KR100519660B1 - Pharmaceutical composition comprising genistein for treating nk/t cell lymphoma - Google Patents

Abstract

The present invention relates to a composition containing genistein as an active ingredient for the treatment of NK / T-cell lymphoma, wherein the composition according to the invention is an NK / T-cell lymphoma cell line. Treating NK / T-Cell Lymphomas by Inducing Hank-1 Induced Mitochondrial Dysfunction and Loss of Bcl-xL Proteins by Inhibiting the Growth of NK / T-Cell Lymphomas in a Caspase 3 Independent Method It can be usefully used for the manufacture of pharmaceuticals, processed foods, functional foods, beverages and alcohol with this purpose.

Description

PHARMACEUTICAL COMPOSITION COMPRISING GENISTEIN FOR TREATING NK / T CELL LYMPHOMA}

The present invention induces mitochondrial dysfunction and loss of Bcl-xL protein in Hank-1, a NK / T-cell lymphoma cell line, thereby inhibiting the growth of NK / T-cell lymphoma in a Kespase-3 independent manner. A genistein containing composition that can be usefully used to treat T-cell lymphoma.

Angiocentric T-cell lymphomas occur mainly in the nasal region and are common in Asians (Jaffe et al., Am. J. Surg. Pathol . 20: 103-111, 1996). These lymphomas are also called NK / T-cell lymphoma because tumor cells often share markers of NK-cell and T-cell in immunostaining. In almost all cases, Epstein-Barr virus (EBV) has been identified, suggesting that EBV is one of the most important causes. In addition to lymph nodes, it invades skin, soft tissues, testes, upper respiratory tract, and gastrointestinal tract. There is a characteristic. In the early stage, the prognosis is good because it responds well to radiation therapy, but the prognosis is known to be very poor because it does not respond to chemotherapy in the case of systemic disease (Liang et al., J. Clin. Oncol . 13: 666-670 , 1995; Cheung et al., J. Clin. Oncol. 16: 70-77, 1998).

The research on the molecular biological characteristics of these NK / T-cell lymphomas has not progressed much since then, because the concept of the disease has only recently been established and there are not many established cell lines for this tumor. Hank-1 cell line among these cell lines is interleukin-2 (IL-2) dependent nasal NK / T-cell lymphoma established in 1998 by Dr. Kagami et al. (Kagami et al., Br. J. Haematol 103: 669-677, 1998). Immune marker tests showed that Hank-1 is positive for CD56, CD2, CD3ε, TIA-1, granzyme B, and HLA-DR, and negative for other T-cell markers. Genetically, the T cell receptors β and γ are not rearranged and EBV is observed monoclonal and exhibits a latent type of type 2 antigen for EBV, identical to native NK / T-cell lymphoma. Therefore, Hank-1 is a useful subject for studying the properties of NK / T-cell lymphoma.

Plant-derived genistein is the major isoflavone component of soybeans and has been widely used in estrogen replacement therapy.Many epidemiological and clinical findings suggest that high levels of breast cancer, prostate cancer, And inhibiting the development and growth of many other tumors (Paterson et al., Biochem. Biophys. Res. Commun. 179: 661-667, 1991; Barnes et al., J. Cell Biochem . 221: 81 -87, 1995; Shao et al., Anticancer Res. 18: 435-439, 1998). At the cellular level, genistein inhibits tyrosine kinase, inhibits the function of DNA topoisomerase II (Constantinou et al., Cancer Res . 50: 2618-1624, 1990; Morris et al., Mutat. Res . 405: 41-56, 1998), inhibiting cell cycle progression, inhibiting angiogenesis (Matsukawa et al., Cancer Res . 53: 1328-1331, 1993; Fotsis et al., Cancer Res . 57: 2916-292 , 11997; Fotsis et al., Baillieres Clin. Endocrinol. Metab . 12: 649-666, 1998). Genistein's ability to inhibit tumor growth has been shown in particular in the G2 / M arrest, induction of p21 and induction of apoptosis in the cell cycle (Bergamaschi et al., Leukemia 7: 2012-2018, 1993; Edwards et al., Int. J. Oncol . 3: 583-588, 1999). However, the molecular biological mechanism by which genistein induces apoptosis is only partially known in some cells. Apoptosis is a very important mechanism for maintaining cell homeostasis. The control of apoptosis is known as an important process before tumor development and is also used as a therapeutic target.

Apoptosis is morphologically characterized by cell contraction, chromatin enrichment, and oligonucleosomal DNA segments (Thompson, Science 267: 1456-1462, 1995; Cohen, Immunol. Today 14: 126- 130, 1993). A key step in this apoptosis process is the activation of kephases, a member of the cysteine protein enzyme (Salvesen et al., Cell 91: 443-446, 1997; Raff, Nature 396: 119-122, 1998; Green, Cell 94: 695-698, 1998). Two distinct mechanisms are known for the activation of these cascades and the induction of apoptosis (Hsu et al., Cell 81: 495-504, 1995; Hsu et al., Cell 84: 299-308, 1996; Feinstein et al., Trends Biochem.Sci. 20: 342-344, 1995; Green et al., Science 281: 1309-1312, 1998; Yin et al., Nature 400: 886-891, 1999). One is the activation of caspase 8 by complementary binding to death receptors, the activation of this initiator caspase then activates effector caspases such as caspase 3. On the other hand, many types of cell damage stimuli cause changes in mitochondria, leading to depolarization of mitochondrial membranes and release of cytochrome C, and thus the release of cytochrome C activates caspase 9 to activate other effector cascades. In addition, the bcl-2 family gene plays a very important role in regulating apoptosis (Adams et al., Science 281: 1322-1326, 1998). This bcl-2 family gene usually promotes apoptosis ( It consists of genes that act as pro-apoptotic and anti-apoptotic. The genes are structurally very similar to each other, but functionally complementary to play a role in regulating apoptosis. In addition, it has recently been reported that the bcl-2 family genes are involved in the mechanism of resistance to anticancer drugs (Minn et al., Blood 86: 1903-1910, 1995).

The present inventors searched for a natural food or plant-derived compound that may be useful for the treatment of NK / T-cell lymphoma that is resistant to many drugs, while Genistein is present in the NK / T-cell lymphoma cell line Hank-1. It can be usefully used to treat NK / T-cell lymphoma by inducing mitochondrial dysfunction and loss of Bcl-xL protein to inhibit the growth of NK / T-cell lymphoma in a caspase 3 independent manner. The present invention was completed by confirming the presence of the present invention.

It is an object of the present invention to provide a novel use of genistein for the treatment of NK / T-cell lymphomas exhibiting multiple drug resistance.

In accordance with this object, the present invention provides pharmaceutical compositions and foodstuffs that inhibit the growth of NK / T-cell lymphoma comprising genistein as an active ingredient together with a pharmaceutically acceptable carrier.

Hereinafter, the present invention will be described in detail.

Genistein of the present invention induces mitochondrial dysfunction and loss of Bcl-xL protein in Hank-1, a NK / T-cell lymphoma cell line, which inhibits the growth of NK / T-cell lymphoma in a caspase-3 independent manner. It can be usefully used.

Genistein induces apoptosis in Hank-1 and Jurkat more than the control group, respectively, and showed better apoptosis than other conventional anticancer drugs (see FIG. 1 ). In particular, NK or T lymphoma cell lines induce more apoptosis than B cell lines. (See FIG. 2 ). In addition, induction of apoptosis by genistein was observed to increase with increasing concentration of genistein and with increasing time (see FIGS. 3A and 3B ).

Western blot analysis was performed to determine whether the treatment of genistein caused changes in the expression of the bcl-2 family protein known to promote or inhibit apoptosis. As a result, the expression of bcl-xL was treated by the genistein treatment of Hank-1 and Jurkat. It was reduced in all, from which it can be seen that the reduction of bcl-xL expression that plays a role in inhibiting apoptosis plays an important role in the induction of apoptosis by genistein (see Figure 4 ).

In addition, since both Hank-1 and Jurkat are CD95 / Fas-expressing cell lines, we investigated whether the induction of apoptosis by genistein is related to the interaction of CD95-CD95 ligand. In Jurkat, the expression of CD95 ligand increased with increasing expression of CD95, as is known, while the expression did not increase (see FIG. 5 ). In addition, apoptosis was induced by directly stimulating CD95 by treating anti-Fas antibodies to Hank-1 and Jurkat cells. As a result, apoptosis was induced in Jurkat, but apoptosis was not induced in Hank-1 (see FIG. 6 ). Induction of apoptosis by genistein was confirmed to be independent of CD95-CD95 ligand interaction in Hank-1, unlike Jurkat.

To investigate whether the induction of apoptosis by genistein is accompanied by a change in mitochondria, the changes in mitochondrial membrane potential and the release of mitochondrial cytochrome C were investigated . Accompanied by (see FIG. 7B ) it can be seen that apoptosis caused by genistein occurs through the change of the mitochondria.

In order to identify the cases of apoptosis induced by genistein, a treatment of genistein followed by extraction of proteins over time followed by Western blot analysis of the cases 3, 8, and 9 resulted in Hank-1. No change of caspase 8 was observed, indicating that the apoptosis induction mechanism by the death receptor did not work in the case of Hank-1 with no increase in expression of CD95 ligand (see FIG. 8 ). In addition, few changes in caspase 9 were observed in Hank-1, and pro-kephase 3 decreased with time and the truncated form was smaller than that of Jurkat. This could be confirmed by the fact that PARP, which is known as one of the substrates of kease phase 3, decreased in pro-form with time after treatment with genistein.

As a result of the direct measurement of the activity of the case phase 3, Hank-1 showed a lower increase in the activity of the case phase 3 by genistein treatment compared to Jurkat (see FIG. 9A ). In addition, when the z-DEVD, an inhibitor specific to the case 3, was treated, the activity of the case 3 was suppressed, but apoptosis by genistein was not inhibited (see FIGS. 9A and 9B ). However, treatment with z-VAD, a pan-casease inhibitor, moderately inhibited apoptosis. As a result, it was found that apoptosis by genistein treatment in Hank-1 was related to the case phase, but the case 3 was weakly known as an effective effector case phase.

Genistein of the present invention can be prepared into pharmaceutical compositions for the treatment of NK / T-cell lymphoma by mixing with a suitable pharmaceutically acceptable carrier or excipient or dilution with a diluent according to conventional methods.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulation may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. A typical daily dose of Genistein of the present invention is a range in which the Genistein concentration is maintained at 10 μM in the blood of the patient, preferably in the range of 10 mg / kg body weight to 1 g / kg body weight, administered once or in several doses. can do. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage should in any way be regarded as the present invention. It does not limit the scope of.

In addition, the present invention provides a functional food or beverage composition that can inhibit the growth of NK / T-cell lymphoma by inducing apoptosis in NK / T-cell lymphoma resistant to many drugs, including an effective amount of Genistein do. Examples of foods to which the Zenithine of the present invention can be added to exhibit the above effects include, for example, various foods, meats, beverages, chocolates, snacks, confectionery, pizzas, ramen noodles, other noodles, gums, ice creams, alcoholic beverages, Vitamin complexes and other health supplements, but are not limited thereto.

Zenithine of the present invention can be added to the raw material during the manufacture of food or mixed appropriately in the cooked food to prepare the above-mentioned health-promoting food or drink, in which case the content of the zenistein in the finally prepared food or drink after The range in which the genistein concentration is maintained at 10 μM in the blood of the patient is preferably in the range of 0.1 to 5 wt%.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Induction of Apoptosis by Genistein in NK / T-Cell Lymphoma Cell Line Hank-1

Hank-1, a human NK / T-cell lymphoma cell line used to confirm apoptosis induction, was provided by Dr. Kagami (Kagami et al., Br. J. Haematol. 103: 669-677, 1998), which was 5%. Incubated in Cos medium (COSMO BIO CO. Japan) containing additional human plasma and 100 U / ml recombinant human IL-2 (Chiron-France, France) and then humidified at 37 ° C. with 5% carbon dioxide. Cultured in the environment. Human T-lymphocytic leukemia cell lines, Jurkat (Korea Cell Line Bank) and Burkitt's lymphoma cell lines, Jiyoye (Korea Cell Line Bank) and Ramos (Korea Cell Line Bank), 10% fetal bovine serum (Gibco, USA) and 2 After inoculation into RPMI 1640 (Life Technologies, USA) medium containing additional mM L-glutamine and an anticancer agent, the cells were cultured in a humid environment at 37 ° C. containing 5% carbon dioxide. NK leukemia cell line NK-L (Korea Cell Line Bank) was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 200 U / ml of recombinant human IL-2.

First, Hank-1 and Jurkat cell lines were treated with various known anticancer agents and genistein, and then the apoptosis induction ability of genistein was examined by comparing the degree of apoptosis.

Cells cultured as above (1 × 10 ⁶ cells / ml) were treated with etoposide (etoposide, Sigma, USA), dexamethasone (Sigma, USA), and actinomycin D (actinomycin D, BioVison, USA) was treated with 10 μM and Genistein (Sigma, USA) was treated with 100 μM.

The degree of apoptosis was analyzed by flow cytometry through annexin V staining. About 2 × 10 ⁵ cells were washed twice with phosphated buffered saline (PBS) and the cells were resuspended in Annexin V binding buffer (BD Clontech). After adding 2.5 μg / ml of fluorescein isothiocyanate (FITC) -conjugated Annexin V (Pharmingen) and 5 μg / ml of propidium iodide (PI, Pharmingen) to the suspension for 15 minutes at room temperature Analysis was performed with a FACScan flow cytometer (Epics XL, Coulter, Marseille, France). At this time, cells that were Annexin V positive and PI negative were regarded as cells inducing apoptosis.

As shown in FIG. 1 , genistein induced apoptosis by 45.9% and 44.8%, respectively, in Hank-1 and Jurkat, compared to the control group, which showed superior ability to induce apoptosis than other conventional anticancer drugs.

On the other hand, in order to determine how the induction of apoptosis by genistein affects other NK and B cell lines other than Hank-1 and Jurkat cell lines, NK leukemia cell line NK-L and Burkitt lymphoma cell line Jiyoye and Ramos The same treatment with Genistein and confirmed the degree of apoptosis. As a result, genistein induced more apoptosis in NK or T lymphoma cell lines than in B cell lines ( FIG. 2 ).

In addition, Hank-1 and Jurkat cell lines were treated with various concentrations of genistein, and the degree of apoptosis with time was evaluated through the annexin V-PI staining as described above. As a result, it was observed that the induction of apoptosis by genistein increases with increasing concentration of genistein and with increasing time ( FIGS. 3A and 3B ).

Example 2 Expression Changes of bcl-2 Family Protein Following Genistein Treatment

Western blot analysis was performed to determine if the treatment of genistein caused a change in the expression of the bcl-2 family protein known to promote or inhibit apoptosis.

Treating Hank-1 and Jurkat cell lines cultured as in Example 1 with 100 μM concentration of Genistein, 4, 8 and 24 hours later, the cells were taken out and washed with PBS, and dissolved in lysis buffer (20 mM Tris (pH 7.4), Resuspended in 100 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 5 mM MgCl ₂ , Protease Inhibitor Mixture (Invitrogen) The suspension was suspended at 4 ° C. for one hour. After the reaction, centrifugation was performed at 15,000 rpm for 10 minutes at 4 ° C., and the supernatant was taken to measure protein concentration.30 μg of protein was separated through SDS-PAGE, followed by nitrocellulose membrane (Strohl, Schleicher and Schuell, Dassel, Germany) Blocked with TTBS (0.5 M NaCl, 20 mM Tris-HCl pH 7.4, 0.2% Tween20) containing 5% skim milk powder at room temperature for 2 hours and reacted with each primary antibody overnight at 4 ° C. Each secondary antibody was reacted for one hour at room temperature. And then visualized with an ECL detection kit (Amersham, Buckinghamshire, England) The primary antibodies for the Western blot analysis were bax, bid, bcl-2, bcl-x (from Santa Cruz Biotechnology) and β-actin. Anti-chlorine, anti-rabbit antibody (Santa Cruz Biotechnology) and anti-mouse antibody (Amersham) were used as secondary antibodies.

As shown in FIG. 4 , the expression of bcl-xL was reduced in both Hank-1 and Jurkat by genistein treatment, from which the reduction of bcl-xL expression, which serves to inhibit apoptosis, induces apoptosis by genistein It plays an important role in However, no changes in the other bcl-2 family protein genes, bcl-2 and bax, were observed. As shown in Jurkat, bid was cut and expression was reduced, but in Hank-1, no specific bid was observed. .

Example 3 Interaction of Genistein with Apoptosis and CD95-CD95 Ligand

Since Hank-1 and Jurkat are both cell lines expressing CD95 / Fas, we investigated whether induction of apoptosis by genistein is associated with CD95-CD95 ligand interaction.

The cell surface expression of CD95 and CD95 ligand was analyzed as follows: FITC-conjugated CD95 antibody (Pharmingen) for CD95 on Hank-1 and Jurkat cells (approximately 2 × 10 ⁵ ) washed twice with PBS. ) Was reacted by shading on ice for 30 minutes, and then fixed with 4% paraformaldehyde (PFA). For CD95 ligand, CD95 ligand antibody (Pharmingen) was reacted for 1 hour on ice, followed by PE-conjugation 2 The reaction was carried out by shading 30 minutes on ice with a primary antibody (Pharmingen) and fixed with 4% PFA. Fixed cells were analyzed by FACScan flow cytometer within one week.

As a result, the expression of CD95 ligand was not increased by genistein treatment in Hank-1, whereas the expression of CD95 ligand was increased with the increase of CD95 expression as known in Jurkat ( FIG. 5 ) (Friesen et al., Nat. Med . 2: 574-577, 1996; Fulda et al., Blood 95: 301-308, 2000).

In addition, 1 × 10 ⁶ Hank-1 and Jurkat cells were treated with 50 ng / ml of anti-Fas antibody (Pharmingen), respectively, to stimulate CD95 directly to induce apoptosis. The degree of apoptosis was determined by Annexin V-PI staining. As a result, apoptosis was induced in Jurkat but apoptosis was not induced in Hank-1 ( FIG. 6 ). From the above results, it can be seen that induction of apoptosis by genistein is not related to CD95-CD95 ligand interaction in Hank-1, unlike Jurkat.

Example 4 Changes in Mitochondria Associated with Induction of Apoptosis by Genistein

In order to confirm the induction of apoptosis by genistein, the change of mitochondrial membrane potential and the release of mitochondrial cytochrome C were investigated.

The change in mitochondrial membrane potential (Δψm) was achieved after adding 40 nM of 3,3'-dihexylocarbocyanine iodide (3,3'-dihexylocarbocyanine iodide, DiOC6, Molecular Probes, Eugene, USA) to cells. The reaction was carried out at 15 ° C. for 15 minutes and immediately analyzed by flow cytometry.

In order to investigate the release of mitochondrial cytochrome C by genistein treatment by Western blot analysis, Hank-1 and Jurkat cells cultured as in Example 1 were washed twice with cold PBS, followed by extraction buffer (20 mM HEPES-KOH ( pH 7.0), 10 mM KCl, 1 mM NaEGTA, 2 mM MgCl ₂ , 1 mM EDTA, 1 mM DTT, 250 mM sucrose, 1 mM PMSF, protein inhibitor). After reaction on ice for 30 minutes, it was homogenized with a glass rod. The homogenate was centrifuged at 1,000 g at 4 ° C. for 10 minutes to remove nuclei. The supernatant was further centrifuged at 100,000 g for 20 minutes, and the supernatant (cytoplasmic fraction) and the precipitate (mitochondrial fraction) were taken and used for Western blot analysis. An antibody against cytochrome C (Pharmingen) was used as the primary antibody, and an anti mouse antibody (Amersham) was used as the secondary antibody.

As a result, it can be seen that the genistein treatment was accompanied by depolarization of the mitochondrial membrane potential ( FIG. 7A ) and release into the cytoplasm of cytochrome C ( FIG. 7B ), resulting in apoptosis caused by the change of mitochondria.

Example 5 Treatment of PARP and Changes of Kephas Following Induction of Apoptosis by Genistein

In order to identify the kephases that act when apoptosis is induced by genistein, Western blot analysis was performed on the kease phases 3, 8 and 9 by treating the genistein and extracting the protein over time.

Hank-1 and Jurkat cells cultured as in Example 1 were washed twice with cold PBS followed by extraction buffer (20 mM HEPES-KOH, pH 7.0), 10 mM KCl, 1 mM NaEGTA, 2 mM MgCl ₂ , 1 mM EDTA, 1 mM DTT, 250 mM sucrose, 1 mM PMSF, protein inhibitor). After reaction on ice for 30 minutes, it was homogenized with a glass rod. The homogenate was centrifuged at 1,000 g at 4 ° C. for 10 minutes to remove nuclei. The supernatant was further centrifuged at 100,000 g for 20 minutes, and the supernatant (cytoplasmic fraction) and the precipitate (mitochondrial fraction) were taken and used for Western blot analysis. The primary antibodies used in the Western blot analysis include antibodies against caspase 3, 8 and 9 (from Santa Cruz Biotechnology), antibodies against poly- (ADP-ribose) polymerase (PARP) (Upstate Biotechnology) and β Antibodies against actin (Sigma) were used, respectively, and anti-mouse antibodies (Amersham) were used as secondary antibodies.

As shown in FIG. 8 , no change of caspase 8 was observed in Hank-1, which was not observed in FIG. 5B , but increased expression of CD95 ligand was observed. In this case, it does not work.

In Jurkat, the expression of caspase 8 decreased at 24 hours, which is consistent with the increased expression of CD95 ligand in FIG. 5B . In addition, in Jurkat, the expression of caspase 9 was decreased and the caspase 3 was cut. It was found that the changes of mitochondria observed in FIGS. 7A and 7B cause not only the activity of caspase 9 but also the activity of caspase 3. Could. In Hank-1, however, little change in caspase 9 was observed, and pro-kezephase 3 decreased over time and the truncated form was less than Jurkat. This could be confirmed by the fact that PARP, which is known as one of the substrates of kease phase 3, decreased in pro-form with time after treatment with genistein. Of particular note, in Hank-1, a slightly smaller form of caspase 3 was observed along with a previously known form of 32 kD, which was also reduced by genistein treatment, and PARP was also cut from the beginning. Observed together.

In addition, the effects of pan-casease inhibitor Z-VAD and the caspase 3 inhibitor z-DEVD on ketase 3 activity and apoptosis were investigated in the Hank-1 and Jurkat cell lines treated with Genistein.

Hank-1 and Jurkat cells (Calbiochem Co.) Z-VAD (Calbiochem Co.) and z-DEVD (Calbiochem Co.) were cultured as in Example 1 for 1 hour in advance at a concentration of 100 μM, respectively, and then Genistein 100 Treated at μM concentration. Approximately 1 × 10 ⁶ cells were taken from the cells, washed with PBS, resuspended in 50 μl of lysis buffer (200 mM HEPES (pH 7.5), 0.1% CHAPS, 0.1 mM PMSF, protein inhibitor) and iced for 10 minutes. I left it on. Determination of enzyme activity was performed by adding 50 μl cytoplasmic protein and 20 μM specific fluorescent substrate to a buffer containing 50 mM HEPES (pH 7.0), 10% PEG, 0.1% CHAPS and 10 mM DTT. After reacting for a period of time, the result was analyzed by a fluorometer. For caspase 3, Z-Asp-Glu-Val-Asp-AMC (z-DEVD-AMC; BioVision) was used as substrate, which was excited at 400 nm to emit a fluorescent signal at 505 nm.

As a result of the direct measurement of the activity of the case phase 3, the increase in the activity of the case phase 3 was lower in the Hank-1 by Genistein treatment compared to Jurkat ( FIG. 9A ). In addition, when the z-DEVD, an inhibitor specific to the case 3, was treated, the activity of the case 3 was suppressed, but apoptosis by genistein was not inhibited ( FIGS. 9A and 9B ). However, treatment with z-VAD, a pan-casease inhibitor, moderately inhibited apoptosis. As a result, it was found that apoptosis by genistein treatment in Hank-1 was related to the case phase, but the case 3 was weakly known as an effective effector case phase.

Genistein of the present invention can be used in the form of preparations such as powders, tablets, capsules, injections, solutions and the like according to the methods commonly used pharmaceutically or in combination with excipients used alone or pharmaceutically.

Formulation examples are illustrated below.

Production Example 1 Powder

Genistein 2 g

1 g lactose

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Tablet

Genistein 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components and tableting according to the conventional tablet manufacturing method to produce a tablet.

Preparation Example 3 Capsule

Genistein 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components to fill a gelatin capsule in accordance with the conventional capsule preparation method to prepare a capsule.

Production Example 4 Injection

Genistein 100 mg

Suitable amount of distilled water for injection

pH adjuster

According to a conventional injection preparation method, the active ingredient is dissolved in distilled water for injection, the pH is adjusted to about 7.5, and the whole is filled with 2 ml of ampoule with injection distilled water and sterilized to prepare an injection.

In addition, the health food is prepared by the following method.

<Manufacture example 5> Wire type

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to make a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then ground to a powder having a particle size of 60 mesh.

Zenithine of the present invention was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain an extract dry powder.

The grains, seeds and pine needle extract dry powder prepared above were blended in the following ratio to make granules.

Cereals: Brown rice 30% by weight, barley 15% by weight, barley 20% by weight

Seeds: Perilla 7% by weight, Black beans 8% by weight, Black sesame 7% by weight,

Genistein 3% by weight, ganoderma lucidum 0.5%

In addition, health drinks and alcohol are prepared in the following manner.

Production Example 6 Health Drinks

Genistein 0.15 to 0.5%, sugar 5 to 10%, citric acid 0.05 to 0.3%, caramel 0.005 to 0.02%, vitamin C 0.1 to 1% of the additives are mixed with 79 to 94% purified water to make a syrup, the The syrup was sterilized at 85 to 98 ° C. for 20 to 180 seconds, mixed with cooling water at a ratio of 1: 4, and 0.5 to 0.82% of carbon dioxide was injected to prepare a carbonated beverage containing Zenistein.

Instant sterilization by homogeneously mixing subsidiary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and genistein (0.3%) Healthy drinks were prepared by packaging in small packaging containers such as glass bottles and plastic bottles.

<Manufacture example 7> Shochu

Genistein 0.1% ~ 0.6% mixed with alcohol concentration 20-25% shochu to make an emulsion and then separated for 15 minutes in a vacuum centrifuge at 7,000 rpm or a high-speed mixer at 9,000 rpm to prepare a liquor containing Zenithine It was.

<Manufacture example 8> Fruit wine

Genistein 0.15 ~ 0.6% is mixed with fruit alcohol of 4.5-15% alcohol concentration to make an emulsion state, and separated by vacuum centrifuge for about 15 minutes at 9,000 rpm to prepare a fruit wine containing genistein.

As described above, genistein according to the present invention induces dysfunction of mitochondria and loss of Bcl-xL protein, thereby inhibiting the growth of NK / T-cell lymphoma in a ketase-3-independent manner to prevent NK / T-cells. Lymphoma can be effectively treated.

1 is a result of comparing the induction of apoptosis of genistein in the Hank-1 and Jurkat cell line with various conventional anticancer agents,

2 is a result of observing the apoptosis inducing ability of genistein in various cell lines,

Figure 3a is a result of observing the apoptosis-inducing ability of genistein with concentration in Hank-1 and Jurkat cell line

Figure 3b is the result of observing the apoptosis induction ability of genistein over time in Hank-1 and Jurkat cell line,

FIG. 4 shows the results of Western blot analysis of expression changes of bcl-2 family proteins by genistein treatment in Hank-1 and Jurkat cell lines.

5a shows the results of observing the expression changes of CD95 by genistein treatment in Hank-1 and Jurkat cell lines,

Figure 5b is the result of observing the expression changes of CD95 ligand by Genistein treatment in Hank-1 and Jurkat cell lines,

6 is a result of observing the induction of apoptosis by anti-Fas antibody treatment in Hank-1 and Jurkat cell line,

Figure 7a is a result of observing the change in mitochondrial membrane potential by genistein treatment in Hank-1 and Jurkat cell lines,

FIG. 7B is a result of Western blot analysis of the release of mitochondrial cytochrome C by genistein treatment in Hank-1 and Jurkat cell lines,

FIG. 8 shows Western blot analysis of genistein-induced processing of caspase 3, 8, 9 and PARP over time in Hank-1 and Jurkat cell lines, FIG .

9A is a result of investigating the effect of z-VAD and z-DEVD on the caspase 3 activity in the Hank-1 and Jurkat cell lines treated with Genistein;

9b is a result of investigating the effect of z-VAD and z-DEVD on apoptosis in Hank-1 and Jurkat cell lines treated with Genistein.

Claims (4)

A pharmaceutical composition for treating NK / T-cell lymphoma by inducing apoptosis, comprising genistein as an active ingredient together with a pharmaceutically acceptable carrier. The method of claim 1, A pharmaceutical composition, characterized by inducing mitochondrial dysfunction and loss of Bcl-xL protein, thereby inhibiting growth of NK / T-cell lymphoma in a caspase-3 independent manner. The method of claim 1, Pharmaceutical composition, characterized in that the effective amount of Genistein is 10 to 1,000 mg / kg / day. delete