WO2002076456A1

WO2002076456A1 - Method for inhibiting apoptosis of stem cells

Info

Publication number: WO2002076456A1
Application number: PCT/NL2002/000198
Authority: WO
Inventors: Belinda Van't Land; Laura M'rabet
Original assignee: Nutricia, N.V.
Priority date: 2001-03-27
Filing date: 2002-03-27
Publication date: 2002-10-03

Abstract

The invention relates to a method for inhibiting apoptosis of stem cells. More in particular, the invention relates to such a method wherein the activity of p53 in said cells is suppressed.

Description

Title: Method for inhibiting apoptosis of stem cells

The invention relates to a method for inhibiting apoptosis of stem cells. More in particular, the invention relates to such a method wherein the activity of p53 is modulated.

Dividing cells in living tissue are very sensitive to the influence of stressors, such as the presence of cytotoxins or gamma radiation (see e.g. Sonis, S.T., Oral Oncol. 34 (1998) 39-43). As a consequence, undesired presence of stressors, e.g. as a result of poisoning or a bacterial infection, may result in damage to the cells. A typical complication of this type is mucosal barrier injury. Mucosal barrier injury is an inflammation of the outer mucosa layer of the epithelial cells of the intestines or respiratory system. It is a complex biological process that may occur in periods wherein the immunity system is deficient and/or when excessive burden on epithelial cells is exerted.

Also, in some cases, the above-mentioned stressors are applied deliberately, as is the case in chemotherapy or radiotherapy, in which cases rapidly dividing cells, such as tumors, are intended to be destroyed. As an undesired side-effect, other cell types with a high rate of division, such as epithelial cells in the gastrointestinal tract, the lungs and the skin, are affected by the therapy as well, which leads to complications. Typically, these complications comprise mucosal barrier injury, particularly mucosal barrier injury of the epithelial cells of the gastrointestinal tract. These side effects usually occur in a later phase of therapy and limit the dose of drug that can be administered. Mucosal barrier injury leads to pain, reduced food uptake, malabsorption and diarrhea. These effects may further harm the condition of the patient. Also, mucosal barrier injury reduces the barrier function of the intestine, which may cause systemic infection and sepsis, which puts a further burden on the condition of the patient's immune system, which is usually already in a bad condition to begin with.

Without wishing to be bound to any theory, it is believed that the undesired cell death of e.g. epithelial cells is caused by apoptosis of these cells, which is triggered by stressors resulting from e.g. radio- or chemotherapy. In the broadest sense, it is an object of the present invention to provide for a method which may be used as a therapy to inhibit apoptosis of cells, in particular of stem cells. Stem cells are cells that occur in many tissues where there is a recurring need to replace differentiated cells that cannot themselves divide. Stem cells are not themselves terminally differentiated, can divide without limit and produce daughter cells which can either remain stem cells or embark to a terminal differentiation. Stem cells of particular relevance are those occurring in the epidermis of gut and skin or in bone marrow.

In the art, it has been described that radio- and chemotherapy increase DNA damage and thereby the functional level of p53 in the intestinal crypts (see Wilson et al., Am. J. Pathol. 153(3), 1998, 899-909). This increase in the level of functional p53 appeared to be sufficient to induce apoptosis, because inhibition of p53 could inhibit the side effects of chemotherapy in mice (see Komarov et al., Science, 285(5434), 1999, 1733-7).

As a possible mediator of p53 induced apoptosis, NF-κ-B has recently been identified (see Ryan et al, Nature 404(6780), 2000, 892-7). These authors continue to suggest that this process plays a major role in the development of cancer. They did not comment on the role of NF-K-B in radio- and chemotherapy induced apoptosis.

It is known that NF-K-B can be activated after DNA damage by radiation (see Bender et al, Embo. J. 17(17), 1998, 5170-81; Li et al., Proc. Natl. Acad. Sci. USA, 95(22), 1998, 13012-7; and Granville et al, Blood, 95(1), 2000, 256-62), suggesting that NF-κ-B might play a role in the induction of apoptosis. However, most of the publications state that NF-K-B is essential for cell proliferation and cell survival after immune related responses or DNA damage (see Rothwarf et al., Science Signal Transduction, 5, 1999, 1-16; and Schmid et al., Gastroenterology, 118(6), 2000, 1208-28), and references cited therein (see Jones et al., J. Exp. Med., 191(10), 2000, 1721-34; Kane et al., Curr. Biol., 9(11), 1999, 601-4; Madrid et al., Mol. Cell Biol., 20(5), 2000, 1626- 38; Romashkova et al., Nature, 401(6748), 1999, 86-90; and Van Knethen et al., J. Immunol, 163(5), 1999, 2858-66). In addition, cell survival, achieved through the inhibition of p53, is only possible by the activation of NF-K-B (see Kawakami et al., Blood, 94(11), 1999, 3847-54; and Pei et al., J. Biol. Chem., 274(49), 1999, 35240-6) and inhibition of NF-κ-B is essential for the induction of apoptosis (see Kawai et al., Cancer Res., 59(24), 1999, 6038-41; Levkau et al., Nat. Cell Biol, 1(4), 1999, 227-33; and Philips et al., Mol. Cell, 4(5), 1999, 771-81). In conclusion, most publications state that the balance between active NF-K-B and functional p53 determines cell fate (see Scheme 1). Therefore, the role of NF-κ-B activity in radio- and chemotherapy induced apoptosis remains inconclusive.

The present inventors now surprisingly have found that, of the various mechanisms that give rise to apoptosis of cells (e.g. after radiation), the activation of the p53 protein plays a prominent role. Based on this insight, the inventors have developed a method wherein apoptosis, particularly of stem cells, may be inhibited by suppression of this route.

It is preferred to suppress the p53 by administration of a chemical compound. Compounds that are very suitable for this purpose are berberine (5,6-dihydro-9,10-dimethoxybenzo(g)-l,3-benzodioxola-[5,6-a]quinolizinium, CAS# 2086-83-1), curcumin (diferuloylmethane or tumeric yellow, CAS# 458- 37-7) or functional analogues thereof. The salts of these compounds, such as the chloride, hydroxide or sulfate salts, or other derivates may be used as well. For example, desmethoxy-and bidesmethoxycurcumin, as well as dihydrocurcumin may be used equally well. In addition to being effective suppressers of p53 activation, these compounds remain intact after digestion in the stomach, which makes them suitable for oral administration.

US-A-5,891,924 discloses a method of inhibiting the activation of the NF-K-B transcription factor by administering a pharmacologically effective dose of curcumin. The medical conditions mentioned in this publication are all immunodeficiency related. p53 induced NF-K-B activation is not disclosed, nor is its role in the apoptosis mechanism. The activation of NF-K-B by immune related responses is different from p53 mediated NF-K-B activation (see also Li et al., supra; Bender et al., supra; Ryan et al., supra; and Jobin et al., J. Immunol, 163(6), 1999, 3474-83). In addition, the present inventors have shown (see Control Example 3, below) that CAPE, a known NF-κ-B inhibitor, does not protect epithelial stem cells from chemotherapy induced apoptosis, while curcumin does provide this protection. Preferred functional analogues of curcumin and berberine are capsaicin (CAS# 404-86-4), apocynin (CAS# 498-2-2) or chlorogenic acid (CAS# 327-97-9).

Curcumin may be obtained e.g. by extraction using a polar solvent (such as water or supercritical CO₂) by boiling rhizomes of Curcuma species (such as C. Longa, Xanthorriza, Zedoaria or Zingiber officinale) prior to drying. By this treatment the lignide structures are broken and the curcumin as well as the above-mentioned curcuminoids are released from the cell structures.

Berberine may be obtained from various plants, such as Adonis vernalis, Argemone mexicana, Berberis vulgaris, Chelidonium majus, Corydalis spp, Macleaya cordata, Menispermum canadense, Papaver somniferum, Podophyllum peltatum, some Coccinia species and Zanthoxylum americanum or from the species Andira inermis, Phellodendri (e.g. amurense) or Zanthoxylumalatum, in particular the bark thereof, and from the species Coptis spp (e.g. Chinensis or japonica) or Podophyllum hexandrum, in particular the rhizomes thereof, and from the species Hydrastis canadensis, Mahonia aquifolia and Sanguinaria canadensis, in particular from the roots thereof and from the Eschscholzia California, in particular from the shoots thereof. Isolation may carried out by applying the above-mentioned methods. Extraction of finely divided cells may be carried out using a polar solvent such as water or supercritical CO2. Purification is carried out by known methods, including filtration and/or chromatography. Subsequently the solution may be concentrated by means of evaporation or precipitation.

It is preferred to administer the active compound such that a concentration of about 10 μg/ml locally in the cell's environment is obtained. For this reason, preferred concentrations for oral administration are 0.02 - 0.2 mg/ml. It is preferred to limit the amount of the active compound to about 0.2 mg per dose for topical administration. For topical administration (e.g. subcutane injection preferred concentrations are 0.005 - 0.02 mg/ml. The total dose of active compound per day is preferably kept below 70 mg. The method of the invention finds particular use in suppressing the undesired side-effects of therapies, such as chemo- and radiotherapy, while the desired results thereof, i.e. death of harmful cells, are substantially maintained. The method of the invention is particularly suitable for the treatment of mucosal barrier injury, which frequently occurs as a side-effect of chemotherapy or radiation therapy.

Although it has been suggested to administer capsaicin for relieving pain associated with mucosal barrier injury (see Berger et al., Journal of Pain and Symptom Management, 10(3), 1995, 243-8), it has never been reported that it can actually prevent mucosal barrier injury. In addition, Shimizu et al. (Journal of Toxicological Sciences, 24(5), 1999, 433-9) have suggested that chlorogenic acid may inhibit gastric acid protection. However, gastric acid is of no relevance to the prevention of mucosal barrier injury.

The active compounds can also very suitably be added to a food compound, which may serve as a dietary supplement. The compounds may also be used as a prophylactic for the above-mentioned disorders associated with apoptosis.

The invention will now be further elucidated by the following, non- restrictive examples. Materials and Methods

Cells

The Human squamous epithelial cell line (FaDu) and the non- transformed small intestinal rat epithelial cell line (IEC-6) were obtained from ATCC (Rockville, MD). FaDu cells were grown in Minimal Essential Medium Eagles (MemE) containing 0.1 mM non-essential amino acids (NEAA), and 1.0 mM sodium pyruvate. The IEC-6 cells were cultured in Dulbecco's modified Eagle's medium adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose. In addition, all media were supplemented with 50 IU/mL penicillin, 50 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). Media and supplements were obtained from Gibco BRL. The cell lines were kept in a continuing culture in a humidified atmosphere at 37°C and 5% CO₂.

Cell viability and cytokine detection Cells in the exponential phase of growth were harvested and plated in 96-well microtiter plates (costar) cultured form 24h to obtain a 80% confluence monolayer. Various concentrations cytostatics as indicated were added and after another 48h incubation at 37°C and 5% CO₂, 50 μL supernatant was harvested and stored at -20°C for cytokinase analyses. To the remaining culture 50 μL XTT labeling mixture (Boehringer Mannheim) was added and after 4h incubation at 37°C and 5% CO2, formation of the formazan dye by metabolic active cells was measured spectrophotometrically at 490 nm. For cytokine analyses, supernatants from triplicate wells were pooled and measured in a human IL-6 or IL-8 specific Elisa (kits were obtained from CLB Amsterdam). Assay was performed according to manufactory protocols.

Cell death /cell cycle arrest

Cells were cultured in a 6-well plate, under the same conditions as described above. After incubation for 48h with Ara-C, the cells were harvested and prepared for FACS analysis as described by Fraker et al., Methods Cell Biology, 46, 1995, 57-76. Shortly, cells were washed twice with PBS containing 0.1% EDTA (PBS-E) before fixation in 70% ethanol for 45 minutes at -20°C. To label the DNA with propidium iodide, the 70% ethanol was washed away and the cells were incubated with PBS-E containing propidium iodide (5 μg/mL) and RNA-se (1 μg/mL) for 45 minutes in the dark. Afterwards the cells were analyzed on a flowcytometer (coulter).

Example 1

This example demonstrates the effectiveness of berberine and curcumin in protecting against chemically induced apoptosis.

Epithelial rat cells (IEC-6 cells) were incubated with different concentrations of the chemotherapeutic compound Ara-C. Figure 1 demonstrates that this leads to cell death.

The incubation in the presence of Ara-C was repeated, however, the incubation was carried out in the presence of different concentrations (0, 5, 10 and 20 μg/ml) of the active compounds berberine and curcumin. The applied concentrations of Ara-C were 0, 0.6 and 1.25 μg/ml. The experiments were repeated using the same compounds which had been incubated for 1 h at pH=3 in the presence of active pepsin, as to simulate digestion in the stomach. Figure 2 shows the vitality of cells subjected to Ara-C induced apoptosis, treated with curcumin and berberine, respectively after 48 h.

From Figure 2 it follows that the viability of the cells, as reflected by the extinction at 490 nm, increased considerably due to the presence of the active compound.

Control Example 1

Example 1 was repeated using transformed epithelial mouth cells (FaDu's) of which the p53 activation can not be regulated. It was found that these cells were not protected by the above-mentioned active compounds, as is shown in Figure 3. This proves that protection against chemotherapy induced apoptosis is by controlling the p53 of the apoptosis.

Control Example 2 In order to check that the protective effect of the active compounds mentioned above, was not due to a possible anti-oxidative nature of these compounds, a control experiment was carried out using different concentrations of Vitamin E (0, 18.8, 37.5, 75, 150 and 300 μg/mL), which is a known radical scavenger. The results, expressed as the viability of the cells compared to that of a control (no Ara-C treatment) are given in Figure 4. It follows that treatment with Vitamin E does not give rise to increased protection against damage resulting from the chemotherapy. Therefore, the results obtained in the previous experiment, using the active compounds in accordance with the invention, are not to be attributed to a possible anti-oxidant effect.

Control Example 3

Small epithelial cells, IEC-6 cells were pre-incubated with CAPE (a specific NF-κB inhibitor) at concentrations as indicated in Figure 6 or with curcumin at concentrations as indicated in Figure 6. Both components were supplemented 24h before treatment with Ara-C (2.5 μg/mL) for an additional 48 hours. Afterwards cells were harvested, fixed and coloured with propidium iodide (PI) staining before analysis on flowcytometry. PI colours DNA and fragmentation of DNA can easily be detected. Fragmentation of DNA only takes place when cell death/apoptosis is occurring. Percentages of apoptotic cells are indicated as Mean +/- SD of two independent experiments. As can be seen CAPE does enhance Ara-C induced apoptosis, whereas curcumin inhibits Ara-C induced apoptosis, indicating that curcumin does not protect IEC-6 cells from Ara-C induced apoptisis in a NF-K-B dependent manner.

Claims

1. Method for inhibition of apoptosis of stem cells comprising suppressing p53 activity in said cells.

2. Method according to claim 1, wherein p53 activity is suppressed by administering a compound selected from berberine, curcumin or functional analogue thereof, or the salts of these compounds.

3. Method according to claim 2, wherein said compound is a functional analogue or a salt thereof selected from capsaicin, apocynin or chlorogenic acid.

4. Method according to any of the claims 2-3, wherein said compound is administered orally in a concentration of 0.02 - 0.2 mg/ml.

5. Method according to any of the claims 2-3, wherein said compound is administered topically in a concentration 0.005 - 0.02 mg/ml.

6. Method according to any of the previous claims, wherein said apoptosis of cells is associated with mucosal barrier injury.

7. Method according to any of the previous claims, wherein said apoptosis of stem cells results from chemotherapy or radiation therapy.

8. Use of a compound that suppresses p53 activity in the preparation of a medicament or dietary supplement for the treatment of an apoptosis related disorder.

9. Use according to claim 8, wherein said treatment is prophylactic.