JP5742050B2 - NK cell activator and NK cell activation method - Google Patents

Description

  The present invention relates to a natural killer cell activator, a method for producing activated natural killer cells, and a screening method.

  Polymethoxyflavone is a component that is abundant in the citrus peel. Several techniques using polymethoxyflavone as an active ingredient for the prevention or treatment of cancer or infectious diseases are known.

  Patent Document 1 describes a composition for preventing or treating cancer, which contains citrus flavonoids.

  In Patent Document 2, 4 ′, 5,6,7,8-pentamethoxyflavone and 3 ′, 4 ′, 5,6,7,8-hexamethoxy are used to inhibit the growth of tumor cells in animals. Methods have been described for administering orange peel extracts containing flavones to animals.

  Patent Document 3 describes a method of administering a flavin such as polymethoxyflavone in order to treat a virus or parasitic infection.

Special table 2003-510240 gazette (published March 18, 2003) Special table 2003-509447 gazette (published March 11, 2003) Special table 2003-504327 gazette (published February 4, 2003)

  However, in the prior art as described above, since the compound is directly administered, not only cancer cells and infected cells but also normal cells may be affected. Therefore, development of a method for selectively inhibiting or suppressing cancer cells and infected cells is desired.

  This invention is made | formed in view of the problem which said prior art has, The objective is to provide the technique which selectively inhibits or suppresses a cancer cell and an infected cell.

  In order to solve the above-mentioned problems, the present inventors have also called natural killer cells (hereinafter referred to as “NK cells”) which are a kind of immune cells having a function of selectively damaging and eliminating cancer cells and infected cells. )). And earnest examination was carried out, it discovered that the polymethoxyflavone which has a predetermined structure activated NK cell directly, and increased the damaging activity with respect to the cancer cell of NK cell, and completed this invention.

  The natural killer cell activator (hereinafter also referred to as “NK cell activator”) according to the present invention is characterized by comprising a compound represented by the following formula (1) as an active ingredient.

(In said formula (1), R < ₁ > -R < ₁₀ > is a hydrogen atom, a hydroxyl group, a C1-C5 alkyl group, or a C1-C4 alkoxy group each independently, R < ₁ > -R < ₁₀ > (At least 4 of them are alkoxy groups having 1 to 4 carbon atoms)
Moreover, in the natural killer cell activator which concerns on this invention, in said formula (1), R < ₁ >, R < ₃ >, R < ₅ >, R < ₇ >, R <_9> , R < ₁₀ > is respectively independently a hydrogen atom, C1-C1. 5 or an alkoxy group having 1 to 4 carbon atoms, and at least two of R ₁ , R ₃ , R ₅ , R ₇ , R ₉ and R ₁₀ are alkoxy groups having 1 to 4 carbon atoms. , R ₄ and R ₈ are each an alkoxy group having 1 to 4 carbon atoms, and R ₆ is preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

In the natural killer cell activator according to the present invention, in the above formula (1), R ₁ , R ₃ , R ₅ , R ₇ , R ₉ and R ₁₀ are each independently a hydrogen atom or a methoxy group. , R ₁ , R ₃ , R ₅ , R ₇ , R ₉ and R ₁₀ are methoxy groups, R ₄ and R ₈ are methoxy groups, and R ₂ is a hydrogen atom, hydroxyl group or methoxy group And R ₆ is preferably a hydrogen atom.

  Further, in the natural killer cell activator according to the present invention, the compound is synencetin, tangeretin, nobiletin, gardenine, 3,7,3 ′, 4 ′, 5′-pentamethoxyflavone, 5,7,3 ′, Selected from the group consisting of 4 ', 5'-pentamethoxyflavone, 3', 4 ', 5', 5,6,7-hexamethoxyflavone, and 3,7,8,2 ', 4'-pentamethoxyflavone It is preferred that

  The natural killer cell activation method according to the present invention includes an activation step of bringing a natural killer cell into contact with any of the natural killer cell activators described above.

  The method for producing a natural killer cell according to the present invention includes an activation step of bringing a natural killer cell into contact with any of the natural killer cell activators described above.

  The method for screening a compound having the ability to activate natural killer cells according to the present invention comprises a test compound treatment step in which a natural killer cell is contacted with a test compound for treatment, and a natural killer cell treated in the test compound treatment step. A mixing step for obtaining a mixed solution with cancer cells, and a measuring step for measuring lactate dehydrogenase activity in the mixed solution.

  According to the present invention, a technique for selectively inhibiting or suppressing cancer cells and infected cells can be provided.

It is a bar graph which shows the screening result of the compound which accelerates | stimulates the cytotoxic activity of NK cell. (A) to (D) are nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′. It is a figure which shows structural formula of 4,4'-pentamethoxyflavone (D). (A) to (D) are nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′. It is a graph which shows the cytotoxic activity of the KHYG-1 cell processed by 4,4'-pentamethoxyflavone (D). Nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′, 4′-pentamethoxyflavone (D ) Is a graph showing the cytotoxic activity of itself. Nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′, 4′-pentamethoxyflavone (D It is a graph which shows the cytotoxic activity of the culture supernatant of the KHYG-1 cell processed. (A) to (c) are nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′. , 4′-pentamethoxyflavone (D) is a graph showing the expression levels of IFN-γ gene (a), perforin gene (b) and granzyme B gene (c) in KHYG-1 cells treated.

[NK cell activator]
The NK cell activator according to the present invention comprises a compound represented by the above formula (1) as an active ingredient.

In the above formula _{(1), R} 1 ~R ₁₀ are each independently a hydrogen atom, a hydroxyl group, an alkyl group or an alkoxy group having 1 to 4 carbon atoms of 1 to 5 carbon atoms. At least four of R _{1 to} R ₁₀ are alkoxy groups. It is preferable that four to seven of the R ₁ to R ₁₀ is an alkoxy group, and more preferably 5 or 6 alkoxy group.

The alkoxy group in R _{1 to} R ₁₀ preferably has 1 to 2 carbon atoms, and more preferably a methoxy group. In other words, each of R _{1 to} R ₁₀ is preferably independently a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 2 carbon atoms, and more preferably independently a hydrogen atom, a hydroxyl group, or a methoxy group. .

The alkyl group in R _{1 to} R ₁₀ preferably has 1 to 3 carbon atoms, and more preferably has 1 to 2 carbon atoms.

R ₁ , R ₃ , R ₅ , R ₇ , R ₉ , and R ₁₀ are each independently preferably a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms, It is more preferably independently a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or an alkoxy group having 1 to 2 carbon atoms, and further preferably each independently a hydrogen atom or a methoxy group.

R ₄ and R ₈ are preferably each independently an alkoxy group having 1 to 4 carbon atoms, more preferably each independently an alkoxy group having 1 to 2 carbon atoms, and preferably a methoxy group. Further preferred.

R ₆ is preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, more preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and even more preferably a hydrogen atom.

R ₁ , R ₃ , R ₅ , R ₇ , R ₉ and R ₁₀ are each independently a hydrogen atom, an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and R ₁ , At least two of R ₃ , R ₅ , R ₇ , R ₉ and R ₁₀ are an alkoxy group having 1 to 4 carbon atoms, R ₄ and R ₈ are an alkoxy group having 1 to 4 carbon atoms, and R ₆ is preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

R ₁ , R ₃ , R ₅ , R ₇ , R ₉ , R ₁₀ are each independently a hydrogen atom or a methoxy group, and R ₁ , R ₃ , R ₅ , R ₇ , R ₉ , R ₁₀ More preferably, at least two of them are methoxy groups, R ₄ and R ₈ are methoxy groups, R ₂ is a hydrogen atom, hydroxyl group or methoxy group, and R ₆ is a hydrogen atom.

Further, R ₂ , R ₃ , R ₄ , R ₅ , R ₈ are each independently an alkoxy group having 1 to 4 carbon atoms, and R ₁ , R ₆ , R ₇ , R ₉ , R ₁₀ are each independently And preferably a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 5 carbon atoms.

R ₂ , R ₃ , R ₄ , R ₅ , R ₈ , R ₉ are each independently an alkoxy group having 1 to 4 carbon atoms, and R ₁ , R ₆ , R ₇ , R ₁₀ are independently And preferably a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 5 carbon atoms.

R ₁ , R ₄ , R ₇ , R ₈ , R ₉ are each independently an alkoxy group having 1 to 4 carbon atoms, and R ₂ , R ₃ , R ₅ , R ₆ , R ₁₀ are each independently And preferably a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 5 carbon atoms.

R ₂ , R ₃ , R ₄ , R ₇ , R ₈ , and R ₉ are each independently an alkoxy group having 1 to 4 carbon atoms, and R ₁ , R ₅ , R ₆ , and R ₁₀ are independently And preferably a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 5 carbon atoms.

R ₁ , R ₄ , R ₅ , R ₈ and R ₁₀ are each independently an alkoxy group having 1 to 4 carbon atoms, and R ₂ , R ₃ , R ₆ , R ₇ and R ₉ are each independently And preferably a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 5 carbon atoms.

  The compound represented by the above formula (1) is preferably a so-called polymethoxyflavone. The polymethoxyflavone may be a flavone having a plurality of methoxy groups, and may be a natural compound or a non-natural compound.

  Examples of the compound represented by the above formula (1) include sinensetin, tangeretin, nobiletin, gardenine, 3,7,3 ′, 4 ′, 5′-pentamethoxyflavone, 5,7,3 ′, 4 ′, 5. '-Pentamethoxyflavone, 3', 4 ', 5', 5,6,7-hexamethoxyflavone, 3,7,8,2 ', 4'-pentamethoxyflavone, Natsudaidyne, 3,5,6,7 , 3 ′, 4′-hexamethoxyflavone, 3,5,6,7,8,3 ′, 4′-heptamethoxyflavone, and metabolites thereof. Among them, sinensetin, tangeretin, nobiletin, gardenine, 3,7,3 ′, 4 ′, 5′-pentamethoxyflavone, 5,7,3 ′, 4 ′, 5′-pentamethoxyflavone, 3 ′, 4 ′ , 5 ′, 5,6,7-hexamethoxyflavone and 3,7,8,2 ′, 4′-pentamethoxyflavone.

  The metabolite may be, for example, a demethylated compound, and examples thereof include 4'-demethylnobiletin, 3 ', 4'-didemethylnobiletin and the like.

  The compound represented by the above formula (1) may be artificially synthesized. As a synthesis method, a known method can be used. Further, the compound represented by the above formula (1) may be obtained from nature. For example, some of polymethoxyflavones can be extracted from citrus peel and the like. As an extraction method, a known method can be used. The NK cell activator may contain the compound represented by the above formula (1) as an extract from citrus.

  In the NK cell activator according to the present invention, the compound represented by the above formula (1) may be dissolved in a solvent. As the solvent, for example, dimethyl sulfoxide (DMSO), ethanol or the like can be used.

  The NK cell activator according to the present invention may further contain a physiologically active substance (cytokine or the like), serum, buffer, medium, additive and the like. NK cell activators include excipients, disintegrants, emulsifiers, plasticizers, buffering agents (such as HEPES), saccharides, vitamins (such as ascorbic acid), pH adjusters, fragrances, amino acids, and known NK cells. An activator or the like may further be included.

  The NK cell activator according to the present invention can enhance and activate the cytotoxic activity of NK cells. By using this activated NK cell, cancer cells and infected cells can be selectively damaged. Therefore, cancer cells and infected cells can be selectively inhibited or suppressed by utilizing the present invention.

  The present invention is useful, for example, for immune cell therapy (particularly activated NK cell therapy). In addition, the NK cell activator according to the present invention can be suitably used for medicines, foods, beverages, supplements and the like for enhancing immune function. In addition, the NK cell activator according to the present invention can be suitably used for experimental reagents such as activators and inhibitors.

〔Composition〕
The present invention also provides a novel composition. The composition according to the present invention contains at least a compound represented by the above formula (1).

In the above formula (1), R _{1 to} R ₁₀ may be the same as those exemplified in the item of the NK cell activator. Moreover, the compound illustrated by the item of the NK cell activator can be used suitably as a compound represented by the said Formula (1).

  The composition may further contain a solvent for dissolving the compound represented by the above formula (1). As the solvent, those exemplified in the item of NK cell activator can be used. The composition may further contain a physiologically active substance (cytokine or the like), serum, buffer, medium, additive and the like. In addition, the composition includes excipients, disintegrants, emulsifiers, plasticizers, buffers (such as HEPES), saccharides, vitamins (such as ascorbic acid), pH adjusters, fragrances, amino acids, and known NK cell activators. Etc. may be further included.

  With the above configuration, the composition according to the present invention contains the compound represented by the above formula (1), and therefore can be suitably used for the activation of NK cells. By using this activated NK cell, cancer cells and infected cells can be selectively damaged. Therefore, cancer cells and infected cells can be selectively inhibited or suppressed by utilizing the present invention.

  The present invention can be suitably used for immune cell therapy, pharmaceuticals for enhancing immune functions, foods, beverages, supplements, etc., and experimental reagents.

[NK cell activation method]
The present invention also provides a method for activating NK cells. The NK cell activation method according to the present invention includes an activation step of bringing NK cells into contact with the NK cell activator according to the present invention. That is, in the activation step, NK cells are brought into contact with the compound represented by the above formula (1).

  The method for bringing the NK cell into contact with the NK cell activator is not particularly limited. For example, a method of culturing the NK cell in a medium to which the NK cell activator is added can be used.

  When the NK cell is brought into contact with the NK cell activator in the activation step, the concentration of the compound represented by the formula (1) in the reaction solution is preferably 1 μM to 30 μM. The contact time is preferably 4 hours to 24 hours. When NK cells are cultured in a medium to which an NK cell activator is added, known conditions can be suitably used as the culture conditions.

  The activation step may be performed outside the body.

  By using the NK cell activation method according to the present invention, NK cells can be activated efficiently. Therefore, it is possible to efficiently provide NK cells that can selectively inhibit or suppress cancer cells and infected cells.

[Method for producing activated NK cells]
The present invention also provides a method for producing activated NK cells comprising the activation step described above. The activation step may be performed outside the body.

  An “activated NK cell” refers to an NK cell that has a higher cytotoxic activity than an untreated NK cell. The activated NK cells preferably have a cytotoxic activity 1.5 times or more that of untreated NK cells.

  According to the method for producing activated NK cells according to the present invention, activated NK cells can be produced efficiently. Therefore, cancer cells and infected cells can be selectively inhibited or suppressed by utilizing the present invention.

[Screening method]
The present invention also provides a method for screening a compound having the ability to activate NK cells. The screening method according to the present invention includes a test compound treatment step, a mixing step, and a measuring step.

  The test compound treatment step is a step in which NK cells are contacted with a test compound for treatment. Although it does not specifically limit as a method to contact, For example, the method etc. which culture | cultivate NK cell in the culture medium which added the test compound can be used.

  The test compound treatment step is preferably performed in a system that does not contain cells other than NK cells. For example, it is preferable to use established NK cells. This makes it possible to select a compound that can directly activate NK cells.

  The mixing step is a step of obtaining a mixed solution of NK cells and cancer cells treated in the test compound treatment step. The method of mixing is not particularly limited, and for example, a method of mixing each cell suspension may be used. The ratio of the number of NK cells and cancer cells in the mixing step is preferably 20: 1 to 5: 1.

  A measurement process is a process of measuring LDH activity in a liquid mixture. Thereby, the activity of LDH released from damaged cancer cells can be measured. From this LDH activity, the cytotoxic activity of the test compound can be determined. For example, the ratio of the LDH activity measured in the measurement step to the LDH activity when almost all cancer cells in the mixed solution are damaged can be obtained and used as the cytotoxic activity.

  The LDH activity when almost all cancer cells in the mixture are damaged is, for example, a cytotoxic reagent (for example, a surfactant) on the same number of cancer cells used in the mixing step. It is good also as LDH activity measured after adding.

  In the present invention, the activity of LDH released from damaged cells is used as an index of cytotoxic activity. Therefore, it is not necessary to perform various procedures necessary for the isotope experiment, to prepare facilities necessary for the isotope experiment, or to manage the isotope, as compared with the case of using the method using the isotope. Therefore, it is possible to measure the cytotoxic activity more easily.

  The screening method according to the present invention may further include a selection step. In the selection step, the cytotoxic activity was calculated from the LDH activity measured in the measurement step, and when the cytotoxic activity was larger than the cytotoxic activity of untreated NK cells, it was used in the test compound treatment step. This is a step of selecting test compounds. For example, when the calculated cytotoxic activity is 1.5 times or more of the cytotoxic activity of untreated NK cells, the test compound used in the test compound treatment step may be selected.

  The test compound selected in the selection step is a compound having NK cell activation ability. Therefore, according to the present invention, a useful compound that can be an active ingredient of an NK cell activator can be screened. By using this useful compound, cancer cells and infected cells can be selectively inhibited or suppressed.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail.

[Example 1: Screening]
First, compounds that promote the cytotoxic activity of natural killer (NK) cells were screened. As test components, 71 types of compounds shown in Table 1 below were used. The source of the test component is shown in parentheses in Table 1.

(Table 1 continued)

(material)
KHYG-1 was used as the NK cell. In addition, a promyelocytic leukemia cell line K562 was used as a cancer cell. KHYG-1 and K562 were purchased from Human Science Research Resource Bank. RPMI-1640 medium, penicillin / streptomycin solution, human IL-2, and RNA extraction reagent (ISOGEN) were purchased from Wako Pure Chemical Industries, Ltd.

  Cytotoxicity Detection Kit (LDH) and a master mix for real-time PCR (FastStart Universal SYBR Green Master (Rox)) were purchased from Roche. Advanced RPMI 1640 medium was purchased from Life Technologies. Fetal bovine serum (FBS) was purchased from Biological Industries. A cDNA synthesis reagent (PrimeScript (trade name) RT reagent Kit) was purchased from Takara Bio Inc.

(Cell culture)
KHYG-1 cells are maintained in RPMI-1640 medium containing 10% FBS, 50 ng / ml human IL-2, 100 units / ml penicillin G, and 100 μg / ml streptomycin at 37 ° C., 5% CO _2. Passed every other day.

K562 cells were maintained in RPMI-1640 medium containing 10% FBS, 100 units / ml penicillin G, and 100 μg / ml streptomycin at 37 ° C., 5% CO ₂ and passaged every 3 days.

  KHYG-1 cells and K562 cells were cultured using a 60 mm culture dish.

(Cytotoxic activity test)
Next, KHYG-1 cells were treated with each test compound, and cytotoxic activity was evaluated. In this example, the activity of LDH released from damaged cells was used as an index of cell damage.

  Each test compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution. This stock solution was added to the medium to a final concentration of 30 μM at the time of passage of KHYG-1 cells. The concentration of DMSO contained in the medium after addition was set to 0.1% or less.

KHYG-1 cells cultured for 3 days in a medium containing each test compound were collected by centrifugation, and the test compound contained in the medium was removed. The collected cells were suspended in Advanced RPMI-1640 medium containing 1% FBS to 1 × 10 ⁶ cells / ml to prepare a KHYG-1 cell solution.

On the other hand, K562 cells were collected by the same method as KHYG-1, and suspended in Advanced RPMI-1640 medium containing 1% FBS to 5 × 10 ⁴ cells / ml to prepare a K562 cell solution.

  In this experiment, lactate dehydrogenase (LDH), which is an index of cytotoxicity, is also contained in the serum, and it is desirable to carry out under conditions where the serum concentration is suppressed in order to suppress the background. In this example, by using Advanced RPMI-1640 medium, low serum conditions could be achieved and stable results could be obtained.

  A solution (ETCmix: effect-target cell mix) containing KHYG-1 cells and K562 cells was placed in a well of a 96-well plate (U bottom). The ratio of KHYG-1 cells to K562 cells in ETCmix was 20: 1.

  Moreover, each control solution was put into the other well of this 96-well plate. As a control solution, a medium alone (BC: background control), a surfactant added to the K562 cell solution (HC: high control), a K562 cell solution only (LC: low control), and a KHYG-1 cell solution only (ECC: The effector cell control was used.

  The control (HC) obtained by adding a surfactant to the K562 cell solution is for measuring the activity when almost all K562 cells are damaged.

Specifically, the prepared KHYG-1 cell solution was dispensed in 100 μl (1 × 10 ⁵ cells) into ETCmix and ECC wells. In addition, 100 μl (5 × 10 ³ cells) of the K562 cell solution was dispensed into ETCmix, HC, and LC wells. A medium containing 2% of Triton X-100 was added to a well for HC (100 μl (final concentration of Triton X-100 of 1%)). Further, 100 μl of the medium alone was added to the LC well and 200 μl was added to the BC well.

Experiments on ETCmix and each control were performed in triplicate. The plate was allowed to stand in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 4 hours, centrifuged, and then 80 μl of the supernatant was transferred to a 96-well assay plate (flat bottom). Add 80 μl of reaction solution (including diaphorase, NAD ⁺ , iodotetrazolium chloride, and sodium lactate components) prepared according to the instructions attached to Cytotoxicity Detection Kit (LDH) to each well from 15 minutes in the dark. Let stand for 30 minutes. After confirming the color development, the absorbance was measured at 490 nm using a visible light plate reader. The reference wavelength was 600 nm.

  And the value which deducted background (BC) from the light absorbency of each well was calculated | required. Based on this value, cytotoxic activity (%) = ((ETCmix−ECC) −LC) / (HC−LC) × 100 was determined. This cytotoxic activity (%) is a percentage when the activity when almost all K562 cells are damaged is defined as 100.

  In this screening, about 10 kinds of test compounds were tested per 96-well plate. In each test (each plate), the cytotoxic activity of untreated KHYG-1 cells was simultaneously evaluated.

  FIG. 1 is a bar graph showing the results of screening for compounds that promote the cytotoxic activity of NK cells. In FIG. 1, the vertical axis represents the relative value of the cytotoxic activity for each test compound when the cytotoxic activity of untreated KHYG-1 cells is 1. The white bar indicates the result of the test compound belonging to polymethoxyflavone.

  First, the test compounds Nos. 1 to 60 were tested, and the test compounds having a relative value of the cytotoxic activity of KHYG-1 cells of 1.5 or more were citral (16), sinensetin (27), tangeretin (35), and Four were nobiletin (52). Among these, reproducibility was observed for three of synencetin, tangeretin and nobiletin, which promote the cytotoxic activity of KHYG-1 cells. On the other hand, with respect to citral, reproducibility for promoting cytotoxic activity was not observed.

  Three compounds, sinensetin, tangeretin and nobiletin, are compounds belonging to polymethoxyflavone. Therefore, next, other polymethoxyflavones (61-71) were similarly tested. As a result, it was shown that many polymethoxyflavones have the activity of promoting the cytotoxic activity of KHYG-1 cells. Polymethoxyflavones that showed particularly high activity are gardenine (63), 3,7,3 ′, 4 ′, 5′-pentamethoxyflavone (64), 5,7,3 ′, 4 ′, 5′-penta. Methoxyflavone (65), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (66), and 3,7,8,2 ′, 4′-pentamethoxyflavone (69).

  From the above results, it was shown that many polymethoxyflavones have the activity of promoting the cytotoxic activity of NK cells, that is, the ability to activate NK cells.

[Example 2: Examination of concentration dependence on NK cell activation by polymethoxyflavone]
Examples 2 to 5 were carried out for several compounds among polymethoxyflavones that were found to have the activity of promoting the cytotoxic activity of NK cells in the screening in Example 1.

  In Example 2, the concentration dependence on NK cell activation was examined for these polymethoxyflavones. In Example 1, the final concentration of the test compound was 30 μM, but it was examined whether NK cells could be activated even at a lower concentration.

  In this example, nobiletin (52), tangeretin (35), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (66), and 3,7,8,2 ′, 4′- Pentamethoxyflavone (69) was studied. 2 (A)-(D) show nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8, It is a figure which shows the structural formula of 2 ', 4'-pentamethoxyflavone (D).

  The final concentration of these compounds was 30 μM or 10 μM, and cell culture and preparation, treatment of NK cells, and cytotoxic activity test were performed in the same manner and conditions as in Example 1.

  The results are shown in FIGS. 3 (A) to (D) show nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5, 6, 7-hexamethoxyflavone (C), and 3, 7, 8, It is a graph which shows the cytotoxic activity of the KHYG-1 cell processed by 2 ', 4'-pentamethoxyflavone (D). The vertical axis | shaft of FIG. 3 (A)-(D) shows a percentage when the activity when almost all K562 cells are damaged is set to 100. FIG.

  As shown in FIGS. 3 (A) to (D), these compounds enhanced the cytotoxic activity of NK cells in a concentration-dependent manner.

[Example 3: Examination of cytotoxic activity against K562 cells by polymethoxyflavone itself]
In this example, it was decided to examine whether polymethoxyflavone itself has cytotoxic activity against K562 cells.

KHYG-1 cells cultured for 3 days under conditions not containing polymethoxyflavone were collected by centrifugation. The collected cells were suspended in Advanced RPMI-1640 medium containing 1% FBS to 1 × 10 ⁶ cells / ml to prepare a KHYG-1 cell solution. Further, K562 cells cultured for 3 days were similarly collected by centrifugation, and suspended in Advanced RPMI-1640 medium containing 1% FBS to 5 × 10 ⁴ cells / ml to prepare a K562 cell solution. Also, an Advanced RPMI-1640 medium containing 30 μM polymethoxyflavone was prepared.

The cytotoxic activity test was carried out under the same conditions as in Example 1 with ETCmix and the same controls as in Example 1. Furthermore, a well for adding polymethoxyflavone directly to K562 cells was provided. To this well, medium containing 100 μl of K562 cell solution (5 × 10 ³ cells) and 100 μl of polymethoxyflavone (final concentration 15 μM) was added.

The 96-well plate was allowed to stand in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 4 hours, centrifuged, and 80 μl of the supernatant was transferred to a 96-well assay plate (flat bottom). To each well, 80 μl of the prepared reaction solution was added, and allowed to react for 15 to 30 minutes in the dark. After confirming the color development, the absorbance was measured at 490 nm using a visible light plate reader. The reference wavelength was 600 nm.

  And the value which deducted background (BC) from the light absorbency of each well was calculated | required. Based on this value, cytotoxic activity (%) = ((ETCmix−ECC) −LC) / (HC−LC) × 100 was determined. For evaluation of wells in which polymethoxyflavone was directly added to K562 cells, cytotoxic activity (%) = (absorbance of target well-LC) / (HC-LC) × 100 was determined.

  FIG. 4 shows nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′, 4′-penta. It is a graph which shows the cytotoxic activity of methoxyflavone (D) itself. The vertical axis of FIG. 4 shows the percentage when the activity when almost all the K562 cells are damaged is 100. The control (con) in FIG. 4 shows the LC results. Although not shown, the cytotoxic activity of ETCmix was about 40%.

  As shown in FIG. 4, under the conditions of this example (incubation time: 4 hours), nobiletin, tangeretin, 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone, and 3,7 , 8,2 ′, 4′-pentamethoxyflavone itself did not show cytotoxic activity. Therefore, it was suggested that polymethoxyflavone itself has no cytotoxic activity.

[Example 4: Examination of cytotoxic activity against K562 cells by conditioned medium of KHYG-1 cells]
In the cytotoxic activity test in Examples 1 and 2, the KHYG-1 cells after the test compound treatment were used after washing by medium exchange. However, it is also possible that the culture supernatant (ie, conditioned medium (CM)) of KHYG-1 cells remained. Therefore, it was decided whether or not any humoral factor contained therein was toxic to K562 cells by treating K562 cells with CM of KHYG-1 cells.

  The culture supernatant of KHYG-1 cells cultured for 3 days under conditions containing 30 μM polymethoxyflavone or not containing polymethoxyflavone was collected by centrifugation. The collected culture supernatant contains humoral factors released from KHYG-1 cells. The culture supernatant containing polymethoxyflavone also contains polymethoxyflavone.

As a control for cytotoxic activity, KHYG-1 cells cultured for 3 days under conditions not containing polymethoxyflavone were collected by centrifugation. The collected cells were suspended in Advanced RPMI-1640 medium containing 1% FBS to 1 × 10 ⁶ cells / ml to prepare a KHYG-1 cell solution.

On the other hand, K562 cells cultured for 3 days were collected by centrifugation in the same manner as KHYG-1 cells, suspended in Advanced RPMI-1640 medium containing 1% FBS to 5 × 10 ⁴ cells / ml, and KHYG-1 Cell fluid was prepared.

The cytotoxic activity test was performed under the condition where ETCmix and the same control as in Example 1 were arranged. Furthermore, a well for adding CM of KHYG-1 cells to K562 cells was provided. To this well, 100 μl of K562 cell solution (5 × 10 ³ cells) and 100 μl of CM were added.

The 96-well plate was allowed to stand for 4 hours in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for reaction. After centrifugation, 80 μl of the supernatant was transferred to a 96-well assay plate (flat bottom). 80 μl of the prepared reaction solution was added to each well and allowed to react for 15 to 30 minutes in the dark. After confirming the color development, the absorbance was measured at 490 nm using a visible light plate reader. The reference wavelength was 600 nm.

  And the value which deducted background (BC) from the light absorbency of each well was calculated | required. Based on this value, cytotoxic activity (%) = ((ETCmix−ECC) −LC) / (HC−LC) × 100 was determined. For evaluation of wells in which CM of KHYG-1 cells was added to K562 cells, cytotoxic activity (%) = (absorbance of target well-LC) / (HC-LC) × 100 was determined.

  FIG. 5 shows nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8,2 ′, 4′-penta. It is a graph which shows the cytotoxic activity of the culture supernatant of the KHYG-1 cell which processed methoxyflavone (D). The vertical axis in FIG. 5 shows the percentage when the activity when almost all the K562 cells are damaged is 100. The control (con) in FIG. 5 shows the cytotoxic activity of the culture supernatant under conditions that do not contain polymethoxyflavone. Although not shown, the cytotoxic activity of ETCmix was about 40%.

  As shown in FIG. 5, the culture supernatant of KHYG-1 cells did not show cytotoxic activity. From the results of Example 3, it is clear that polymethoxyflavone itself has no cytotoxic activity. In addition to this, it was shown in this example that the humoral factor released from the KHYG-1 cells contained in the culture supernatant also has no cytotoxic activity. Therefore, it was shown that activated KHYG-1 is necessary for exerting cytotoxic activity by polymethoxyflavone.

[Example 5: Expression analysis of genes related to cytotoxic activity in KHYG-1 cells]
KHYG-1 cells were cultured in a medium containing 30 μM polymethoxyflavone for 2 days. A 24-well plate was used for culture, and 4 wells were used for each compound. An RNA extraction reagent was added to the cells collected by centrifugation, and total RNA was extracted according to the instruction manual of the reagent. Using cDNA synthesized based on this as a template, real-time PCR was performed to analyze the expression of cytotoxic activity-related genes.

  IFN-γ gene, perforin gene, and granzyme B gene were analyzed as genes related to cytotoxic activity. The GAPDH gene was used as an internal standard. The primer sequences used for PCR of each gene are as follows.

GAPDH forward: SEQ ID NO: 1
GAPDH reverse: SEQ ID NO: 2
IFN-γ forward: SEQ ID NO: 3
IFN-γ reverse: SEQ ID NO: 4
perforin forward: SEQ ID NO: 5
perform reverse: SEQ ID NO: 6
granzyme B forward: SEQ ID NO: 7
granzyme B reverse: SEQ ID NO: 8.

  The expression level of each gene was expressed as a relative value with respect to the GAPDH gene used as an internal standard, and an average value and a standard deviation of the relative values in four independent wells of each test compound were calculated.

  6 (a)-(c) shows nobiletin (A), tangeretin (B), 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone (C), and 3,7,8, It is a graph which shows the expression level of IFN-γ gene (a), perforin gene (b) and granzyme B gene (c) in KHYG-1 cells treated with 2 ′, 4′-pentamethoxyflavone (D). Each expression level shown in FIGS. 6A to 6C is expressed as a relative value when the expression level in KHYG-1 cells not treated with polymethoxyflavone is 1. Moreover, control (con) in FIG. 6 (a)-(c) shows the expression level in the KHYG-1 cell which is not processing polymethoxyflavone.

  From the above results, nobiletin, tangeretin, 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone and 3,7,8,2 ′, 4′-pentamethoxyflavone are different from each other. However, it was found that the expression of the IFN-γ gene, perforin gene and granzyme B gene was increased in KHYG-1 cells. In particular, 3 ', 4', 5 ', 5,6,7-hexamethoxyflavone significantly increased the expression of each gene.

  The IFN-γ gene, perforin gene and granzyme B gene are genes encoding effector factors characteristic of NK cells that induce cytotoxicity. From this example, nobiletin, tangeretin, 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone and 3,7,8,2 ′, 4′-pentamethoxyflavone activated NK cells. It was further proved that it was possible.

  In addition, nobiletin, tangeretin, 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone and 3,7,8,2 ′, 4′-pentamethoxyflavone are NF cell IFN-γ genes. May increase Th2 cells. That is, these compounds may also have an effect of alleviating allergic symptoms.

  In addition, since these compounds, particularly 3 ′, 4 ′, 5 ′, 5, 6, 7-hexamethoxyflavone, remarkably enhance the expression of the IFN-γ gene, It can be used as a lead compound.

  The present invention can be suitably used for immune cell therapy (particularly activated NK cell therapy), pharmaceuticals for enhancing immune function, foods, beverages, supplements, reagents, and the like.

Claims (4)

A compound selected from the group consisting of 3 ', 4', 5 ', 5,6,7-hexamethoxyflavone and 3,7,8,2', 4'-pentamethoxyflavone as an active ingredient A natural killer cell activator characterized by activating natural killer cells outside the body . 2. The natural killer cell activator according to claim 1, comprising 3 ′, 4 ′, 5 ′, 5,6,7-hexamethoxyflavone as the compound. A natural killer cell activation method comprising an activation step of contacting a natural killer cell with the natural killer cell activator according to claim 1 or 2 outside the body . The manufacturing method of the activated natural killer cell characterized by including the activation process which makes a natural killer cell contact the natural killer cell activator of Claim 1 or 2 in vitro .

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