JP3899462B2 - Cancer cell apoptosis inducer - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
[0002]
The present invention relates to a cancer cell apoptosis inducer, and more particularly to a cancer cell apoptosis inducer containing an oligosaccharide-containing substance as a component.
[0003]
[Prior art]
[0004]
Conventionally, as described in JP-A-2001-86999, which is a patent document 1, as a cancer cell apoptosis inducer containing an oligosaccharide-containing substance, β-1,3 xylan obtained from a cell wall of seaweed It has been known that the main component is β-1,3 xylo-oligosaccharide obtained by treating with β-1,3 xylanase.
The cancer cell apoptosis-inducing agent is obtained from, for example, Sorigota, which is an algae of the moss family, deionized water, washed with 1% hydrochloric acid solution and 23% sodium hydroxide solution, decolorized with sodium chlorite solution, 10% sodium hydroxide. Β-1,3 xylan used as a raw material was obtained by performing extraction with a solution and fractional purification by ethanol precipitation.
[0005]
[Patent Document 1]
Japanese Patent Laid-Open No. 2001-86999 [0006]
[Problems to be solved by the invention]
[0007]
However, in order to obtain β-1,3 xylan used as a raw material, the cancer cell-inducing apoptosis-inducing agent containing β-1,3 xylo-oligosaccharide as a main component is obtained from deionized water such as sorghum mosquito, which is an algae of the moss family. It is necessary to perform a multi-stage process including washing with 1% hydrochloric acid solution and 23% sodium hydroxide solution, decolorization with sodium chlorite solution, extraction with 10% sodium hydroxide solution, and fractional purification by ethanol precipitation. Therefore, there are problems that the refining operation is very complicated and the yield is low, and it is very difficult to manufacture efficiently at a low cost.
[0008]
Therefore, the problem to be solved by the present invention is to induce apoptosis of cancer cells, which contains an oligosaccharide-containing component, which is easy to purify, has a high yield, and can be produced inexpensively and efficiently. It is to obtain an agent.
[0009]
[Means for Solving the Problems]
[0010]
Means for solving the problems of the present invention are as follows.
1stly, the cancer cell apoptosis inducer which uses an arabino oligosaccharide containing material as a component.
2ndly, the cancer cell apoptosis inducer which uses the arabino oligosaccharide containing material whose average degree of polymerization is 10 or less as a component.
Third, the arabinan-containing fiber is hydrolyzed using an arabino-oligosaccharide-producing enzyme by Penicillium sp. GALA22 deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center as FERMP-18894. A cancer cell apoptosis inducer comprising the arabino-oligosaccharide-containing product obtained as described above.
Here, as the arabinan-containing fiber, in addition to beet juice lees, beet fiber, beet pulp, apple juice lees, apple fiber, any one of peanut ginger, citrus peel or flesh, okara, soybean fiber More than one can be used.
Fourth, the cancer cell apoptosis-inducing agent according to the third aspect, wherein beet juice is used as the arabinan-containing fiber.
[0011]
The arabino-oligosaccharide composition used in the present invention preferably has an average sugar polymerization degree of 10 or less, and the binding mode is not particularly limited.
The average degree of polymerization is a value defined by the total sugar amount (in terms of arabinose) / reducing sugar amount (in terms of arabinose). The total sugar amount is determined by measuring the phenol / sulfuric acid method and the reducing sugar amount by the Nelson / Somogi method. It is done.
[0012]
The arabino oligosaccharide in the present invention is an oligosaccharide composed of 2 to 10 sugars containing 1 to 10 molecules of arabinose. The bonding mode is not limited to a straight chain composed of α-1,5 bonds, but includes those having various branched structures including α-1,2 and α-1,3 bonds. This includes those in which at least one sugar other than arabinose, such as xylose, glucose, galactose, mannose and galacturonic acid, is bonded to the reducing end, non-reducing end, or in the molecule.
[0013]
In addition, the arabino-oligosaccharide-containing material used in the present invention may be any of powder, liquid, crystal or granule.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
[0015]
The arabino-oligosaccharide-containing material used in the present invention is hydrolyzed to beet juice lees or apple juice lees using enzymes such as endoarabinanase and arabinofuranosidase to obtain an oligosaccharide mixture. It can be obtained by separating and recovering the arabino-oligosaccharide from the sugar mixture.
Any enzyme agent can be used.
When a commercially available enzyme is used, it is difficult to obtain a purified product, and therefore a fiber degrading enzyme may be used.
Examples thereof include Pectinase G Amano (trade name) manufactured by Amano Enzyme, Sumiteam AC (trade name) manufactured by Shin Nippon Chemical Industry Co., Ltd., and the like.
[0016]
The hydrolysis treatment of beet juice lees or apple juice lees with enzymes may use free enzymes or enzymes immobilized on a carrier.
In addition, the hydrolysis treatment with an enzyme may be performed by a continuous method or a batch method, and various conditions such as the type and origin of the enzyme, the amount of the enzyme added, the temperature at the time of treatment, pH, temperature, etc. It is good to choose and process according to the situation.
[0017]
The sugar mixture solution containing the oligosaccharide obtained as described above is separated by a conventional method.
That is, in such a case, it is necessary to perform well-known means usually used in the field, such as filtration, centrifugation, ion exchange or adsorption chromatography, membrane concentration, membrane separation, solvent extraction, vacuum concentration, crystallization and the like. It is used in combination accordingly.
As an example, residues such as fibers are removed from the sugar mixture solution by filtration, centrifugation, etc., then this solution is treated with activated carbon to remove colored substances, and further desalted with an ion exchange resin, and then concentrated. It can be made into syrup or powder.
[0018]
In addition, by using an arabino-oligosaccharide-producing enzyme by Penicillium sp. GALA22 deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center as FERMP-18894, the arabinan-containing fiber content is hydrolyzed. Next, a method for producing the resulting arabino-oligosaccharide-containing product will be described.
[0019]
First, Penicillium sp. GALA22 is cultured to prepare an arabino oligosaccharide producing enzyme.
The strain is cultured under an aerobic condition using a liquid medium or a solid medium containing a carbon source, a nitrogen source, inorganic salts and the like.
As the carbon source, arabinan-containing fibers such as beet juice lees and apple juice lees are used.
These fibers can be used after pulverization.
As the nitrogen source, nitrogen compounds that can be used by microorganisms such as yeast extract, peptone, malt extract, corn steep liquor and the like are used.
As the inorganic salts, for example, salts such as magnesium sulfate, sodium nitrate, potassium dihydrogen phosphate, calcium hydroxide are used.
In addition to these carbon source, nitrogen source and inorganic salts, various organic substances and inorganic substances necessary for the growth of filamentous fungi can be added as necessary.
The culture method using the above medium may be either solid culture or liquid culture, but aeration and agitation culture using a shaking culture or a fermenter is preferable.
The culture temperature is not particularly limited as long as it is within the range where it can grow, but it is preferably around 30 ° C.
Although culture | cultivation pH will not be specifically limited if it is in the range which can be grown, pH around 3-5 is desirable.
The culture time is not particularly limited, but is preferably about 24 to 168 hours.
After culturing as described above, in order to collect and purify the target arabino-oligosaccharide-producing enzyme from the culture, well-known means usually used, for example, filtration, centrifugation, ion exchange or gel filtration Operations such as chromatography, desalting, membrane concentration, membrane separation, etc. can be used in combination as appropriate.
As an example, an enzyme solution obtained by removing cells from a culture solution by filtration, centrifugation, or the like and performing a desalting operation using a dialysis membrane can be used.
Here, the enzyme solution can be recovered and used repeatedly.
The arabino-oligosaccharide is produced by saccharification by adding the enzyme solution obtained by the above method to an arabinan-containing fiber such as beet juice lees or apple juice lees.
The reaction temperature is within the range where the enzyme is not inactivated, that is, 20 to 70 ° C, preferably 40 to 60 ° C.
The enzyme reaction pH is desirably the reaction under the optimum conditions of the enzyme, and is in the range of pH 3 to 9, preferably pH 4 to 7.
Although the reaction time depends on the composition and shape of the arabinan-containing fiber to be used and the amount of enzyme added, it is usually preferable to set the reaction time to 3 to 72 hours.
As the reaction proceeds, the arabinan-containing fiber is hydrolyzed and arabino-oligosaccharides are produced and released. After the reaction is completed, the arabinan-containing fiber is separated from the reaction solution by a conventional method.
That is, in such a case, it is necessary to carry out well-known means usually used in the field such as filtration, centrifugation, ion exchange or adsorption chromatography, membrane concentration, membrane separation, solvent extraction, vacuum concentration, crystallization and the like. It can be used in combination as appropriate.
For example, residues such as fibers are removed from the reaction solution by filtration, centrifugation, etc., and then this solution is treated with activated carbon to remove coloring substances, and further deionized with an ion exchange resin, and then concentrated. Syrup or powder.
[0020]
The arabino-oligosaccharide used in the present invention is not limited to those produced by the above method, and any arabino-oligosaccharide can be used. For example, an arabino-oligosaccharide obtained by a chemical method without using an enzyme can also be used. it can.
[0021]
The assay for induction of apoptosis using these arabino-oligosaccharide-containing substances is the proliferation of cancer cells (HL60 human leukemia cells) in a culture system to which the arabino-oligosaccharide-containing substance is added, the morphological changes of the cells and the cell nuclei, and It is possible to investigate by observing fragmentation of nuclear DNA.
The proliferation of HL60 human leukemia cells in a culture system to which an arabino-oligosaccharide-containing product is added can be easily examined by measuring the number of viable cells exhibiting trypan blue dye exclusion ability using a white sphere calculator.
In the culture system in which the concentration of the arabino-oligosaccharide-containing material in the culture system was adjusted to 0.5 mg / ml or more, it was found that the proliferation of HL60 cells was clearly inferior to the system without the addition of the arabino-oligosaccharide-containing material. ing.
As shown in Test Example 1 below, the decrease in HL60 cell proliferation in the cell line containing this arabino-oligosaccharide-containing material is clearly observed from the observation result of nuclear DNA by electrophoresis with a microscope and agarose gel. It turned out to be due to death.
[0022]
[Example 1]
[0023]
EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited to this.
[0024]
a) Preparation of enzyme solution From 20.0 g of beet fiber (manufactured by Nippon Beet Sugar), processed from beet juice lees, 3 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 0.5 g of corn steep liquor Put 5ml of sterilized water and a few defoamers into the slant in which Penicillium sp. GALA22 strain (FERMP-18894) is cultured in a jar fermenter (Sakura Seiki, 1.5 liter culture tank capacity) containing 1 liter of the resulting medium. The suspension was aseptically inoculated and cultured with aeration and stirring at a temperature of 30 ° C., pH 5.0, aeration volume of 0.5 L / min, and rotation speed of 300 rpm for 72 hours.
After completion of the culture, bacterial cells and medium residues were removed by centrifugation (5000 G, 10 minutes) and a filter having a pore size of 0.45 μm.
Subsequently, a solution concentrated up to 20-fold amount with an ultrafiltration membrane having a molecular weight cut-off of 10,000 was used as an enzyme solution.
[0025]
b) Preparation of arabino-oligosaccharide solution 1.4 kg of ground beet juice koji was suspended in 35 liters of 25 mM acetate buffer (pH 5.0).
After heating this to 50 ° C., 1 liter of the prepared enzyme solution was added, and hydrolysis was carried out for 24 hours while maintaining the same temperature.
Thereafter, the solution temperature was raised to 90 ° C., and the temperature was maintained for 30 minutes to inactivate the enzyme.
This was centrifuged (5000 G, 5 minutes), and then filtered through a filter having a pore size of 0.45 μm to obtain a solution containing useful carbohydrates.
[0026]
c) Purification of arabino-oligosaccharide solution After adding this solution to a column (diameter 12 cm, length 100 cm) packed with 4 kg of activated carbon (calgon granular activated carbon), 100 liters of distilled water and 50% ethanol 50 at a flow rate of 170 ml / min. Monosaccharides were removed by washing with liters.
Next, 50% of 10.0% ethanol, 50 liters of 20.0% ethanol, 50 liters of 30.0% ethanol, and 50 liters of 50.0% ethanol were sequentially flowed to obtain an arabino oligosaccharide fraction.
These were concentrated to 1 liter with an evaporator and then applied to a column (diameter 6 cm, length 100 cm) packed with 850 g of cation exchange resin (DOWEX88) and 1500 g of anion exchange resin (Purolite A-10312).
Galacturonic acid was removed and desalted by passing 80 liters of distilled water at a flow rate of 150 ml / min.
[0027]
d) Preparation of cancer cell apoptosis inducer Fractions from which galacturonic acid was removed and desalted were concentrated to 200 ml with an evaporator, and then lyophilized to obtain 52.6 g of an arabino-oligosaccharide composition as a cancer cell apoptosis inducer. Obtained.
The average degree of polymerization of the arabino-oligosaccharide composition obtained as described above was 2.8.
[0028]
[Test Example 1]
[0029]
Next, a cancer cell apoptosis induction test was performed by the following method using the cancer cell apoptosis inducer containing the arabino-oligosaccharide having an average polymerization degree of 2.8 obtained in Example 1 as a component of the present invention.
[0030]
Here, HL60 human leukemia cells used were distributed from the Human Science Research Resource Bank.
The HL60 cells were cultured using RPM11640 medium (GIBCO) containing 10% fetal calf serum at 37 ° C. in the presence of 5% CO _{2 at} a relative humidity of 100%.
HL60 cells are seeded in a 24-well plate at 1 × 10 ⁵ cells / ml, and the arabino-oligosaccharide sample of the present invention is added to the culture solution to a final concentration of 5 mg / ml, followed by incubation for 24 hours. Thereafter, the number of viable cells exhibiting trypan blue dye exclusion ability was counted using a hemocytometer.
[0031]
As a control test, saccharides other than arabino-oligosaccharides (arabinose, galacturonic acid, glucose, cellobiose, α-1,4 xylo-oligosaccharide, arabinan) were also tested under the same conditions and procedures as in the present invention.
[0032]
The results are shown in FIG. 1 as a graph showing the survival rate of cancer cells in each carbohydrate.
As shown in FIG. 1, the arabino-oligosaccharide of the present invention group suppresses the strongest number of viable cells of HL60 cells 24 hours after addition, about 45%, compared with the saccharide of the control group.
[0033]
Therefore, in order to clarify the HL60 cell growth inhibitory effect of the arabino-oligosaccharides of the present invention, the number of viable HL60 cells was counted for 6 to 48 hours after the addition of each concentration of arabino-oligosaccharide samples.
The results are shown in FIG. 2 as a graph showing the measurement results of the number of viable HL60 cells.
As shown in FIG. 2, arabino-oligosaccharide clearly inhibits the growth of viable HL60 cells from a concentration of 0.5 mg / ml.
[0034]
Next, DNA fragmentation specific to apoptosis was observed in order to test whether the inhibition of proliferation of HL60 cells was cell death based on apoptosis.
First, cells were lysed in 20 μl of lysate [50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 0.5% SDS], 1 μl of RNase A (10 mg / ml lysate) (SIGMA) was added, and 50 ° C. Incubated for 30 minutes.
Thereafter, 1 μl of Proteinase K (10 mg / ml lysate) (SIGMA) was further added and incubated at 50 ° C. for 60 minutes.
This cell lysate was electrophoresed on a 2% agarose gel containing 0.5 μl / ml ethidium bromide (Wako Pure Chemical Industries), and DNA fragmentation was observed on a UV illuminator.
FIG. 3 shows a photograph showing the results of agarose electrophoresis.
In FIG. 3, from the left to the lane, the first is a marker, the second is a control group without addition, the third to sixth are arabino-oligosaccharide added groups of the present invention, and the third is arabino-oligosaccharide 0.5 mg / ml added. The arabino-oligosaccharide 1.0 mg / ml addition group, the arabino-oligosaccharide 5.0 mg / ml addition group, the sixth shows the arabino-oligosaccharide 10.0 mg / ml addition group.
As shown in FIG. 3, arabino-oligosaccharides induced DNA fragmentation in nucleosome units characteristic of apoptosis in HL60 cells at a concentration of 0.5 to 10 mg / ml.
[0035]
Further, in the group of the present invention, the nuclear DNA of HL60 cells was fluorescently stained with Hoechst 33258 (Wako Pure Chemical Industries) and observed with a fluorescence microscope for the group with arabino-oligosaccharide 5.0 mg / ml added and the control group without addition.
In FIG. 4, the photograph which shows the observation result of the HL60 cell by the fluorescence microscope of a control group is shown.
Moreover, the photograph which shows the observation result of the HL60 cell with the fluorescence microscope of invention zone in FIG. 5 is shown.
As shown in FIG. 5, nuclear condensation and fragmentation characteristic of apoptosis were observed by addition of arabino oligosaccharide.
[0036]
【The invention's effect】
[0037]
The cancer cell apoptosis inducer of the present invention is easy to purify, has a high yield, and can be produced efficiently at low cost.
[Brief description of the drawings]
FIG. 1 is a graph showing the survival rate of cancer cells in each carbohydrate. FIG. 2 is a graph showing the measurement results of viable HL60 cells. FIG. 3 is a photograph showing the results of agarose electrophoresis. FIG. 5 is a photograph showing the observation results of HL60 cells with a fluorescence microscope in the invention section.

Claims (4)

A cancer cell apoptosis inducer comprising an arabino-oligosaccharide-containing substance as a component. A cancer cell apoptosis inducer comprising an arabino-oligosaccharide-containing product having an average polymerization degree of 10 or less. A cancer cell apoptosis inducer comprising, as a component, an arabino-oligosaccharide-containing product obtained by hydrolyzing an arabinan-containing fiber using an arabino-oligosaccharide-producing enzyme by Penicillium sp. GALA22 deposited as FERMP-188941. The cancer cell apoptosis inducer according to claim 3, wherein beet juice is used as the arabinan-containing fiber.

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