JPWO2005099736A1 - Antitumor treatment - Google Patents

Abstract

An object of the present invention is to provide an antitumor therapeutic agent that has a low apoptosis effect on normal cells and has an enhanced antitumor effect. By using a combination of a specific antitumor drug, particularly an antitumor drug having an apoptotic effect, and a component derived from corn. Antitumor drugs that have an apoptotic effect alone have side effects due to apoptosis of normal cells, whereas the combined use of components derived from corn reduces the apoptotic effect on normal cells and the apoptotic effect of antitumor drugs on tumor cells. Can be maintained or enhanced, and exhibits an excellent antitumor effect.

Description

  The present invention relates to an anti-tumor therapeutic agent having an excellent combined effect, more specifically, an anti-tumor therapeutic agent having few side effects when used in combination, and more effective anti-tumor effect when combined. It relates to an anti-tumor therapeutic agent.

  This application claims the priority from Japanese Patent Application No. 2004-114173, which is incorporated herein by reference.

  Antitumor drugs include alkylating agents (cyclophosphamide, ifosfamide, carbocon, etc.), antibiotics that affect nucleic acids (doxorubicin, bleomycin, etc.), platinum complexes (cisplatin, etc.), alkaloid antitumor drugs (vincristine, vinblastine) Etc.), antimetabolites (5-fluorouracil, etc.), topoisomerase inhibitors (etoposide, camptothecin, etc.), biological response modifiers (interferon, etc.), hormone therapy agents (tamoxifen, etc.) .

  In addition to the mechanism of action that causes damage to DNA and causes necrosis in cancer / tumor cells, antitumor drugs also have a mechanism that causes apoptosis. It induces apoptosis in cancer cells / tumor cells, and selectively kills cancer cells / tumor cells to bring about an antitumor effect. However, when a sufficient amount of an antitumor drug is used to kill cancer / tumor cells, apoptosis occurs in normal cells, and there is a problem of side effects due to cell damage.

  Early therapeutic drugs that were introduced with chemotherapy with antitumor drugs were mainly treated with a single drug, and the goal was to administer blood levels to maintain a constant level. However, single-agent administration of antitumor drugs increases the toxicity to normal cells with increasing dose and limits the dose, so combined therapy with two or more antitumor drugs has been attempted. .

  For example, chemotherapy combined with antitumor drugs for leukemia was tried in the late 1950s and later applied to solid cancer. Since then, chemotherapy with antitumor drugs has been centered on multi-drug combinations that are expected to have additive or synergistic effects. Against this background, attempts have been made to combine drugs with different mechanisms of action, ara-C and etoposide, in the treatment of leukemia (Non-patent Document 1).

  The above-mentioned various anti-tumor drugs are intended to kill cancerous cells, so if an anticancer agent sufficient to kill cancerous cells is administered, it will have a non-negligible adverse effect on normal cells in the body, causing strong side effects There was a problem.

In recent years, it has been reported that vitamins contained in vegetables and the like, for example, vitamins C and E or β-carotene have a carcinogenesis-inhibiting action, and are attracting attention as being highly safe.
In addition, drugs such as N-acetylcysteine, retinoid, piroxicam, and aspirin have been reported to have anticarcinogenic activity. Focusing on herbal medicine, which is a compound of various compounds with pharmacological action, and herbal medicine, which is a mixture of herbal medicines, specific herbal medicine, specifically Daihuang, Yamakiko, Huangren, or a kind of yellow rice It has been reported that a combination of these can be used as a carcinogenesis inhibitor (Patent Document 1). In addition, there is a report on an anti-carcinogenic promoter agent having a particularly remarkable anti-carcinogenic promoter action from flavonoids contained in yellow corn known as a crude drug (Patent Document 2).

Further, there is immunity as a biological reaction for the living body to identify and protect the non-self such as cancer and antibody-producing cells. For example, it is known to produce heat shock proteins, which are a group of proteins that activate immunity, in order to protect the cells themselves when invasion harmful to cell survival is added. This heat shock protein is involved in the cell cycle and cell differentiation even in normal cells, and plays an important role in maintaining physiological functions of cells and tissues.
It has been reported that extract of yellow rice and Indian snakewood as herbal medicines can greatly increase stress protein production in cells (Patent Document 3).

However, there is no report on an anti-tumor therapeutic agent using a crude drug-derived component and an anti-tumor drug in combination.
YAKUKAGAKU ZASSHI, 122 (7), 471-480 (2002) JP-A-8-337535 Japanese Laid-Open Patent Publication No. 5-25041 Japanese Patent Laid-Open No. 2001-39875

  An object of the present invention is to provide an anti-tumor therapeutic agent in which the apoptosis effect on normal cells is suppressed to a low level and the apoptosis effect on tumor cells is maintained or enhanced.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors, as a single agent administration of an antitumor drug having an apoptotic effect, give side effects due to apoptosis to normal cells when administered in an effective amount against the tumor. In contrast, the combined use of herbal medicines, especially components derived from yolk, can reduce the apoptotic effect on normal cells and can be an excellent anti-tumor therapeutic agent that maintains or enhances the apoptotic effect on tumor cells. The headline and the present invention were completed.

That is, this invention consists of the following.
1. An antitumor therapeutic agent comprising a combination of an ingredient derived from corn and an antitumor agent having an apoptotic action.
2. 2. The antitumor therapeutic agent according to item 1, wherein the antitumor agent having an apoptotic action is a topoisomerase inhibitor, an alkaloid antitumor agent, a platinum complex, an antibiotic, or an alkylating agent.
3. 3. The antitumor therapeutic agent according to 2 above, wherein the topoisomerase inhibitor is etoposide, camptothecin or a derivative thereof.
4). 3. The antitumor therapeutic agent according to item 2, wherein the alkaloid antitumor agent is vinblastine or a derivative thereof.
5. 3. The antitumor therapeutic agent according to 2 above, wherein the platinum complex is cisplatin or a derivative thereof.
6). 3. The antitumor therapeutic agent according to item 2 above, wherein the antibiotic is doxorubicin or a derivative thereof.
7). 3. The antitumor therapeutic agent according to 2 above, wherein the alkylating agent is carbocon or a derivative thereof.
8). 8. The antitumor therapeutic agent according to any one of 1 to 7 above, wherein the yellow rice-derived component is a component containing a flavonoid.
9. 9. The antitumor therapeutic agent according to 8 above, wherein the flavonoid-containing component is ougonin.
10. 10. The antitumor therapeutic agent according to any one of 1 to 9 above, which is used in combination with an amount of a component derived from corn that can prevent side effects caused by apoptosis on normal cells caused by an antitumor agent having an apoptotic action .
11. 11. The antitumor therapeutic agent according to item 10 above, wherein the ratio of the yellow corn-derived component and the antitumor agent having an apoptotic action is 10: 1 to 1:10 in terms of molar ratio.
12 12. A pharmaceutical composition comprising the antitumor therapeutic agent according to any one of 1 to 11 above.
13. 13. The pharmaceutical composition according to item 12, which is used for blood cancer or solid cancer.
14 13. The pharmaceutical composition according to the above item 12, which is used for leukemia or lung cancer.

  It is confirmed that the combination of a yellow corn-derived component and an antitumor agent having an apoptotic effect reduces the apoptotic effect on normal cells and maintains or enhances the apoptotic effect on tumor cells. Can be provided.

It is a figure which shows the DNA fragmentation rate when YO-2 or etoposide and ougonin are used together in normal cells. Example 1 It is a figure which shows caspase-3-like activity when using together etoposide and ougonin to a normal cell. (Example 2) It is a figure which shows the DNA fragmentation rate when using etoposide or a cisplatin, and ougonin together with a Jurkat cell. (Example 3) It is a figure which shows the DNA fragmentation rate when etoposide or cisplatin and ougonin are used together in HL-60 cells. Example 4 It is a photograph which shows the form of the nucleus when etoposide and ougonin are used together in Jurkat cells. (Example 5) It is a figure which shows the effect which acts on a cell viability when doxorubicin and ougonin are used together in A549 cells. (Example 6) It is a figure which shows the effect which acts on a cell survival rate when using together cisplatin and ougonin to A549 cell. (Example 7) It is a figure which shows the effect which acts on a cell viability when carbocon and ougonin are used together in A549 cells. (Example 8) It is a figure which shows the effect which acts on a cell survival rate when using vinblastine and ougonin together with A549 cell. Example 9 It is a figure which shows the effect which acts on a cell survival rate when using together camptothecin and ougonin to A549 cell. (Example 10) It is a figure which shows the effect which acts on a cell survival rate when using together etoposide and ougonin to A549 cell. (Example 11) It is a figure which shows the effect which acts on a cell survival rate when using etoposide and ougonin together with NCI-H226 cell. (Example 12)

  The antitumor therapeutic agent of the present invention is based on the combined use of a component derived from corn and an antitumor agent having an apoptotic action. In the present specification, an antitumor drug having an apoptotic action may be simply referred to as an “antitumor drug”, and is distinguished from an “antitumor drug” by the combination of the present invention.

  Ingredients derived from yellow rice and conventionally used as crude drugs include flavonoids, and the main components thereof include ougonin, baicalin, baicalein and the like. The components used in the anti-inflammatory therapeutic agent according to the combination of the present invention are also components derived from yellow rice, and include flavonoids. Specifically, ougonin, baicalin, baicalein and the like can be used, preferably ougonin is used. be able to. In addition, the components derived from the yellow rice of the present invention are not limited to components extracted from natural products, and include flavonoids and components having substantially the same effect as the components derived from yellow rice as crude drugs. As long as it is such a component, a component obtained by synthesis may be used.

A compound showing the above-mentioned component containing flavonoid is represented by a structure containing a basic skeleton represented by the following (Chemical Formula 1).

Specific examples of the compound containing flavonoids include compounds represented by the following (Chemical Formula 2) to (Chemical Formula 6). (Formula 2) for each compound to (Formula 6), wherein, R ₁ - R ₅ represents respectively the hydrogen atom, a hydroxyl group, a methoxy group or a methylenedioxy group. Among these compounds, preferred compounds are ougonin and its derivatives represented by (Chemical Formula 2), and particularly preferred is ougonin. Ougonin is a compound in which all of R ₁ to R ₅ are hydrogen atoms in (Chemical Formula 2).

  The antitumor drug having an apoptotic action of the present invention can be selected from antitumor drugs classified into topoisomerase inhibitors, alkaloid antitumor drugs, platinum complexes, antibiotics, alkylating agents, etc., preferably platinum Complexes and topoisomerase inhibitors can be selected.

Topoisomerase inhibitors have the mechanism of action by indirect DNA strand breakage through topoisomerase II inhibition or DNA synthesis inhibition by topoisomerase I inhibition. An example of a topoisomerase II inhibitor is etoposide, and an example of a topoisomerase I inhibitor is camptotecin.
Topoisomerase II is a dimeric enzyme that forms a covalent bond at the 5'-end of DNA, binds to DNA in the nucleus in an ATP-dependent manner, causes transient DNA strand breaks, and changes the structure of the DNA strand. Later, by recombining the cleaved DNA, the three-dimensional structure is changed.
Etoposide is thought to exert an antitumor effect by forming a complex consisting of DNA and topoisomerase II, and inhibiting re-binding of DNA strands that have been transiently cleaved.

  Alkaloid antitumor drugs bind to tubulin to prevent mitosis, act specifically in the M phase of the cell cycle, and are used for the treatment of Kaposi's sarcoma and malignant lymphoma. An example of an alkaloid is vinblastine.

Platinum complex is a non-cell cycle specific anticancer drug that binds to guanine and distorts the DNA structure, including testicular, bladder, and ovarian tumors, non-small cell lung cancer, esophageal cancer, cervical cancer, and neuroblastoma It is used to treat various solid malignant tumors such as gastric cancer. Specific examples of the platinum complex include cisplatin.
When cisplatin enters a Cl ⁻ -rich cell from a Cl ⁻ -rich extracellular environment, the Cl ⁻ group of cisplatin is replaced by a hydroxyl group of water, and the two hydroxyl groups are covalently bound to guanine of DNA in each cell. The DNA structure is distorted to form As a result, DNA replication and transcription are inhibited, and it is thought to exert an antitumor effect.

  Specific examples of antibiotics having an antitumor effect include doxorubicin. Doxorubicin is an antitumor antibiotic extracted from the anthracycline, Streptomyces products. Doxorubicin has a mechanism of action that binds to DNA in tumor cells, prevents DNA cleavage during cell division, and causes DNA replication and RNA synthesis inhibition. It also acts to suppress DNA-dependent RNA polymerase and deoxyribonuclease.

  The alkylating agent inhibits tumor cell DNA and RNA biosynthesis by alkylating DNA and RNA. In particular, it inhibits DNA biosynthesis. A specific example of the alkylating agent is carboquone.

  In the antitumor therapeutic agent of the present invention, the amount of the component derived from corn and the antitumor agent having an apoptotic action used in combination can be determined based on the apoptotic action on tumor cells and normal cells.

For example, when used alone, an antitumor agent having an apoptotic effect can be used even in an amount that exhibits an apoptotic effect on tumor cells and normal cells or an amount that does not exhibit an apoptotic effect.
When using an antitumor agent alone and using an amount that exhibits an apoptotic effect on tumor cells and normal cells, the component derived from yellow corn maintains or enhances the apoptotic effect of the antitumor agent on tumor cells. And an amount that can reduce the apoptotic effect of the antitumor drug on normal cells can be used.
In addition, when using an antitumor agent alone in an amount that does not show an apoptotic effect on tumor cells and normal cells, the combined yellow-derived component enhances the apoptotic effect of the antitumor agent on tumor cells. An amount of the antitumor drug that does not show an apoptotic effect on normal cells can be used.

  In this way, the combined use amount of an ingredient derived from corn and an antitumor agent that can exert the apoptotic effect on tumor cells to the maximum and reduce the side effects due to the apoptotic effect on normal cells as much as possible is an antitumor agent. Or it can select suitably by the kind of tumor cell made into the treatment objective. For example, the ratio of the component derived from yellow rice: antitumor drug can be selected from the range of 10: 1 to 1:10, preferably from the range of 2: 1 to 1: 2, in terms of molar ratio. can do.

  Specifically, the antitumor drug can be used so as to be 0.01 to 100 μM, preferably 0.1 to 50 μM, more preferably 1 to 30 μM under cultured cell conditions. On the other hand, the component derived from yellow rice is an amount that satisfies the above conditions, and can be selected from the range of 100 to 0.1 μM, preferably 50 to 0.1 μM, more preferably 30 to 1 μM.

The apoptotic effect can be confirmed biochemically and morphologically. For example, apoptosis can be confirmed by measuring DNA fragmentation. Measurement of DNA fragmentation can be performed according to known methods (McConkey, DJ, et al., Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca ²⁺ concentration. Arch. Biochem. Biophys., 269: 365-370, 1989).

  In addition, when cells are destroyed by apoptosis, caspase-3-like activity is observed, so that apoptosis can be confirmed by measuring the activity. Caspase-3-like activity can be measured by known methods (Takahashi, A., et al., Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8. Oncogene, 14: 2741-2752, 1997).

  Morphological effects on cells due to apoptosis can be observed according to known methods (Lee, E. et al., A novel DNA cleaving agent, 2,2'-bis (2-aminoethyl) -4, 4'-bithiazole, induces thymocyte apoptosis. Biochem. Mol. Biol. Int., 40: 151-157, 1996).

  The antitumor therapeutic agent of the present invention effectively exhibits an antitumor effect on malignant tumors by an apoptotic effect. The type of tumor is not particularly limited and can be used for blood cancer and solid cancer. For example, blood cancer can be used for leukemia, and solid cancer can be used for lung cancer.

  The present invention relates to an antitumor therapeutic agent comprising a combination of a component derived from corn and an antitumor agent having an apoptotic action. Further, the present invention extends to a pharmaceutical composition containing an anti-tumor therapeutic agent and exerting an effect on the malignant tumor. The pharmaceutical composition can include a pharmacologically acceptable salt. Examples of the “pharmacologically acceptable salt” include conventional non-toxic salts, that is, acid addition salts and salts with various bases. More specifically, inorganic acid salts such as hydrochloric acid, nitric acid and sulfuric acid, organic acid salts such as acetic acid, citric acid, fumaric acid and tartaric acid, sulfonic acid salts such as methanesulfonic acid and p-toluenesulfonic acid, and alanine and leucine Amino acid salts such as glutamic acid, and inorganic base salts such as alkali metal salts (for example, sodium salts, potassium salts), alkaline earth metal salts (for example, magnesium salts, calcium salts), and triethylamine salts, pyridine salts, picoline salts, ethanol Organic amine salts such as amine salts, triethanolamine salts, dicyclohexylamine salts, N, N′-dibenzylethylenediamine salts and the like can be mentioned.

  The pharmaceutical composition containing the antitumor therapeutic agent of the present invention can be administered by selecting an optimal administration method such as oral administration, intravenous injection administration, intramuscular injection administration, etc. according to the properties of various antitumor agents. it can.

  Examples will be described below to deepen the understanding of the present invention. However, these are only specific examples, and the present invention is not limited thereto.

(Example 1) DNA fragmentation when various chemotherapeutic agents and ougonin are used in combination with normal cells Thymocytes are collected from the thymus of young male rats (4-5 weeks old) and contain 10% fetal bovine serum The cell density was 10 × 10 ⁶ cells / ml in RPMI 1640 medium. Cells were subjected to experiments immediately after collection.
As an anti-tumor agent, YO-2 (Lee, E., et al., Biochem. Pharmacol., 63: 1315-23, 2002; Okada, Y., et al., Bioorg. Med. Chem. Lett., 10 : 2217-21, 2000) (30 μM) and etoposide (10 μM), 5% CO ₂ , cultured at 37 ° C for 6 hours, and 100 μM ougonin for each culture The apoptotic effect was confirmed by measuring DNA fragmentation. The DNA fragmentation rate was measured by separating and quantifying fragmented DNA and non-fragmented DNA. The amount of DNA was determined according to the method described in McConkey, DJ, et al., Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca ²⁺ concentration. Arch. Biochem. Biophys., 269: 365-370, 1989. Measured by the method. The ratio of the amount of fragmented DNA to the total amount of DNA (fragmented DNA + non-fragmented DNA) was expressed in%.

  As a result, in the case of the antitumor agent alone, a tendency of DNA fragmentation was observed, and when ougonin was added, reduction of DNA fragmentation was observed. The effect was particularly remarkable with respect to etoposide (FIG. 1). From this, it was confirmed that the antitumor drug has an apoptotic effect on normal cells, but the apoptotic effect on normal cells is reduced by the addition of ougonin. From this, it was suggested that the side effect by an antitumor agent is reduced by the addition of ougonin.

(Example 2) About caspase-3-like activity when etoposide and ougonin are used in combination with normal cells Thymocytes (100 × 10 ⁶ cells / ml) can be added together with etoposide 10 μM in the presence or absence of ougonin 100 μM. After 4 hours of incubation, 50 μl of extraction buffer (50 mM PIPES-NaOH (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl ₂ , 20 μM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride , 1 mM dithiothreitol, 1 μg / ml chymostatin, 1 μg / ml leupeptin, 1 μg / ml pepstatin A, 2.83 μg / ml E-64-d) It was. Activity measurement buffer (100 mM HEPES-KOH, 10% sucrose, 0.1% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS), 10 mM dithiothreitol, 0.1 mg / ml ovo After diluting the enzyme preparation with (albumin), react with 100 μM acetyl-Asp-Glu-Val-Asp-α- (4 methyl-coumaryl-7-amide) (Ac-DEVD-MCA) at 37 ° C, The fluorescence of -amino-4-methyl-coumarin (AMC) was measured.

As a result, under the control conditions in which nothing was added, the caspase-3-like activity did not increase rapidly with the passage of the culture time, and the system in which ougonin was added alone showed almost the same tendency as the control. On the other hand, when cultured in a system with 10 μM etoposide alone, caspase-3-like activity increased over the course of the culture time, but in the system with 10 μM etoposide and 100 μM ougonin, etoposide alone Compared to the case, the increase in caspase-3-like activity was suppressed (FIG. 2).
This result suggests that the caspase-3-like activity was suppressed lower in the system containing etoposide 10 μM and ougonin 100 μM than in the system using etoposide alone, suggesting that the apoptotic effect of etoposide on normal cells is reduced. .

(Example 3) DNA fragmentation when etoposide and ougonin are used in combination with tumor cells (Jurkat cells)
Jurkat cells (peripheral blood-derived cells from patients with acute lymphoblastic leukemia) were prepared using RPMI 1640 medium containing 10% fetal bovine serum to a cell density of 1 × 10 ⁵ cells / ml. The cells were cultured under conditions of 5% CO ₂ and 37 ° C. and passaged every 4 days. What was prepared using RPMI 1640 medium containing 10% fetal bovine serum to a cell density of 3 × 10 ⁶ cells / ml was prepared in the presence or absence of each concentration of ougonin at 5 μM etoposide or 30 cisplatin. After culturing with μM for 6 hours, the DNA fragmentation rate of the Jurkat cells was examined. The measurement of DNA fragmentation followed the method described in Example 1.

The results are shown in FIG. From FIG. 3, DNA fragmentation was not observed with ougonin alone, and no apoptotic effect on the cells was observed.
In addition, as a result of examination with the etoposide 5 μM system, DNA fragmentation by etoposide was not observed when ougonin was not added, whereas in the system cultured with each concentration of ougonin added to etoposide, Depending on the concentration, an increase in DNA fragmentation was observed and was found to have an apoptotic effect.
From these results, it was suggested that even when etoposide alone does not show an apoptotic effect on Jurkat cells, an antitumor effect due to the apoptotic action can be obtained by using ougonin together.

  On the other hand, as a result of examination in the system of cisplatin 30 μM, DNA fragmentation by cisplatin was not observed when ougonin was not added, and DNA fragmentation did not change even when each concentration of ougonin was added. This suggests that for Jurkat cells, this concentration of cisplatin does not show antitumor effects due to apoptosis even with cisplatin alone or in combination with cisplatin and ougonin.

(Example 4) DNA fragmentation when etoposide and ougonin are used in combination with tumor cells (HL-60 cells)
HL-60 cells (peripheral blood-derived cells from patients with acute promyelocytic leukemia) were prepared using RPMI 1640 medium containing 10% fetal bovine serum to a cell density of 1.5 × 10 ⁵ cells / ml. The cells were cultured under conditions of 5% CO ₂ and 37 ° C. and passaged every 4 days. What was prepared using RPMI 1640 medium containing 10% fetal bovine serum to a cell density of 4 × 10 ⁶ cells / ml was prepared in the presence or absence of each concentration of ougonin at 5 μM etoposide or 30 cisplatin. After culturing with μM for 6 hours, the DNA fragmentation rate of the HL-60 cells was examined. The measurement of DNA fragmentation followed the method described in Example 1.

The results are shown in FIG. From FIG. 4, DNA fragmentation was not observed with ougonin alone.
In addition, as a result of studying the system of etoposide 5 μM, the DNA fragmentation rate by etoposide was about 10% when ougonin was not added, whereas in the system cultured with each concentration of ougonin added to etoposide, Depending on the concentration of ougonin, an increase in DNA fragmentation was observed, and an enhanced apoptotic effect was observed.
From these results, it was suggested that antitumor effect by apoptotic action can be obtained more effectively by using ougonin in combination with HL-60 cells than by apoptotic action by etoposide alone.

On the other hand, as a result of examination in the system of cisplatin 30 μM, the DNA fragmentation rate by cisplatin was 12% when ougonin was not added, and the DNA fragmentation rate slightly increased when each concentration of ougonin was added to cisplatin.
This suggested that for HL-60 cells, the antitumor effect due to the apoptotic action of cisplatin tends to be enhanced by the combined use of ougonin.

(Example 5) Combined effect of etoposide and ougonin on tumor cells (Jurkat cells) (morphological observation)
Jurkat cells were incubated with 5 μM etoposide in the presence or absence of 10 μM ougonin for 6 hours, and then fixed with phosphate buffered saline (PBS) containing 5% formaldehyde (4 ° C., 15 minutes or longer) . Cells were collected by centrifugation at 300 × g for 10 minutes and resuspended in PBS. To the cell suspension, 12.3 μg / ml Hoechst 33342 was added and reacted at room temperature for 10 minutes to stain the nucleus and washed with PBS using a confocal laser microscope (LSM GB 200, Olympus Corporation) The cell nuclei were observed (40 times).
As a result, normal nuclei were observed in each cell when etoposite and oregonin were not added, and when ogonin alone was added, whereas apoptotic morphological characteristics were observed when etoposide alone was added. Slight apoptotic bodies were observed (FIG. 5). When etoposide and ougonin were added, condensed nuclei and apoptotic bodies were markedly increased compared to etoposide alone. This suggests that the addition of ougonin enhances the apoptotic effect of etoposide.

(Example 6) Combined effect of doxorubicin and ougonin on human lung cancer cells (A549 cells) (measurement of cell viability)
Cells after culturing A549 cells (distributed by Human Science Foundation) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM doxorubicin in the presence or absence of 25 μM ougonin Survival was examined.
A MEM medium supplemented with 10% fetal calf serum (FCS) was used as a culture solution and used for culturing A549 cells.
A549 cells are suspended in the above culture solution so as to be 1 to 1.5 × 10 ⁵ cells / ml, and 10 ml of the cell suspension is seeded on a culture dish having a diameter of 100 mm, and the condition is 37% under 5% CO ₂ condition. Incubated at 0 ° C. The culture medium was changed every 2-3 days, and the cells were grown to 80-90% (subconfluent). The cells grown to the sub-confluent were treated with a 0.25% trypsin solution to detach the cells from the culture dish, washed with the culture solution, and then a cell suspension having a cell number of 5 × 10 ⁴ cells / ml was prepared. .

A cell suspension having a cell number of 5 × 10 ⁴ cells / ml was seeded in each well of a 96-well microplate in an amount of 200 μl. At this time, the number of cells in each well is 1 × 10 ⁴ cells / well.
After culturing at 37 ° C. for 24 hours under 5% CO ₂ , the culture medium in each well was removed by aspiration and replaced with a new culture medium of 200 μl / well. Furthermore, after culturing for 24 hours, the culture medium in each well is removed by aspiration and cultured with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM doxorubicin and ougonine (0, 25 μM). The solution was added at 200 μl / well.
After further incubation for 24 hours in culture medium containing doxorubicin and ougonin at each concentration, the culture medium in each well is removed by aspiration, and 100 μl / well of a new culture medium not containing doxorubicin and ougonin is added. Reagent for measurement (Cell Counting kit-8: manufactured by Dojindo Laboratories) was added at 10 μl / well. After 2 hours of reaction at 37 ° C. under 5% CO ₂ , the amount of formazan produced was measured by absorbance at wavelength A ₄₅₀ using a microplate reader. A cell culture that did not contain doxorubicin and ougonin was used as a control, the absorbance of the control was 100%, and the cell viability when each concentration of drug was added was calculated.

  As a result, a decrease in cell viability was confirmed when the amount of doxorubicin added was 100 μM or more (FIG. 6). Moreover, even the system to which ougonin was added did not inhibit the cytotoxic effect of doxorubicin.

(Example 7) Combined effect of cisplatin and ougonin on human lung cancer cells (A549 cells) (measurement of cell viability)
Cells after culturing A549 cells (distributed by Human Science Foundation) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM cisplatin in the presence or absence of 25 μM ougonin Survival was examined.
The concentration of each drug, the A549 cell culture conditions, and the cell viability were calculated in the same manner as in Example 6.
As a result, a decrease in cell viability was confirmed when the amount of cisplatin added was 100 μM or more (FIG. 7). Moreover, even in the system added with ougonin, the cytotoxic effect of cisplatin was not inhibited.

(Example 8) Combined effect of carbocon and ougonin on human lung cancer cells (A549 cells) (measurement of cell viability)
Cells after culturing A549 cells (distributed by Human Science Foundation) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM carbocones in the presence or absence of 25 μM ougonin Survival was examined.
The concentration of each drug, the A549 cell culture conditions, and the cell viability were calculated in the same manner as in Example 6.
As a result, a decrease in cell viability was confirmed when the amount of carbocon added was 5 μM or more (FIG. 8). Moreover, in the system to which ougonin was added, the cytotoxic effect of carbocon was not inhibited, but a tendency to increase was observed.

(Example 9) Combined effect of vinblastine and ougonin on human lung cancer cells (A549 cells) (measurement of cell viability)
Cells after culturing A549 cells (distributed by Human Science Foundation) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM vinblastine in the presence or absence of 25 μM ougonin Survival was examined.
The concentration of each drug, the A549 cell culture conditions, and the cell viability were calculated in the same manner as in Example 6.
As a result, when the added amount of vinblastine was 50 μM or more, a rapid decrease in cell viability was confirmed (FIG. 9). Moreover, in the system added with ougonin, the cytotoxic effect of vinblastine was not inhibited.

(Example 10) Combined effect of camptothecin and ougonin on human lung cancer cells (A549 cells) (measurement of cell viability)
Cells after culturing A549 cells (distributed by Human Science Foundation) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM camptothecin in the presence or absence of 25 μM ougonin Survival was examined.
The concentration of each drug, the A549 cell culture conditions, and the cell viability were calculated in the same manner as in Example 6.
As a result, a decrease in cell viability was confirmed when the amount of camptothecin added was 10 μM or more (FIG. 10). In addition, in the system in which ougonin was added in the presence of 1 to 50 μM camptothecin, a tendency to enhance the cytotoxic effect of camptothecin was confirmed.

(Example 11) Combined effect of etoposide and ougonin on human lung cancer cells (A549 cells) (measurement of cell viability)
Cells after culturing A549 cells (distributed by Human Science Foundation) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM etoposide in the presence or absence of 25 μM ougonin Survival was examined.
The concentration of each drug, the A549 cell culture conditions, and the cell viability were calculated in the same manner as in Example 6.
As a result, a decrease in cell viability was confirmed when the amount of etoposide added was 1 μM or more (FIG. 11). In addition, when the addition amount of etoposide was 1 to 100 μM, it was confirmed that the cytotoxicity effect of etoposide was clearly enhanced in the system added with ougonin.

(Example 12) About the combined effect of etoposide and ougonin on human lung cancer cells (NCI-H226 cells, ATCC No. CRL-5826) (measurement of cell viability)
Cell viability after culturing NCI-H226 cells (purchased from ATCC) with 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μM etoposide in the presence or absence of 25 μM ougonin I investigated.
The RPMI medium supplemented with 10% fetal calf serum (FCS) was used as the culture medium, and was used to culture NCI-H226 cells. Other than the cell culture conditions (temperature, time, etc.), drug concentration, and cell viability The calculation was performed by the same method as in Example 11.
As a result, a decrease in cell viability was confirmed when the amount of etoposide added was 10 μM or more (FIG. 12). In addition, in the system added with ougonin, the tendency to enhance the cytotoxic effect of etoposide was clearly confirmed.

  As described above, the anti-tumor therapeutic agent using the combination of the component derived from corn of the present invention and the anti-tumor agent reduces apoptosis for normal cells, and can maintain or enhance apoptosis for tumor cells. It was confirmed that side effects caused by tumor drugs were reduced. As a result, an antitumor therapeutic agent that exhibits a more effective antitumor effect and has reduced side effects on normal cells and a pharmaceutical composition containing the antitumor therapeutic agent can be provided. These anti-tumor therapeutic agents can be applied not only to blood cancer but also to solid cancer such as lung cancer.

Claims (14)

An antitumor therapeutic agent comprising a combination of an ingredient derived from corn and an antitumor agent having an apoptotic action. The antitumor therapeutic agent according to claim 1, wherein the antitumor agent having an apoptotic action is a topoisomerase inhibitor, an alkaloid antitumor agent, a platinum complex, an antibiotic, or an alkylating agent. The antitumor therapeutic agent according to claim 2, wherein the topoisomerase inhibitor is etoposide, camptothecin or a derivative thereof. The antitumor therapeutic agent according to claim 2, wherein the alkaloid antitumor agent is vinblastine or a derivative thereof. The antitumor therapeutic agent according to claim 2, wherein the platinum complex is cisplatin or a derivative thereof. The antitumor therapeutic agent according to claim 2, wherein the antibiotic is doxorubicin or a derivative thereof. The antitumor therapeutic agent according to claim 2, wherein the alkylating agent is carbocon or a derivative thereof. The antitumor therapeutic agent according to any one of claims 1 to 7, wherein the yellow corn-derived component is a component containing a flavonoid. The antitumor therapeutic agent according to claim 8, wherein the component containing flavonoid is ougonin. 10. An ingredient according to any one of claims 1 to 9, wherein an amount of a component derived from corn that can prevent side effects caused by apoptosis on normal cells caused by an antitumor drug having an apoptotic action is used in combination. The antitumor therapeutic agent described. 11. The antitumor therapeutic agent according to claim 10, wherein the ratio of the yellow tumor-derived component and the antitumor agent having an apoptotic effect used is 10: 1 to 1:10 in terms of molar ratio. A pharmaceutical composition comprising the antitumor therapeutic agent according to any one of claims 1 to 11. The pharmaceutical composition according to claim 12, which is used for blood cancer or solid cancer. The pharmaceutical composition according to claim 12, which is used for leukemia or lung cancer.