HK1067985B - Lactic acid bacteria and their use for treating and preventing cancer - Google Patents

Description

The field of invention

The present invention relates to the demonstration of the usefulness of the lactic acid bacterium strain Lactobacillus acidophilus I-1492 in the prevention and treatment of cancer, and in particular to the use of the lactic acid bacterium to facilitate the induction of cancer cell apoptosis.

Description of the art of the past

The bacteria Lactobacillus acidophilus I-1492 present in the product Bio-K+ and covered by the patent application WO 98/23727 are known to have a beneficial effect on blood cholesterol levels in mammals.

International application No WO 98/23727 concerns a lactic acid which includes a strain of Lactobacillus acidophilus I-1492 and is therefore mainly used for the preparation of a lactate ferment to reduce blood cholesterol in mammals.

In addition, these bacteria are also known to have properties that have the effect of strengthening the immune system, facilitating the absorption of nutrients and stimulating the intestinal flora. It is known that lactic acid bacteria have a positive effect on the intestinal flora and also on the immune system.

In the United States, a large number of people die each year from colon cancer. Cancer is the third leading cause of death each year. In Canada, in the year 2000, the cancer death rate was 6,500 and there are more than 17,000 new cases.

Nutraceutical use involving the administration of yogurt and/or fermented milk as an adjunct to cancer treatment is also known.

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

Chemotherapy is also available. 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. 5 fluorouracil is a drug that is commonly used to treat colon cancer. This drug can be administered orally or intraperitoneally (closest to the cancer target) due to its high instability in serum. It is known to cause the death of colon cancer cells and does so by various means.

Cell death, also called apoptosis, can be accomplished through the mediation of different proteins. For example, cell apoptosis can result from the activation of a membrane receptor or cytoplasmic expression of different proteins that promote this phenomenon. In this regard, 5FU is known to increase the expression of the p53 protein as well as the expression of the membrane receptor Fas.

1 General information on apoptosis 1.1 Definition of apoptosis

In 1972, the term apoptosis was finally adopted and coined by Currie and colleagues, to describe a common pattern of programmed cell death that the authors repeatedly observed in several tissues and cell types. It was observed that these dying cells shared several morphological characteristics, which are different from those seen on cells with a common pathology and necrosis cells, and they suggest that these shared morphological characteristics could be the result of a programmed cell death and preservation.

1.2 Role of apoptosis

Researchers have discovered that cells in our body can die, they know that this cell suicide called apoptosis is essential to the body. Apoptosis is as fundamental to the physiology of cells and tissues as cell division and differentiation. Apoptosis is the most common form of physiological cell death that occurs at different times such as during embryonic development, during tissue reorganization, during immune regulation and tumor regression.And the cell is involved in maintaining cell and tissue homeostasis in a way that is opposite to mitosis. It's the innate mechanism by which the body eliminates unwanted cells. Each cell carries within it the genetic mechanism of its own destruction. Cells only survive if they receive survival signals from their environment. If the cell receives signals telling it to kill itself, then it triggers the death program. A disruption in the protein balance between the cells that are alive and the proteins that are now leading to cell death can be associated with a wide spectrum of diseases including cancer,neurodegeneration, autoimmune diseases, diabetes and other disorders.

2 Morphological features of apoptosis and necrosis 2.1 Apoptosis

A key feature of cell apoptosis is cell shrinkage. As cell shrinkage occurs, the cytoplasm compresses and nuclear chromatin condenses and forms aggregates, in the nucleus, which then stick against the nuclear membrane. Cell organelles, including the mitochondria, appear relatively unchanged. Subsequently, the nucleus becomes fragmented. The formation and emission of buds are observed on the cell surface.The apoptotic cell fragments are rapidly swallowed up by the surrounding phagocytic cells, such as the macrophage. Apoptosis is a so-called clean death since the cell fragments are quickly eliminated. There is no inflammatory phase, no damage to the surrounding tissue and this is partly because their cell membrane remains intact. In summary, the characteristic morphological changes at apoptosis are the shrinkage of the cytoplasm, the condensation and fragmentation of DNA and finally, the formation of apoptotic bodies contains the nucleus fragments surrounded by the cytoplasm and cell membrane.

2.2 Necrosis

Necrosis refers to sudden death that occurs as a result of extreme physical or chemical stress. It is marked by different morphological criteria. During necrosis, this uncontrolled cell death, there is rapid loss of control of ion flow resulting in water penetration and increased ion influx, cells swell and its organelles such as mitochondria and endoplasmic reticulum until membranes burst and non-specific DNA fragmentation in the nucleus.

Distribution tissulaire	regroupement cellulaire	cellule isolée
Réaction tissulaire	La lyse et le relâchement du contenu cellulaire aboutissant à l'inflammation des tissus environnants	Phagocytose des corps apoptotiques par les macrophages ou les cellules environnantes et aucune inflammation
Morphologie Cellule	Gonflement	Rétrécissement, perte de contact avec les cellules environnantes, "blebbing", formation de corps apoptotique
Organelles	Endommagées	Intactes
Noyau	Désintégré	Condensé et fragmenté
Lysosomes	Endommagés	Intactes
Mitochondries	Défectueuses, épuisées en ATP, enflées et endommagées	Enflées, changement peuvent se rompre, relâchement de cytochrome C
Biochimie ADN	dégradation non spécifique	clivage à l'intérieur du noyau
Protéines	dégradation non spécifique	Activation des caspases

3 Different Stages of the Apoptotic Process

Apoptosis can have three different stages (Figure 2). First the cell must receive an apoptotic signal, so the triggering or engaging phase, then there is the regulating or controlling phase and finally the execution phase during which the intracellular enzyme cascade would occur inducing apoptotic death.

3.1 Step of initiation of apoptosis

A variety of internal and external stimuli can activate the cells to become apoptotic. Among the different stimuli, we can list biological agents (membrane receptors, transcription factors, oncoproteins, viral infection, bacterial toxins, ...), suppression of factors essential for cell growth (cytokines, growth factors and nutrients, ...), genomic DNA damage (spontaneous or induced), exposure to chemicals (anti-cancer agents), exposure to physical inducers (UV rays, X-rays, microwaves, heat, ...).

3.2 Decision or execution stage of apoptosis

Following these different stimuli, the cell receives the different signals and decides to become apoptotic or not. This step includes different transduction signal pathways including activation (or inactivation) of serine/threonine and tyrosine kinases and phosphatases, synthesis of second messengers, alteration of gene expression, and activation of specialized proteases known as caspases. The final decision to become apoptotic depends on several factors including the balance between pro-apoptotic and anti-apoptotic proteins (the Bcl-2 protein family), the metabolic status of the cell, and also the stage of the cell cycle in which the cell is.The Bcl-2 family of proteins is divided into two groups, one inhibiting apoptosis (Bcl-2, Bcl-XL, Bcl-w, CED-9,...) and the other promoting apoptosis (Bax, Bid, Bad, Bak, Bcl-Xs, ...). Whether pro- or anti-apoptotic, they have the ability to control ion flow between various cell compartments, especially between the mitochondria and the cytoplasm.The intermembrane space of the mitochondria includes several proteins involved in the activation of apoptosis, such as pro-caspases-2, -3, -7 and -9, AlF and cytochrome c. The activation of these caspases will lead to a point of no return since by their activation, they will split their different targets, among others, the proteins necessary for the survival of the cell.

3.3 Degradation stage of apoptosis

The cell is now irreversibly engaged in the cell death program which consists of presenting the morphological characteristics of the apoptotic signature. So, by activating the different caspases, several proteins necessary for cell survival are cleaved and become non-functional, such as poly (DNA) ribose polymerase. Other caspase targets may be activated by these, including those of DNases which will themselves cut chromatin into high molecular weight fragments.

Proteins involved in the regulation of apoptosis 4.1.1 The Bcl-2 family of proteins

Bcl-2 and its family members are important modulators of apoptosis. In this family of proteins, there are two classes including anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bfl-1/A1, Brag-1) and pro-apoptotic proteins (Bax, Bak, Bad, Bik, Bim, Bcl-xs) (Reed, 1996). Bid members of the Bcl-2 family are classified according to their number of homology domains with Bcl-2 (BH: Bcl-2-homology).The protein is therefore formed of two central α-helices surrounded by five amphipathic α-- helices. Interestingly, our three-dimensional structure is homologous to bacterial toxins that form pores in membranes, such as diphtheria toxin and colicin, which could suggest a potential mechanism of action for these proteins at the mitochondrial level. Another structural characteristic would be the ability of these proteins to homo and dimerize with each other,The BH3 domain is the BH3 domain, and therefore they may favor or antagonize their functions.

4.1.2 Roles of anti- and pro-apoptotic agents A- On the mitochondria

Experimentally, MPT is characterized by a spike in the permeability of the inner membrane to particles with a molecular weight ≤ 1500 Da. This permeability transition has several consequences including collapse of the Δψm, osmotic swelling, relaxation of the Ca2+ nucleus, creation of oxygen-adenoscopic matrix species, and rupture of the outer membrane of the Ca2+ nucleus, which allows for the interspacing of cytochrome-transferases between the inner membrane and the mitochondrial membrane. This allows for direct communication between the mitochondrial membrane and the pores (known as the PT-V and PT-V) and thus allows for the direct control of the pores.

Bcl-2 family proteins have different cytoplasmic distributions. Bcl-2 and Bcl-xL have a hydrophobic c-terminal tail containing a membrane insertion sequence, and most of these proteins are known to be associated with mitochondrial, endoplasmic reticulum and nuclear membrane membranes. In their inactive form, pro-apoptotic proteins, Bad, Bax and Bid, have a primary cytoplasmic location. However, when activated, they reside on the mitochondria. When Bid is cleaved, its terminal part COOH resides on the surface of the mitochondria.The Bcl-2 family is closely involved in the release of cytochrome c, the electron transporter protein inside the mitochondria. In addition to being involved in oxidative phosphorylation in the mitochondria, Bcl-2 is involved in the production of the Bcl-2 protein, which is a protein that is involved in the release of the cytochrome c.Cytochrome c is one of the constituents (along with the adaptor protein Apaf-1) that is required for caspase-9 activation in the cytosol. How do members of the Bcl-2 family regulate the release of cytochrome c? Several hypotheses have been put forward, but none is definitively proven. There are three basic models that can be suggested. 1- Bcl-2 family members form a channel that facilitates protein transport. Based on similarity in structure of Bcl-XL to the pore-forming subunit of diphtheria toxin, it has been suggested that Bcl-2 proteins could be inserted into the outer membrane of mitochondria,The Bcl-2 family members can insert themselves into a synthetic bi-lipid layer, oligomerize, and form a channel with discrete conduction. Bid and Bik proteins can directly induce mitochondria to release cytochrome c without interacting with VDAC or ANT suggesting they act outside of PT.2-pores.A particularly interesting candidate for such a protein would be the voltage-dependent anion channel (VDAC), several members of the Bcl-2 family can bind to it and regulate its channel activity. Since the pore size characteristic of the VDAC channel is too small to allow proteins to pass through, this model must assume that the VDAC undergoes a conformational change following binding of Bcl-2 family members. Bcl-2 and Bcl-XL proteins have been shown to promote PT pore closure, while pro-apoptotic Bax protein has the opposite effect, it interacts with ANT and VDAC to promote pore opening and cytochrome c re-latency.3- The Bcl-2 family members induce rupture of the outer membrane of the mitochondria. It is possible that the Bcl-2 family controls mitochondrial homeostasis. In this model, the apoptotic signal would alter mitochondrial physiology (e.g., ion exchange or oxidative phosphorylation) such that the organelle swells, resulting in the physical rupture of the outer membrane and the re-release of proteins located between the mitochondrial membranes, into the cytosol. The need to pass through a channel thick enough to allow cytochrome c to form is now more necessary since proteins would simply diffuse by tearing into the lipid bilayer.

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

The AIF protein (for apoptosis-inducing factor) that has been identified and its gene cloned, is capable, on its own, of inducing apoptosis in isolated nuclei. This molecule is synthesized in the cytosol as a precursor and then imported into the mitochondria. Like cytochrome c, it is a phylogenetically ancient molecule, with a dual function: oxidative reduction and apoptosis factor. However, unlike the cytochrome c pathway, which requires the activation of other pathways to induce apoptosis, the AIF pathway is instead independent of the cases and does not require any intermediate factors to induce apoptosis.

Hypotheses regarding the mechanisms of inhibition of apoptosis and in particular the sequestration of Apaf-1 by Bcl-2 and its anti-apoptotic agonists still seem to be highly debated. Apaf-1 is likely an important target of Bcl-2 family members since the deficient Apaf-1 cells are refractory to various pro-apoptotic signals and are themselves inhibited by Bcl-2. In addition, over-expression of Apaf-1 showed that this protein was associated with survival proteins such as Bcl-XL and Bcl-2. However, it was shown that there is no co-immunoprecipitation between the Bcl-2 family members and Apaf-1. Apaf-1 was also found at sites where Bcl-2 resides on the outer membrane, such as the nucleus and the endoplasmic membrane, and at the level of Bcl-2 receptors such as the Bcl-2 and Bcl-2 nucleoplasts.

4.1.3 Mechanism of modulation of Bcl-2 family proteins

Several different mechanisms exist to modulate the functions of pro- and anti-apoptotic proteins. First, the dimerization state of Bcl-2 family members affects their activity. One of the functions of anti-apoptotic proteins Bcl-2 and Bcl-XL is to dimerize with pro-apoptotic protein Bax to neutralize its activity.For example, when the number of Bcl-2 is greater than or equal to the number of Bax, the cells in question are protected from apoptosis. However, when the number of Bax exceeds the number of Bcl-2, the cell is more likely to become apoptotic. Third, the proteins of the Bcl-2 family can be modified by phosphorylation. The best example of this would be the pro-apoptotic protein Bad. In its non-phosphorylated state, it dimerizes with Bcl-2 and Bcl-XL, thus neutralizing their anti-apoptotic activity.Bid is another protein in the Bcl-2 family that is activated by caspase cleavage. While the protein at its full length is inactive, following caspase 8 cleavage, Bid induces mitochondrial re-release of cytochrome c. Finally, conformation of Bcl-2 proteins alters their activity. The best evidence for this mechanism comes from studies done on Bax.In its inactive state, Bax exists under a conformation under which it resists proteolytic cleavage, but following its activation and re-location on the mitochondria, the N-terminal region of the protein becomes susceptible to cleavage, suggesting that a conformational change is indeed occurring.

In summary, mitochondria play an important role in the initiation of apoptosis. Their intermembrane space contains several proteins (cytochrome c, caspase-2, -3, -7, and -9, AIF) which, once released into the cytoplasm, participate in the degradation phase of apoptosis. The riddle of the mechanisms of induction and control of apoptosis by mitochondria is based on four essential points: namely, the molecules of the Bcl-2/Bcl-xL family which could contribute to the formation of ion channels at the intracellular membrane level.The Bcl-2 family of anti-apoptotic molecules may also act by titrating endogenous activators (such as Apaf-1) of apoptosis. A final point is that some pro-caspases also have a mitochondrial localization. Thus, apoptotic promoters or Bci-2 family inhibitors regulate apoptosis through multiple effects on caspase activation cascades, redox potential, and the permeability barrier function of mitochondrial membranes.

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 only cleave after aspartic acid residues. This unusual specificity of having an aspartate as a substrate is found only in another protease, granzyme B, however this enzyme uses a serine as an active site. Caspases are synthesized as a simple chain of polypeptides and they are inactive zymogens. These zymogens are composed of three domains: one N-terminal pro-domain, and two other domains, p10 and p20, which are found in the mature enzyme.When activated, each polypeptide chain is cleaved into two subunits, a large (p20) and a small (p10) which subsequently dimerize. Thus, the mature enzymes that have been observed are hetero-tetramers composed of two p20/p10 hetero-dimers and two active sites. The N-terminal peptide, is cleaved and released upon activation. This N-terminal peptide is not required for enzymatic activity, its role is known on caspase 8 and 10, where it acts as an interaction domain with other proteins to modulate their activation.whereas caspase 2 and 9 contain a caspase activation and recruitment domain (CARD).

There are at least 14 different caspases identified in mammalian tissues to date. It is possible to divide caspases into three different groups based on their substrate specificity, that is, their recognition of the three amino acids that precede aspartic acid. The first group, contains caspases involved in the inflammatory process, therefore pro-cytokine activation, which includes caspases 1, 4 and 5. These enzymes are sometimes known as caspases ICE-like, because another name for caspukinase-1 is Interle-1-converting enzyme (ICE).The second group of caspases contains caspases 6, 8, 9, and 10. These enzymes are considered to be signaling caspases because they can activate other caspases and thus initiate the cascade. Their recognition motif is (LV) EXD. The last group contains caspases 2, 3 and 7. These enzymes are known as effector because they split multiple cell targets resulting in the morphological appearance of apoptosis.Effector enzymes are the most specific, with a need for an aspartic acid in the first and fourth positions prior to the cleavage site. They have their recognition motif DEXD. The most recent caspases, caspases 12-14, have not yet been characterized sufficiently to be classified in one of the three groups.

4.2.2 Activation of caspases

There are three different mechanisms for caspase activation. The first mechanism is activation of the caspase by another caspase that has been previously activated. Most caspases are activated following a proteolytic cleavage of the zymogen between the p20 and p10 domains, and usually another cleavage between the pro-domain and the p20 domain. Interestingly, all of these cleavage sites occur after an aspartate, the caspase substrate, which suggests the possibility of autocatalysis activation.

As shown in Figure 4, the first cleavage is between the p20 and p10 domains (within 12 kDa) to separate the two subunits.

Caspase cascade is a very useful method for amplifying the pro-apoptotic signal, but it cannot explain how the first, most downstream caspase 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 rapprochement. Caspase-8 is known to be the initiating caspase during death receptor-induced apoptosis. When the ligand binds to its receptor, the CD95/Fas death receptor trimerizes and forms membrane-bound signaling complexes.The final model of caspase activation is the association of pro-caspase with a regulatory subunit. Take for example caspase-9 which requires association with cofactors for activation. The cofactor apoptoticase activating factor-1 (Apaquef-1) has been identified by a protective biochemical approach as one of two proteins needed for caspase activation.The complex that these three proteins form, requiring ATP, gives the active form of caspase-9 often called apoptosome. So Apaf-1 is not only a caspase-9 activating protein but is a vital subunit for its functioning. In summary, effectors are usually activated by caspases downstream, while initiators are activated by regulated protein-protein interactions.

4.2.3 Victims of the caspases

Caspases cleavage a large number of cell proteins, and the process of proteolysis is limited as there are a small number of cuts that are made. Sometimes cleavages result in protein activation, and other times inactivation but never degradation since their substrate specificity distinguishes caspases as the strictest endopeptidases. Caspases cleavage several cell proteins whose number is constantly increasing. Structural, nuclear and signaling proteins are all targeted by the caspases (Table 1). - What?

Signalisation	autres caspases	Activation
	PKC δ	Activation, fragmentation nucléaire
		Activation
		Formation de fragment pro-apoptotiques
	Bid	Activation
	ICAD	Activation de l'endonucléase CAD
Nucléaire	Facteur de fragmentation de l'ADN	Fragmentation de l'ADN
	Polymérase poly (ADP-ribose)	Inactivation
	Protéine kinase ADN-dépendante	Inactivation
	U1 (70 kDa)-snRNP	Diminution de la synthèse des ARNs
	Lamine A et B	Désassemblage de la lamine nucléaire
Structurale	Actine	Réarrangement du cyto-squelette
	Gelsoline	Réarrangement du cyto-squelette
	α-Fodrin	Changement membranaire

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 made at an activated checkpoint when DNA is damaged. Depending on the stimulus and the phase in which the cell is, activation of p53 may lead to cell proliferation stopping and DNA repair or apoptosis. While the first stimulus to activate p53 is damaged DNA, other cellular stresses such as metabolite deprivation, physical damage, heat, and oxygen can also activate p3.5

The p53 increases dramatically within a few minutes of the cell's sudden damage. This increase is possible by post-translational changes in the p53 polypeptide, without obvious dramatic induction of the p53 mRNA level following DNA damage. The change that is made to the p53 polypeptide level following DNA damage results in phosphorylation. In a cell that has undergone no stress, the p53 protein has an extremely short stable half-life but becomes much longer following cell damage.Thus, p53 is marked by ubiquitins with the help of a protein called Mdm2, a protein that plays a role in the negative regulation of p53. It is shown that the protein Mdm2 interacts with p53 so that it becomes the perfect target of proteases by ubiquitins. The protein Mdm2 also causes p53 to be translocated from the cell nucleus to the cytoplasm, where it will undergo ubiquitin-induced proteolysis. So, by phosphorylating the regulatory domain in the C-terminal part of p53, it is shown that the activation of its DNA binding depends on specific sequences at the site.In addition, phosphorylation of serine-15 and serine-20 at the N-terminus of p53 causes inhibition of the interaction between p53 and Mdm2 and therefore increases the level of p53 and converts it to a form capable of transcriptional activity. A large number of p53 kinases phosphorylate p53, including casein kinase, extracellular signal-linked kinases, protein kinase C, and kinase Raf-1. Once phosphorylated, p53 acts as a transcription factor to increase and decrease transcription of several genes involved in apoptosis.

Several cell cycle regulatory proteins are induced by p53, e.g. p21, GADD45 and family members 14-3-3. The ability of p53 to induce cell cycle arrest in the G1 phase following DNA damage is well known and may be explained by the fact that p53 when stimulated has transcriptional activity that allows the cyclin-dependent kinase (Cdk) inhibitor gene, protein p21 to be transcribed. A high number of p21s will then inhibit cyclin E/dkc2 and cyclin A/dkc2 kinases, thereby preventing these kinases from promoting cell cycle progression.In addition, p53 is also involved in cell cycle arrest in the G2 phase in part because p53 induces the expression of the protein sigma 14-3-3 which will cause the sequestration of the cyclin B/Cdc2 complex. p53 can lead to apoptosis by activating the transcription of different genes giving proteins involved in the apoptotic process. The proteins that are induced are Bax protein, Fas receptor and DR5 (receptor for the death ligand TRAIL), all of which are involved in the process of apoptosis.The p53 protein is a protein that is activated by the axon axon of the axon, which is the axon of the axon, and is a protein that is activated by the axon axon of the axon, which is the axon of the axon.

The end results of DNA damage can be cell cycle arrest, growth, or apoptosis. DNA damage results in the accumulation and activation of the p53 protein (Figure 5). Once activated, p53 has transcriptional 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 p21, which will inhibit cyclin-dependent kinases (cld), the cell cycle is then stopped at G1.

4.4 Death receptors

Death receptors are receptors located on the surface of the cell and are so named because when they bind to their ligand, they can initiate the process of apoptosis. These receptors are part of the TNF receptor family, specifically the TNF-R1 receptor itself, the Fas receptor (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-bound such as tumor necrosis factor-α (TNF-α), Fas-L and TNF-related apoptosis inducing ligand (TRAIL).The ligand-receptor interaction induces receptor trimerization, which allows the physical association of adaptive proteins with the interacting protein death domain (RIP-DD) domains, promotes recruitment and activation of proximal caspases such as pro-caspases-8,-10 and -2, which are then able to transmit the death signal inside the cell.TRADD can also recruit FADD and RIP leading to the apoptotic process and NF-κB activation, respectively. RIP can also recruit RAIDD RIP-associated ICH-1/CED-3-homologous protein with a death domain which then recruits caspase-2 and induces apoptosis. When FasCase is taken as the model for apoptosis activation, the Fas complex and FADD will recruit pro-pase-8 which will induce the death signal complex, the death signal-inducing complex (DISC).Once assembled, the DISC will cause rapid auto-activation of caspase-8 which will activate caspase-3 and cause cell apoptosis. Thus, this first pathway of action of the Fas receptor is a fast pathway that short-circuits the mitochondria and does not require the input of new molecules because it is based on the interaction of pre-existing molecules.

However, it has recently been shown that activation of caspase-8 following trimerization of the Fas receptor can also cause cleavage of Bid, a pro-apoptotic protein of the Bcl-2 family. This cleavage results in the penetration of a truncated form of Bid into the mitochondria with the consequent release of cytochrome c, followed by depolarization of mitochondrial membrane potential and apoptosis. It has also been shown that when there is activation of DR-4 and DR5 by TRAIL, caspase-8 is activated.It is possible that by binding TRAIL to the DR4 or DR5 receptors, caspase-8 is activated by recruitment by an adaptor molecule, which both activates caspase-3 and clive Bid, so that the latter becomes active and allows the re-release of cytochrome c which will have the effect of activating caspase-9 which in turn will activate caspases-3 which will cause an amplification of the caspase cascade and cause cell death.

In summary, there are at least two pathways for transduction of the apoptotic signal by some death receptors, one direct, fast, and the other slower, involving mitochondrial relay. For this reason, some authors classify cells (type I or II) according to their mode of induction of apoptosis by Fas. For example, activation of Fas in some cells leads almost exclusively to the caspase pathway only (type I cells). These cells usually show no involvement from mitochondria, and cell death is not usually inhibited by B-2 or B-L-x. In other cells, activation of the pathway is largely by mitochondria using the cytochrome B-8 relay, and in the case of Fas-L-x, activation of the cytochrome B-2 relay can occur in the cytochrome B-8 relay, as a result of the action of cytochrome B-L-x.

Several stimuli can initiate apoptosis, but common morphological and biochemical alterations are observed regardless of the initial stimulus.

The abbreviations mentioned in this figure have the following meanings: DD, death domain; TRADD, TNF-receptor associated death domain; FADD, Fas-associated death domain, DISC, death-inducing signaling complex; RIPNF, death-interacting protein, TRAF-12, T-receptor associated factor-2; NF-BIP, nuclear factor B, BCH-B, BCH-B, BKNK, TNK, TNF-related factor; TNF-related domain, TNF-related factor, TNF-binding factor; RIPNF, death-interacting protein, TRAF-12, T-receptor associated factor-2; RIPNF, death-related death receptor, TRAF-45, TNF-related factor, TNF-related factor; RIPNF, death-related death receptor, TRAF-12, T-receptor-related factor-2; NF-BIP, nuclear factor B, BCH-B, BCH-B, BCH-B, BCH-B, BKNK, TNF-related factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor, TNF-binding factor; TNF-binding factor, TNF-binding factor, TNF-binding factor; TNF-binding factor, TNF-binding factor; TNF-binding factor, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding, TNF-binding

The process of apoptosis requires the participation of several pathways in order to activate caspases (Figure 7). The two best known and best characterized are the transduction of the apoptotic signal by death receptors and the other pathway more internally in the cell is apoptosis induced by changes in mitochondrial integrity, particularly the release of apoptotic factors such as cytochrome c and AIF. There are interconnections between these two signaling pathways and signal amplification loops.

4.5 Protein kinase C and Nurr77

Protein kinase C (PKC) belongs to the serine/threonine kinase family. There are at least 11 different PKC isoenzymes that can be divided into three subgroups based on their structure and mechanism of response to regulatory factors. Conventional PKCs (α, βI, βII, γ) are Ca2+-dependent and are activated by either diacylglycerol (DAG) or 12-o-tetradecanoylphorbol-3-acetate (PMA) in vivo.PKCs are responsible for the transduction of multiple cell signals during a variety of cell processes such as cell growth, differentiation, malignant transformation, and apoptosis. PKCs are also known to modulate the activity of different membrane proteins such as transporter proteins, channels, and cytoskeleton-bound membrane proteins. Since they have different roles, their activation has been shown to give different results that may even be opposite results.PKC has also been shown to inhibit Fas-receptor-induced apoptosis by modulating potassium (K+) loss and inhibiting the activity of caspase-8 and caspase-3.[3] It has been shown by a different researcher that PKCs have a role in transcriptronal regulation of the expression of the Fas and FasL genes. Park's team has shown that the ability of PKCs to induce Fas expression is possible only through the T-cell death-associated gene (TDAG51) in wild cells.Nurr77 plays a role in cell growth through its role as a nuclear transcription factor. It has been shown that by adding Nurr77 in transgenic form to thymocytes, an increase in Fas ligand is obtained which suggests that Nurr77 may lead the cell to become apoptotic by inducing Fas ligand expression. Another role was identified for Nurr77. Nurr77 is said to be able to regulate apoptosis by a means independent of its transcriptional re-regulation activity, including by its re-location from the nucleus to the mitochondria causing cytochrome c to be released.Nurr77 can thus cause a cell to become apoptotic.

5 Colon Cancer and Its Defense

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 among the cells that can contribute to tumor formation. Several causes can lead to cancer formation, both a failure in the growth process and a failure in the apoptosis process. These faults are responsible for many diseases including cancer. A cell accumulation can occur when the rate of cell death is normal but the growth rate is abnormally high or when the rate of growth is normal but the death rate is abnormally low.

Normally, tumor cells are eliminated by presenting cytotoxic T-cell (CTL) and natural killer (NK) antigens on the membrane surface by the major histocompatibility complex class I (MHCI) on their surface, which activates the immune response.Cytotoxic cells can recognize tumor cells by the expression on their surface of viral (non-self) antigens, neonegens (from mutated self protein), non-mutated but over-expressed self antigens, oncofoetal antigens (expressed during embryo re-transcription).

Sometimes, tumor cells develop alternatives in order to escape the immune system. Several mechanisms are created by the tumor cell, some causing an aberration of the antigen presentation on the surface of the cancer cell: decreased expression of CMH I (tumor cell is no longer recognizable by CTL, but can be destroyed by NK) and alterations in CMH I structure (recognition of neither CTL nor NK). Then, those causing poor interaction between cytotoxic cells and the cancer cell: decreased secretion of co-stimulating or adhesion molecules (essential in antigen presentation, can cause anemia),A study shows that certain colon cancers, IL-10 production (which inhibits the production of Th1-type CD4+ T cells and inhibits the function of macrophages) is regulated by local production of pro-inflammatory cytokines such as IL-6 and IFN-γ. A second study on 9 colon cancer cell lines showed that those producing immunosuppressant factors,It inhibits T cell proliferation.

The concept of cell immunity is based on the ability of cytotoxic T cells to kill tumor cells. The mode of action of CTLs is to induce apoptosis in cancer cells by two mechanisms: by dead cell receptors and by the perforin/granzyme B pathway. However, in some cases, tumor cells develop ways to counter the surveillance of the immune system and prevent the apoptosis process from being triggered.And the way that we can do this is by causing CTLs to die by binding to the Fas receptor of CTLs and the Fas ligand of cancer cells. But conflicting results have disproved this hypothesis. So you can see that this hypothesis is still a very controversial research topic. Another mechanism that can be developed by cancer cells to prevent apoptosis and neutralize CTLs is to secrete the soluble form of the Fas ligand. So in this scenario, the cancer cell secretes the soluble form of the Fas ligand, causing a mutation in the trans-membrane domain of the Fas protein, so it no longer has a binding point to the cell membrane,In some cases, it has been shown that CTLs can induce apoptosis in Fas-expressing cancer cells by the mechanism of perforin/granzyme B. In other cases, however, tumor cells have developed a mechanism that counteracts the effectiveness of the mechanism of perforin/granzyme B by over-expression of a serine protease inhibitor, PI-9.

Tumor cells can develop many mechanisms to avoid death, but sometimes these mechanisms can cause the cell to commit suicide or cause fratricidal death (death of a neighboring cancer cell). For example, if the cell starts to secrete the Fas ligand on its surface or in soluble form, it could bind to the Fas receptor of a neighboring cell or even to a Fas receptor on the surface of the same cell.

There are other ways that a cancer cell can escape apoptosis, including by mutating a certain protein involved in the apoptosis process, such as the p53 protein, which is one of the targets of most colon cancers. So by mutation, which deprives it of its functions as a genome guardian and activator of the apoptosis process, even if the cell is damaged at the DNA level by various treatments (radiotherapy and/or chemotherapy), it may still escape apoptosis. There is also the over-expression of the protein c-IPFL, which inhibits the expression of the binding protein that recruits different proteins FADD and FADD-8 in vivo. It has been shown that this protein is a frequent factor in tumor transformation.

Of course, the list of escape mechanisms may be long, for cancer cells seem to be developing strategies to overcome a number of obstacles that are continually being discovered.

5.1 Treatments for colon cancer

Colon cancer treatments have a high success rate when it is localized in the colon and has not crossed the colon walls. This high success rate is attributed to early diagnosis of the disease. According to Statistics Canada, colon cancer is the third leading cause of cancer deaths in Canada each year, with 6,500 deaths for the year 2000 and 17,000 new cases. To this end, several treatments are now available to combat the disease.

5.1.1 Stages of colon cancer according to Dukes

The treatment is given according to the stage of the disease, there are two systems to classify what stage a patient's colon cancer is in: the Duke classification and the TNM system. The Duke classification is the most widely used to date, so here's a summary: Stage A represents the stage where colon cancer is limited to the lining or submucosa of the colon. The treatment options at this stage are either colosectomy, in a superficial lesion caused by cancer,The survival rate is 90%. Stage B is measured by the degree of invasion of organs or tissues near the tumor. Treatment in these cases is naturally excision of the tumor and consideration for the use of chemotherapy and/or radiation therapy. Survival rate is between 70-80%. Stage C involves invasion of lymph nodes and metastasis formation in major blood vessels. Treatments to be prescribed are excision of diseased parts, chemotherapy with combination of adjuvants.Stage D, the last stage, is characterized by distant metastases. Treatments include removal of the individual metastases (liver, lung, ovaries), as well as chemotherapy and/or palliative radiation therapy. Survival after surgery is generally less than 5 years.

5.1.2 Chemotherapy

The most commonly used drug for chemotherapy is 5-fluorouracil (5FU), which is given intravenously.Studies have shown that using 5FU after excision of the tumor is more beneficial for patients in stage C of Dukes than excision alone.Then, in the desire for better results in defeating colon cancer, combined treatments are prescribed to different patients with stage B and C colon cancer.Several studies show that by combining 5FU with an adjuvant, levamisole or leucovorin, better results are obtained for stage C of Dukes.Leucovorin is a folic acid that is given to prevent hematopoietic negative effects, thus helping to keep healthy cells alive and leaving cancer cells prone to the cytotoxic action of 5FU. In general, studies agree that treatments with the adjuvant combination have a positive effect on the rate of recurrence as well as the time of tumor recurrence following cancer removal compared to 5FU alone.The mechanism of action of 5FU is to bind to an enzyme inside the cell, which allows the synthesis of thymine during DNA replication, and inhibit it. Therefore, the cell unable to divide will die. Other drugs that may work against colon cancer are currently in clinical trials or will soon be. The different drugs are: Irinotecan (Camptosar, CPT-11), Oxaliplatin and Ralitrexed, Xeloda (Capecitabine). Irinotecan works by inhibiting toposomase I, which is needed to give a certain shape to DNA during translation, transcription, and replication.Oxaliplatin acts on DNA by forming bridges in DNA thus inhibiting its synthesis and replication Ralitrexed has a role similar to 5FU as it interferes with the DNA synthesis phase by blocking the enzyme that synthesizes thymine Xeloda is an oral tablet that turns into 5FU upon ingestion.

5.1.3 Mechanism of destruction of cancer cells by chemotherapy

When the effect of the drug on the cells is known, it is possible to understand the mechanism that causes the cell to become apoptotic. Since 5FU is an inhibitor of thymine synthase, it causes DNA damage during cell division by depriving it of one of the four pyramidal bases that make up DNA. By this DNA damage, 5FU is responsible for activating the genome's guardian protein, p53. It is shown that this protein, through its transcription activity, can modulate the expression of various proteins involved in the process of apoptosis such as Baxter protein, Bakthalon and B-2.53, when the expression of Baxter is increased and B-2 is decreased.

It is also suggested that by inducing the expression of the Fas receptor and the Fas ligand on the surface of the treated cancer cells, autocrine, paracrine or fratricidal death may follow. Then, as the cells express the receptor and the ligand, there could be a cross-link between the receptor and the ligand of the same cell (autocrine), and the cells may be able to express the receptor and ligand.It is suggested that in type I cells, both pathways of initiation of apoptosis are taken during chemotherapy. Thus, the treatment promotes the aggregation of the Fas receptor which causes activation of caspase-8 which will directly activate caspase and caspase-8-3, and can also activate the cleavage protein which will activate the mitochondrial pathway of optosis.In type II cells, apoptosis is controlled by the mitochondrial pathway since the use of a FADD inhibitor did not decrease chemotherapy-induced apoptosis in this type of cell.

Other studies have shown that DNA damage caused by chemotherapy or irradiation increases expression of the DR5 death receptor in a p53-dependent and independent manner.

Several proteins are involved in chemotherapy, and it is important to understand the mechanisms of this treatment, as many cancers develop different ways of preventing death.

6 The Lactic Bacterium

It was the scientist E. Metchnikoff (1845-1919) who proposed that the longevity and health of the Bulgarian people is due to their ingestion of fermented dairy products. It was well known that certain bacteria are pathogenic to the body, so it was proposed to replace these bacteria with yogurt bacteria since they had been used for a long time without any fear. Several characteristics exist to define good lactic acid bacteria: they must maintain their activity and viability before consumption, they must survive the gastrointestinal tract, they must be able to survive and grow in the intestines and eventually produce pathological effects.

Since then, several attempts have been made to improve health by modifying the gut flora with live lactic acid bacteria. Today, the beneficial effects of these lactic acid bacteria are well identified and have now attempted to explain the mechanism (s) related to these benefits. Salminen's team has summarized the most important beneficial effects, supported by scientific evidence, such as immune modulation and strengthening of the intestinal mucosa barrier. Other teams have shown, in mice, that the growth of cancer as well as microflora metastases can be inhibited by the pathogenic bacteria Bactobacillus casei. Different mechanisms suggested to explain these beneficial effects: the alteration of the ability of the intestinal mucosa to adhere to the enzyme, or the alteration of the ability of the intestinal proteins, are ultimately related to the alteration of the ability of the intestinal mucosa to bind to the intestinal proteins, and the alteration of the ability of the intestinal enzyme, especially those associated with the alteration of the intestinal mucous membrane, to alter the pathology of the intestinal enzyme, or to alter the pathology of the intestinal enzyme.

Most studies indicate a therapeutic potential of lactic acid bacteria and yogurt which is mainly due to the change in gastrointestinal micro-ecology. The effectiveness of lactic acid bacteria is increased by their ability to adhere to the intestinal wall since the adhering strains have a competitive advantage, important for maintaining their place in the gastrointestinal tract. However, no strains have yet been shown to adhere permanently. By increasing the amount of lactic acid bacteria in the intestines, it is possible to suppress the growth of pathogenic bacteria, which in turn contribute to a reduction in infections. An intact intestinal epithelium with an optimal flora represents a barrier against invasion or colonization by pathogens, and is resistant to the growth of pathogens and bacteria in the intestinal tract.

In general, consumption of lactic acid bacteria acts by enhancing the nonspecific immune response or acts as an adjuvant in the antigen-specific immune response. Animal studies have shown that lymphoid tissue associated with the intestines, is stimulated by live lactic acid bacteria, resulting in production of cytokines and antibodies (IgA) and an increase in mitogenic activity of Peyer's plaque-forming cells and splenocytes. In human cell studies, cytokine production, phagocytic activity, antibody production, T cell functions, and NK cell activity, are increased by consumption of yurts or NK cells when exposed in vitro to the plaques.

Some evidence suggests that yogurt that boosts the immune system may be associated with decreased incidence of pathological conditions such as cancer, gastrointestinal disorders and allergy symptoms.

6.1 Anti-cancer properties

Lactic bacteria are believed to have antineoplastic properties in a variety of human and animal cancer cell lines. In short, lactic bacteria reduce the viability of tumor cells, decrease induced carcinogenesis in the colon and liver, inhibit mutagenic activity, and bind to potentially mutagenic compounds. Although no mechanism is known, it is suggested that inactivation or inhibition of cancer formation in the intestinal tract is induced.

There is considerable interest in the metabolic activity of the gut microflora, especially in relation to the etiology of colon cancer. Studies have included measurement of key enzymes β-glucuronidase, azo-reductase, and nitro-reductase. These enzymes catalyze the conversion of indirect carcinogens to carcinogens in the intestines. By absorbing lactic acid bacteria, these would decrease the activity of these key enzymes and thus prevent tumor formation.The activation of DMH occurs in the large intestine and these the bacterial enzyme β-glucuronidase which turns it into a potential carcinogen. Suppression of this enzyme can reduce DMH activation and subsequently tumor formation. These studies show that the addition of lactic acid bacteria can delay colon cancer formation by prolonging induction, indicating that the model lactobacillus can slow tumor development in the experimental animal.

In summary, several conclusions are suggested as to what attracted the inhibitory functions of lactic acid bacteria to colon cancer. Among other things, increasing or stimulating immune function could help to reduce the risk of cancer development or recurrence. Also, lactic acid bacteria could replace pathogenic bacteria that would cause the formation of mutagenic compounds that cause colon cancer. There is therefore a need to find methods that are more effective or have fewer side effects than the treatments already available to treat the disease.

The invention is summarized in the following table:

As mentioned above, new properties of these bacteria have been discovered, in that it has been surprisingly found that the effect of lactic acid bacteria, particularly those in the product sold under the trade name Bio-K+ International, in combination with an anticancer agent can cause cell apoptosis.

Several mechanisms may be at the origin of this phenomenon, for example, lactic acid bacteria can prevent the mutation that causes cancers, or they can prevent the progression of tumors by strengthening the immune system.

The complainant was surprised to find that the consumption of lactic acid bacteria would prevent the development of cancer, particularly colon cancer.

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

Thus, the present invention relates to the use of a lactic bacterial strain to enhance an immune response in a mammal for the purpose of preventing or treating cancer, the said strain being Lactobacillus acidophilus I-1492 registered with the CNCM.

Another purpose is the use of a lactic bacterial strain to facilitate the induction of apoptosis in cancer cells, that strain being Lactobacillus acidophilus I-1492 registered with the CNCM.

Another subject is the use of a lactic bacterial strain for the manufacture of a medicinal product for the treatment or prevention of cancer, that strain being Lactobacillus acidophilus I-1492 registered with the CNCM.

Depending on the preferred method of manufacture, the bacterial strain is in a live or irradiated form.

Another purpose of the present invention is to provide a composition to treat or prevent a cancer, such as colon cancer. The composition of the present invention includes an effective amount of a lactic acid bacterial strain as defined above and a pharmaceutically acceptable vehicle. According to a preferred embodiment of the invention, the composition also includes an anticancer agent, such as 5-fluorouracil.

Another object of the present invention is a method to facilitate the apoptosis of cancer cells in a mammal, characterized by the fact that it includes the administration to the mammal of a composition as defined above.

Another subject matter of the present invention concerns the use of lactic acid bacteria to increase cancer cell apoptosis, the said bacteria being Lactobacillus acidophilus I-1492 filed with the CNCM.

Another subject matter of the present invention concerns the use of a combination of lactic acid bacteria and an anticancer agent, such as 5FU, for the treatment of colon cancer, the bacteria being Lactobacillus acidophilus I-1492 as registered with the CNCM.

Another object of the invention is to provide a kit for the prevention or treatment of cancer in a mammal, characterized by the fact that it includes a container containing a composition as defined above.

A brief description of the drawings

Figure 1 is a diagram illustrating a typical analysis of the percentage of apoptosis by flow cytometry.Figure 2 is a diagram illustrating a model of apoptotic process.Figure 3 is a diagram illustrating assumptions of release by the Bcl-2 protein family.Figure 4 is a diagram illustrating the proteolytic depiction of pro-caspase-3.Figure 5 is a diagram illustrating the recapitulation of the effects of p53 activation.Figure 6 is a diagram illustrating the structure of the members of the membrane-dead receptor and their interactions with the major cutoplasmic effectors involved in the apoptotic pathways.Figure 7 is a diagram illustrating the interconnections between the two different transduction pathways of apoptosis.Figure 8 is a graph showing the optimal 5-fluorouracil dose to achieve 50% cell mortality.Figure 9 shows a visual presentation of flow cytometry-derived apoptosis patterns.Figure 10 is a graph showing the effect of the preferred embodiment of the invention on the viability of LS 513.Figure 11 is a graph showing the effect of the preferred embodiment of the invention on the apoptosis of LS 513.Figures 12a, 12b, 12c and 12d are diagrams illustrating the flow cytometry-derived apoptosis measurement.Figure 12a illustrates cells that do not undergo any treatment.Figure 12b shows the cells in the presence of 5FU at a concentration of 100 μg/ml. Figure 12c shows the cells in the presence of lactic acid at a concentration of 108. Figure 12d shows the combination of cells, lactic acid (108) and 5FU (100 μg/ml).Figure 13 shows a western blot illustrating the activation of caspase 3 by cells in the presence of a composition in a preferred mode of the invention.Figure 14 is a graph showing the measure of apoptosis of LS 513 cells in the presence of compositions in a preferred mode of the invention.Figure 15 is a graph showing the measure of viability of LS 513 cells by the MTT.Figure 16 is a graph showing the effect of live lactic acid bacteria versus heated bacteria on apoptosis of LS 513.Figure 17 shows western blots illustrating the effect of live lactic acid bacteria versus heated bacteria on caspase activation 3.Figure 18 is a graph showing the measure of apoptosis inherent in the lactic acid bacterial strains of the present invention.Figure 19 is a graph showing the apoptotic effect of mixing live bacteria and heated bacteria.Figure 20 is a graph showing the effect of adding butyric acid and butyric acid to the heat of LS 513.Figure 21 is a graph showing the effect of the composition in a preferred mode of the invention on the expression of the Fas receptor.Figure 22 is a graph showing the effect of the composition in a preferred mode of the invention on the expression of the Fas ligand.Figure 23 shows western blots illustrating the effect of the composition in a preferred mode of the invention on the expression of a protein involved in apoptosis, the p53 protein.Figure 24 shows western blots illustrating the effect of the composition in a preferred mode of the invention on the expression of a protein involved in apoptosis, the p21 protein.Figure 25 is a graph showing the effect of PKCoptosis on apoptosis.Figure 26 is a graph showing the effect of PKC inhibition on apoptosis.Figure 27 is a graph illustrating the effect of the invention's lactic acid surfactants on the induction of caspase-3 activity in LS 513.Figure 28 shows photographs illustrating the effect of the invention's lactic acid surfactants on the fluorescent staining of the LS 513 cell nucleus.Figure 29 is a graph illustrating the effect of the invention's lactic acid surfactants on the viability of LS 513.Figure 30 is a graph illustrating the effect of the invention's lactic acid surfactants on the gel cell of LS 513.

The device is designed to be used in a wide range of applications.

The present invention therefore aims to demonstrate the use of novel properties of the lactic acid bacterium strain Lactobacillus acidophilus I-1492 in the prevention or treatment of cancer, in particular the use of these lactic acid bacteria to facilitate the induction of cancer cell apoptosis.

The invention also relates to the use of this lactic acid strain in methods and compositions useful in the treatment or prevention of cancer, such as colon cancer.

According to a first embodiment, the present invention is intended to use the lactic acid bacterium strain Lactobacillus acidophilus I-1492 as a candidate for use in the CNCM to enhance the immune response in a mammal for the purpose of preventing or treating cancer.

According to a second embodiment, the present invention is intended to use the said lactic acid bacteria to facilitate the induction of apoptosis of cancer cells. Facilitate the induction of apoptosis means a process by which the presence of the lactic acid bacterial strains of the invention positively modulates the cell death of a tumor, and preferably a colon tumor.

mammal means any living organism that may be susceptible to cancer, and this includes vertebrates such as humans, domestic and wild animals.

Treatment refers to a process by which the symptoms of cancer, especially colon cancer, are alleviated or completely eliminated.

prevention is a process by which cancer, especially colon cancer, is stopped or delayed.

Depending on a preferred embodiment of the invention, the bacterial strain is in a live or irradiated form.

According to a third embodiment, the present invention relates to the use of this lactic acid bacterial strain for the preparation of compounds useful in the treatment or prevention of cancer, such as colon cancer. A composition according to the present invention includes an effective amount of this lactic acid bacterial strain and a pharmaceutically acceptable vehicle. Preferably, the composition of the invention includes a mixture of this strain of L. acidophilus and a strain of L. casel.

Err1:Expecting ',' delimiter: line 1 column 47 (char 46)

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

The amount or concentration of lactic bacteria that is present in the composition of the invention is a therapeutically effective amount. A therapeutically effective amount of lactic bacteria is the amount necessary to achieve positive results without causing excessively negative side effects in the host in which the lactic bacteria or composition is administered. Moreover, an effective amount of lactic bacteria to treat a particular cancer is an amount that is sufficient to alleviate or reduce in any way the symptoms associated with the cancer. Such a quantity may be administered in a single dose or may be administered according to a regimen, which is effective. The amount of bacteria the composition contains may treat the present invention, but typically the amount of such a composition is administered in a different manner to treat each type of cancer, such as the age of the breast and the exact amount of bacteria and the composition of the other ingredients.

The compositions of the present invention may be in any solid or liquid form which is usual for pharmaceutical administration, i.e. for example in liquid, gel or other medium known to the professional. The compositions which may be used include, in particular, oral compositions. In this case, the composition of the present invention may be administered as food or food supplements. Injectable compositions, particularly intended for injection into the human bloodstream, may also be used.

A person who is well versed in the field will be able to prepare pharmaceutically acceptable formulations and determine, on the basis of several factors, the preferred method of administration and the amount to be administered.

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

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

Example

The following example is intended to illustrate the extent of use of the present invention and not to limit its scope. Modifications and variations may be made to it without escaping the spirit and scope of the invention. Although other methods or products equivalent to those described below may be used to test or realize the present invention, the preferred materials and methods are described.

The Commission has

In the context of the present invention, in order to determine how lactic acid bacteria aid in cancer apoptosis, tests were performed on the human colon cancer cell line LS-513. The lactic acid bacteria used are a mixture of Lactobacillus acidophilus and Lactobacillus casei. The anticancer agent is fluorouracil-5 (5FU). This compound acts as an inhibitor of the enzyme that synthesizes thymine.

Materials and Methods 1 Lactic acid 1.1 Origin of the name

The mixture of bacteria used for the various experiments is supplied by Bio-K (Laval, Qc, Canada) and includes a combination of Lactobacillus acidophilus I-1492, which is the subject of international application WO 98/23727, and Lactobacillus casei.

1.2 Preparation

Bacteria received in 9 mL of MRS complex medium (Difco laboratories, Detroit, USA) are immediately multiplied into 100 mL of the same medium by taking 100 μL of the bacterial suspension. After an 18-hour incubation in a 37°C incubator, 10 mL of glycerol is added to the 100 mL mixture which is then divided into aliquot parts of 1 mL in several sterile plastic vials up to 1.5 mL. These vials are stored in a freezer at -80°C.

After the various stimulation protocols, a vial is thawed and placed in 9 mL of MRS and incubated for 18 hours at 37°C. After this incubation time, a graft is performed. A volume of 100μL is taken 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 collected 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 berovin serum and are then ready to be used for cell stimulation. For experiments, the bacteria used are then irradiated in different, live and live forms.

1.3 Heating

Err1:Expecting ',' delimiter: line 1 column 94 (char 93)

1.4 Irradiation

To produce the irradiated bacterial mixture, tubes of live bacteria are irradiated at a minimum dose to achieve a 100% lethality, i.e. 5 kGy. The mixture is irradiated in a Gammacell-220 (MDS Nordion, Laval, Qc, Canada) using Cobalt-60 (60Co) as the gamma-ray emitting source.

To make a bacterial count to obtain a concentration of bacteria per1 mL of this suspension, an isotonic solution containing 0.1% bactopeptone is added to 9 mL of peptone water (Difco laboratories, Detroit, USA). Dilutions are made in batches. Then 1 mL of these dilutions is taken and deposited in a petri dish to which 10 mL of MRS with 1.5% agar is added (Difco Laboratories, Detroit, USA) to allow a count to be made after 48 hours incubation in an incubator at 37°C. Each sample is made in duplicate.

2 Cancer cells 2.1 Origin of the name

The LS 513 cell line (ATCC, Rockville, MD, USA) is a continuous line of human colon cancer.

The European Union

Err1:Expecting ',' delimiter: line 1 column 308 (char 307)

3 Co-culture of cancer cells and bacteria 3.1 Addition of bacteria

Err1:Expecting ',' delimiter: line 1 column 324 (char 323)

3.2 Experience with the addition of butyrate

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

3.3 Harvesting and study of the survivals

The purpose of this experiment is to verify 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 of the different stimulation products. Following this incubation, the survival factors are harvested and added to a new culture of newly adhered cells.

4 Measuring the viability of cancer cells 4.1 Proliferation 4.1.1 MTT

Err1:Expecting ',' delimiter: line 1 column 286 (char 285)The solution is then settled and another solution is added to dissolve the crystals formed in the 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 the crystals. Following this incubation, the plate is read in a spectrophotometer (Mandel Scientific company, Bio-Tek instruments, Microplate EL309 Autoreader) at 540M. Each sample is taken in triplicate,The average value of the control is 100% of living cells. To get the percentage of other samples, just cross-produce.

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 shown above. The various products are added to the wells, and a 48-hour incubation is done.

After incubation, the cell surfactants are collected in separate 15 mL tubes and centrifuged at 1500 RPM for 5 minutes. The cell coils are then collected by settling the surfactants and placed on ice. This step consists of recovering the suspended cells. The adhered cell sheet is then washed with 0.5 mL of trypsin-EDTA, then 0.2 mL of trypsin-EDTA is added to each well to allow the cells to detach when incubated for about 8 minutes in the incubator at 37°C. The cells are suspended in 3 mL of full 10% SVF RPI to stop the enzymatic activity of trypsin.The cell suspension is centrifuged at 1500 RPM for 5 minutes in the flow cytometry tubes. The two cell clots (suspension cells and adhered cells) are then combined and washed twice with cold PBS supplemented with 0.25% EDTA to prevent the formation of cell aggregates. Following the washings, 0.5 mL of propidium iodide solution is added to the cell clot. The solution is composed of 0.1% sodium citrate (Fisher Scientific, New Jersey, USA), 0.1% TritonX-100 (Sigma, St-Louis, USA), 50μg/mL of RN (Sigma, St-Louis, USA) and 20μg/mL of propidium iodide (Sigma, St-Louis, USA)The program measures the percentage of fluorescence that is formed by the large peak below the peak corresponding to G0-G1, which is a fragmented piece of DNA, resulting from the cleavage of DNA by a different enzyme activated during apoptosis.

Measurement of the expression of proteins involved in apoptosis (p53, p21, caspase 3, Bax) 5.1 Western-style gossip 5.1.1 Stimulation and extraction of proteins

The following day, as the cells become adhesive, the stimulation products are added. The different stimulation products are 5-Fluorouracil (100μg/mL) and bacteria, live or heated, at a concentration of 1X108 bacteria per mL. These products will cause the cells to become apoptotic by modulating different proteins involved in the process. It is this modulation, increased expression or activation of protein, that the technique allows to check. Following different stimulation times (since this is kinetics), the cells are harvested and centrifuged for 1500 RPM for 5 minutes.Err1:Expecting ',' delimiter: line 1 column 580 (char 579)The final sample is aliquoted by volume of 20 μl and stored at -20°C.

5.1.2 Separation and identification of proteins

Err1:Expecting ',' delimiter: line 1 column 306 (char 305)Err1:Expecting ',' delimiter: line 1 column 149 (char 148)The second marking is performed with a second antibody that recognizes the first antibody and is coupled to the peroxidase. An incubation of one hour is performed with this second antibody, which is also diluted in the blocking solution at a concentration indicated by the supplier. As soon as this incubation is completed, a 15 minute wash, as well as two 5 minute washes, are performed with a solution of PBS-Tween 80 to 0.1% without skimmed milk powder.The markings are then revealed on photographic paper (hyperfilm ECL, Amersham Pharmacia Biotech Inc, Baie d'Urfé, Qc. Canada) which will be marked with the activated peroxidase. The photographic paper is then developed. Table 1 summarises the proteins sought and the reagents used. Tableau 1 : Les anticorps utilisés pour les différentes protéines à identifier.

Premier anticorps
Protéine	Description	Dilution	Compagnie
p53		1:1000	BD PharMingen, Mississauga, Ontario
p21		2 :1000	BD PharMingen, Mississauga, Ontario
Caspase 3	Anti-capase 3 lapin polyclonal	1 :1000	BD PharMingen, Mississauga, Ontario
Bax	Anti-Bax	1 :1000	Santa Cruz California, USA

Tableau 1 : Les anticorps utilisés pour les différentes protéines à identifier.

Deuxième anticorps
IgG	Anti-IgG souris	3 :10 000	Sigma, St-Louis, USA
	Couplé péroxidase
	Développé dans chèvre
IgG	Anti-IgG lapin	2.5 :10 000	Sigma, St-Louis, USA
	Couplé péroxidase
	Développé dans chèvre

6 Measurement of the expression of margurs on the cell membrane (Phase, Phase L) 6.1 Flow cytometry

A total of about 5x105 cells are used per sample. The cells are prepared as mentioned above. A 24-hour incubation is carried out after the addition of the various stimulation products (5FU, live or heated bacteria and others depending on the experiments that are performed).

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7 Measurement of expression of the Nurr77 gene 7.1 Extraction of RNA

A number of 3x105 cells is used per sample. A 3-hour incubation is performed following the addition of the various stimulation products. The cells are then harvested using a scraper and then centrifuged at 1500 RPM for 5 minutes. The total RNA of each sample is extracted and purified using the High Pure RNA kit from Roche Diagnostics (Laval, Qc, Canada), as indicated by the manufacturer. The RNA concentration is then measured with the machine (Pharmacia Biotech, Gene RNA/DNA Calculator) and then adjusted to 92 ηg/μL.

The following information is provided:

The LightCycler RNA amplification kit SYBR Green 1 from Roche Diagnostics (Laval, Qc, Canada) is used to perform the reverse transcription reaction (RT) and polymerase chain reaction (PCR). The principle of the LightCycler is very similar to that of the Thermocycle. The major difference is that the LightCycler can observe amplification at each cycle, thanks to a fluorescent molecule called SYBR Green 1 that fits into each double strand formed.

The amplification is performed at a frequency of approximately 0.25 μm, and the amplification is performed at a frequency of approximately 0.25 μm. The amplification is performed at a frequency of approximately 0.25 μm. The amplification is performed at a frequency of approximately 0.25 μm. The amplification is performed at a frequency of approximately 0.25 μm. The amplification is performed at a frequency of approximately 0.25 μm.Two other parameters vary in the amplification program: for example in the hybridization segment, the temperature to be used varies according to the starter. The temperature (set at 5°C less than the starter hybridization temperature (Tm) is calculated by the following formula: Tm = 2°C (A+T) + 4°C (C+G). For the starter used the Tm is 64°C. The second parameter is the incubation time for elongation, still located in the amplification program; it is determined by the following formula t = (number of base pairs of the amplification product ÷ 25) seconds. In our case, the base number is 658,So the amplifications are subject to a gradual increase in temperature and at each degree the fluorescence is measured and recorded. By increasing the temperature gradually, the double strands formed break off when the temperature gets high enough, and so it's at this temperature that we see a decrease in fluorescence.The machine does not allow the number of base pairs to be visualized, so an amplifier, a migration on 2% agarose gel with a base pair marker, allows the verification after staining with ethidium bromide at a concentration of 0.5 μg/mL for 15 minutes.

8 Treatment with PKC inhibitors or stimulators

The stimulation with PKC inhibitors and stimulators is carried out in the same way as the stimulation with bacteria and 5FU. First, the cells are prepared the day before to allow them to adhere, and on the day of the stimulation, the different stimulation products are added together with the PKC inhibitor GÖ 6976 (Sigma) and/or the PKC inhibitors ionomycin (Sigma) and PMA (phorbol 12-myristate 13-acetate) (Sigma).

9 Cytokine dosage 9.1 TNF dosage by bioassay

The amount of TNF in a surfactant can be measured using the L929 cell line, which is a line of mouse fibroblasts sensitive to the cytotoxic action of TNF. The principle of this bioassay is simple: the more TNF in the surfactant added to the L929 cell sheet, the more cell death will occur. The rate of surviving cells can then be measured. First, the cells are cultured in full RPMI 1640 + 5% SVF. The cells are removed from the vial using trypsin-EDTA (Gibco, Burlington, ON, Canada) by incubating for about 1 minute at 37°C. A computer is prepared to perform a bioassay of 3.3 x 105 mL/cell suspension for the cell.

A volume of 75 μL is deposited in each well of a 96-well plate. All lines except the first line are given this volume, which is used as an empty control. Following a 24-hour incubation, a volume of 25 μL of actinomycin D, at a concentration of 2 μg/mL, is added to all wells in all lines except the second line which is used as an actinomycin D control, in order to stop cell growth.The third row is empty as it will serve as a positive control, i.e. it will represent the maximum number of cells since there is no cytotoxic agent added. With the samples added, successive dilutions are made from the 100 μL which are diluted in series in the following 8 rows. When the samples are diluted, incubation is done for 16-20 hours at 37°C + 5% CO2.The plates are emptied and rinsed 3 times with running water. The remaining fixed cells are then stained with purple crystal at a rate of 50 μL per well for 5 minutes. Subsequently, the plates are emptied and excess dye is removed by 3 rinses with running water. Once the plates are dry, 100 μL of a 33% acetic acid solution is added to each well to dissolve purple crystal absorbed by the fixed cells. The absorbance is read at a wavelength of 540 nm in a plate spectrophotometer, using column 1 as a reference ( white ).

To calculate the number of TNF units, one TNF unit is considered to be the inverse of the dilution factor giving 50% cytotoxicity. To calculate the percentage of cytotoxicity for each sample, the following equation is used. $\%\hspace{0.17em}\mathrm{cytotoxicité}=\frac{D.O.\hspace{0.17em}\mathrm{échantillon}}{D.O.\hspace{0.17em}\mathrm{positif}}\times 100$

The D.O. of the sample is the average of the three absorbances obtained for a dilution following the plaque reading. The D.O. of the positive control is the average of the wells in line 3. The percent cytotoxicity is calculated for each dilution. Next, a linear regression line is drawn for the dilutions of a sample, the dilution factor (= x) and the percent cytotoxicity (= y) and the 50% point is found using the equation on the right. The inverse of the dilution (2x) is equal to the number of TNF units in the original undiluted sample. The results are expressed in U/mL.

The results 1) Finding the optimal dose of 5 Fluorouracil

Figure 8 shows the measurement of apoptosis by flow cytometry following exposure to increasing doses of 5-Fluorouracil (5FU) to achieve an ideal concentration giving 50% mortality. A total of 3x105 cells are exposed to different concentrations of 5FU for 48 hours. Then the DNA content of the cells is marked with propidium iodide and analyzed by flow cytometry to obtain the percentage of apoptosis.

Figure 9 illustrates a diagram of one of two experiments to obtain the ideal concentration of 5FU. The apparatus measures the number of events of the sub-G1 peak relative to the number of events in the sample, giving a percentage result. Since the sub-G1 peak is known to be DNA cleavage, a sign of apoptosis, the apoptosis value is given as a percentage.

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

The colonic cancer cells are dosed by the MTT following a 48-hour incubation in the presence or absence of 5-Fluorouracil (5FU) (2.5ug/mL) and live bacteria (B) at different concentrations (106-108). A total of 3.3 X 104 cells per well in a 96-well tray are brought into contact with the different stimulation products. The staining produced by the reaction of the MTT with the live 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 live bacteria and 5FU on LS 513 apoptosis. Live lactic acid (B) bacteria at different concentrations (106-109) and 5-Fluorouracil (5FU) (100ug/mL) are added to LS 513 cells. Flow cytometry measurement of apoptosis is performed after 48 hours of incubation. DNA marking with propidium iodide solution allows the percentage of cleaved DNA (sub-G1) cells produced following incubation to be observed. The test is performed on cells that have not undergone any treatment.

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

The protein was extracted and migrated onto polyacrylamide gel, then transferred to nitrocellulose membrane and labeled with a specific antibody for caspase 3 at a concentration of 1:1000.

3) Action of the state of the bacteria 3.1) Living bacteria versus irradiated bacteria

Figure 14 shows the measurement of apoptosis of LS 513 cells in the presence of live bacteria, irradiated bacteria and 5FU. This Figure shows more specifically the measurement of apoptosis by percentage of sub-G1 by flow cytometry using a propidium iodide marker (20 ug/mL). Colon cancer cells are tested in the presence or absence of 5-Fluoro-Uracil (5FU) (100 ug/mL) and live bacteria (B) or irradiated bacteria (C) at different concentrations (106-109) over a 48-hour period. The control cells are untreated.

3.2) Living bacteria versus heated bacteria

Figure 15 shows the measurement of viability of LS 513 cells by MTT. More specifically, this Figure shows the effect of live (B) and heated (C) bacteria 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 48 hours of incubation. The values are obtained by reading at the spectrophotometer (540nm), by staining due to MTT which therefore stains the functional mitochondria only those of living cells. The cell concentration used is 3.310 x 4 cells per well.

Figure 16 shows the effect of live bacteria versus heated bacteria on apoptosis of LS513 cells. The analysis of the percentage of sub-G1 by DNA marking was obtained with propidium iodide. A total of 10,000 events are treated per sample. A 48-hour incubation in the presence of live (B) and heated (C) bacteria at different concentrations with or without 5 Fluoro-Uracil (100ug/mL) are the different samples shown in the figure.

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

4) Possible mechanisms inherent in bacterial cultures

Figure 18 shows the measure of apoptosis inherent in the lactic acid strains of the invention. A first stimulation was performed for 48 hours. Some wells did not contain cells (F). The various surnages (D) were harvested and added onto a fresh cell culture (E). The 5-Fluorouracil (5FU) concentration is 100 ug/mL and two different concentrations are used for live bacteria (B) and heated bacteria (C) which are 1X107 and 108.

Figure 19 shows the apoptotic effect of mixing live and heated bacteria. The flow cytometry measurement of apoptosis was taken after 48 hours incubation. The LS 513 cell line is placed in the presence of a determined concentration of 5FU (100ug/mL) and the presence of live (B) and heated (C) bacteria at two different concentrations (107 and 108). The test is made up of cells without any treatment.

Figure 20 shows the effect of the addition of butyric acid and 5FU on apoptosis of LS 513 cells. A dose of 5FU (100ug/mL) is added to colon cancer cells along with different doses (2mM and 4mM) of butyric acid (ab).

5) Possible mechanism inherent in tumor cells

Figure 21 shows the effect of composition according to a preferred mode of embodiment on the expression of the Fas receptor.

Figure 22 shows the effect of the expression of the Fas ligand.

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

Figure 24 illustrates the effect of composition in a preferred mode of the invention on the expression of 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 tumour cells LS 513

The apoptotic potential of bacterial surfactants on the LS 513 lineage, a tumor lineage, was analysed. Results on caspase-3 activity show that surfactants, even in the presence of 5-FluoroUracil (2.5 μg/ml and 100 μg/ml), do not significantly activate this caspase (Figure 27). On the contrary, in the presence of 100 μg/ml of 5FU, surfactants inhibit caspase-3 activity. Fluorescent staining of the nucleus of cancer cells using the DAPI flow technique failed to identify nucleuses where bacteroproteins are disrupted, a fundamental characteristic of living cells. Results from studies on apoptosis in cells typically inducing cell death and LF 51 (Figure 28), LF 30 (Figure 30), and LF 630 (Figure 29) show that cell staining is not a significant factor in the size of cells.

The Commission has

The action of intact bacteria, that is, those that are alive and irradiated on cancer, is the same, whereas uninfected bacteria, for example those destroyed by heat, seem to have an opposite action to intact bacteria.

In addition, it has been observed from the work done in the context of the present invention that the efficacy of an anticancer agent such as 5FU is considerably increased in the presence of intact bacteria, which would be dose-dependent.

The presence of bacteria heated with 5FU would increase expression of p21 protein, so there could be modulation via an unknown receptor of apoptosis/cell cycle regulatory proteins (p21). An increase in p21 protein has been observed when there is less apoptosis, and a decrease in 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 colon cancer cells in vitro. There will be a synergy between butyrate and 5FU on colon cancer. It has also been noted that butyric acid inhibits the in vivo growth of human colon cancer in mice.

In view of the above, live lactic acid bacteria synergistically work with 5FU to reduce the number of cancer cells in culture (MTT) or to increase apoptosis in cancer cells (cytosine flow).

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

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

Claims (16)

1. Use of a lactic acid bacterial strain for the manufacture of a medicament for strengthening an immune response in a mammal for the purpose of preventing or treating a cancer, said strain being Lactobacillus acidophilus I-1492 deposited in the CNCM.
2. Use according to Claim 1, characterized in that said mammal is a human being.
3. Use of a lactic acid bacterial strain for the manufacture of a medicament for facilitating the induction of apoptosis in the cells of a cancer, said strain being Lactobacillus acidophilus 1-1492 deposited in the CNCM.
4. Use of a lactic acid bacterial strain for the manufacture of a medicament for the treatment or prevention of cancer, said strain being Lactobacillus acidophilus I-1492 deposited in 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 but intact form.
6. Use according to any one of Claims 1 to 5, characterized in that a strain of the species Lactobacillus casei is also used.
7. Use according to any one of Claims 1 to 6 with an anticancer agent.
8. Use according to Claim 7, characterized in that the anticancer agent is 5-fluorouracil.
9. Use according to any one of Claims 1 to 8, characterized in that said cancer is a colon cancer.
10. Composition for treating or preventing a cancer, comprising an effective amount of a lactic acid bacterial strain and a pharmaceutically acceptable vehicle, said strain being Lactobacillus acidophilus 1-1492 deposited in the CNCM.
11. Composition according to Claim 10, characterized in that the bacterial strain is in a living or non-living but intact form.
12. Composition according to Claim 10 or 11, characterized in that it also comprises a strain of the species Lactobacillus casei.
13. Composition according to any one of Claims 10 to 12, characterized in that it comprises an anticancer agent.
14. Composition according to Claim 13, characterized in that the anticancer agent is 5-fluorouracil.
15. Composition according to any one of Claims 10 to 14, characterized in that said cancer is a colon cancer.
16. Kit for preventing or treating a cancer in a mammal, characterized in that it comprises a receptacle containing a composition as defined in Claims 10 to 14.