WO2002102400A1

WO2002102400A1 - Use of macrocyclic peptide compound as anticancer agents and method for diagnosing cancer

Info

Publication number: WO2002102400A1
Application number: PCT/KR2002/001122
Authority: WO
Inventors: Sung-Chun Kim
Original assignee: GENOPROT CO Ltd
Current assignee: GENOPROT CO Ltd
Priority date: 2001-06-18
Filing date: 2002-06-14
Publication date: 2002-12-27
Anticipated expiration: 2003-12-18

Abstract

A use of the macrocyclic peptide compounds micrococcin and thiostrepton as anticancer agents which exhibit selective toxicity against cancer cells is disclosed. When the compounds micrococcin and thiostrepton are treated to cancer cells and normal cells, they exhibit selective toxicity only against cancer cells to induce apoptosis of the cancer cells. The anticancer agent comprising at least one compound selected from the group consisting of the macrocyclic peptide compounds micrococcin and thiostrepton as an active ingredient, did not show toxicity against normal cells, but showed selective toxicity against cancer cells, thereby inducing selective apoptosis of cancer cells. Therefore, the macrocyclic peptide compounds micrococcin and thiostrepton are useful as anticancer agents. Further, from the finding that the eukaryotic mitochondrial L11 gene or its analogue on which micrococcin and thiostrepton act is differently expressed between healthy persons and cancer patients, it is expected that the gene will be used to diagnose cancers.

Description

Use of Macrocyclic Peptide Compound as Anticancer Agents and Method for Diagnosing Cancer

Technical Field The present invention relates to an anticancer agent, and more particularly to a use of the macrocyclic peptide compounds micrococcin and thiostrepton, as anticancer agents which exhibit selective toxicity against cancer cells, and a method for diagnosing cancers comprising detecting the differences in expression patterns, between healthy persons and cancer patients, of the eukaryotic mitochondrial genes on which micrococcin or thiostrepton acts.

Background Art

The incidence of cancers has been rapidly increasing by 5% per year throughout the world. This is partly because of the increased age of populations and environmental pollution. In Korea, it was reported that there were approximately 100,000 newly-diagnosed cases of cancers per year, and 50,000 people died of cancers. Now, the number of cancer patients amounts to approximately 120,000. Among them, gastric cancer, liver cancer and lung cancer account for 21 %, 12% and 11 % of male cancer patients, respectively, and uterine cervical cancer, gastric cancer and breast cancer account for 20%, 16% and 13% of female cancer patients, respectively. The rate of yearly increase of such cancer patients is about 10% in Korea.

Representative clinical treatments for cancers in modern medicine include surgical resection, chemotherapy, radiotherapy, biotherapy, and a combination of two or more therapies, etc. However, these therapies have a significant failure rate. Therefore, it is recognized that cancer has become one of the leading diseases that should be overcome in order to prolong the human life.

Anticancer agents refer to all drugs exhibiting cytotoxicity or cytostatic effect against cancer cells by interfering in various metabolic pathways of cancer cells. Anticancer agents developed so far can be categorized into the following classes, depending on their action mechanisms and chemical structures: metabolic antagonists, plant alkaloids, topoisomerase inhibitors, alkylating agents, antitumor antibiotics, hormones, and other drugs. Each anticancer agent has different intracellular targets, and blocks DNA replication, transcription and translation in cells or inhibits the functions of proteins indispensable for cell survival. These functions of anticancer agents kill cancer cells via necrosis or apoptosis. Generally, since the metabolic pathways on which the anticancer agents act are not specific only to cancer cells, damage (i.e. toxicity) to normal cells by the anticancer agents is unavoidable.

However, since there are significant quantitative differences between the metabolisms of cancer cells and normal cells, the anticancer agents exhibit more toxicity against cancer cells than against normal cells. Using the selective toxicity of the anticancer agents makes it possible to clinically treat cancer through chemotherapy. Therefore, the greater the specific therapeutic index of the anticancer agent, the more preferable it is in terms of safety as an anticancer agent. For example, since cancer cells show a higher protein synthesis rate than normal cells, agents inhibiting protein synthesis can show anticancer effects including selective toxicity against cancer cells.

Examples of antibiotics that can inhibit protein synthesis include kanamycin, tetracycline, chloramphenicol, erythromycin, streptomycin, lincomycin, clindamycin, thiostrepton, micrococcin, etc.

Micrococcin (C48H49N1309S6. 9H2, C: 49.51 %, H: 5.97%, N: 15.64%, O: 12.36%, S". 16.52%), a macrocyclic peptide antibiotic, represented by Fig. 1 , can be isolated from microorganisms such as Staphylococcus sciuri, Bacillus pumilus, Staphylococcus equorum WS2733, Micrococcus varians, etc., or can be organicaiiy synthesized. It has been reported that micrococcin shows antibiotic activity against gram-positive bacteria. Also, the methods for synthesizing micrococcin are disclosed in Okumura K., Suzuki T., Nakamura Y., and Shin C. 1999. Total synthesis of a Macrocyclic antibiotic, Micrococcin Pi . Bull. Chem. Soc. Jpn., 72, 2483-2490 and Ciufolini M.A., and Shen Y.C. 1999. Synthesis of the Bycroft-Gowland structure of micrococcin P1. Org Lett. 1 (11), 1843-1846. Thiostrepton (C72H85N19018S5 16H2, C: 51.24%, H: 5.59%, N: 15.65%, O: 16.83%, S: 8.37%, H: 0.12%), a macrocyclic peptide antibiotic, represented by Fig. 4, can be isolated from microorganisms including Streptomyces species such as Streptomyces laurentii (e.g., ATCC NO. 31 ,255), Streptomyces azureus (e.g., ATCC NO. 14,921 ), Streptomyces hawaiiensis (e.g., ATCC NO. 12,236), etc. It has been reported that thiostrepton shows antibiotic activities against gram-positive bacteria.

Micrococcin and thiostrepton act on the GTPase center which is well conserved in prokaryotic and eukaryotic cells. Ribosomes, the site of protein biosynthesis, consist of two subunits, and the GTPase center is located on the larger subunit (LSU) of the two subunits. The GTPase center is located at the double hairpin structure within domain II of the 23 S-like rRNA, and the pentameric protein complex of proteins L1 1 and L10(L12)4 binds to the double hairpin structure to perform biological functions.

Micrococcin and thiostrepton act as antibiotics by binding to the GTPase center in prokaryote and interfering the GTPase reaction occurring at the ribosome in the presence of EF-G to inhibit protein synthesis (Porse BT and Garrett RA. 1999. Ribosomal mechanics, antibiotics, and GTP hydrolysis. Cell 97, 423-426). Also, the antibiotic micrococcin and thiostrepton inhibit protein synthesis at the plastid— like organelle of the malaria parasite to inhibit the growth of the parasite (Rogers MJ, Cundliffe E, McCutchan TF. 1998. The antibiotic micrococcin is a potent inhibitor of growth and protein synthesis in the malaria parasite. Antimicrob Agents Chemother 42(3): 715-6). However, recent studies have revealed that micrococcin and thiostrepton do not exhibit said inhibitory effects against eukaryote (Porse BT and Garrett RA. 1999. Ribosomal mechanics, antibiotics, and GTP hydrolysis. Cell 97, 423-426). Proteins corresponding to the protein L1 1 constituting the GTPase center in prokaryotes exist in the mitochondria of eukaryotes, and the stereostructure of the GTPase center is well conserved (Koc EC, Burkhart W, Blackburn K, Moyer MB, Schlatzer DM, Moseley A, Spremulli LL 2001 . The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present. J. Biol. Chem 276: 43958-43969).

It is expected that the L1 1 analogue located at the chromosome in eukaryotic cells is expressed as a protein, which is transferred to mitochondria to constitute the GTPase center of the ribosome in mitochondria, thereby being involved in protein synthesis in the mitochondria. However, there has been no reliable report about the association between the L1 1 analog activity and cancers.

Mitochondria provide energy sources necessary for cell growth, i.e. ATP and intermediate metabolic products, and play a key role in apoptosis. The toxicity of compounds directly or indirectly inhibiting oxidative phosphorylation in mitochondria, such as oligomycin, antimycin A, rotenone, etc., are not reflective for normal cells or cancer cells.

Cancer cells in the outer part of tumor are in the state of normpoxia, but cells in the inner core are in the state of hypoxia (anaerobic state). The cells in the inner core obtained more ATP through glycolysis than from mitochondrial synthesis. Glycolytic inhibitors, such as 2-deoxyglucose, oxamate, iodoacetate, etc., are highly active against enzymes associated with glycolysis. These glycolytic inhibitors inhibit glycolysis in cancer cells and inhibit cancer cell growth (Liu H, Hu YP, Savaraj N, Priebe W, Lampidis TJ. 2001 . Hypersensitization of tumor cells to glycolytic inhibitors. Biochemistry 40(18): 5542-7).

The present inventors have first found that the macrocyclic peptide compounds micrococcin and thiostrepton, which inhibit the function of ribosomes involving in the protein synthesis in cancer cell lines, have anticancer effects and that these compounds exhibit none of the side effects or problems occurring in the clinical use of conventional chemotherapies, and as a result, they accomplished the present invention.

Disclosure of Invention Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide an anticancer agent which comprises at least one compound selected from the group consisting of the macrocyclic peptide compounds micrococcin and thiostrepton as an active ingredient. In accordance with the present invention, there is provided a use of the macrocyclic peptide compounds micrococcin and thiostrepton, as anticancer agents, that is, an anticancer agent which comprises micrococcin or thiostrepton as an active ingredient.

Micrococcin used as an anticancer agent in the present invention can be extracted from various microorganisms. For example, micrococcin is obtained by a method comprising the steps of: collecting cells by centrifuging a culture of Staphylococcus sciuri, a species of Staphylococcus; washing the collected cells with phosphate buffer and treating with methanol to extract crude micrococcin, followed by centrifuging the crude micrococcin to remove the cells; drying the methanol extract and dissolving in a mixture of dichloromethane and methanol, and loading onto a silica gel chromatography column; and purifying by chromatography (eluent: a mixture of dichloromethane and methanol) to obtain micrococcin. Micrococcin can be obtained from other microorganisms such as

Bacillus pumilus, Staphylococcus equorum WS2733, Micrococcus varians, etc., and can be organically synthesized in accordance with well-known processes.

Thiostrepton used as another anticancer agent in the present invention can also be obtained through the extraction from microorganisms or a synthesis process. For example, thiostrepton is obtained by a method comprising the steps of: collecting mycelia after separating supernatant from the culture of Streptomyces laurentil (ATCC No. 31 ,225), a kind of actinomyces, by centrifugation; obtaining a mycelium extract by treating the collected mycelia with chloroform; concentrating the chloroform extract on a vibrator, eliminating impurities from the extract with a small amount of methanol, and re-dissolving the extract in chloroform; and adding a small of alcohol to obtain thiostrepton as a crystal. Thiostrepton can be obtained from Streptomyces species such as Streptomyces azureus (e.g., ATCC No. 14,921 ), Streptomyces hawaiiensis (e.g., ATCC No. 12,236), etc.

When a solution of micrococcin and/or thiostrepton in a pharmaceutically acceptable solvent such as dimethyl sulfoxide is treated to cancer cells and normal cells, these compounds exhibit selective toxicity against cancer cells to induce apoptosis of the cancer cells, thereby exhibiting anticancer effects. When the macrocyclic peptide compounds used in the present invention are mixed with other glycolytic inhibitors, and then the mixture is applied to cancer cells, the anticancer effects can be further improved. The reason is that the protein encoded by the target gene of the macrocyclic peptide compounds is a component of ribosomes in mitochondria, and the macrocyclic peptide compounds inhibit the functions of mitochondria. That is, since mitochondria provide energy sources for cells through oxidative phosphorylation processes, a mixture of thiostrepton and/or micrococcin and a glycolytic inhibitor, another route providing energy for cells, can exhibit superior anticancer effects to thiostrepton or micrococcin.

The anticancer agent comprising micrococcin and/or thiostrepton as an active ingredient according to the present invention, or the anticancer agent composition comprising micrococcin and/or thiostrepton and the glycolytic inhibitor can be administered orally or parenterally according to a desired purpose.

The anticancer agent according to the present invention can be prepared by mixing micrococcin and/or thiostrepton with pharmaceutically acceptable carriers. The anticancer agent composition according to the present invention can further comprise excipients, disintegrating agents, lubricants, etc. In particular, since the macrocyclic peptide compounds micrococcin and thiostrepton, are highly hydrophobic and thus have low water solubility, they are preferably formulated with a liposome in order to deliver an effective amount of the compounds. For example, when thiostrepton formulated with the liposome is administered to a nude mouse implanted with liver cancer cell line, death, clinical symptoms and changes in body weight in the tested mouse are not observed, and the tumor growth is 50% less, compared with a negative control.

Until now, it has been found that micrococcin has little or no toxicity to mononuclear cell cultures from human blood at concentrations lower than 625nmol. The present inventors have found that the IC50 value of micrococcin against cancer cell line is in the range of from about O.δnmol to about 5nmol.

It has also been found that thiostrepton has little or no toxicity to mononuclear cell culture from human blood at concentrations lower than 10μ mol. The present inventors have found that the IC50 value of micrococcin against cancer cell line is in the range of from about 1 u mol to 5u mol.

The anticancer agent according to the present invention can be formulated as tablets, capsules, powders, granules, suspensions, emulsions, syrups or formulations for non-oral administration in accordance with conventional processes. These formulations can be administered in a single dosage form or in separate forms. The effective amount of micrococcin administered is in the range of from about 0.6μ g to about 500mg/Kg of body weight per day, and preferably from 0.5 to 15mg/Kg of body weight per day. The effective amount of thiostrepton administered is in the range of from 0.5 to 15mg/Kg of body weight per day.

The present inventors have investigated the expression pattern of the gene corresponding to the L1 1 , which binds to thiostrepton and micrococcin in prokaryote. It was found that the L1 1 exists as a complete gene and two partly-deleted genes. The expression of the complete gene in normal cells is weak, but the expression in cancer cells is strong. In particular, the complete gene and partly-deleted gene all are expressed in cancer patients. From this finding, the present inventors have found that the L11 has an association with cancerization. Therefore, it is expected that cancer will be diagnosed by investigating the expression pattern of genes similar to the L1 1 gene, which is an element of ribosomes interacting with the macrocyclic peptide compounds in prokaryote, in normal and cancer sites of gastric cancer patients and tissues of healthy men.

Brief Description of Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 shows the chemical structure of micrococcin;

Figs. 2a to 2k are graphical representations showing the effect of micrococcin on the growth of normal cells (MNC), cancer cell line (AGS), SNU-638, SNU-601 , Hep G2, SNU 368, SK-Hep1 , KM1214, LoVo, Jurkat and NCI-H23, respectively;

Fig. 3 is a photograph showing apoptosis of cancer cell line (AGS) induced by micrococcin; Fig. 4 shows the chemical structure of thiostrepton;

Figs. 5a to 5k are graphical representations showing the effect of thiostrepton on the growth of normal cells (MNC), cancer cell line (AGS), MKN-74, SNU-638, Hep G2, SK-Hep1 , Hep 3B, LoVo, KM 1214, SK-BR-3 and Jurkat, respectively;

Fig. 6 is a photograph showing apoptosis of cancer cell line (AGS) induced by thiostrepton;

Figs. 7a to 7c are diagrams showing the effect of micrococcin on ATP synthase subunit I, cytochrome c oxidase subunit I and II, all of which are mitochondrial proteins, relative to increasing time, respectively;

Figs. 8a to 8c are diagrams showing the expression patterns of the human mitochondrial L1 1 gene in healthy human tissues, cancer cell line, and cancer tissue and normal tissue of a gastric cancer patient, respectively;

Fig. 9 is a graph showing the effect on the growth of a nude mouse implanted with SK-Hep1 , which is a liver cancer cell line, with increasing concentration of micrococcin formulated with a liposome; and

Figs. 10a and 10b are graphs showing the effect on the growth of AGS, a gastric cancer cell line, with increasing concentration of a complex of 2-deoxyglucose or oxamate, and micrococcin, respectively.

Best Mode for Carrying Out the Invention 1. Extraction of micrococcin

After culturing Staphylococcus sciuri by a well-known standard culturing process, cells were collected by centrifugation. 1 g of the cells were washed with phosphate buffer (pH 7.0) and treated with 3ml of methanol to extract crude micrococcin. The crude micrococcin was centrifuged to remove the cells. The methanol extract was dried on a rotary evaporator (Rotavap TM) and dissolved in a mixture of dichloromethane and methanol (95:5). The solution was loaded onto a silica gel chromatography column and purified by chromatography using dichloromethane/methanol (95:5) as an eluent to obtain micrococcin.

2. Effect of micrococcin on the growth of cancer cell line

Mononuclear cells (MNC) separated from the blood of a healthy person,

gastric cancer cell line (AGS, SNU638, SNU 601 ), liver cancer cell line (Hep G2, SNU 368, SK-Hep1 ), large intestine cancer cell line (KM 1214, LoVo), leukemia cell line (Jurkat) and lung cancer cell line (NCI-H23), respectively, were treated with a solution of micrococcin in DMSO (dimethyl sulfoxide) at various

concentrations lower than 10nmol, as shown in Fig. 2. By examining the growth rates of cancer cells over 72 hours of incubation, the effect of micrococcin on the growth of normal cells and cancer cell lines was evaluated.

First, after normal cells (MNC) and cancer cell lines were cultured in DMED

(Hep G2, SK-Hep1 ) and RPMI (MNC and other cancer cell lines) supplemented

with 10% fetal bovine serum, the respective cultures were spread on 6-well plates in an amount of 104-105 cells, and incubated in a 5% C02 humidified incubator at 37^°C for 24 hours. Each medium was treated with micrococcin at various concentrations, as shown in Fig. 2, further incubated in a 5% C02 humidified incubator at 37^°C for 72 hours, and then the cell proliferation was evaluated by performing the MTT assay. After each cancer cell line was treated with 5mg/ml MTT up to 1/10 of culture volume and stored at a temperature of 37^°C for 4 hours, each medium was removed. After MTT dye (violet) was dissolved in a solution of 0.05M HCI in isopropanol, only living cells were stained using the MTT dye. The absorbance of the MTT dye was read at 570nm. Data were obtained by subtracting the absorbance at 630nm (background value) from the absorbance at 570nm.

The results are shown in Fig. 2. As shown in Fig. 2, micrococcin exhibited cytotoxicity against various cancer cell lines in proportion to the concentration of micrococcin added. However, micrococcin exhibited no toxicity against mononuclear cells (MNC) isolated from the blood of a healthy person.

3. Mechanism of micrococcin action on the growth of cancer cell line The mechanism of micrococcin' s inhibition of the growth of cancer cell line was studied by performing apoptosis analysis. The apoptosis analysis was performed in accordance with the protocol of the manufacturer (Apoptosis Detection System, Fluorescein protocol, Promega).

First, cancer cell line (AGS) was cultured in RPMI medium supplemented with 10% fetal bovine serum, spread on Lab-Tek chamber slides, and incubated in a 5% C02 humidified incubator at 37^°C for 24 hours. After the cancer cells were treated with O.δnmol of micrococcin and further incubated for 6 hours, the culture was washed twice with PBS (phosphate-buffered saline) and the cells were fixed by treatment with methanol-free 4% formaldehyde in PBS solution at a temperature of 4°C for 30 minutes, and then washed three times with PBS. Next, after the culture was treated with 0.2% Triton X- 0 in PBS solution at room temperature for 15 minutes to induce permeabilization, the culture was washed three times with PBS, treated with 100u I of equilibration buffer, and then left at room temperature for 10 minutes. The cells were treated with TdT incubation buffer (45μ I of equilibration buffer, 5μ I of nucleotide mix and 1 μ I of TdT enzyme), stored in a humidified chamber at 37°C for 1 hour, treated with 2^χSSC and then stored at room temperature for 15 minutes. Finally, the culture was washed three times with PBS, treated with 1 g/ml propidium iodide, stained in a dark place for 15 minutes, and then washed three times with deionized water for 5 minutes. Apoptosis was observed as a green fluorescence by the use of a fluorescent microscope. The results are shown in Fig. 3. As shown in Fig. 3, it can be seen that micrococcin apparently induces apoptosis of the cancer cell line.

4. Extraction of thiostrepton

After culturing Streptomyces laurentii (ATCC No. 31 ,255) for 65 hours by a well-known standard culturing process, mycelia were collected after separating supernatant from the culture by centrifugation. Thereafter, the mycelia were extracted with chloroform, and the chloroform extract thus obtained was concentrated under vacuum at a temperature of 40 ^°C. Impurities were eliminated from the extract using methanol, and the extract was re-dissolved in chloroform. Finally, the extract was crystallized from methanol to obtain thiostrepton as a crystal.

5. Effect of thiostrepton on the growth of cancer cell lines Mononuclear cells (MNC) separated from the blood of a healthy person, gastric cancer cell line (AGS, MKN-74, SNU 638), liver cancer cell line (Hep G2, SK-Hep1 , Hep 3B), large intestine cancer cell line (LoVo, KM 1214), breast cancer cell line (SK-BR-3), and leukemia cell line (Jurkat), respectively, were treated with a solution of thiostrepton in DMSO (dimethyl sulfoxide) at various concentrations O Onmol to 10μ mol), as shown in Fig. 5. By examining the growth rates of cancer cells over 72 hours of incubation, the effect of thiostrepton on the growth of normal cells and cancer cell lines was evaluated.

First, after normal cells (MNC) and cancer cell lines were cultured in DMED (Hep G2, SK-Hep1) and RPMI (MNC and other cancer cell lines) supplemented with 10% fetal bovine serum, the respective cells were spread on 6-well plates in an amount of 104-105 cells, and incubated in a 5% C02 humidified incubator at 37 °C for 24 hours. Each medium was treated with thiostrepton at various concentrations, as shown in Fig. 5, further incubated in a 5% C02 humidified incubator at 37°C for 72 hours, and then the cell proliferation was evaluated by performing the MTT assay.

After each cancer cell line was treated with 5mg/ml MTT up to 1/10 of culture volume and stored at a temperature of 37^°C for 4 hours, each medium was removed. After MTT dye (violet) was dissolved in a solution of 0.05M HCI in isopropanol, only living cells were stained using the MTT dye. The absorbance of the MTT dye was read at 570nm. Data were obtained by subtracting the absorbance at 630nm (background value) from the absorbance at 570nm.

The results are shown in Fig. 5. As shown in Fig. 5, thiostrepton exhibited cytotoxicity against various cancer cell lines (Figs. 5b to 5k) in proportion to the concentration of thiostrepton added. However, thiostrepton exhibited no toxicity against mononuclear cells (MNC) separated from the blood of a healthy person.

6. Mechanism of thiostrepton on the growth of cancer cell line The mechanism of thiostrepton' s inhibition of the growth of cancer cell lines was observed by performing apoptosis analysis. Apoptosis analysis was performed in accordance with the protocol of the manufacturer (Apoptosis Detection System, Fluorescein protocol, Promega).

First, cancer cell line (AGS) was cultured in RPMI medium supplemented with 10% fetal bovine serum, spread on Lab-Tek chamber slides, and incubated in a 5% C02 humidified incubator at 37^°C for 24 hours. After the cells were treated with O.δnmol of thiostrepton and further incubated for 6 hours, the culture was washed twice with PBS (phosphate-buffered saline) and the cells were fixed by the treatment with methanol-free 4% formaldehyde in PBS solution at a temperature of 4^°C for 30 minutes and then washed three times with PBS. Next, after the culture was treated with 0.2% Triton X-100 in PBS solution at room temperature for 15 minutes to induce permeabilization, the culture was washed three times with PBS, treated with 100μ I of equilibration buffer, and then left at room temperature for 10 minutes. The cells were treated with TdT incubation buffer (45μ I of equilibration buffer, 5g I of nucleotide mix and 1 μ I of TdT enzyme), stored in a humidified chamber at 37^°C for 1 hour, treated with 2^χSSC and then stored at room temperature for 15 minutes. Finally, the culture was washed three times with PBS, treated with 1 g/ml propidium iodide, stained in a dark place for 15 minutes, and then washed three times with deionized water for 5 minutes. The apoptosis of the cancer cell line was observed as a green fluorescence by the use of a fluorescent microscope. The result is shown in Fig. 6. As shown in Fig. 6, it can be seen that thiostrepton apparently induces apoptosis of the cancer cell line.

7. Effect of micrococcin on mitochondrial protein synthesis in cancer cell line

After cancer cell line treated with 1 μ mol of micrococcin were collected by centrifugation, mitochondrial protein was separated using ApoAlert Cell Fraction kit (BD Biosciences Clontech, USA), in accordance with the standard protocol of the manufacturer. The collected cells were washed with wash buffer, suspended in fraction buffer mix, cooled on ice for 10 minutes, ground using an ice-cold dounce tissue grinder, transferred to a centrifuge tube, and centrifuged at 10,000 xg at a temperature of 4^°C for 25 minutes. After the mitochondrial fraction was

dissolved in fraction buffer mix and quantified, 10μ g of protein was mixed with a

loading buffer for electrophoresis. After the mixture was electrophoresed in 12% SDS-PAGE, Western hybridization was performed on mouse anti-ATP synthase subunit I, and mouse anti-cytochrome C oxidase subunits I and II (Molecular Probes, Inc. USA), in accordance with a standard protocol.

As shown in Fig. 7, the treatment time with 1 u mol of micrococcin had no affect on levels of ATP synthase subunit I, a protein coded on nuclear DNA, but decreased levels of cytochrome c oxidase subunits I and II, proteins coded on mitochondrial DNA. Therefore, it was confirmed that micrococcin inhibits mitochondrial protein synthesis.

8. Effect of a mixture of micrococcin and glycolytic inhibitor on the growth of cancer cell line

In order to assess the cytotoxicity of oxamate, 2-deoxyglucose, and their mixtures with micrococcin, the MTT assay was performed. Oxamate and 2-deoxyglucose are glycolytic inhibitors. Gastric cancer cell line (AGS) were treated with solutions of micrococcin and glycolytic inhibitors in DMSO (dimethyl sulfoxide) at various concentrations

OOnmol to 10μ mol), as shown in Fig. 5. By examining the growth rates of cancer cells over 72 hours of incubation, the effect of micrococcin and the glycolytic inhibitors on the growth of the cancer cells was evaluated.

First, after the cancer cells were cultured in RPMI supplemented with 10% fetal bovine serum, the respective cultures were spread on 6-well plates in an amount of 104-105 cells, and incubated in a 5% C02 humidified incubator at 37°C for 24 hours. Each medium was treated with micrococcin and the glycolytic inhibitors at various concentrations, as shown in Fig. 5, further incubated in a 5% C02 humidified incubator at 37 ^°C for 72 hours, and then the cell proliferation was evaluated by performing the MTT assay.

All cells were treated with 5mg/ml MTT up to 1/10 of culture volume and stored at a temperature of 37^°C for 4 hours, medium was removed. After MTT dye (violet) was dissolved in a solution of 0.05M HCI in isopropanol, only living cells were stained using the MTT dye. The absorbance of the MTT dye was read at 570nm. Data were obtained by subtracting the absorbance at 630nm (background value) from the absorbance at 570nm.

The results are shown in Fig. 8. As shown in Fig. 8, the glycolytic inhibitors exhibited cytotoxicity against the cancer cell lines in proportion to the concentration of glycolytic inhibitors added, at a constant concentration of micrococcin.

9. Effect of a complex of micrococcin and a liposome on nude mouse implanted with liver cancer cell line

Since micrococcin is highly hydrophobi it was formulated with a liposome to facilitate in vivo delivering. A mixture of micrococcin and egg phosphatidylcholine at a ratio of 1 :100 (w/w) were diluted in PBS to obtain various desired concentrations. The preparation of the liposome was carried out in an extrusion process in accordance with the standard protocol of the manufacturer (Northern Lipids Inc. Canada). That is, the liposome was prepared by feeding N2 gas through filters having pore sizes of 1.2μ m and 0.4μ m, respectively. The anticancer activity of a complex of micrococcin and liposome was identified using xenograft regression model, which uses a liver cancer cell line-implanted BALB/C nude mouse. Test groups were divided into groups administered with doses of 1.6, 8, 16 and 32mg/kg of the complex, respectively. A negative control group was administered with physiological saline for injection. The test groups and negative control group were administered intraperitoneally every day over 4 weeks, during which time clinical symptoms, changes in body weight and tumor growth were observed.

The results are shown in Fig. 9. As shown in Fig. 9, there were no clinical symptoms and changes in body weight. However, the group administered with a dose of 1.6mg/kg showed decreased inhibitory rate of tumor growth (30%) and the group administered with a dose of 32mg/kg also showed decreased inhibitory rate of tumor growth (60%), compared with the control group.

10. Structure and expression of gene encoding target protein

The present inventors have blast searched the NCBI database (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) using the amino acid sequences of the target protein L11 of thiostrepton and micrococcin in prokaryote, and identified a protein corresponding to the target protein and a gene coding for the protein in humans. As a result, the present inventors have found that the gene has a high similarity with EST clones of Genbank Accession Nos. NM-016050, AF151871 , etc, and was identified as MRPL11 , constituting an element of human mitochondrial ribosomes. Primers specific to the base sequences of the identified cDNA were designed, and then RT-PCR was performed in accordance with a standard procedure. After the PCR products were electrophoresed in a 2% agarose gel, and stained with EtBr, DNA was isolated at a desired band and cloned into pGEM T-vector to determine base sequences of the DNA. Based on the determined base sequences, the present inventors have searched the NCBI database, and found that the base sequences correspond to GenBank Accesion Nos. AP001 107 and AC005740. The former is located at chromosome 11q, has 5 exons and codes for a complete gene; whereas the latter is located at chromosome 5p, has 2 exons and codes for a gene having 160 bases (between 392 and 551 base) deleted.

In order to investigate the expression of the above genes, cDNAs derived from tissues of healthy persons and cancer cell lines were prepared by isolating RNAs using MTC panel I and MTC panel II (BD Biosciences Clontech, USA), followed by reverse transcribing the RNAs. cDNAs derived from cancer patients were prepared by isolating RNAs from normal sites and cancer sites of 1 1 gastric cancer patients in accordance with a standard procedure, followed by reverse transcribing the RNAs. Using primer s (5'-GGACAGCCCACTGTTTCCTAC-3';

5'-CA GTCATGAAAACCATCATCA-3') designed to distinguish mRNAs of the complete gene and deleted gene, PCR was performed in accordance with a standard procedure. The results are shown in Fig. 10. Fig. 10 reveals that the PCR product derived from the complete gene of MRPL1 1 is a fragment of 437 bp, and the PCR products derived from the deleted gene is a fragment of 277 bp. The expression of the MRPL1 1 gene varied widely in tissues of healthy persons, but is over-expressed in all cancer cell lines. The MRPL1 1 gene from the cancer sites and normal sites in stomach tissues of gastric cancer patients was expressed in the full form and deleted form, and its expression patterns varied depending on the specimens. Therefore, it can be seen that the expression pattern of the MRPL1 1 gene is very different between healthy persons and cancer patients.

As described above, the macrocyclic peptide compounds micrococcin and thiostrepton, according to the present invention, did not show toxicity against

normal cells, but showed selective toxicity against cancer cells, thereby inducing the apoptosis of cancer cells. Therefore, the macrocyclic peptide compounds micrococcin and thiostrepton, according to the present invention can be used as anticancer agents.

Further, from the finding that the L1 1 gene or its analogue constituting ribosome which interacts with the macrocyclic peptide compounds micrococcin and thiostrepton, according to the present invention, has different expression pattern between healthy persons and cancer patients, it is expected that the gene will be used to diagnose cancers. Although the preferred embodiments of the present have been disclosed purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

What is claimed is:

1. An anticancer agent comprising at least one compound selected from the group consisting of the macrocyclic peptide compounds micrococcin and thiostrepton as an active ingredient.

2. The anticancer agent as set forth in claim 1 , wherein the effective amount of micrococcin is in the range of from 0.6μ g to 500mg/Kg of body weight of a patient per day.

3. The anticancer agent as set forth in claim 1 , wherein the effective amount of thiostrepton is in the range of from about 0.5 to 15mg/Kg of body weight of a patient per day.

4. The anticancer agent as set forth in any one of claims 1 to 3, further comprising a mitochondrial glycolytic inhibitor.

5. The anticancer agent as set forth in any one of claims 1 to 3, wherein the macrocyclic peptide compound is formulated with a liposome.

6. A eukaryotic mitochondrial protein inhibitor comprising at least one compound selected from the group consisting of the macrocyclic peptide compounds micrococcin and thiostrepton as an active ingredient.

7. A method for diagnosing cancers, comprising detecting the differences in expression patterns, between healthy persons and cancer patients, of the eukaryotic mitochondrial L1 1 gene or its analogue on which at least one compound selected from the group consisting of the macrocyclic peptide compounds micrococcin and thiostrepton acts.