JP2004182628A - Apoptosis inducer of cancer cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a cancer cell apoptosis inducer which has ready purification operation and a high yield and is inexpensively and efficiently produced. <P>SOLUTION: The cancer cell apoptosis inducer comprises an arabinooligosaccharide-containing substance as a component which is obtained by hydrolyzing an arabinan-containing fiber component using an arabinooligosaccharide-forming enzyme by Penicillium sp. GALA22 deposited as FERM P-18941 and has ≤5 average polymerization degree. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
[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 a cancer cell apoptosis inducer containing an oligosaccharide-containing substance as a component, β-1,3 xylan obtained from a cell wall of a seaweed as described in Japanese Patent Application Laid-Open No. 2001-86999 (Patent Document 1). Is known, which comprises β-1,3 xylo-oligosaccharide as a main component, obtained by treating E. coli with β-1,3 xylanase.
The cancer cell apoptosis-inducing agent is prepared from scotch ivy, which is an algae of the family Crocodidae, by washing with deionized water, 1% hydrochloric acid solution and 23% sodium hydroxide solution, decolorizing with sodium chlorite solution, 10% sodium hydroxide By performing extraction with a solution and fractional purification by ethanol precipitation, β-1,3 xylan used as a raw material was obtained.
[0005]
[Patent Document 1]
JP 2001-86999 A
[Problems to be solved by the invention]
[0007]
However, the cancer cell-induced apoptosis inducer containing β-1,3 xylo-oligosaccharide as a main component is prepared from deionized water such as Srikkogita, which is an algae of the Ipaceae family, in order to obtain β-1,3 xylan used as a raw material. Washing with 1% hydrochloric acid solution and 23% sodium hydroxide solution, decolorization with sodium chlorite solution, extraction with 10% sodium hydroxide solution, fractional purification by ethanol precipitation, and multi-step treatment must be performed. Therefore, there is a problem in that the purification operation is very complicated and the yield is low, and it has been very difficult to produce inexpensively and efficiently.
[0008]
Therefore, the problem to be solved by the present invention is to induce cancer cell apoptosis containing oligosaccharide-containing components as components, which are easy to purify, have a high yield, and can be produced inexpensively and efficiently. To obtain an agent.
[0009]
[Means for Solving the Problems]
[0010]
Means for solving the problems of the present invention are as follows.
First, a cancer cell apoptosis inducer containing an arabino-oligosaccharide-containing component as a component.
Second, a cancer cell apoptosis-inducing agent comprising an arabino-oligosaccharide-containing substance having an average degree of polymerization of 10 or less.
Third, arabinonan-containing fiber is hydrolyzed using an arabino-oligosaccharide-forming enzyme derived from Penicillium sp. GALA22 deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology as FERMP-188941. A cancer cell apoptosis inducer comprising the arabino-oligosaccharide-containing material obtained as a component.
Here, as the arabinan-containing fiber, besides beet juice lees, beet fiber, beet pulp, apple juice lees, apple fiber, peanut lees, citrus peel or pulp, okara, soy fiber More than one can be used.
Fourth, the cancer cell apoptosis-inducing agent according to the third above, wherein beet juice lees is used as the arabinan-containing fiber component.
[0011]
The arabino-oligosaccharide composition used in the present invention preferably has an average degree of polymerization of saccharide of 10 or less, and the binding mode is not particularly limited.
The average degree of polymerization is a value defined by the amount of total sugar (converted to arabinose) / the amount of reducing sugar (converted to arabinose). The total amount of sugar is determined by measuring the amount of reducing sugar by the phenol-sulfuric acid method and the amount of reducing sugar by the Nelson-Somogi method. Can be
[0012]
The arabino-oligosaccharide in the present invention is an oligosaccharide composed of 2 to 10 saccharides containing 1 to 10 molecules of arabinose. The bonding mode is not limited to a linear one composed of α-1,5 bonds, but also 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 or non-reducing end, or in the molecule.
[0013]
The arabino-oligosaccharide-containing material used in the present invention may be any of powder, liquid, crystal and granules.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
[0015]
The arabino-oligosaccharide-containing material used in the present invention is obtained by hydrolyzing beet juice cake or apple juice cake with an enzyme such as endoarabinase or arabinofuranosidase to obtain an oligosaccharide mixture. It can be obtained by separating and collecting arabino-oligosaccharide from the saccharide mixture.
In this case, any enzyme agent can be used.
When a commercially available enzyme is used, it is difficult to obtain a purified product. Therefore, a fibrinolytic enzyme may be used.
Examples thereof include Pectinase G Amano (trade name) manufactured by Amano Enzyme Co., Ltd., and Sumiteam AC (trade name) manufactured by Shin Nippon Chemical Industry Co., Ltd.
[0016]
In the hydrolysis treatment of beet juice lees and apple juice lees with an enzyme, a free enzyme or an enzyme immobilized on a carrier may be used.
In addition, the hydrolysis treatment with the 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 select and process according to the situation.
[0017]
The oligosaccharide-containing saccharide mixture solution obtained as described above is separated by a conventional method.
That is, in such a case, well-known means usually used in the art, such as filtration, centrifugation, ion exchange or adsorption chromatography, membrane concentration, membrane separation, solvent extraction, concentration under reduced pressure, crystallization and the like become necessary. They are used in combination as appropriate.
For example, filtration, centrifugation or the like is used to remove residues such as fibers from the sugar mixture solution, and then the solution is treated with activated carbon to remove coloring substances and the like, and further desalted with an ion exchange resin, and then concentrated. Into a syrup or a powder.
[0018]
Further, by using arabino-oligosaccharide-forming enzyme produced by Penicillium sp. GALA22 deposited at National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary as FERMP-18941, the arabinan-containing fiber is hydrolyzed. The method for producing the resulting arabino-oligosaccharide-containing material is described below.
[0019]
First, Penicillium sp. GALA22 is cultured to prepare an arabino-oligosaccharide-forming enzyme.
The culture of the strain is carried out under aerobic conditions 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 fiber such as beet juice lees and apple juice lees is used.
These fibers may be used after being subjected to a pulverizing treatment or the like.
As the nitrogen source, nitrogen compounds usable by microorganisms, for example, 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, and calcium hydroxide are used.
In addition to these carbon sources, nitrogen sources, and inorganic salts, various organic and inorganic substances necessary for the growth of filamentous fungi can be further added, if necessary.
As a method of culturing in the above-mentioned medium, either solid culturing or liquid culturing may be used, but shaking cultivation or aeration and stirring cultivation using a fermenter or the like is preferable.
The culture temperature is not particularly limited as long as it is within the range in which it can grow, but is preferably around 30 ° C.
The culture pH is not particularly limited as long as it can be grown, but is preferably around pH 3-5.
The culture time is not particularly limited, but is preferably about 24 to 168 hours.
After culturing as described above, the target arabino-oligosaccharide-forming enzyme can be collected and purified from the culture by well-known means commonly used, for example, filtration, centrifugation, ion exchange or gel filtration. Operations such as chromatography, desalting, membrane concentration, and membrane separation can be appropriately combined and used as necessary.
For example, a culture solution obtained by removing bacterial cells from a culture solution by filtration, centrifugation, or the like, and performing a desalting operation using a dialysis membrane can be used as the enzyme solution.
Here, the enzyme solution can be collected and used repeatedly.
In the production of arabino-oligosaccharide, saccharification is carried out by adding the enzyme solution obtained by the above method to arabinan-containing fiber such as beet juice cake and apple juice cake.
The reaction temperature is within a range where the enzyme is not inactivated, that is, 20 to 70 ° C, preferably 40 to 60 ° C.
It is desirable that the enzyme reaction is carried out under the optimum conditions for the enzyme, and the pH is in the range of pH 3 to 9, preferably pH 4 to 7.
The reaction time depends on the composition and shape of the arabinan-containing fiber used and the amount of enzyme added, but it is usually preferable to set the reaction time at 3 to 72 hours.
As the reaction proceeds, the arabinan-containing fiber component is hydrolyzed, and arabino-oligosaccharide is generated and released. After completion of the reaction, the arabino-oligosaccharide is separated from the reaction solution by a conventional method.
That is, in such a case, it is necessary to use well-known means commonly used in the art, such as filtration, centrifugation, ion exchange or adsorption chromatography, membrane concentration, membrane separation, solvent extraction, vacuum concentration, crystallization, and the like. They can be used in combination as appropriate.
For example, the reaction solution is filtered, centrifuged to remove residues such as fibers, and then the solution is treated with activated carbon to remove coloring substances and the like, 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 as long as it is an arabino-oligosaccharide.For example, it is also possible to use arabino-oligosaccharide obtained by a chemical method without using an enzyme. it can.
[0021]
Assays for apoptosis induction using these arabino-oligosaccharide-containing substances include the proliferative properties of cancer cells (HL60 human leukemia cells) in culture systems to which arabino-oligosaccharide-containing substances have been added, and the morphological changes of the cells and the cell nuclei. It is possible to check by observing the fragmentation of nuclear DNA.
Proliferation of HL60 human leukemia cells in a culture system to which an arabino-oligosaccharide-containing substance has been added can be easily examined by measuring the number of viable cells exhibiting trypan blue dye exclusion ability using a leukocyte 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.
The decrease in the HL60 cell proliferation in the cell line containing the arabino-oligosaccharide-containing material was clearly observed by the observation of nuclear DNA by electrophoresis with a microscope and agarose gel, as shown in the following Test Example 1. It turned out to be due to death.
[0022]
Embodiment 1
[0023]
Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
[0024]
a) Preparation of Enzyme Solution From beet fiber (manufactured by Nippon Sugar Beet Sugar Co., Ltd.) obtained by processing beet juice lees into powder, 20.0 g, potassium dihydrogen phosphate 3.0 g, magnesium sulfate 0.5 g, and corn steep liquor 0.5 g Into a jar fermenter (manufactured by Sakura Seiki Co., Ltd., culture tank capacity: 1.5 liters) containing 1 liter of medium, 5 ml of sterilized water and several drops of an antifoaming agent are added to a slant in which Penicillium sp. The suspension was aseptically inoculated, and cultured under aeration and stirring at a temperature of 30 ° C., a pH of 5.0, an aeration rate of 0.5 L / min, and a rotation speed of 300 rpm for 72 hours.
After completion of the culture, the 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 to a 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 pulverized beet juice cake was suspended in 35 L 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 at the same temperature for 24 hours.
Thereafter, the temperature of the solution was raised to 90 ° C., and the temperature was maintained for 30 minutes to inactivate the enzyme.
After centrifugation (5000 G, 5 minutes), the solution was filtered with a filter having a pore size of 0.45 μm to obtain a solution containing a useful saccharide.
[0026]
c) Purification of arabino-oligosaccharide solution This solution was added to a column (diameter 12 cm, length 100 cm) packed with 4 kg of activated carbon (calgon granular activated carbon), and then 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 liters 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 passed to obtain an arabino-oligosaccharide fraction.
After concentrating these to 1 liter with an evaporator, they were applied to a column (diameter 6 cm, length 100 cm) packed with 850 g of cation exchange resin (DOWEX 88) 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 The fraction from which galacturonic acid was removed and desalted was 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 polymerization degree 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 carried out by the following method using the cancer cell apoptosis-inducing agent containing arabino-oligosaccharide having an average degree of polymerization of 2.8 obtained in Example 1 as the present invention.
[0030]
Here, HL60 human leukemia cells used were purchased from the Human Science Research Resource Bank.
The HL60 cells were cultured in RPM11640 medium (GIBCO) containing 10% fetal bovine serum at 37 ° C. in the presence of 5% CO _{2 at} a relative humidity of 100%.
HL60 cells are seeded on a 24-well plate at 1 × 10 ⁵ cells / ml, and a sample of arabino-oligosaccharide of the present invention is added to the culture solution to a final concentration of 5 mg / ml and cultured for 24 hours. After that, 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-oligosaccharide (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 has the most viable cell number of HL60 cells 24 hours after addition, about 45%, as compared with the carbohydrate of the control group.
[0033]
Then, in order to clarify the inhibitory effect of the arabino-oligosaccharide of the present invention on the growth of HL60 cells, the number of viable HL60 cells was counted for 6 to 48 hours after the addition of the sample of each concentration of arabino-oligosaccharide.
The result is shown in FIG. 2 as a graph showing the measurement result of the number of viable HL60 cells. As shown in FIG. 2, arabino-oligosaccharide clearly inhibits the growth of living HL60 cells at a concentration of 0.5 mg / ml.
[0034]
Next, in order to examine whether the growth suppression of HL60 cells was cell death based on apoptosis, fragmentation of DNA specific to apoptosis was observed.
First, cells were lysed in 20 μl of a lysis solution [50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 0.5% SDS], and 1 μl of RNaseA (10 mg / ml lysis solution) (SIGMA) was added. Incubated for 30 minutes.
Thereafter, 1 μl of Proteinase K (10 mg / ml solution) (SIGMA) was further added, and the mixture was incubated at 50 ° C. for 60 minutes.
The cell lysate was electrophoresed on a 2% agarose gel containing 0.5 μl / ml of 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 lane left, 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 fourth, the arabino-oligosaccharide 1.0 mg / ml addition section, the fifth, the arabino-oligosaccharide 5.0 mg / ml addition section, and the sixth, the arabino-oligosaccharide 10.0 mg / ml addition section.
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]
In addition, in the group of the present invention in which 5.0 mg / ml of arabino-oligosaccharide was added and in the control group where no arabino-oligosaccharide was added, nuclear DNA of HL60 cells was fluorescently stained with Hoechst 33258 (Wako Pure Chemical Industries) and observed with a fluorescence microscope.
FIG. 4 shows a photograph showing the results of observation of HL60 cells by a fluorescence microscope in the control section.
FIG. 5 shows a photograph showing the results of observation of HL60 cells by a fluorescence microscope in the invention section.
As shown in FIG. 5, nuclear addition and fragmentation characteristic of apoptosis were observed by the addition of arabino-oligosaccharide.
[0036]
【The invention's effect】
[0037]
The cancer cell apoptosis-inducing agent of the present invention is easy to purify, has a high yield, can be inexpensively and efficiently produced.
[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 results of measuring the number of viable HL60 cells. FIG. 3 is a photograph showing the results of agarose electrophoresis. FIG. FIG. 5 is a photograph showing a result of observation of HL60 cells by a microscope. FIG. 5 is a photograph showing a result of observation of HL60 cells by a fluorescence microscope in an invention section.

Claims (4)

An agent for inducing apoptosis of cancer cells, comprising an arabino-oligosaccharide-containing component. An agent for inducing apoptosis of cancer cells, comprising an arabino-oligosaccharide-containing substance having an average degree of polymerization of 10 or less. A cancer cell apoptosis-inducing agent comprising, as a component, an arabino-oligosaccharide-containing substance obtained by hydrolyzing an arabinan-containing fiber using an arabino-oligosaccharide-forming enzyme deposited with Penicillium sp. GALA22 deposited as FERMP-18941. The cancer cell apoptosis-inducing agent according to claim 3, wherein beet juice lees is used as the arabinan-containing fiber component.

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