CA2467449C - Lactic acid bacteria and their use for treating and preventing cancer - Google Patents

Abstract

The present invention relates to the demonstration of new properties of lactic bacterial strains. These new properties are advantageously useful in the prevention and treatment of cancer. More particularly, the present invention relates to the use of lactic acid bacteria to facilitate induction of cellular apoptosis of cancer. The present invention also proposes the use of lactic bacterial strains, such as Lactobacillus acidophilus and Lactobacillus casei in methods and composition for preventing and treating cancer, especially colon cancer.

Description

LACTIC ACID BACTERIA AND THEIR USE IN TREATMENT
AND THE PREVENTION OF CANCER

FIELD OF THE INVENTION

The present invention relates to the demonstration of the utility of strain bacterial lactic in the prevention and treatment of cancer. More particularly, the present invention relates to the use of bacteria lactic to facilitate the induction of cellular apoptosis of cancer.

DESCRIPTION OF THE PRIOR ART

Lactobacillus acidophilus I-1492 bacteria present in the product Bio-K + and which are the subject of patent application PCT / CA97 / 00915 are recognized to have a beneficial effect on blood cholesterol levels in the mammals.

International application no. WO 98/23727 relates to a ferment lactic acid which comprises a strain of Lactobacillus acidophilus 1-1492. So, these bacteria are mainly used for ferment preparation lactic to reduce blood cholesterol levels in mammals.

In addition, these bacteria are also known to have which have the effect of strengthening the immune system, facilitating absorption of nutrients and stimulate the intestinal flora. It is known that bacteria lactic acid have a positive effect on the intestinal flora and also on the system immune. Indeed, the lactic acid bacteria allow a stimulation of the immune system thus having the effect of giving a better defense to the level of the digestive system. These bacteria are also known to neutralize the side effects caused by various antibiotics.

Many people in the country die every year from cancer of the colon. Cancer is the third leading cause of death year.
SUBSTITUTE SHEET (RULE 26)

2 In Canada, in the year 2000, the mortality rate caused by cancer was 6500 and there are more than 17,000 new cases.

Nutraceutical use involving the administration of yogurt and / or milk fermented as a complementary treatment against cancer is also known.

The treatment already used as therapy in humans, is the removal of the cancerous mass by surgery and then there may be irradiation of the area where was cancer to avoid leaving traces of cells undesirable.

There is also chemotherapy. This treatment involves the administration of anticancer agents such as 5-fluorouracil (5FU). This compound is combined with an adjuvant to limit the negative effects of chemotherapy. 5Fluorouracil is a drug that is commonly in use to treat colon cancer. This medicine can be administered in a oral intraperitoneal (closest to the cancer target) because of its great instability in the serum. It is known to cause the death of cancer cells from colon and by different means.

The death of a cell, which is also called apoptosis, can be performed by through different proteins. For example, apoptosis cell can result from activation of a membrane receptor or expression cytoplasmic of different proteins promoting this phenomenon. On this subject, he is known that 5FU increases the expression of the p53 protein as well as expression of the Fas membrane receptor.

1 General on apoptosis 1.1 Definition of apoptosis Discovered and rediscovered several times by different cytologists and biologists, programmed cell death to acquired different name in the short of last two centuries. In 1972, the term apoptosis was finally adopted and invented by Currie and it's colleagues, to describe a frequent pattern of death

3 programmed cell that the authors have repeatedly observed in many tissues and cell types. It has been observed that these dying cells shared several morphological characteristics, which are different of the characteristics observed in cells with a pathology and necrotic cells, and they suggest that these morphological features shared could be the result of a common cell death program and retained.

1.2 Role of apoptosis The researchers discovered that the cells of our body can be give death, they know that this cell suicide, called apoptosis is indispensable to the body. Apoptosis is also fundamental for the physiology of cells and tissues as division and differentiation cellular.
Apoptosis is the most common form of physiological cell death that himself produced in different moments such as when developing embryonic, during tissue reorganization, during regulation immune and tumor regression. So, cell death physiological is a spontaneous cellular elimination process, ensuring the cell renewal, which is involved in the maintenance of homeostasis cellular and tissue opposite to mitosis. It is the mechanism innate by which the body eliminates unwanted cells. Each cell carries in it the genetic mechanism of its own destruction. The cells do not stay in life only if it receives life signals from its environment.
If the cell receives signals telling it to commit suicide, then it sets off the program of death. A disturbance in the balance between protein now the cell alive and between the proteins leading to the death of cell can be associated with a broad spectrum of diseases including cancer, neurodegeneration, autoimmune diseases, diabetes and other orders.

2 Morphological characteristics of apoptosis and necrosis

4 2.1 Apoptosis One of the key features of apoptosis is cell shrinkage.
As cell shrinkage occurs, the cytoplasm compresses and the nuclear chromatin condenses and forms aggregates, in the nuclei, which then stick against the nuclear membrane. Cellular organelles, whose mitochondria, on the other hand, appear relatively unchanged. Subsequently, the nuclei becomes fragmented. The formation and the emission of buds are observed on the surface of the cell. The integrity of the plasma membrane, even if the permeability increases, however, is preserved throughout this process. During the final stage of apoptosis, the cell breaks down into many vesicles containing a variety of intact organelles and fragments nuclear.
The fragments of apoptotic cells are quickly engulfed by the surrounding phagocytic cells, for example the macrophage.
Apoptosis is a so-called clean death since the fragments cell are quickly eliminated. There is no inflammatory phase or tissue lesion and partly because their cell membrane remains intact.
In In summary, the morphological changes characteristic of apoptosis are narrowing of the cytoplasm, condensation and fragmentation of the DNA and finally, the formation of apoptotic bodies contain fragments of core surrounded by the cytoplasm and the cell membrane.

2.2 Necrosis Necrosis refers to a sudden death occurring as a result of stress extreme physical or chemical It is marked by morphological criteria different. During necrosis, this uncontrolled cell death, there is quickly loss of control of the ionic flux resulting in the penetration of water and a increased ionic influx, the cells swell as well as its organelles as the mitochondria and the endoplasmic reticulum until the bursting of membranes and non-specific fragmentation of DNA in the nucleus. The release of cytoplasmic content on the outside to the addition of others events, most often causes lesions in the tissues located at proximity and triggers a very pronounced local inflammatory response.

WO 03/04540

5 PCT / CA02 / 01826 Characteristics Apoptosis Necrosis Tissue distribution isolated cellular cell grouping Tissue Reaction Lysis and relaxation of body phagocytosis cell content resulting in apoptotic inflammation of macrophage tissues or cells surrounding surroundings and no inflammation Morphology Swelling Shrinking, loss of contact with the cells Surrounding cell, "blebbing", body training apoptotic Intact Damaged Organelles Disintegrated Core Condensed and Fragmented Intactes Damaged Lysosomes Defective mitochondria, depleted in ATP, swollen, change swollen and damaged can break, cytochrome release VS

Biochemistry nonspecific degradation cleavage within the nucleus DNA

Nonspecific degradation proteins Activation of caspases Table 1- The main differences between necrosis and apoptosis.

3 Different stages of the apoptotic process Apoptosis can comprise three different steps (Figure 2). It takes everything first that the cell receives an apoptotic signal so the phase of trigger or commitment. Then there is the regulation or control then finally the execution phase during which would take place the intracellular enzymatic cascade inducing apoptotic death.

6 3.1 Apoptosis triggering stage A variety of stimuli, both internal and external, can activate the cells to become apoptotic. Among the different stimuli, one can enumerate biological agents (membrane receptors, transcription factors, oncoproteins, viral infection, bacterial toxins, etc.), the removal of essential factors for cell growth (cytokines, growths and nutritive, ...), genomic lesions of DNA (spontaneous or provoked), exposure to chemicals (anticancer agents), exposure to of the physical inducing agents (UV rays, X-rays, microwaves, heat, ...).
This phase continues with many biochemical changes.

3.2 Decision or execution stage of apoptosis Following these different stimuli, the cell receives the different signals and decide to become apoptotic or not. This step includes different paths of transduction signals among other things the activation (or inactivation) of serine / threonine and tyrosine kinases and phosphatases, the second synthesis messengers, modification of gene expression and activation of proteases specialized known as caspases. The final decision to become apoptic depends on several factors including the balance between protein pro-apoptotic and anti-apoptotic (the Bcl-2 protein family), the state metabolic of the cells and also the cell cycle stage in which the cell is.
Several arguments suggest that the course of this second stage is controlled by the family of Bcl-2 proteins, which can be found in general associated with the outer membrane of mitochondria, endoplasmic reticulum and the nucleus. The family of Bcl-2 proteins is divided into two groups of proteins, some inhibit apoptosis (Bcl-2, BCI-XL, Bcl-w, CED-9, ...) and the others promotes apoptosis (Bax, Bid, Bad, Bak, Bcl-Xs, ...). Whether pro or anti-apoptotic, they have the ability to control the ionic flow between various cell compartments, especially between the mitochondria and the cytoplasm.
At this same stage, caspases are activated as a system amplification by self-activation by themselves and between them but also by the release of activation factors of apoptosis, such as the AIF
(apoptosis-

7 inducing factor) and cytochrome c, by mitochondria. The international space membrane of the mitochondria comprises several proteins involved in activation of apoptosis, such as pro-caspase-2, -3, -7 and -9, AIF and cytochrome c. Activation of these caspases will lead to a point of no return since by their activation, they will cleave their different targets, among other things the proteins necessary for the survival of the cell.

3.3 Step of degradation of apoptosis The cell is now irreversibly engaged in the program of cell death which consists of the presentation of characteristics morphological features of the apoptotic signature. So by activating different caspases, several proteins necessary for the survival of the cell are cleaved and become non-functional, such as poly (DNA-ribose) polymerase. other targets of caspases can be activated by them, among others DNase which themselves will cut chromatin into high-weight fragments molecular.

4. Proteins involved in regulatory mechanisms of apoptosis 4.1.1 The family of Bcl-2 proteins Bcl-2 protein and those of the same family are important modulating apoptosis. In this family of proteins, there are two classes including anti-apoptotic proteins (Bcl-2, Bcl-xL, McI-1, Bcl-w, Bfl-1 / A1, Brag-1) and pro-apoptotic proteins (Bax, Bak, Bad, Bid, Bik, Bim, Bci-xs) (Reed 1996). Members of the Bcl-2 family are classified according to their number of homology domain with Bcl-2 (BH: Bcl-2-homology). Bcl-2 protein contains 4 domains. All anti-apoptotic proteins have all four domains, while pro-apoptotic proteins can be divided into three categories. One group contains the domains BH1, BH2 and BH3 (Bax, Bak), while that the other group contains only the BH3 domain (Bad, Bid, Bik). Bcl-xs forms its own group, containing domains BH3 and BH4. A study by

8 Crystallographic study made it possible to determine the structure of the BCI-XL protein.
The protein is therefore formed of two central helix-a surrounded by five helix-a amphipathic. It is interesting to us that the structure three-dimensional is homologous to bacterial toxins forming pores in membranes, such as diphtheria toxin and colicin, which could suggest a mechanism potential action for these proteins at the level of the mitochondria. Another structural feature would be the ability of these proteins to s'homo and is straight dimerize with each other, by their domain BH3, because of this, they could favor or antagonize their functions among themselves.

4.1.2 Roles of anti- and pro-apoptotics A- On the mitochondria First, a description of the changes that the mitochondria undergo in a situation of apoptosis. Isolated mitochondria in a situation apoptosis undergo what is referred to as a mitochondrial permeability transition (MPT).
Experimentally, MPT is characterized by a sharp rise in the Permeability of the inner membrane to particles with a molecular weight of <1500 Da. This permeability transition has several consequences including the collapse of the Dylm, osmotic swelling, relaxation of the matrix Ca 2+, the creation of oxygen-reactive species, and rupture of the outer membrane of the mitochondria leading to the release of cytochrome c from membrane of the mitochondria. A biochemical characterization of the Mitochondria has identified voltage-sensitive pores and Ca2 +
control the MPT, which will be named PT pores. The pores are located at the junction of the inner and outer membranes of the mitochondria, and thus, the opening pores allow direct communication between the matrix of the mitochondrion and its environment. The voltage-of during anion channel (VDAC) and the adenine nucleotide translocator (ANT) are part of PT pore.

Proteins of the Bcl-2 family have different cytoplasmic distributions.
Bcl-2 and Bcl-xL proteins have a c-terminal hydrophobic tail containing a membrane insertion sequence, and the majority of these protein

9 are known to be associated with mitochondrial membranes, endoplasmic reticulum and nuclear membrane. In their inactive form, the pro-apoptotic proteins, Bad, Bax and Bid, have a primary location cytoplasmic. On the other hand, when they are activated, they are mitochondria. When Bid is cleaved, its terminal portion COOH resets to the surface of the mitochondria. The action of these proteins, either to prevent to initiate apoptosis is at the level of the mitochondria, whereas the mechanism of these actions remains controversial and uncertain. It has been shown that by adding protein Bax, a pro-apoptotic protein, at isolated mitochondria induction of relaxation of cytochrome c, while the expression of Bci-2 or Bci-XL protein prevents the release of cytochrome c therefore blocks apoptosis. It is clear that the Bcl-2 family is closely involved in the release of cytochrome c, the electron transport protein inside of the mitochondria. In addition to being involved in oxidative phosphorylation in the mitochondria, cytochrome c is one of the constituents (as well as the protein Apaf-1 adapter) which is required for activation of caspase-9 in the cytosol.
How members of the Bcl-2 family regulate the release of cytochrome vs? Several hypotheses have been put forward, but none is proven final. There are three basic models that can be suggested.

1- Members of the Bci-2 family form a channel that facilitates transportation proteins. Based on the similarity of the structure of Bcl-XL to the sub-unit of the diphtheria toxin that forms pores, it has been suggested that protein Bcl-2 could fit into the outer membrane of the mitochondria, where they could form a channel or even a large hole. The members of the Bci-2 family indeed can fit into a bi lipid layer synthetic, to oligomerize, and to form a channel with a discrete conduction. The proteins Bid and Bik can directly induce mitochondria to relax cytochrome c without interact with VDAC or ANT suggesting that they act outside the PT pores.
2- The members of the Bcl-2 family interact with other proteins for form channels. The Bci-2 protein family interacts with several proteins. One possibility would be that members of the protein family pro-apoptotic is recruiting other proteins from the outer membrane of the mitochondrion to form a pore large enough to make a channel. A candidate particularly interesting for such a protein would be the anion channel voltage dependent (VDAC), several members of the Bcl-2 family can bind to it and 5 regulate its channel activity. Since the size of the pore characterized channel VDAC is too small to let the proteins through, this model must assume that the VDAC undergoes a conformational change as a result of members of the Bcl-2 family. It has been shown that BcI-2 proteins and Bcl XL promote PT pore closure, while protein pro-apoptotic

10 Bax have the opposite effect, it interacts with ANT and VDAC to favor the opening of these pores and the releasing of cytochrome c.

3- The members of the Bcl-2 family induce a rupture of the membrane exterior of the mitochondria. It is possible that, the Bcl-2 family controls the homeostasis of the mitochondria. In this model, the apoptotic signal altèrerais the physiology of mitochondria (eg, ion exchange or oxidative phosphorylation) such as organelle swells, which gives as result physical rupture of the outer membrane and re-release of proteins located between the membranes of the mitochondria, in the cytosol. The need to train a channel big enough to pass the cytochrome c is now more necessary since the proteins would simply diffuse through the tears in the lipid bilayer.

Pro-apoptotic proteins (Bid) can homogenize dimerize to form a pore to let out cytochrome c. Anti-apoptotic proteins (Bcl-2) have the ability to bind with PT pores and thus prevent re-release of the inter-membrane proteins, however, pro-apoptotic proteins (Bax), will allow the opening of PT pores.

The protein AIF (for apoptosis-inducing factor) that has been identified and its cloned gene, is capable, on its own, of inducing apoptosis in nuclei isolated.
This molecule is synthesized in the cytosol as a precursor and then is imported into the mitochondria. Like cytochrome c, this is a phylogenetically old molecule, with a dual function:

11 redox and apoptogenic factor. Nevertheless, unlike the path of cytochrome c, which requires the activation of other factors to induce apoptosis, the AIF pathway is, on the contrary, independent of caspases and does not require any intermediate to induce apoptosis. It would, among other things, constitute prototype apoptosis pathways independent of caspases.

The hypotheses concerning the mechanisms of inhibition of apoptosis and particularly the sequestration of Apaf-1 by Bcl-2 and its anti-agonists apoptotic still seem very controversial. Apaf-1 is probably an important target of members of the Bcl-2 family, since Apaf-1 deficient cells are refractory to various pro-apoptotic signals and which are themselves inhibited by Bcl-2. In addition, over-expression of Apaf-1 showed that this protein was associated with survival proteins like Bcl-xL and Bcl-2. On the other hand, he at has been shown that there is no co-immunoprecipitation between the the Bcl-2 family and Apaf-1. Apaf-1 was also found at sites where the survival proteins like Bcl-2 and BCI-XL resident such as membranes external mitochondria, nuclear envelope and reticulum endoplasmic.
4.1.3 Mechanism of modulation of Bcl-2 family proteins Several different mechanisms exist to modulate the functions of pro- and anti-apoptic proteins. First, the dimerization state of members of the Bcl-2 family affect their business. One of the functions of protein anti-apoptotic Bcl-2 and Bcl-XL is to dimerize with protein pro Bax apoptotic to neutralize its activity. Being a heterodimer, Bax is inactive, but once free to dimerize with itself, Bax is able to induce apoptosis. Bid, Bik and Bad can act by inhibiting apoptotic Bcl-2 and BCI-XL forming heterodimers. Second, in altering the expression level of the pro and anti-apoptotic limbs of the family Bcl-2, can either initiate apoptosis or inhibit it. For example, when the number of Bcl-2 is greater than or equal to the number of Bax, the cell in question is protected from apoptosis. On the other hand, when the number of Bax exceeds the number of Bcl-2, the cell is more prone to become apoptotic. In third place place, the Bcl-2 family proteins can be modified by phosphorylation. The

12 best example for this purpose would be the pro-apoptotic protein Bad. In his unphosphorylated state, it dimerizes with Bcl-2 and BCI-XL, neutralizing so their anti-apoptotic activity. On the other hand, when Bad is phosphorylated, she is sequestered and therefore can not interact and neutralize Bcl-2 and Bcl-XL. In Fourth, the Bci-2 family can be modified by cleavage. During a apoptosis caused by Fas, caspases have been shown to cleave Bcl-2 and Bcl-XL
and that cleaved products are no longer protective and even they become apoptotic. Bid is another protein in the Bcl-2 family that is activated by the cleavage of caspases. While the protein at its full length is inactive following the cleavage caused by caspase 8, Bid induces the re-release of the cytochrome c by the mitochondria. Finally, the conformation of Bcl-2 proteins modifies their activity. The best evidence for this mechanism comes from the studies done on Bax. In its inactive state, Bax exists under a conformation in which she resists proteolytic cleavages. However, following its activation and re localization on the mitochondria, the N-terminal region of the protein becomes susceptible to cleavages, suggesting that there is indeed a change in conformation that happens.

In summary, mitochondria have an important place in the triggering apoptosis. Their inter-membrane space contains several proteins (cytochrome c, caspase-2, -3, -7, and -9, AIF) which once released in the cytoplasm participate in the phase of degradation of apoptosis.
The riddle mechanisms of induction and control of apoptosis by mitochondria based on four essential points: namely the molecules of the Bcl-2 / Bcl Which could contribute to the formation of ion channels at the level of intracellular membranes. Pro-apoptotic members of this family (like Bax, Bid, ...) could also intervene in the permeability of PT pores of the mitochondrial membrane, especially as proteins activators of apoptosis. Finally, the anti-apoptotic molecules of Bcl- family 2 could also act by titrating endogenous activators (like Apaf-1) of Apoptosis. One last point is that some pro-caspases also have a mitochondrial localization. Thus, apoptotic promoters or inhibitors of the Bcl-2 family regulate apoptosis through multiple effects on

13 caspase activation cascades, on the redox potential, and on the function of barrier of permeability of mitochondrial membranes. All in all observation therefore suggest an implication of the permeability transition in the regulation of mitochondria-induced apoptosis.

4.2 Role of caspases in apoptosis 4.2.1 Definition and classification of caspases Caspases are specialized proteases that are essential for apoptosis. They are different from other proteases because they use a cysteine for catalysis and they cleave only after acid residues aspartic. This unusual specificity of having an aspartate as a substrate is found only in another protease, granzyre B, however this enzyme uses a serine as an active site. Caspases are synthesized as a single chain of polypeptides and they are inactive zymogens.
These zymogens are composed of three domains: an N-terminal pro-domain, and two other domains, p10 and p20, which are found in the mature enzyme.
then of their activation, each polypeptide chain is cleaved into two subunits, a large (p20) and a small (p10), which later dimerize. So the mature enzymes that have been observed, are hetero-tetramers composed of two p20 / p10 hetero-dimer and two active sites. The peptide in N-terminal, is cleaved and released during activation. This peptide in N-terminal is not) required for enzymatic activity, are role is known on Caspase 8 and 10, where it is as a field of interaction with other proteins to modulate their activation.
Caspases 8 and 10 contain a death-effector domain (DED), while caspase 2 and 9 contain a caspase activation and recruitment domain (GARD).

There are at least 14 different caspases identified in the tissues of mammals to date. It is possible to divide caspases into three groups different by their specificity of substrate, ie, by their recognition of three amino acids that precede aspartic acid. The first group, contains the caspases involved in the inflammatory process, therefore activation of

14 pro-cytokines, which includes caspases 1, 4 and 5. These enzymes are sometimes known as the ICE-like caspases, because another name for caspase-1 is Interleukin-1-converted ng enzyme (ICE). Their tetra-peptide motif they recognize and prefer is WEHD, however the ICE-like caspases are the more tolerant in terms of amino acid substitution to compare the signaling and effector caspases. The second group of caspases contains caspases 6, 8, 9 and 10. These enzymes are considered as caspases signaling because they can activate other caspases and so start the cascade. Their motive for recognition is (LV) EXD. The last group contains caspases 2, 3 and 7. These enzymes are known as effector since they cleave several cellular targets which gives as a result the morphological appearance of apoptosis. Activation of these caspases resulted usually in a point of no return in cell death. The effector enzymes are the most specific, with a need to have a acid aspartic in the first and fourth position preceding the site of cleavage.
Their motive for recognition is DEXD. The most recent caspases, caspases 12-14, have not yet been sufficiently characterized to be able to classify into one of the three groups.

4.2.2 Activation of caspases There are three different mechanisms for activating caspases. The first mechanism is the activation of caspase by another caspase previously activated. Most caspases are activated following cleavage proteolytic of the zymogen between the domains p20 and pl 0, and usually another cleavage between the pro-domain and the p20 domain. It is interesting to note that all these cleavage sites occur after an aspartate, the substrate of caspases, this who suggests the possibility of activation by auto catalysis. Indeed, the way most easy to activate a caspase is to put it in the presence of another caspase already activated. This caspase cascade strategy is widely used by the cell for the activation of three important caspases, caspase-3, -6 and -7. These three effector caspases are considered to be the most family of caspases, and are usually more numerous and active than the other.

As shown in Figure 4, the first cleavage is between domains p20 and p10 (here 12 kDa) to separate the two subunits. The second cleavage Proteolytic is carried out between the pro-domain and the large subunit and then there a formation of a heterotetramer which leads to mature caspase in its active.

The caspases cascade is a very useful method to amplify the signal pro-apoptotic, but he can not explain how the first, the most downstream 10 caspases is activated. There are at least two other models that could explain the activation of the very first caspases. The first is induction of activation by reconciliation. It is known that caspase-8 is the caspase initiator during apoptosis induced by death receptors. When link from ligand to its receptor, the CD95 / Fas death receptor trimerize and form

Signaling complexes bound to the membrane. These complexes recruit, by adapter proteins, several pro-caspase-8 molecules, giving as a result a large local concentration of zymogens. This model activation by reconciliation stipulates that under this crowd condition, the low intrinsic proteolytic activity of pro-caspase-8 is sufficient to to permit to the pro-enzymes to separate each other and to activate each other.
The latest model of caspase activation is the association of pro-caspase with a regulatory subunit. Take as an example caspase-9 which requires an association with cofactors for its activation. The cofactor apoptotic protease activating factor-1 (Apaf-1) has been identified by an approach biochemical as being one of the two proteins necessary for the activation of the caspase-9, the other being cytochrome c. The complex that forms these three proteins, requiring ATP, gives the active form of caspase-9 often called apoptosome. So, Apaf-1 is not just an activator protein caspase-9 but is an essential subunit for the functioning of this.
In summary, effector caspases are usually activated by

16 caspases downstream, whereas the initiating caspases are activated by regulated protein-protein interactions.

4.2.3 The victims of caspases Caspases cleave a good number of cellular proteins, and the proteolysis process is limited since there is a small number of cuts who are realized. Sometimes the cleavages cause the activation of the protein, and at other times, inactivation but never degradation since their specificity of substrates distinguishes caspases as being the most endopeptidases strict. Caspases cleave several cellular proteins whose number born ceases to increase. Structure, Nuclear and Signaling Proteins are all targets of caspases (Table 1). There are different proteins of cytoskeleton cleaved by caspases, for example, lamin, a-fodrin and actin. Cleavage of these proteins is probably responsible for morphological changes observed during apoptosis, eg cleavage nuclear lamins is necessary for shrinkage and the budding nuclear. The fragmentation of the DNA is due to the activation of the caspase-activated Dnase (CAD) by caspase-3. This DNase exists in the form of Inactive complex with an inhibitory subunit, ICAD. Activation of CAD
is therefore carried out by cleavage of the inhibitory subunit by caspase-3 resulting in the relaxation and activation of the catalytic subunit.

Category Target Effect Signaling other caspases Activation PKC 6 Activation, fragmentation nuclear Phospholipase A2 Activation Bcl-2, Bcl-xL Fragment Formation apoptotic Bid Activation ICAD Activation of the endonuclease CAD
Nuclear Fragmentation Factor DNA DNA Fragmentation Polymerase poly (ADP-ribose) Inactivation

17 DNA-dependent protein kinase Inactivation U1 (70 kDa) -snRNP Decrease in synthesis ARNs Lamine A and B Disassembly of lamina nuclear Structural Actin Rearrangement of cyto-skeleton Gelsoline Rearrangement of cyto-skeleton a-Fodrin Membrane change Table 2- Some examples of victims of caspases.

4.3 The p53 protein The p53 protein is a transcription factor that plays a critical role in cancer prevention. The p53 protein is considered to be the guardian of the genome. This protein is a good example of how the decision between apoptosis or life can be done at an activated verification point when DNA
is damaged. Depending on the stimulus and the phase in which the cell is, the activation of p53 can lead to a stop of cell proliferation and at the DNA repair or apoptosis. While the first stimulus for activate the p53 is the damaged DNA, other cell stress like the deprivation metabolite, physical damage, heat, and the oxygen can also activate the p53.

The level of p53 increases dramatically in a few minutes according to the damage that the cell undergoes. This increase is possible by post-translational modifications of the p53 polypeptide, without induction dramatic evidence of p53 mRNA level following damage to DNA.
The modification which is carried out on the polypeptide of p53 following a damage to DNA results in phosphorylation. In a cell that has not experienced any stress, the p53 protein has an extremely short half-life but becomes much more stable due to damage to the cell. The instability of the

18 p53 protein under normal conditions for the cell is connected to the fact that p53 is the target of proteolysis induced by small peptides, ubiquitins.
So p53 is marked by ubiquitins with the help of a protein bearing the last name of Mdm2, a protein that plays a role in the downregulation of p53. he is demonstrated that the Mdm2 protein interacts with p53 so that it becomes the target perfect proteases by ubiquitins. The Mdm2 protein also causes the p53 translocation from the nucleus of the cell to the cytoplasm, where it will undergo ubiquitin-induced proteolysis. So, by the phosphorylation of field in the C-terminal part of p53, it is shown that activating its DNA binding according to specific sequences takes place. In addition, the phosphorylation of serine-15 and serine-20, in p53 N-terminal, cause inhibition of the interaction between p53 and Mdm2 therefore, increase the level of p53 and converting it into a form capable of an activity of transcription. A large number of phosphorylent p53 kinases, including the kinase casein, kinases related to extracellular signals, protein kinase C and the Raf-1 kinase. Once phosphorylated, p53 acts as a transcription to increase and decrease the transcription of several genes involved in apoptosis.

Several cell cycle regulatory proteins are induced by p53, for example p21, GADD45 and members of the 14-3-3 family. The skill p53 to induce a cell cycle arrest in phase G1 following injury at DNA is well known and can be explained by the fact that the p53 protein a times stimulated has a transcription activity that makes it possible to transcribe the uncomfortable of cyclin-dependent kinase inhibition (Cdk), the p21 protein. A
high number of p21 will then inhibit cyclin kinases E / cdk2 and cyclin A / cdk2, thus preventing these kinases from promoting the progression of the cycle cellular. In addition, the p53 protein is also involved in stopping the cycle in G2 phase in part because p53 induces the expression of the protein sigma 14-3-3 which will cause sequestration of the cyclin B / Cdc2 complex. The p53 protein can lead to apoptosis by activating the transcription of different genes giving proteins involved in the process apoptotic.
The proteins that are induced are Bax protein, Fas receptor and DR5

19 (receptor for the TRAIL death ligand), all of which are involved in the process of apoptosis. It also causes the decrease of the expression of Bcl-2 mRNA, thus promoting apoptosis. There seems to be a way p53-induced apoptosis that does not require relaxation of cytochrome c but which always requires the activation of caspases. Although the expression of the protein bax is increased, it is rather in the cytosol and no translocation on mitochondria is detectable. So, it could exist another pathway through which the p53 protein induces apoptosis without there being relaxation of cytochrome c.

The final results following DNA damage may be the stopping of the cycle cell growth, or apoptosis. DNA damage given accumulation and activation of the p53 protein (Figure 5). Once activated, p53 has a transcription activity that will increase the transcription of different genes (GADD45, 14-3-3, Mdm2, p21, Bax, Fas, DR5). It can also downregulate different genes (Bcl-2). By increasing the p21, which will go inhibit dependent cyclin kinases (cdk), the cell cycle is then stop in Gl.
The cell cycle can also be stopped in G2 phase by increasing of the GADD45 and 14-3-3 proteins by p53. The process of apoptosis is achieved by different proteins (Bax, Fas, DR5) that are upregulated by p53.
A
regulation loop of the p53 protein is possible through the increase of Mdm2, a protein binding to p53 and promoting its degradation.

4.4 The death receptors The death receptors are receptors located on the surface of the cell and are named so because at the bond with their ligand they can initiate the process of apoptosis. These receivers are part of the family of TNF receptor, in particular the TNF-R1 receptor itself, the receptor Fas (also called CD95 or Apo-1) and also the DR-3, DR-4 and DR-5 receptors.
These receptors are activated by their ligand which are soluble or membrane like tumor necrosis factor- (x (TNF-a), Fas-L and TNF-related apoptosis inducing ligand (TRAIL) The ligands of death receptors make gone of the TNF-α cytokine family, and are homotrimeric molecules. of the crystallographic analyzes indicate that each monomer of the ligand binds to a receptor, indicating that binding of a ligand involves trimerization of these receptors. The ligand-receptor interaction induces the trimerization of receiver, which 5 allows the physical association of adapter proteins with the domains interacting protein death domain (RIP-DD), promote recruitment and activation of proximal caspases such as pro-caspases-8, -10 and -2, then able to transmit the death signal inside the cell.
take as an example the activation of the TNF-R1 receptor. By TNF binding to 10 TNF-R1 receptor, the latter trimerizes, giving as a result aggregation domains of death, allowing the recruitment of TRADD which in turn recruits an adapter molecule, TRAF2 TNF receptor-associated factor 2, who leads to the activation of the JNK and NF-KB channels. TRADD can also recruit FADD and RIP leading to the apoptotic process and the activation of NF-KB, 15 respectively. RIP can also recruit RAIDD RIP-associated ICH-1 / CED-3-homologous protein with a death domain which subsequently recruits the caspase-2 and induces apoptosis. When have it takes the Fas receiver as activation model of apoptosis, the Fas and FADD complex will recruit the caspase-8 which will form the complex that will induce the death signal, inducing

Complex signal (DISC). Once assembled, the DISC will cause a rapid activation of caspase-8 which will activate caspase-3 and cause apoptosis of the cell. Thus, this first course of action of the Fas receptor is a way fast bypassing the mitochondria and that does not require the contribution of new molecules because it is based on the interaction of molecules preexisting.

However, it has recently been shown that activation of caspase-8, continued trimerization of the Fas receptor, could also cause cleavage of Bid, a pro-apoptotic protein of the Bcl-2 family. This cleavage leads to penetration of a truncated form of Bid into the mitochondria with consequence, the output of cytochrome c, then the depolarization of the potential of mitochondrial membrane and apoptosis. It is also shown that when there

21 DR-4 and DR5 activation by TRAIL, caspase-8 is activated. The way in use is not yet well understood but many confirm that there is activation of the caspases-3 and -9 following activation of caspase-8 and observe that Bid is cleft following the activation of caspase-8. It is possible that by the link of TRAIL to DR4 or DR5 receptors, there is activation of caspase-8, by recruitment to ugly of an adapter molecule, which both activates caspase-3 and cleaves Bid, so that the latter becomes active and allow the releasing of cytochrome c which will have as an effect of activating caspase-9 which in turn will activate caspase-3 this who will cause an amplification of caspases cascade and will give death to the cell.

In summary, there would be at least two signal transduction pathways apoptotic by some death receptors, one fast forward, the other slower putting into play the mitochondrial relay. That's why some authors rank cells (type I or II) according to their mode of induction of apoptosis by Fas.
As for example, the activation of Fas, in some cells, leads nearly exclusively to the caspase pathway only (type I cells). These cell usually shows no involvement from mitochondria, and the cell death is not inhibited by Bcl-2 or Bci-xL. In other cells, activation of Fas triggers the pathway using mitochondria in large part, and this after the activation of caspase-8. Bcl- anti-apoptotic proteins 2 or Bcl-xL can inhibit apoptosis only in type II cells, by their action to prevent the release of cytochrome c in the cytosol.

Several stimuli can initiate apoptosis, but alterations morphological and biochemical characteristics are observed independently initial stimulus. Research suggests that most of the signals apoptotic converges on a limited number of pathways leading to apoptosis.

Figure 6 shows the structure of the limbs of the dead receptors membrane and their interactions with major cytoplasmic effectors involved in apoptotic pathways. The red arrows indicate a activation of caspase-8, and black arrows an inhibition of apoptosis with some intermediate steps (activation of kinases / transcription factor). The

22 abbreviations in this figure have the following meaning: DD, death domain; TRADD, TNF-receptor associated death domain; FADD, Fas-associated death domain; DISC, death-inducing signaling complex; RIP, receptor interacting protein; TRAF2, TNF-receptor associated factor-2; NF-KB, nuclear factor kappa B
I-xB, inhibitory kappa B; JNKK, JNK kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; Fas L, Fas ligand; TRAIL, tumor necrosis factor-related apoptosis inducing ligand; DR4-5, death receptor 4-5; DED, death-effector domain; RAIDD, RIP-associated ICH-1 / CED-3-homologous protein with a death domain;

The process of apoptosis requires the participation of several to activate the caspases (Figure 7). The two best known and best characterized are the transduction of the apoptotic signal by the death and the other pathway more internal to the cell is the apoptosis induced by the changes in mitochondrial integrity, particularly the release of factors apoptogens such as cytochrome c and AIF. There are interconnections between these two signaling channels and signal amplification loops.
Abbreviations: AIF, apoptosis-inducing factor; tBid, truncated protein.

4.5 Protein Kinase C and Nurr77 Protein kinase C (PKC) is part of a serine / threonine family kinase. There are at least 11 different PKC isoenzymes that can be to divide into three sub-groups, based on their structure and mechanism of response to regulatory factors. Conventional PKCs (a, Pl, (3II, y) are Ca2 + -dependent and are activated by diacylglycerol (DAG) or 12-o-tetradecanoylphorbol-3-acetate (PMA) in vivo. The second subgroup (S, s, it, 0, p), the new iso-types, do not respond to Ca 2+ but it is activated by the DAG and PMA. The last subgroup (a ,, Ç, i), the atypical PKCs, are insensitive to both Ca2} and DAG. PKC are responsible for the transduction of multiple cellular signals in a variety of processes cell growth, differentiation, transformation malignancy and apoptosis. PKCs are also known to modulate the activity different membrane proteins such as transport proteins,

23 channels, and membrane proteins related to the cytoskeleton. since they have different roles, we find that their activation can give different results that may even be opposite results. It has been shown that activation of PKC induces apoptosis on a cell line cancerous treated with PMA via caspase-3 and of serine proteases. PKC has also been shown to inhibit apoptosis induced by the Fas receptor and this by modulating the loss of potassium (K}) and inhibition of caspase-8 and -3 activity. It is demonstrated by different researcher that PKC have a role in the transcriptional regulation of the expression of Fas and FasL genes. Park's team demonstrated that skill PKC to induce Fas expression is possible thanks to the gene TDAG51 (T-cell death-associated gene) in cells with wild type only. he there is another mediator in the expression of the Fas / FasL system which is a member of the superfamily of orphan nuclear steroid receptors, Nurr77. Nurr77 plays a role in cell growth by its role of postman.
nuclear transcription. It has been shown that by adding Nurr77 under form transgenic in thymocytes, an increase of Fas ligand is obtained suggesting that Nurr77 can lead the cell to become apoptotic by induction of Fas ligand expression. Another role was identified for Nurr77.
Nurr77 would be able to regulate apoptosis by means independent of its transcriptional regulation activity, among other things by its re-location of the core to mitochondria causing relaxation of cytochrome c. So, by his role of transcription factor and its role as a protein that can cause the release of cytochrome c, Nurr77 can thus cause a cell to become apoptotic.

5 Colon cancer and its defenses Within a tissue, homeostasis is maintained by the balance between cell growth and programmed cell death. A cancer cell can be defined as a cell that has survived the process of apoptosis and is now part of the cells that can contribute to the formation of tumor.
Several causes can lead to the formation of cancer, as a fault in

24 the growth process as a flaw in the process of apoptosis. These Faults are responsible for many diseases including cancer. A
cell accumulation can occur when the death rate is normal But that the growth rate is abnormally high or when the rate of growth is normal but the mortality rate is abnormally low. The malignant transformation and tumor progression are complex processes requiring a good number of genetic alterations.

Normally, tumor cells are removed by presentation, at the membrane surface, antigens to cytotoxic T lymphocytes (CTLs) and natural killer (NK) by the major class I histocompatibility complex (CMH
I) on their surface which allows to activate the immune response. Cells cytotoxic agents can recognize tumor cells by expressing their surface area, viral antigens (non-self), neo-antigens (derived from self mutated), unmutated but over-expressed self antigens, oncofoetal antigens (gene extinguished during embryogenesis which would be spontaneously re-transcribed).
Sometimes tumor cells develop alternatives to escape to the immune system. Several mechanisms are created by the cell tumor, some causing an aberration of the antigen presentation to the surface of the Cancer cell: The decrease in MHC I expression (tumor cell is more recognizable by CTL, but can be destroyed by NK) and alterations of the MHC I structure (recognition of neither CTL nor NK). Then, those causing a bad interaction between cytotoxic cells and the cancer cell : the decreased secretion of costimulatory molecules or adhesions (essential in the antigenic presentation, can cause anergy), decrease or mutation of the Fas membrane receptor and the DR4 or DR5 receptor (tumor cell less susceptible to CTL or NK attack). The tumor cells can also secrete cytokines that promote their growth. A study demonstrates that some colon cancer, that the production of IL-10 (which has for effect to inhibit the production of Th1 type CD4 + T cells and to inhibit functions of macrophages) is regulated by local production of pro-inflammatory cytokines as IL-6 and IFN-γ. A second study of 9 cell lines Cancer of the colon, demonstrates that these produce immunosuppressive factors, inhibiting the proliferation of T cells.

The concept of cellular immunity is based on the ability of T lymphocytes cytotoxic to eliminate tumor cells. The mode of action of CTL is To induce apoptosis in cancer cells by two mechanisms:
the receptors and by the perforin / granzyme B pathway.
certain case, the tumor cells develop ways to counter-attack the monitor the immune system and make sure that the process apoptosis is not triggered. Teams of researchers have demonstrated that the cells 10 colon cancer patients can defend themselves against CTL by expressing Fas ligand on their membrane surface, causing the death of CTLs by bond CTL Fas receptor and Fas ligand of cancer cells. By against, contradictory results have contradicted this hypothesis. So, we can note that this hypothesis remains a controversial subject of research.
A
15 other mechanism can be developed by the cancer cells so to avoid apoptosis and neutralize CTL: secretion in soluble form of ligand Fas. So in this scenario, the cancer cell secretes the Fas ligand under soluble form, causing a mutation in the trans-membrane domain of the protein so it no longer has inking point to the membrane Cell, and thus the Fas ligand binds to its ligand on the surface CTL and thus causing apoptosis in CTL. It has been shown that in some cases, CTL can induce apoptosis in cancer cells expressing the ligand Fas by the mechanism of perforin / granzyme B. In contrast, in other cases the tumor cells have developed a mechanism counteracting the effectiveness of

Perforin / granzyme B mechanism by overexpression of an inhibitor of serine protease, PI-9.

Tumor cells can develop many mechanisms to avoid death, but sometimes these mechanisms can lead the cell to self-suicide or to cause fratricidal death (death of a neighboring cancer cell).
By for example, if the cell secretes the Fas ligand on its surface or under form

26 soluble, it could bind to the Fas receptor of a cell neighbor or even on a Fas receiver located on the surface of the same cell.

There are other ways that a cancer cell can escape apoptosis among other things by the mutation of certain protein implicate in the apoptosis process such as p53 protein. This protein is one of the targets of most cancers of the colon. So by mutation, this who deprives it of these functions of guardian of the genome and activation of the process of apoptosis, even if the cell is damaged in its DNA by different treatments (radiotherapy and / or chemotherapy) it risks when even to escape apoptosis. There is also over-expression of the protein c-FLIP
this protein inhibits the binding step between the recruiting proteins FADD
and the caspase-8 by its binding to FADD. It has been shown that over-expression of this protein is common in different colon cancer which could contribute to tumor transformation in vivo.

Naturally, the list of escape mechanisms can be long because the cancer cells seems to develop strategies allowing them to to circumvent several obstacles that are constantly being discovered.

5.1 Colon Cancer Therapies Colon cancer treatments get a high rate of when it is localized in the colon and has not crossed the walls of colon. This high success rate is attributed to the diagnosis obtained early in the development of the disease. According to Statistics Canada, colon cancer is the third cancer causing the most deaths in Canada per year, 6500 deaths for the year 2000 and 17,000 new cases. To this end, several treatments are now available to counter this disease.

5.1.1 Stages of colon cancer according to Dukes Treatments are given according to the stage of the disease, there are two systems to classify in which stage of a patient's colon cancer is the Duke classification and the TNM system (tumor characteristics, nodal

27 involvelment and amount of metastasis). Duke's classification is the more used so far, here is a summary: Stage A represents the stage where the Colon cancer is limited to the lining or submucosa of the colon. The Treatment options at this stage are either colosectomy, during an injury superficial cause of cancer, excision of the affected portion, then of a deeper lesion. The postoperative survival rate is 90%. The Stage B is measured by degree of invasion of organs or tissue located near the tumor. The treatment to be given in these cases is naturally excision of the tumor and consideration for the use of the chemotherapy and / or radiotherapy. The survival rate is between 70-80%. Stage C involves invasion of lymphatic nodules and the formation of metastases in the major blood vessels. The treatments to be prescribed are the excision of sick parts, chemotherapy with adjuvant combination. Stage D, the last, distant metastases are present. The treatments are removal different isolated metastases (liver, lung, ovaries), as well as chemotherapy and / or palliative radiotherapy. The survival rate following the operation is usually less than 5 years old.

5.1.2 Chemotherapy The most used drug for chemotherapy is 5-fluorouracil (5FU), it is administered intravenously. Studies have shown than the use of 5FU, following excision of the tumor, is more beneficial for the Dukes phase C patients that excision alone. Then the desire to get of best results to defeat colon cancer, combined treatments are prescribed to different patients with colon cancer in phase B and C.
Several studies show that by combining 5FU with an adjuvant, levamisole or leucovorin, better results are obtained for stage C
of Dukes. Levamisole is known to improve the efficiency of 5FU by combination, it is shown that the cases of recurrences are decreased, and the mechanism is probably connected by macrophage activation that destroy the remaining tumor cells since it seems to be modulating the system immune positively. Leucovorin is a folic acid that is given to prevent the

28 hematological negative effects, so help keep healthy cells alive and leaving cancer cells subject to the cytotoxic action of 5FU. In Generally, studies agree that treatments with combination of adjuvant has a positive effect on the survival rate as well as the time recurrence of the tumor following removal of the cancer in comparison with the use of 5FU alone. The mechanism of action of 5FU is to bind to a enzyme inside the cell, which allows the synthesis of thymine during of the DNA replication, and inhibit it. Therefore, the cell unable to divide will die. Other drugs may work against colon cancer and are currently in clinical study, or will be soon. The different drugs are: Irinotecan (Camptosar, CPT-1 1), Oxaliplatin and Ralitrexed, Xeloda (Capecitabine). Irinotecan works by inhibiting the topoisomerase I, necessary to give some form to the DNA during the translation, transcription, and replication. Oxaliplatin, acts on DNA
forming bridges in the DNA thereby inhibiting its synthesis and replication. The Ralitrexed to a role similar to 5FU since it interferes with the synthesis phase of DNA
by blocking the enzyme synthesizing thymine. Xeloda, -is an oral tablet which turns into 5FU after ingestion.

5.1.3 Mechanism of destruction of cancer cells by chemotherapy When the effect of the drug on cells is known, it is possible to understand the mechanism that causes the cell to become apoptotic. The 5FU
being an inhibitor of thymine synthetase, it causes harm to DNA
during cell division by depriving her of one of the four bases pyrimidine constituting the DNA. By this damage to DNA, 5FU is responsible for activation of the guardian protein of the genome, p53. It is shown that this protein, by their transcription activity, can modulate the expression of different proteins involved in the process of apoptosis like for example the Bax protein, Bak and Bcl-2. So when the expression of Bax is increased and that the expression of Bcl-2 is diminishing, the chances that the potential membrane mitochondrial be lost is increased which leads to trigger the process

29 of apoptosis by the mitochondrial pathway. These proteins being regulated by the protein p53, determines the sensitivity of the cell to chemotherapy since in the majority of colon cancers, p53 is mutated. Although p53 is mutated, it is possible to observe the death of these cells following the addition of 5FU.

It has also been shown that the treatment of cancer cells 5FU induces the expression of Fas receptor and Fas ligand on the surface of cell cancerous. The induction of Fas receptor expression thus allows a better chance of cells being eliminated from the cells Immunity. It is also proposed that by inducing the expression of Fas receptor and Fas ligand on the surface of the treated cancer cells, a autocrine, paracrine or fratricidal death may follow. Since the cells express the receptor and the ligand, there could be cross-linking between the receptor and the ligand of the same cell (autocrine), or the receptor of a cell and the ligand of another cell (paracrine). Different teams of researchers have demonstrated that apoptosis induced by chemotherapy treatment, involves activation of caspase-3 and -8 whether it be a type I or II cell. he is suggested that in cells of type 1, both pathways of initiation of apoptosis are borrowed during chemotherapy. So the treatment promotes aggregation of Fas receptor which causes the activation of caspase-8 which will be activated directly caspase-3, and caspase-8 can also activate the protein cleavage which will activate the mitochondrial pathway of apoptosis. With regard to cell type II, apoptosis is controlled by the mitochondrial pathway since use of FADD inhibitor did not decrease apoptosis caused by chemotherapy in this type of cell. So, activation of caspase-8 in cells of type It occurs following mitochondrial signaling events.

Other studies have shown that damage to DNA by chemotherapy or irradiation, increases the expression of the death receptor DR5 dependent and independent of p53.

The involvement of several proteins is observed following a treatment of chemotherapy. It is important to understand the mechanisms used by this treatment since several cancers develop different tips for avoid death.

6 Lactic acid bacteria It was the scientist E. Metchnikoff (1845-1919) who proposed that the 5 longevity and health of the Bulgarian people is attributable to their ingestion of product fermented milk. It was well known that some bacteria are pathogenic for the organism, so it was proposed that these bacteria be replaced by bacteria yogurt since they have been used for a long time without any fears.
Several features exist to define good bacteria lactics:
10 they must keep their activity and viability before they consumption, they must survive the gastrointestinal tract, they must be able to to survive and grow in the intestines and eventually produce effects beneficial.
In addition, microorganisms must not be pathological or toxic.

Since then, several attempts have been made to improve health through Modification of the intestinal flora by live lactic acid bacteria.
Today, the beneficial effects of these lactic acid bacteria are well identified and have attempted now to explain the mechanism (s) related to these benefits.
The team Salminen summarized the most important benefits, supported by of the scientific evidence, such as immune modulation and enhancement of the 20 mucosal barrier of the intestines. Other teams have demonstrated, in the mouse, that growth as well as cancer metastasis can be inhibited by the strain of lactobacillus casei. Different mechanisms are proposed to to explain what these benefits would be: changing the flora intestinal, adhesion to the intestinal mucosa with the ability to prevent adherence of Pathogenic bacteria or the activation of pathogens, the modification of protein by the intestinal microflora, the modification of the capacity enzyme especially those suggested to be related to the induction of cancer, and finally the influence on the permeability of the mucosa intestinal.

Most studies indicate a therapeutic potential of bacteria

30 lactic and yogurt that is due mainly by the change of the microphone-

31 gastrointestinal ecology. The efficiency of lactic acid bacteria is increased by their ability to adhere to the intestinal wall since adherent strains have a competitive advantage, important to maintain their place in the tract gastrointestinal intestinal. However, no strains have yet been demonstrated for adherence permanently. By increasing the amount of lactic acid bacteria in intestines, it is possible to suppress the growth of pathogenic bacteria, who contribute in return to a reduction of infections. Intestinal epithelium intact with optimal intestinal flora represents a barrier against invasions or colonization by pathogenic microorganisms, antigens and compounds harmful to the intestinal tract.

In general, the consumption of lactic acid bacteria acts by a strengthening the non-specific immune response or acting as an adjunct in the antigen-specific immune response. Animal studies have demonstrated that lymphoid tissue associated with the intestines is stimulated by living lactic acid bacteria, resulting in cytokine production and of antibodies (IgA) and an increase in the mitogenic activity of cells forming Peyer's plates and splenocytes. In studies on human cells, cytokine production, phagocytic activity, production antibodies, T cell functions and NK cell activity, are increased by yogurt consumption or when cells are exposed to lactic acid bacteria in vitro.

Some evidence indicates that yogurt stimulates the system Immune may be associated with pathological decline in incidence like cancer, gastro-intestinal disorders and the symptoms of allergies.

6.1 Anti-cancer properties Lactic acid bacteria have antineoplastic properties in a variety of cancer cell line of human and animal origin. In in short, lactic acid bacteria reduce the viability of tumor cells, decrease the induced carcinogenesis in the colon and liver, inhibits mutagenic activity and this binds to potentially mutagenic compounds. Although no mechanism

32 is known, it is suggested that inactivation or inhibition of formation of cancer in the intestinal tract is induced.

There is considerable interest in the metabolic activity of the intestinal microflora, especially in relation to the etiology of cancer of the colon.
Studies have focused, among other things, on the measurement of key enzymes:
glucuronidase, azo-reductase and nitro-reductase. These enzymes catalyze the indirect carcinogen conversion to carcinogen in the intestines. By the absorption of lactic acid bacteria, these would reduce the activity of these key enzymes and thus prevent the formation of tumors. A supplement oral of lactic acid bacteria (L. acidophilus) of human origin causes a decrease significant of these three key enzymes. These results were partially confirmed by the Hammer team which recorded a decrease only the nitro-reductase in 9 subjects who ingested lactic acid bacteria (L.
acidophilus, B. bifidum) for 3 weeks. Searches continued on a model of animal colon cancer chemically induced by 1,2-dimethylhydrazine dihydrochloride (DMH). The activation of the DMH is done in the large intestine and these bacterial enzyme R-glucuronidase that transforms it in a potential carcinogen. Suppression of this enzyme can reduce DMH activation and subsequent tumor formation. These studies demonstrate that the addition of lactic acid bacteria can delay the formation of colon cancer by prolonging induction, indicating that lactobacillus can to slow the development of the tumor in the experimental animal model.

In summary, several conclusions are suggested with respect to Inhibitory functions of lactic acid bacteria against colon cancer.
Enter other, the increase or stimulation of immune functions, could help reduce the risk of cancer development or recurrence.
Also, lactic acid bacteria could take the place of bacteria pathogens that may be responsible for the formation of mutagenic compounds causing colon cancer.

33 There is therefore a need to find methods that are more effective or have less side effects that the treatments already available to treat the sickness.

SUMMARY OF THE INVENTION

As mentioned above, new properties of these bacteria have been discoveries. Indeed, it has been surprisingly discovered that the effect of lactic acid bacteria, in particular those contained in the product sold under trademark of Bio-K + International, in combination with an agent anticancer drugs can cause cellular apoptosis.

Several mechanisms can be at the origin of such a phenomenon. By example, lactic acid bacteria can avoid mutation that are at the origin of cancers. They can also prevent the progression of tumors by strengthening the immune system.

The applicant has surprisingly discovered that the consumption of lactic acid bacteria would prevent the formation of a Cancer, including colon cancer.

The Applicant has also surprisingly discovered that the use of lactic acid bacteria in combination with an agent anticancer effect of increasing the susceptibility of cancer cells to apoptosis.

Thus, the present invention relates to the use of at least one strain lactic bacterial to enhance an immune response in a mammal for the purpose of preventing or treating cancer.

Another object is the use of at least one lactic bacterial strain to facilitate the induction of apoptosis of cancer cells.

Another object is the use of at least one lactic bacterial strain for the manufacture of a medicament for the treatment or prevention of Cancer.

34 According to a preferred embodiment, the bacterial strain is under a living or irradiated form. More particularly, this bacterial strain is of genus Lactobacillus and more preferably of the species Lactobacillus acidophilus such as the strain 1-1492 deposited at the CNCM and / or Lactobacillus casei.

Another object of the present invention is to provide a composition for treat or prevent cancer, such as colon cancer. The composition of the present invention comprises an effective amount of at least one bacterial strain lactic acid as defined above and a pharmaceutically acceptable vehicle acceptable. According to a preferred embodiment of the invention, the composition also includes an anti-cancer agent, such as fluoro-uracil.

According to another object, the present invention proposes a method for preventing or treating cancer in a mammal, characterized in that comprises administering to said mammal a composition such as defined previously.

Another object of the present invention is a method for facilitating apoptosis of cancer cells in a mammal, characterized in that what comprises administering to said mammal a composition such as defined previously.

Another object of the present invention is the use of bacteria lactic acid to increase cancer cell apoptosis.

Another object of the present invention is the use in combination of lactic acid bacteria and an anticancer agent, such as FU
for the treatment of colon cancer.

Another object of the invention is to provide a kit for preventing or treating a cancer in a mammal, characterized in that it comprises a container containing a composition as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram illustrating a typical percentage analysis of apoptosis by flow cytometry.

Figure 2 is a diagram illustrating an apopotic process model.

Figure 3 is a diagram illustrating hypotheses of relaxation by the family of Bci-2 proteins.

Figure 4 is a diagram illustrating the proteolytic processing of the caspase-3.

Figure 5 is a diagram illustrating the summary of the effects of activation 10 of p53.

Figure 6 is a diagram illustrating the structure of the members of the receivers of membrane deaths and their interactions with major effectors cutoplasmics involved in apoptotic pathways.

Figure 7 is a diagram illustrating the interconnections between two different 15 pathways for transduction of apoptosis.

Figure 8 is a graph showing the optimal dose of 5-fluoro-uracil for get 50% cell death.

Figure 9 shows a visual presentation of apoptosis patterns obtained by flow cytometry.

Fig. 10 is a graph showing the effect of the composition in one mode.
preferred embodiment of the invention on the viability of LS 513 cells.

Figure 11 is a graph showing the effect of compostion in one mode preferred embodiment of the invention on the apoptosis of LS 513 cells.

Figures 12a, 12b, 12c and 12d are diagrams illustrating the measurement Apoptosis by flow cytometry. Figure 12a illustrates the cells having suffered no treatment. Figure 12b illustrates cells in the presence of 5FU to a concentration of 100 μg / ml. Figure 12c illustrates cells presence with lactic acid bacteria at a concentration of 108. The figure 12d illustrates the combination of cells, lactic acid bacteria (108) and 5FU
(100 μg / ml).

Figure 13 shows a western blot illustrating the activation of caspase 3 by cells put in the presence of a composition according to a preferred mode of the invention.

Figure 14 is a graph showing the extent of cell apoptosis LS 513 in the presence of compositions according to a preferred embodiment of the invention.

Figure 15 is a graph showing the measurement of cell viability LS 513 by the MTT.

Figure 16 is a graph showing the effect of living lactic acid bacteria versus bacteria heated on apoptosis of LS 513 cells.

Figure 17 shows western blots illustrating the effect of lactic acid bacteria live versus heated bacteria on activation of caspase 3.

Figure 18 is a graph showing the extent of apoptosis inherent in lactic bacterial strains of the present invention.

Figure 19 is a graph showing the apoptotic effect of the mixture of live bacteria and heated bacteria.

Figure 20 is a graph showing the effect of addition of butyric acid and 5FU on apoptosis of LS 513 cells.

Figure 21 is a graph showing the effect of the composition according to a preferred embodiment of the invention on the expression of the Fas receptor.

Figure 22 is a graph showing the effect of the composition according to a preferred embodiment of the invention on the expression of Fas ligand.

Figure 23 shows western blots illustrating the effect of the composition according to a preferred embodiment of the invention on the expression of a protein involved in apoptosis, the p53 protein.

Figure 24 show western blots illustrating the effect of composition according to a preferred embodiment of the invention on the expression of a protein involved in apoptosis, the p21 protein Figure 25 is a graph showing the effect of PKC on apoptosis.

Figure 26 is a graph showing the effect of PKC inhibition on Apoptosis.

Figure 27 is a graph illustrating the effect of bacteria supernatants of the invention on the induction of caspase-3 activity in the LS 513 cells.

Figure 28 shows photographs illustrating the effect of supernatants lactic acid bacteria of the invention on the fluorescence staining of the core LS 513 cells.

Figure 29 is a graph illustrating the effect of bacteria supernatants of the invention on the viability of LS 513 cells.

Figure 30 is a graph illustrating the effect of bacterial supernatants of the invention on the cell gel of LS 513 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore relates to highlighting the use of properties of lactic bacterial strains in the prevention or control of the treatment of cancer. More particularly, the use of these bacteria lactic aims to facilitate the induction of cellular apoptosis of cancer.

The invention also relates to the implication of these bacterial strains lactic acid in methods and compositions useful in the treatment or prevention of cancer, such as colon cancer.

According to a first embodiment, the present invention aims to use lactic bacterial strains to enhance the immune response in a mammal for the purpose of preventing or treating cancer.

According to a second embodiment, the present invention aims to use of lactic acid bacteria to facilitate the induction of cell apoptosis of a cancer.
By facilitating the induction of apoptosis is meant a process by which the presence of the lactic bacterial strains of the invention modulates positively cell death of a tumor, and preferably a tumor of the colon.

Mammal means any living organism that may be subject to cancer, and this includes vertebrate beings such as particular beings humans domestic and wild animals.

By treating is meant a process by which the symptoms of cancer, and particularly that of the colon, are attenuated or completely eliminated.

Prevent means a process by which cancer, and particularly that of the colon, is stopped or delayed.

According to a preferred embodiment of the invention, the bacterial strain is of the genus Lactobacillus and preferentially in a living form or irradiated. More particularly, the bacterial strain is of the species Lactobacillus acidophilus or Lactobacillus casei. In the case where the chosen species is Lactobacillus acidophilus, the inventors prefer to advantageously use the strain 1-1492 deposited at the CNCM.

According to a third embodiment, the present invention relates to the use of these lactic bacterial strains for the preparation of compositions useful in the treatment or prevention of cancer, such as cancer of the colon. A composition according to the present invention comprises a quantity effective of at least one lactic bacterial strain and a vehicle pharmaceutically acceptable. Preferably, the composition of the invention comprises a mixture of L. acidophilus and L. casei strains.

By "pharmaceutically acceptable" is meant a vehicle that can be administered safely to a mammal, in particular to a human being, and this with little or no negative or toxic side effects. Such a vehicle can be used for different functions. For example, it can be used in so as preservative, solubilizer, stabilizer, emulsifier, softening, dye, odorant, or as an antioxidant. These types of vehicles can be easily prepared using well known methods of the skilled person.

According to a preferred embodiment, the composition of this The invention further comprises an anticancer agent. For this purpose, any agent anticancer drugs that may be useful in this context is included in the scope of the present invention. However, 5 fluoro-uracil is advantageously used as an anticancer agent. The composition of this invention may also be part of a more complex therapeutic formulation, which is useful in the treatment and prevention of cancer.

According to a fourth embodiment, the invention proposes a method to prevent or treat cancer, including colon cancer, in a mammal. According to another related embodiment, the present invention proposes a method to facilitate the apoptosis of cancer cells in a mammal, like a human being. These methods include, in addition, the step administering to the mammal in question a composition according to the present invention.

The amount or concentration of lactic acid bacteria that is administered to a human or animal or who is present in the composition of the invention is a therapeutically effective amount. A therapeutically effective lactic acid bacteria is that amount needed to obtain positive results without causing excessively negative side effects in the host in which the lactic acid bacteria or the composition is administered.
Moreover, an effective amount of lactic acid bacteria to treat a Cancer particular is an amount that is sufficient to mitigate or reduce a in any way the symptoms related to cancer. Such a quantity can be Administered in a single dose or can be administered according to a diet, by which is effective. The amount of lactic acid bacteria according to this invention can treat cancer but typically it is administered so to reduce the symptoms of cancer. The exact amount of lactic acid bacteria or of each of the components of the composition and the amount of the composition Administering varira according to factors such as the type of cancer to be treated, others ingredients in the composition, the method of administration, the age and the weight of mammal, etc ...

The compositions according to the present invention may be in any solid or liquid form usual for administration Pharmaceutical, that is, for example, forms of administration liquid, in gel, or any other medium known to those skilled in the art. Among the compositions usable, mention may be made in particular of the compositions which can be administered by oral. In this case, the composition of the invention can be administered under form of food or dietary supplements. We can also mention Injection compositions more particularly for injection in the blood circulation in humans.

A person versed in the field will know how to prepare compositions pharmaceutically acceptable and determine, according to several factors the preferred mode of administration and the quantity to be administered.
From 25 factors that may influence his choices include: the nature of the treatment, the exact nature of the ingredients, whether active or not, used in the composition; the Stadium of the disease; condition, age and weight of the patient, etc.

The present invention also includes useful pharmaceutical kits, for example example, for the prevention or treatment of cancer, such as cancer colon.
The kits comprise one or more containers containing, in addition, a composition according to the present invention. Such kits can additionally include, if desired, one or more conventional pharmaceutical components, such as for example, containers containing one or more vehicles pharmaceutically acceptable, or any other additional component, which will be obvious to someone in the art. A kit according to the present invention can advantageously include instructions in pamphlet form or on any other printed medium, indicating the quantities of the components to be administered, the guidelines for administration, and / or guidelines for mixing components.

The following example will highlight other features and advantages of the present invention.

EXAMPLE
The following example serves to illustrate the extent of use of this invention and not to limit its scope. Changes and variations can to be there without being beyond the scope and scope of the invention.
Although other methods or products equivalent to those found below for testing or carrying out the present invention, the hardware and the Preferred methods are described.

INTRODUCTION
In the context of the present invention, to determine how the Lactic acid bacteria help apoptosis of cancer, tests have been done on the human colon cancer cell line LS-513. Lactic bacteria used constitute a mixture of Lactobacillus acidophilus and Lactobacillus casei. The anti-cancer agent is 5-fluorouracil (5FU). This compound acts in so inhibitor of the enzyme synthesizing thymine.

MATERIAL AND METHODS
1 Lactic Bacteria 1.1 Origin The mixture of bacteria used for the different experiments is provided by the company Bio-K (Laval, Qc, Canada). The mixture includes a combination of Lactobacillus acidophilus 1-1492, which is the subject of request international no. PCT / CA97 / 00915, and Lactobacillus casei.

1.2 Preparation The bacteria received in 9 ml of MRS complex medium (Difco laboratories, Detroit, USA) are immediately multiplied in 100 mL of the same middle by taking 100 μl of the bacterial suspension. After an incubation of 18 hours in a 37 C incubator, 10 mL of glycerol is added to the mixture of 100 mL which is then divided into 1 mL aliquots in several bottles Sterile plastic can hold 1.5 mL. These bottles are stored in a freezer at a temperature of -80 C.

For the different stimulation protocols, a bottle is thawed and put in 9 mL of MRS and incubated for 18 hours at 37 C. After this time incubation, transplanting is performed. A volume of 100 L is collected and added to 9 mL of MRS which is also incubated for 18 hours at 37 C. After these incubations, the bacteria are washed twice in sterile PBS and harvested by centrifugation at 3500 RPM for 10 minutes. They are then suspended in a final volume of 9 mL of sterile RPMI containing 10%
fetal bovine serum and are then ready to be used for a stimulation cellular. During the different experiments, the bacteria are used under form heated, irradiated and alive.

1.3 Heating Bacteria are heated for 40 seconds at "high" in an oven at Microwave (General Electric, Turntable Microwave Oven, 700 walt) so to obtain a temperature of 100 C and produce the mixture of bacteria that will be used as heated bacteria. This step is done in a container of closed glass.

1.4 Irradiation In order to produce the mixture of irradiated bacteria, the tubes of bacteria are irradiated at a minimum dose to achieve 100% lethality, is of 5 kGy. The mixtures are irradiated in Gammacel-220 (MDS Nordion, Laval, Qc, Canada) using Cobalt-60 (60Co) as the source of gamma rays.

In order to make a bacterial count to obtain a concentration of mL bet bacteria of this suspension is added to 9 mL of peptone water, a isotonic solution containing 0.1% bactopeptone (Difco laboratories, Detroit USA). Serial dilutions are performed. Then 1 mL of these dilutions is removed and placed in a petri dish to which is added 10 mL of 1.5% agar MRS
(Difco Laboratories, Detroit, USA) to enable a count to be made after incubation for 48 hours in a 37 C incubator.
sample is made in duplicate.

2 Cancer cells 2.1 Origin The LS 513 cell line (ATCC, Rockville, MD, USA) is a line continuous colon cancer of human origin.

2.2 Culture Since the cell line is adherent, it is obligatory to take off cells, using a trypsin-EDTA solution (Gibco, Burlingtion, ON, Canada) and then resuspended in RPMI supplemented with L-glutamine and 10% SBF, which can be called "complete RPMI". The trays are made the day before the stimulation to allow the cells to adhere to trays. On the day of the stimulation, the different products are added to the desired concentrations and then the cells are incubated for a period determined for each type of experiment, in a 37 C incubator, 5% CO2 and saturated with moisture.

3 Co-cultures of cancer cells and bacteria 3.1 Addition of bacteria Once the cell trays are ready for use, that is when the cells have had time to adhere, and bacteria primed to be used, this is added according to the stimulation protocol in the wells containing the adhered cells and the medium "RPMI complete". An incubation is performed for a determined time depending on the experiment to be performed.

3.2 Experiment involving the addition of butyrate The plateau of cancer cells containing 3 x 105 cells per well is incubated overnight to allow the cells to adhere. Then, different concentrations of butyric acid (Sigma, St-Louis, USA) are added to well containing the adhered cells and the culture medium, the complete RPMI.
Following this addition, a 48-hour incubation is done in order to measure the percentage of apoptosis according to the technique described below.

3.3 Harvest and studies of supernatants The purpose of this experiment is to check whether the presence of bacteria changes the pharmacological presentation of 5FU. A first stimulation is performed on adhered cells for a period of 48 hours in the presence different stimulation products. Following this incubation, the supernatants are harvested and added to a new cell culture freshly adhered.
Then, a second incubation of 48 hours is carried out for pick up cells and measure the percentage of apoptosis.

4 Measuring the viability of cancer cells 4.1 Proliferation 4.1.1 MTT

The cells are prepared as indicated above. The test is carried out in a 96-well plate. A volume of 100 L with a concentration of 3.3 x 105 cells per mL is deposited in each well except in the first row which will serve as "white". Once the stimulation products have been added, a incubation 48 hours is carried out. Following incubation, supernatants are withdrawn by suction, and the cell sheet is washed, adding in a delicate way 200 L of PBS in each well, and by rapidly decanting the PBS added to the sink. Then a solution of MTT (Sigma, St-Louis, USA) at 5X diluted in of Complete RPMI is added to the wells and incubated for 5 hours at 37 ° C
is 10 done. Subsequently, the solution is decanted and another solution is added for the purpose of dissolving crystals formed in living cells. This solution is composed of 50% dimethyl formamide and 12% sodium dodecyl sulfate (SDS). An 18 hour incubation is required to dissolve all crystals. Following this incubation, the plate is read in a spectrophotometer 15 (Mandel Scientific company, Bio-Tek instruments, Microplate EL309 Autoreader) to 540nm. Each sample is made in triplicate, and an average is made to from the values obtained. The average value of the cookie is 100%
of living cells. To obtain the percentage of the other samples, enough to make a cross product.

4.2 Apoptosis 4.2.1 Flow cytometry A concentration of 5x104 cells per mL, at a rate of 6 mL per well, is used to obtain 3x105 cells per sample. The cells are prepared as indicated above. The different products are added to the wells, and A 48 hour incubation is made.

Following incubation, cell supernatants are recovered from different 15 mL tubes and centrifuged at 1500 RPM for 5 minutes. The Cell pellets are then recovered by decantation of the supernatants and placed aside on ice. This step involves recovering the cells in suspension.
Subsequently, the adhered cell sheet is washed with 0.5 mL of trypsin-EDTA, then 0.2 mL of trypsin-EDTA is added to each well to allow cells to peel off when incubated for about 8 minutes in the incubator at 37 C. The cells are suspended in 3.mL of complete RPMI
10% FCS to stop the enzymatic activity of trypsin. Suspension cell is centrifuged at 1500 RPM for 5 minutes in the tubes serving for flow cytometry. Subsequently, the two cell pellets (cells in suspension and adhered cells) are combined and washed twice with PBS
cold supplemented with 0.25% EDTA in order to avoid the formation of agglomerates cells. After washing, 0.5 mL of propidium iodide solution is added to the cellular pellet. The solution is composed of 0.1% sodium citrate (Fisher Scientific, New Jersey, USA), 0.1% TritonX-100M '(Sigma, St. Louis, USA), 50 g / mL of RNase (Sigma, St. Louis, USA) and 20 g / mL of propidium iodide (Sigma, St. Louis, USA). An incubation of 15 minutes at 4 C is performed for then analyze the samples by flow cytometry (Coulter Epics XL-MCL).
Figure 1 summarizes a typical result of a cytometric analysis of flux.
Different peaks are formed following the differences in fluorescence enter every step of the cell cycle. Plus there is a high content of intact DNA
more the fluorescence is high, and vice versa. The programmed measures the percentage fluorescence, which forms the broad peak below the peak corresponding to G1, this peak represents fragmented pieces of DNA, a consequence of DNA cleavage by different enzyme activated during apoptosis.

Measurement of Expression of Proteins Involved in Apoptosis (p53, p21 Caspase 3, Bax) 5.1 Western-type blotch 5.1.1 Stimulation and extraction of proteins A total of about 6x105 cells per sample is used. The next day, while the cells became adherent, the stimulation products are added. The different products of. stimulation are 5-Fluorouracil (100 g / mL) and bacteria, living or heated, at a concentration of 1X108 bacteria per mL. These products will cause the cells to become apoptotic by the modulation of different proteins involved in the process. It's this modulating, increasing the expression or activation of protein, that the technique allows to check. Continued - at different times of stimulation (since is a kinetics), the cells are harvested and centrifuged at 1500 RPM
for 5 minutes. Subsequently, 50 L of lysis buffer composed of 50 mM of Tris-HCl pH 7.5, 150 mM NaCl, Nonidet P-40 1% (Roche Diagnostics, Laval, Qc) as well as a complete tabletTM (containing the protease inhibitors) (Rock Diagnostics, Laval, Qc), are added to the cell pellet which is then incubated 30 minutes on ice, the volume of 50 L is taken and placed in a micro-tube of 1.5 mL. A centrifugation of 10 minutes at 15,000 RPM is carried out in order to precipitate cell debris. The supernatant is recovered and a volume equal of "sample buffer" is added. The "sample buffer" is composed of 100mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol and 0.006% blue bromophenol. Finally, the samples are aliquoted by volume of 20 L
and stored at a temperature of -200C.

5.1.2 Separation and identification of proteins The protein samples are separated on a polyacrylamide gel-SDS at 4% and 12% using Bio-Rad's Mini-PROTEAN Mc machine. The proteins migrate inside. of an electrical current of 200 volts while minutes. The migration is done in an "electrode buffer" at pH 8.3, composed of 1.5% Tris-Base, 7.2% glycine and 0.5% SDS in the mili-Q water. Over there then the proteins are transferred to a nitrocellulose membrane Hybond ECL (Amersham Pharmacia Biotech Inc., Baie d'Urfe, Quebec) using the Bio-Rad transfer machine and this for one hour at 100 volts in a transfer buffer composed of 0.58% Tris-Base, 0.29% glycine, 0.037% SDS and 20% methanol. Following the transfer, the membrane is "blocked."
with a blocking solution composed of 0.1% PBS-Tween Mc 80 and skimmed milk powder at 5% for one hour at room temperature and under agitation. Then, the first labeling is carried out with the antibody recognizing the desired protein. The antibody is diluted in the solution of blocking according to a concentration indicated by the supplier. After an hour of marking, a washing of 15 minutes followed by two washes of 5 minutes, with the blocking solution. The second marking is done with a second antibody that recognizes the first antibody and that is coupled to the peroxidase. An incubation of one hour is made with this second antibody, which is also diluted in the blocking solution at a specified concentration by supplier. As soon as this incubation is complete, a washing of 15 minutes, as well as two washes of 5 minutes, are carried out with a solution of PBS-0.1% Tween 80 without skimmed milk powder. In order to detect the different proteins, an ECL solution (Amersharn Pharmacia Biotech Inc., Baie d'Urfe, Qc.
Canada), which leads to the activation of peroxidase, is added to the membrane according to the supplier's instructions. Subsequently, the markings are revealed sure photographic paper (ECL hyperfilm, Amersham Pharmacia Biotech inc, Bay d'Urfe, Qc. Canada) which will be labeled with activated peroxidase. The paper photographic is then developed. Table 1 summarizes the proteins researched and the reagents used.

First antibody Protein Description Dilution Company p53 Anti-human p53 1: 1000 BD PharMingen, purified Mississauga, Ontario Iso type: IgG2a mouse p21 Anti-p21 mouse 2: 1000 BD PharMingen, purified Mississauga, Ontario Iso type: IgG1 mouse Caspase 3 Anti-capase 3 rabbit 1: 1000 BD PharMingen, polyclonal Mississauga, Ontario Bax Anti-Bax 1: 1000 Santa Cruz California, USA

Second antibody IgG Anti-IgG mouse 3: 10,000 Sigma, St. Louis, USA
Coupled peroxidase Developed in goat IgG Anti-IgG Rabbit 2.5: 10,000 Sigma, St-Louis, USA
Coupled peroxidase Developed in goat Table 1: The antibodies used for the different proteins to identify.

6 Measurement of expression of markers on the cell membrane (Fas, Fas L) 6.1 Flow cytometry A total of approximately 5x105 cells is used per sample. Cells are prepared as mentioned above. A 24-hour incubation is performed following the addition of the different stimulation products (5FU, bacteria living or heated and others according to the experiments that are performed).

Following incubation, the cell sheets are washed with 0.5 mL of trypsin-EDTA, then 0.2 mL of trypsin-EDTA is added to the cell which are then placed in the incubator at 37 C for about 10 minutes.
Once the cells have taken off, 3 ml of complete RPMI are added, and the Cell suspension is then centrifuged for 5 minutes at 1500 RPM.
The Supernatants are decanted and the cells are placed on ice. Then 20 The Anti-receptor antibody solution (BD PharMingen, Mississauga, Ontario) or Fas ligand (BD PharMingen, Mississauga, Ontario) are added to cell pellet, to which 50 L of buffer had previously been added.
flow cytometry "which consists of 1X PBS, 1% BSA, 0.02% azide sodium and 0.25% EDTA. A half-hour incubation on ice in the dark is required. Then two washes with 4 mL of flux"
are performed by centrifugation at 1500 RPM for 5 minutes. To the cells labeled with Fas ligand antibody, an amount of 0.25. L per tube of streptavidin-PE (BD PharMingen, Mississauga, Ontario) is added following the First incubation and two washes. A second incubation of 30 minutes on ice in the dark is performed, followed by two other washes. At the end a labeling, 250 L of paraformaldehyde solution (1x PBS, 2%
paraformaldehyde) and 250 L of "flow cytometry buffer" are added to to fix the markings. The tubes are wrapped in aluminum foil and 10 placed at 4 C until the analysis of the samples.

7 Measurement of Nurr77 Gene Expression 7.1 Extraction of lRNA

A number 3x105 cells is used per sample. An incubation of 3 hours is done following the addition of different stimulation products.
The 15 cells are then harvested using a scraper and then centrifuged at for 5 minutes. The total RNA of each sample is extracted and purified using Roche Diagnostics High Pure RNA Trolley. (Lavai, Qc, Canada), such as indicated by the manufacturer. The RNA concentration is then measured with the machine (Pharmacia Biotech, Gene Quant RNA / DNA Calculator), then Adjusted to 92 μg / L.

7.2 RT-PCR

The LightCyclerM Kit RNA amplification SYBR Green 1 from Roche Diagnostics (Laval, Qc, Canada) is used to carry out the reaction of reverse transcription (RT) and polymerase chain reaction (PCR). The 25 principle of LightCyclerM. is very similar to that of the Thermocycler. The major difference is the possibility, with the LightCyclerMO, observe amplification at each cycle, thanks to a fluorescent molecule called SYBR
Green 1 that fits into each double strand formed. More there is formation of double strands more one observes, using.the program included with the LightCyclerMO

the increase in fluorescence. Both reactions, RT-PCR, are done in a capillary built specifically for the LightCycler (Roche, Laval, Qc, Canada) and all you need to do is mix the products in the kit and use this set according to the indications of the manufacturer.

Before performing an amplification, it is necessary to develop certain conditions. For example, the concentration of MgCl2, the temperature and the time. An ideal concentration of MgCl2 is to be determined. For With the amplification present, a concentration of 7 mM was the best.
The Primer concentration is 0.5 mM, as suggested by the manufacturer.
In the case of the Nurr77 gene, the sequence of primers used for the positive is 5'-CGACCCCCTGACCCCTGAGTT-3 'and that for the negative strand is 5'-GCCCTCAAGGTGTTGGAGAAGT-3 '(Kang JH, Biol.Pharm.Bull.). amplification by this pair of primer gives 658 base pairs. The programming of the LightCycler machine is described in the instruction manual provided by the manufacturing. Two other parameters vary in the program amplification:
among others in the hybridization segment, the temperature to be used varies according to primers. The temperature (set at 5 C below the hybridization temperature primers (Tm) is calculated by the following formula: Tm = 2 C (A + T) + 4 C
(C + G). For the primers used the Tm is 64 C. The second parameter is the incubation time for elongation, always located in the program amplification; it is determined by the following formula t = (number of pairs of base of the amplification product - 25) seconds. In our case, the number of base being 658, so we get 26 seconds. Following the 35 cycles required to amplify the desired part of the gene, a melting point curve is performed, using a temperature of 10 C higher than the temperature hybridization.
So the amplifications are subject to a gradual increase in the temperature and at each degree the fluorescence is measured and recorded. In gradually increasing the temperature, the double strands formed take off when the temperature is high enough, so it's up to temperature is observed a decrease in fluorescence. The program makes a melting point curve with the recorded fluorescences, and by the after, it measures the derivative of fluorescence as a function of temperature. In knowing the theoretical melting temperature of the amplification product, it is possible to get the value of the area under the curve of the peak formed by the separation strands of the amplification product at this temperature. The machine does not allowing to visualize the number of base pairs amplify, a migration on 2% agarose gel with a base pair marker allows the verification following ethidium bromide staining at a concentration of 0.5 g / mL
for 15 minutes.

8 Treatments with PKC inhibitors or pacemakers Stimulations with PKC inhibitors and stimulators are performed in the same way as stimulations by bacteria and by the 5FU. First, the cells are prepared the day before to allow them to adhere, and the very day of stimulation, the different products of stimulation are added at the same time as the inhibitor GO 6976 (Sigma) and / or stimulators of PKC, ionomycin (Sigma) and PMA (phorbol 12-myristate 13-acetate) (Sigma). Then, a 48 hour incubation is performed, the cell are harvested and the percentage of apoptosis is measured.

9 Cytokine assay 9.1 TNF assay by bioassay It is possible to assay the amount of TNF in a supernatant using the L929 cell line, which is a mouse fibroblast line sensitive to (the cytotoxic action of TNF.) The principle of this bioassay is simple: the more it there is TNF in the supernatant added to the L929 cell sheet, the more there will be death cellular. The level of living cells can then be measured. In firstly the cells are cultured in complete RPMI 1640 + 5% FCS. The cells detach from the flask with trypsin-EDTA (Gibco, Burlington, WE, Canada) by incubating for approximately 1 minute at 37 C. A cell count is performed to prepare a suspension of 3.3 x 105 cells / mL for bioassay.

A volume of 75 L is deposited in each well of a plate of 96 well. All lines receive this volume except the first one that is used as an empty witness. Following a 24 hour incubation, a volume of 25xL
of actinomycin D, at a concentration of 2 g / mL, is added to all wells of all the lines except the second line that serves as actinomycin control D, and this in order to stop cell growth .. Thereafter, the different samples are added from the fourth line at the rate of 100 L per well, and this in triplicate. The third line remains empty since it will serve as a control positive is to say that it will represent the maximum number of cells since there is no cytotoxic agent added. With the added samples, dilutions clear are carried out starting from the 100 L which one dilutes in series in the 8 rows following. When the samples are diluted, incubate from 16 to 20 hours at 37 C + 5% C02. After this incubation, the supernatants are discarded and the cells are attached to the bottom of the well using a formaldehyde solution 5% to 100 L per well for 5 minutes. The plates are emptied and rinsed 3 times under running water. The cells which have remained fixed are then stained with crystal violet at 50 L per well for 5 minutes. Subsequently, plates are emptied and excess dye is removed by 3 rinses with running water. A
times the dry plates, 100 L of a solution of acetic acid at 33% is added at each well to dissolve crystal violet absorbed by the cells fixed.
The absorbance is read at a wavelength of 540 nm in a spectrophotometer for plate, using column 1 as a reference ( White ).

In order to calculate the number of TNF units, one unit of TNF is the inverse of the dilution factor giving 50% cytotoxicity.
In order to calculate the percentage of cytotoxicity for each sample, equation next is used.

% cytotoxicity = OD sample x 100 Positive DO

So, the OD of the sample is the average of the three absorbances obtained for dilution following the reading of the plates. The DO of the control positive is the average of the wells in line 3. The% of cytotoxicity is calculated for each dilution. Subsequently, a linear regression line is drawn for dilutions of a sample, the dilution factor (= x) and the% of cytotoxicity (= y) and the 50% point is found using the equation of the line. The opposite of the dilution (2x) equals the number of TNF units in the initial sample not diluted.
The results are expressed in U / mL.

RESULTS
1) Determining the optimal dose of 5 Fluoro-uracil Figure 8 shows the measurement of apoptosis by flow cytometry following exposure of increasing doses of 5-fluorouracil (5FU) in order to obtain a ideal concentration giving 50% mortality. A total of 3x105 cells is placed in the presence of the different concentrations of 5FU for 48 hours. Then, the DNA content of the cells is labeled with propidium iodide and analyzed by flow cytometry to obtain the percentage of apoptosis. The The following results are the average of two independent experiments.

Figure 9 illustrates a diagram representing one of the two experiments performed to obtain the ideal concentration of 5FU. , The device measures the number of events of the sub-G1 peak compared to the number of events of the sample, which gives a percentage result. Since we know that peak sub-G1 corresponds to cleaved DNA, sign of apoptosis, so we give percentage the value of apoptosis.

2) Action of the combination of 5 Fluoro-Uracil + live bacteria on the viability of LS 513 cells.

Figure 10 illustrates the viability of colon cancer cells is assayed by MTT following a 48-hour incubation in the presence or absence of 5-Fluoro-uracil (5FU) (2.5ug / mL) and live bacteria (B) at different concentrations (106-108). A total of 3.3 X 104 cells per well in one tray 96 wells are brought into contact with the different stimulation products. The staining produced by the reaction of MTT with living cells, is evaluated on a spectrophotometer at a wavelength of 540 nm. Each sample is the average of 3 different wells.

Figure 11 illustrates the effect of living bacteria and 5FU on apoptosis LS 513. Live lactic acid bacteria (B) at different concentrations (106-109) and 5-fluorouracil (5FU) (100 μg / ml) are added to the LS 513 cells.
The measurement of apoptosis by flow cytometry is made following an incubation of 48 hours. The labeling of the DNA with a solution of propidium iodide allows To observe the percentage of cells having cleaved DNA (sub-G1) produced following incubation. The control are cells that have undergone no treatment.

Figure 12 shows examples of flow cytometry schemes for the measure of apoptosis. These 4 diagrams represent 4 different samples produced during an experiment. The number of events in sub-G1 gives a Percentage relative to the rest of the stages of the mitosis cycle. The witness (AT) being the cells having undergone no treatment, the image B is composed of cell put in the presence of 5FU at a concentration of 100 μg / mL, image C is the one where living bacteria, at a concentration of 108, were combined with cell and the last image (D), represents the combination of cells, bacteria Live (108) and 5FU (100 g / mL).

Figure 13 illustrates a western blot of caspase 3 and this in its form proactive. A 48-hour incubation in the presence of live bacteria at a concentration of 108 and 5-Fluoro-Uracil (5FU) at a concentration of 100 ng / mL
was done. From this incubation, the proteins were extracted and migrated on gel Polyacrylamide. Subsequently, they were transferred to membrane nitrocellulose and labeled with an antibody specific for caspase 3 and this to a concentration of 1: 1000.

3) Action of the state of bacteria 3.1) Live bacteria versus irradiated bacteria Figure 14 shows the measurement of apoptosis of LS 513 cells in presence of live bacteria, irradiated bacteria and 5FU. This Figure shows more specifically the extent of apoptosis by the percentage of sub-G1 by flow cytometry using propidium iodide labeling (20).
ug / mL). Colon cancer cells are in the presence or absence of 5-Fluoro-Uracil (5FU) (100 μg / mL) and live bacteria (B) or bacteria irradiated (C) at different concentrations (106-109) for a period of hours. The controls are cells without treatment.

3.2) Live bacteria versus heated bacteria Figure 15 shows the measurement of viability of LS 513 cells by the MTT. More particularly, this figure shows the effect of living bacteria (B) and of heated bacteria (C) at different concentrations (10eX) in the presence or absence of 5-Fluoro-Uracil (5FU) (A) (2.5ug / mL) on the viability of LS 513 after a 48-hour incubation. The values are obtained by reading spectrophotometer (540nm), by staining due to MTT which stains the Functional mitochondria therefore those of living cells only. The cell concentration used is 3.3 x104 cells per well. The results following are the average of three wells of a 96-well tray.

Figure 16 shows the effect of live bacteria versus bacteria heated on apoptosis of LS 513 cells. The analysis of the percentage of sub-G1 by DNA labeling was obtained with propidium iodide. A total 10,000 events are processed per sample. A 48-hour incubation presence of live bacteria (B) and heated bacteria (C) and this at different concentrations with or without 5 Fluoro-Uracil (100 μg / ml) are the different samples shown in the figure.

Figure 17 shows the effect of living or heated bacteria on activation of caspase 3.

4) Possible mechanisms inherent to bacterial cultures Figure 18 shows the extent of apoptosis inherent in lactic acid bacteria of the invention. A first stimulation was performed while 48 hours. Some wells did not contain cells (F). The different supernageas (D) were harvested and added on a fresh cell culture (E). The concentration of 5-Fluoro-uracil (5FU) is 100 μg / mL and two concentrations different are used for live bacteria (B) and bacteria heated (C) which are 1X107 and 108. The measurement of apoptosis is done by cytometry in flow by labeling the DNA with propidium iodide.

Figure 19 shows the apoptotic effect of the mixture of live and heated bacteria. The measurement of apoptosis by flow cytometry has been taking following a 48-hour incubation. The LS 513 cell line is implemented presence of a given concentration of 5FU (100ug / mL) and the presence of live bacteria (B) and heated bacteria (C) at two different concentrations (107 and 108). The control is composed of cells without no treatment.

Figure 20 shows the effect of adding butyric acid and 5FU to apoptosis of LS 513 cells. To colon cancer cells is added a dose of 5FU (100ug / mL) and different doses (2mM and 4mM) of acid butyric (ab). Apoptosis is measured following a 48-hour incubation.

5) Possible mechanism inherent to tumor cells Figure 21 shows the effect of the composition according to one embodiment preferred on the expression of the Fas receptor.

Figure 22 shows the effect of Fas ligand expression.

Figure 23 illustrates the effect of the composition according to a preferred mode of the invention on the expression of the p53 protein.

Figure 24 illustrates the effect of the composition according to a preferred mode of the invention on the expression of the p21 protein.

Figure 25 illustrates the effect of PKC activation on apoptosis.

Figure 26 illustrates the effect of PKC inhibition on apoptosis.

6) Characterization of the supernatant of the lactic acid bacteria of the invention on apoptosis of intestinal tumor cells LS 513 The apoptotic potential of the bacteria supernatants on the LS 513 line, tumor line was analyzed. The results obtained on the activity of the Caspase-3 demonstrate that the supernatants even in the presence of 5-FluoroUracil (2.5 μg / ml and 100 dag / ml), do not significantly activate this caspase (Figure 27). At contrary, in the presence of 100 μg / ml of 5FU, the supernatants inhibit the activity of caspase-3. Fluorescence staining of the nucleus of the cancer cells, at using the DAPI technique, it has not been possible to highlight stones where chromatin is condensed, a fundamental feature of cells apopotitiques. Staining of nuclei is typical of healthy cells and alive (Figure 28, 100X and 630X magnification). In addition, cytometry studies of flux using propidium iodide do not demonstrate cell death (Figure 29) nor significant disturbance of the phases of the cell cycle (Figure 30). All of these results indicate that the supernatants of bacteria do not induce apoptosis in LS 513 cells.

DISCUSSION
The action of intact bacteria, that is to say, those that are alive and irradiated on cancer is the same. On the other hand, non-intact bacteria by example those destroyed by heat seem to have an opposite action to the intact bacteria.

In addition, it was noted according to the work done in the context of the present invention that the efficacy of an anti-cancer agent such as 5 FU
is greatly increased in the presence of intact bacteria. This efficiency would be dependent dose. Faster apoptosis in the presence of bacteria alive is noticed.

The presence of bacteria heated with 5 FU would increase expression of the p21 protein. So there could be modulation via a receiver unknown regulatory protein apoptosis \ cell cycle (p21). A
increased p21 protein was noticed when there is less apoptosis, and a decrease in the protein when there is more apoptosis.

Butyric acid is a product of lactic acid bacteria and is present in the intestine. This product causes apoptosis in cancer cells colon in vitro. There will be synergy between butyrate and 5FU on breast cancer colon. He has It has also been noted that butyric acid inhibits the in vivo growth of human colon cancer on mice.

In view of the above, living lactic acid bacteria enter into synergy with 5FU to decrease the number of cancer cells in culture (MTT) or to increase apoptosis in the latter (cytosine flow).

Irradiated lactic acid bacteria have the same action as bacteria while the heated bacteria would have an opposite action.
So, the intact form of lactic acid bacteria is necessary for their action against tumor cells. In addition, the action of bacteria is also dependent and proportional to the dose. The property of inducing or modulating apoptosis by the lactic acid bacteria of the invention has been corroborated by experience showing the non-apoptotic effect of the supernatant of these bacteria.

The expression of caspase 3 as well as that of the p21 protein is modulated by living lactic acid bacteria in the same way as apoptosis.

Claims (21)

CLAIMS: 1- Use of a lactic acid bacterial strain to strengthen a response immune in a mammal for the purpose of preventing or treating cancer, said strain being Lactobacillus acidophilus I-1492 deposited at the CNCM. 2- Use according to claim 1, characterized in that said mammal is a To be human. 3- Use of a lactic acid bacterial strain to facilitate the induction of apoptosis cancer cells, said strain being Lactobacillus acidophilus I-1492 deposited with the CNCM. 4- Use of at least one lactic acid bacterial strain for the manufacture of a medicament for the treatment or prevention of cancer, said strain being Lactobacillus acidophilus 1-1492 deposited with the CNCM. 5- Use according to any one of claims 1 to 4, characterized in that the bacterial strain is in a living or non-living form but intact. 6- Use according to any one of claims 1 to 5, characterized in this that it also includes the use of a strain of the species Lactobacillus casei. 7- Use according to any one of claims 1 to 6, with an agent anticancer. 8- Use according to claim 7, characterized in that the agent anticancer is fluoro-uracil. 9- Use according to any one of claims 1 to 8, characterized in that said cancer is colon cancer. 10- A composition for treating or preventing cancer, comprising an amount effective of at least one lactic acid bacterial strain and a pharmaceutical vehicle acceptable, said strain being Lactobacillus acidophilus 1-1492 deposited in the CNCM. 11-Composition according to claim 10, characterized in that the strain bacterial is in a living or non-living but intact form. 12-Composition according to one of claims 10 or 11, characterized in that what further comprises a strain of the species Lactobacillus casei. 13- Composition according to any one of claims 10 to 12, characterized in this that it includes an anticancer agent. 14- A composition according to claim 13, characterized in that the agent anticancer is 5 fluoro-uracil. 15- A composition according to any one of claims 10 to 14, characterized in that that said cancer is colon cancer. 16-Use of a composition according to any one of claims 10 to 14 for the manufacture of a medicament for preventing or treating cancer in a mammal. 17-Use of a composition according to any one of claims 10 to 14 for the production of a medicament to facilitate apoptosis of cells of a Cancer in a mammal. 18- Use according to claim 16 or 17, characterized in that said mammal is a human being. 19- Kit for preventing or treating cancer in a mammal, comprising a composition according to any one of claims 10 to 14, and a container for contain said composition. 20- Use of a lactic acid bacterial strain for the manufacture of a drug to enhance an immune response in a mammal for the purpose of prevent or to treat cancer, said strain being Lactobacillus acidophilus I-1492 filed at the CNCM. 21- Use according to claim 20, characterized in that said mammal is a To be human.