butyrate, particularly, has received much attention as a potential chemopreventive agent [1,84]. While acting as an energy source for untransformed cells, butyrate possibly reduces survival of tumor cells by inducing apoptosis and differentiation, as well as by inhibiting proliferation. These mechanisms may play an important role in the reduction and/or inhibition of promotion and progression of cancer [1, 85].

226 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

reduction of potentially pathogenic bacteria was observed.

men who ingested *B. longum* for 5 weeks.

reduced by lowering the pH [83].

acids in the stool.

conjugated to glycine or taurine molecules are released into the small intestine and reabsorbed in the same location. It is believed that the deoxycholic acids may be cytotoxic to the epithelial cells, which could lead to the development of colon cancer [71]. Probiotic modulation of the intestinal microbiota may affect the activity of one of the enzymes (7adehydroxylase) forming these toxic products, but probiotics may also reduce the toxicity of bile salts that bind to them [77]. Lidbeck et al. [68] found that administering *L. acidophilus* to colon cancer patients for 6 weeks resulted in reduction in the concentration of soluble bile

The consumption of fermented milk containing *L. acidophilus* may reduce the population of harmful bacteria, such as coliforms, and increased levels of lactobacilli in the intestine [78], suggesting that supplementation with this microorganism can have a beneficial effect since it inhibits the growth of bacteria that harmful are possibly involved in the production of tumor promoters and pro-carcinogens. Savard et al. [79] assessed the impact of four week's consumption of commercial yoghurt with *Bifidobacterium animalis* subsp. lactis (BB-12) and *Lactobacillus acidophilus* (LA-5) on fecal bacterial counts in healthy adults. The yoghurt had a positive effect on the bacterial population in that a the increase in beneficial bacteria and the

Not all studies show a correlation between the administration of probiotics and the activity of intestinal microbiota. Bartram et al. [80] argued that the fecal microbiota is relatively stable and generally unaffected by the administration of probiotics. In an intervention study, 12 individuals consumed yogurt (500 mL) enriched with *B. longum*. No significant difference was found in fecal weight, pH, concentration of fecal short chain fatty acids, bile acids and neutral sterols after 3 weeks of intervention. Despite the rise in the fecal concentration of *B.* 

Some researchers have suggested that a high intestinal pH may be related to increased risk of colon cancer, whereas acidification of the colon could prevent the formation of carcinogens. Benno and Mitsuoka [73] found a significant reduction of faecal pH in health

Evidence indicates that a high concentration of short chain fatty acids (acetate, propionate and butyrate) can assist in maintaining an appropriate pH in the lumen of the colon for the expression of many bacterial enzymes that probably metabolize carcinogens in the gut [81]. The activity of some dietary carcinogens, such as nitrosamines (resulting from commensal bacterial metabolic activity in individuals who consume a diet rich in proteins) can be neutralized by butyric acid produced by some probiotics [82]. Furthermore, production of ammonia, nitrosamines and secondary bile acids in the intestinal environment can be

Butyrate, particularly, has received much attention as a potential chemopreventive agent [1,84]. While acting as an energy source for untransformed cells, butyrate possibly reduces survival of tumor cells by inducing apoptosis and differentiation, as well as by inhibiting

*longum*, the results suggested litlle or no modulation of resident microbiota.

Studies show that the LAB may be involved in the detoxification of various carcinogens such as polycyclic aromatic hydrocarbons and heterocyclic aromatic amines [86]. The mechanisms of action of these bacteria are poorly known, but it is possible that the LAB bind directly to the carcinogen and catalyze detoxification reactions [62]. It is worth noting that the protective effects conferred by LAB only appear when these are at a high density and when there is a regular intake [87].

Evidence is accumulating that heterocyclic aromatic amines (HCAs), which are derived from amino acids in meat during cooking, might be involved in the etiology of human cancer [88]. Zsivkovits et al. [89] showed that *L. bulgaricus* 291, *S. thermophilus* F4, *S. thermophilus* V3 and *B. longum* BB536 are highly protective against the genotoxic effects of HCAs in rats. Additionally, the inhibition of HCAs induced DNA damage was dose dependent and significant when 1 x 107 cells/animal were administered. Other authors showed that *L. casei* DN 114001 may metabolize or adsorb HCAs and reduce their genotoxicity *in vitro* [89].

*In vivo* evidence that probiotics bond the carcinogens are still not conclusive. Hayatsu Hayatsu (1993) demonstrated the marked suppressive effect of orally administered *L. casei Shirota* (LcS) on the urinary mutagenicity arising from ingestion of fried ground beef by humans. In another clinical trial, the consumption of *L. acidophilus* decreased the urinary and fecal excretion of mutagens [68]. In view of the *in vitro* results, it is possible that the LAB supplements are inuencing excretion of mutagens by simply binding them in the intestine [62]. Even though the binding of carcinogens is a possible mechanism for the inhibition of genotoxicity and mutagenicity by LAB in vitro, some researchers have reported that it does not appear to have any influence *in vivo* [90]. Additionally, the extent of the binding depends on the mutagen and bacterial strain used [71].

Several studies have also reported the effect of probiotics on the promotion phase of carcinogenesis. Rowland et al. [91] found that administration of *B. longum* (6 x 109 CFU/day) inhibited the formation of aberrant crypt foci (ACF) in rats that received an induced of carcinogenesis (azomethane). As the probiotic treatment began 1 week after exposure to the carcinogen, these results indicate an effect on the early promotional phase of carcinogenesis [71].

Goldin et al. [92] observed a lower incidence of colonic tumors in rats who consumed *Lactobacillu*s GG before, during and after chemical induction with dimethylhydrazine (DMH) than in animals that were fed the probiotic after receiving carcinogen. The researchers concluded that probiotics acted by inhibiting the initiation stage of carcinogenesis.

Kumar et al. [93] tested the efficacy of *L. plantarum* AS1 in the suppression of colorectal cancer induced by DMH in rats and formed that AS1 was capable of diminishing colon

tumor through its antioxidant activity. However, long-term administration of this strain was necessary to achieve the maximum inhibitory effect.

Probiotics and Intestinal Microbiota: Implications in Colon Cancer Prevention 229

nitrogen species [102]. Studies have shown that probiotics can activate elements responsible for the formation of cytokines and epitope chemokine's, although that response was weaker for *L. rhamnosus* if than for a Gram-positive pathogen (*Streptococcus pyogenes*) [103]. Some authors have suggested that a possible mechanism of action of probiotics would be the inhibition of NF-kB activation by reducing intestinal inflammation [104]. However, the possible mechanisms of probiotics against carcinogenesis, regarding the modulation of the immune system, are complex and still

An inflammatory immune response produces monocytes and macrophages, activated by cytokines that release cytotoxic molecules capable of the lyzing tumor cells *in vitro* [105]. The cytokines IL-1 and inflammatory TNF (tumor necrosis factor) exert cytotoxic and cytostatic effects on neoplastic cells *in-vitro* [106]. Natural-killer cells (NK) are effective against tumor cells and low activity of this cell type has been linked to a risk of cancer [107]. Matsuzaki and Chin [108] found that in mice, NK cell activity and inflammatory responses increased

Several studies in humans have shown an increase of NK cells in response to the consumption of probiotics [109, 110], and the same has been in animal models. When Takagi et al. [111] administered the strain *L. casei* Shirota, in order to inhibit tumor development induced by methylcholantracene in mice, there were high levels of NK cells in the group treated with the probiotic, which slowed the early development of the tumor, compared to

On the other hand, Berman et al. [112] did not observed any increasing in NK cells in healthy subjects who consumed during 8 weeks a formulation containing 4 species of probiotics (*L. rhamnosus*, *L. plantarum*, *L. salivarus* and *B. bifidum*). However, the researchers

Evidence has shown that the probiotic *Lactobacillus casei* Shirota has anti-tumor effects and antineoplastic action in rodents (biologically or chemically induced). Intrapleural administration of the strain in mice with tumor induced the production of various cytokines, such as interferon IL-1 and TNF in the thoracic cavity, which resulted in tumor inhibition and increased survival [113]. A study on *B. longum* and *B. animalis* showed that these bacteria induce the production of inflammatory cytokines (IL-6 and

In a clinical trial, the effect of *L. casei* Shirota on NK cell activity in humans was investigated. The activity of NK was increased as a likely consequence of *L. casei* Shirota-induced IL-12

According to the results of the various studies mention here, the probiotic microorganisms are capable of modulating the immune system in a strain-specific manner [116]. Therefore, different strains may induce different immune responses that might lead to the inhibition of

did note an increase in phagocytosis by neutrophils and monocytes.

production which was detected in *in vitro* assays [115].

need to be better further elucidated.

with the administration of probiotic strains.

the control group.

TNF-) [114].

carcinogenesis.

On the other hand, not all studies have shown significant effects of probiotic on carcinogeninduced ACF. Gallanger et al. [94] using na ACF promotion protocol together with *B. longum* and *L. acidophilus*, obtained inconsistent results, which they attributed to differences in the ages of rats when DMH was administered.

Several studies have correlated the effect of probiotic on colon cancer with the modulation of the immune system. There is evidences that probiotics may contribute to the development of the mucosal immune system by influencing the innate inflammatory response and reducing mucosal inflammation. Additionally, probiotics also act on dendritic and epithelial cells and native T cells in the lamina propria of the gut and can thus influence adaptive immunity [13, 95].

Probiotics may influence the immune system by the action of products, such as metabolites, cell-wall components and DNA. Thus, immune modulatory effects might even be achieved by dead probiotic microorganisms or just probiotic derived components such as peptidoglycan fragments or DNA. Probiotic products are recognized by host cells sensitive to them these because they are equipped with recognition receptors adhesion. The main target cells in this context are therefore gut epithelial and gut-associated immune cells. The adhesion of probiotics to epithelial cells might itself might already trigger a signaling cascade leading to immune modulation [96].

Recent advances in the understanding of the immunomodulatory activity of probiotics have resulted from the discovery of Toll-like pattern recognition receptors (TLRs). These are transmembrane proteins present on the surface of cells such as macrophages, monocytes, dendritic cells and epithelial cells [97].

The innate immune system recognizes a large number of molecular structures from bacteria, such as, lipopolysaccharides and lipoteichoic acid, and is able to distinguish whether a particular microorganism is part of its microbiota or not. Different structures can activate different TLRs [98]. For example, TLR-2 recognizes the peptidoglycan, lipoteichoic acid, which is a component of the wall of Gram-positive bacteria such as lactobacilli and bifidobacteria [99], whereas TLR-4 is the most important receptor for lipopolysaccharide, the main component of the wall of Gram-negative bacteria [100].

Rachmilewitz et al. [101] using a probiotic mixture of 8 strains of freeze-dried lactic acid bacteria (*Bifidobacterium longum, B. infantis, B. breve, Lactobacillus acidophilus, L. casei, L. delbrueckii subsp. bulgaricus, L. plantarum, Streptococcus salivaris subsp. thermophilus*), reported that the chromosomal DNA of this mixture was responsible, via TLR-9 receptors for an antiinflammatory effect observed in mice with colitis.

The connection of components of microorganisms to these receptors can lead to a cascade of inflammatory reactions via the activation of nuclear factor-kB (NF-kB), with subsequent release of cytokines, epitope chemokines and lipid mediators of reactive oxygen and nitrogen species [102]. Studies have shown that probiotics can activate elements responsible for the formation of cytokines and epitope chemokine's, although that response was weaker for *L. rhamnosus* if than for a Gram-positive pathogen (*Streptococcus pyogenes*) [103]. Some authors have suggested that a possible mechanism of action of probiotics would be the inhibition of NF-kB activation by reducing intestinal inflammation [104]. However, the possible mechanisms of probiotics against carcinogenesis, regarding the modulation of the immune system, are complex and still need to be better further elucidated.

228 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

necessary to achieve the maximum inhibitory effect.

in the ages of rats when DMH was administered.

cascade leading to immune modulation [96].

main component of the wall of Gram-negative bacteria [100].

inflammatory effect observed in mice with colitis.

dendritic cells and epithelial cells [97].

immunity [13, 95].

tumor through its antioxidant activity. However, long-term administration of this strain was

On the other hand, not all studies have shown significant effects of probiotic on carcinogeninduced ACF. Gallanger et al. [94] using na ACF promotion protocol together with *B. longum* and *L. acidophilus*, obtained inconsistent results, which they attributed to differences

Several studies have correlated the effect of probiotic on colon cancer with the modulation of the immune system. There is evidences that probiotics may contribute to the development of the mucosal immune system by influencing the innate inflammatory response and reducing mucosal inflammation. Additionally, probiotics also act on dendritic and epithelial cells and native T cells in the lamina propria of the gut and can thus influence adaptive

Probiotics may influence the immune system by the action of products, such as metabolites, cell-wall components and DNA. Thus, immune modulatory effects might even be achieved by dead probiotic microorganisms or just probiotic derived components such as peptidoglycan fragments or DNA. Probiotic products are recognized by host cells sensitive to them these because they are equipped with recognition receptors adhesion. The main target cells in this context are therefore gut epithelial and gut-associated immune cells. The adhesion of probiotics to epithelial cells might itself might already trigger a signaling

Recent advances in the understanding of the immunomodulatory activity of probiotics have resulted from the discovery of Toll-like pattern recognition receptors (TLRs). These are transmembrane proteins present on the surface of cells such as macrophages, monocytes,

The innate immune system recognizes a large number of molecular structures from bacteria, such as, lipopolysaccharides and lipoteichoic acid, and is able to distinguish whether a particular microorganism is part of its microbiota or not. Different structures can activate different TLRs [98]. For example, TLR-2 recognizes the peptidoglycan, lipoteichoic acid, which is a component of the wall of Gram-positive bacteria such as lactobacilli and bifidobacteria [99], whereas TLR-4 is the most important receptor for lipopolysaccharide, the

Rachmilewitz et al. [101] using a probiotic mixture of 8 strains of freeze-dried lactic acid bacteria (*Bifidobacterium longum, B. infantis, B. breve, Lactobacillus acidophilus, L. casei, L. delbrueckii subsp. bulgaricus, L. plantarum, Streptococcus salivaris subsp. thermophilus*), reported that the chromosomal DNA of this mixture was responsible, via TLR-9 receptors for an anti-

The connection of components of microorganisms to these receptors can lead to a cascade of inflammatory reactions via the activation of nuclear factor-kB (NF-kB), with subsequent release of cytokines, epitope chemokines and lipid mediators of reactive oxygen and An inflammatory immune response produces monocytes and macrophages, activated by cytokines that release cytotoxic molecules capable of the lyzing tumor cells *in vitro* [105]. The cytokines IL-1 and inflammatory TNF (tumor necrosis factor) exert cytotoxic and cytostatic effects on neoplastic cells *in-vitro* [106]. Natural-killer cells (NK) are effective against tumor cells and low activity of this cell type has been linked to a risk of cancer [107]. Matsuzaki and Chin [108] found that in mice, NK cell activity and inflammatory responses increased with the administration of probiotic strains.

Several studies in humans have shown an increase of NK cells in response to the consumption of probiotics [109, 110], and the same has been in animal models. When Takagi et al. [111] administered the strain *L. casei* Shirota, in order to inhibit tumor development induced by methylcholantracene in mice, there were high levels of NK cells in the group treated with the probiotic, which slowed the early development of the tumor, compared to the control group.

On the other hand, Berman et al. [112] did not observed any increasing in NK cells in healthy subjects who consumed during 8 weeks a formulation containing 4 species of probiotics (*L. rhamnosus*, *L. plantarum*, *L. salivarus* and *B. bifidum*). However, the researchers did note an increase in phagocytosis by neutrophils and monocytes.

Evidence has shown that the probiotic *Lactobacillus casei* Shirota has anti-tumor effects and antineoplastic action in rodents (biologically or chemically induced). Intrapleural administration of the strain in mice with tumor induced the production of various cytokines, such as interferon IL-1 and TNF in the thoracic cavity, which resulted in tumor inhibition and increased survival [113]. A study on *B. longum* and *B. animalis* showed that these bacteria induce the production of inflammatory cytokines (IL-6 and TNF-) [114].

In a clinical trial, the effect of *L. casei* Shirota on NK cell activity in humans was investigated. The activity of NK was increased as a likely consequence of *L. casei* Shirota-induced IL-12 production which was detected in *in vitro* assays [115].

According to the results of the various studies mention here, the probiotic microorganisms are capable of modulating the immune system in a strain-specific manner [116]. Therefore, different strains may induce different immune responses that might lead to the inhibition of carcinogenesis.
