KR20010103809A - Use of Thiostrepton as Anticancer Agents - Google Patents

Abstract

The present invention relates to the use of an anticancer agent of thiostrepton, which is a kind of a macrocyclic peptide-based compound that exhibits selective toxicity to cancer cells. Thiostrepton for use as an anticancer agent according to the present invention is obtained by culturing Streptomyces laurentii , which is a kind of actinomycetes, separating water layers and cultures by centrifugation, harvesting mycelium, and treating chloroform on the harvested mycelium mass. To obtain a process, the chloroform extract is concentrated in a vibrating state, a small amount of methanol is treated to remove impurities, and then dissolved in chloroform again, and a small amount of methanol is added to harvest a series of thiostreptone harvested It can be obtained from microorganisms, and from other microorganisms such as Streptomyces azureus , Streptomyces hawaiiensis , Streptomyces species . The anticancer agent using thiostrepton according to the present invention is useful as a cancer treatment agent by inducing selective killing of only cancer cells when applied to various cancer patients without showing toxicity to normal cells without showing toxicity to normal cells. Do.

Description

Use of Thiostrepton as Anticancer Agents

(Technology)

The present invention relates to an anticancer agent, and more particularly, to the use of an anticancer agent of thiostrepton, which is a kind of a macrocyclic peptide-based compound that exhibits selective toxicity to cancer cells.

(Background)

The global cancer incidence is increasing by more than 5% every year due to the increase in elderly population and environmental degradation. According to the 1997 statistics, 6 million people died of cancer, accounting for 12% of the global mortality rate.

In South Korea, statistics show that 100,000 new cancer cases and 50,000 deaths occur each year. In 1997, 120,000 cancer patients were diagnosed with gastric cancer (21%), liver cancer (12%), and lung cancer (11). In women, cervical cancer (20%), gastric cancer (16%), and breast cancer (13%) occurred in the order of cancer patients.

Representative cancer treatments of modern medicine include surgical surgery, chemotherapy, biological therapy, and radiotherapy, and these are applied alone or in combination, but there is no perfect treatment. Therefore, cancer is recognized as one of the top tasks to overcome in order to extend the life span of humans in the 21st century.

An anticancer agent is a generic term for drugs that act on various metabolic pathways of cancer cells and exhibit cytotoxicity or cytostatic effects on cancer cells. The anticancer agents developed so far are metabolic antagonists depending on their mechanism of action and chemical structure. , Plant alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones, and other drugs.

Anticancer drugs vary in intracellular targets, depending on the drug, blocking the DNA replication, transcription, and translation processes of the cell or disrupting the actions of proteins important for cell survival. The effects on these intracellular targets are then necrotic or apoptosis. apoptosis kills cancer cells. In general, the metabolic pathways in which these anticancer drugs work are not specific to cancer cells and are also the same for normal cells. Thus, when the anticancer drug is administered, normal tissue damage (ie, toxicity) is inevitable.

However, there is a significant quantitative difference between the metabolism of cancer cells and normal cells, and based on these differences, anticancer drugs are more toxic to cancer tissues. Thus, by utilizing the selective toxicity of such anticancer drugs, Chemotherapy is possible. Therefore, the greater the specific therapeutic index (therapeutic index) that can eliminate cancer cells while avoiding the toxicity of normal tissue, the safer the cancer drug.

Cancer cells show higher protein synthesis capacity than normal cells, so the inhibitors exhibit anti-cancer effects with selective toxicity to cancer cells.

Antibiotics that inhibit protein synthesis in prokaryotes include kanamycin, tetracycline chloramphenicol, erythromycin, streptomycin, lincomycin, clindamycin, clindamycin, and clindamycin. (thiostrepton), micrococcin and the like.

Thiostrepton, a macrocyclic peptide-based antibiotic having a structural formula represented by FIG. 1 (eg, C ₇₂ H ₈₅ N ₁₉ O ₁₈ S _{5 16} H ₂ , C: 51.24%, H: 5.59%, N: 15.65%, O: 16.83%, S: 8.37%, H ₂ : 0.12%), Streptomyces laurentii (e.g. ATCC NO.31,255), Streptomyces azureus (e.g. ATCC NO. 14,921 ), Streptomyces hawaiiensis (e.g. ATCC NO. 12,236), an antibiotic isolated from microorganisms such as Streptomyces species , and has been widely reported to show antibiotic activity mainly against Gram-positive bacteria.

Thiostrepton is a function point of the GTPase center which is well preserved in prokaryotic and eukaryotic cells. The ribosome, a protein synthesis site, consists of two units, and the GTPase center is located in the rRNA of the larger unit (LSU). The GTPase center is located in the double hairpin structure in the domain II of 23 S-like rRNA, where the L ₁₁ protein and L ₁₀ (L ₁₂ ) ₄ pentameric protein complex bind to the biological function. Do this.

Thiostrepton functions as an antibiotic by binding to the GTPase center in prokaryotes and inhibiting protein synthesis by interfering with the GTPase reaction occurring in ribosomes in the presence of EF-G, and also acting as an anti-pigmentary organ of the Plasmodium falciparum . Inhibits the growth of protozoa by inhibiting protein synthesis in plaidid-like organelles. However, previous studies have shown that thiostrepton does not exhibit the above inhibitory effect on eukaryotes.

The present inventors first identified an anti-cancer effect on thiostrepton, a macrocyclic peptide compound that inhibits the function of ribosomes involved in protein synthesis in cancer cell lines, which is completely different from the existing antibiotic effects. The present invention has been completed with the aim of overcoming the problems of clinical utility.

It is an object of the present invention to provide an anticancer agent using these macromolecular peptide compounds, as identified by the anticancer effect of thiostrepton.

1 is a structural formula of thiostrepton,

Figures 2a-2k show normal cells (MNC: Figure 2a), cancer cell lines AGS (Figure 2b), MKN-74 (Figure 2c), SNU-638 (Figure 2d), Hep G2 (Figure 2e), SK-Hep1 ( 2f), graph showing the effect on growth of Hep 3B (FIG. 2g), Lovo (FIG. 2h), KM 1214 (FIG. 2i), SK-BR-3 (FIG. 2j), and Jurkat (FIG. 2k),

Figure 3 is a photograph showing apoptosis of gastric cancer cell line (AGS) by thiostrepton.

According to the present invention, there is provided the use of thiostrepton as an anticancer agent, and accordingly the present invention provides an anticancer agent of thiostrepton.

Thiostrepton for use as an anticancer agent according to the present invention can be obtained by extracting from a microorganism. For example, thiostrepton is a method of culturing Streptomyces laurentii (eg, ATCC NO. 31,255), which is a type of actinomycetes, separating water layers and cultures by centrifugation, harvesting mycelium, and chloroform on the harvested mycelium masses. Process of obtaining an extract, concentrating the chloroform extract in a vibrating state, treating a small amount of methanol to remove impurities, and then dissolving it in chloroform again, and adding a small amount of methanol to harvest thiostreptone in a crystalline state. Through a series of processes, it can be obtained from Streptomyces laurentii and from other microorganisms such as Streptomyces azureus (eg ATCC NO. 14,921 ), Streptomyces hawaiiensis (eg ATCC 12,236), Streptomyces species .

Dissolving thiostrepton in, for example, DMSO (dimethyl sulfoxide), which is a pharmaceutically acceptable solvent, and treating it with cancer cell lines and normal cells, it shows selective toxicity to cancer cell lines and induces cancer cell death. Will be displayed.

Tiostrepton toxicity on normal cells reported so far has been found to be almost nontoxic at concentrations of 10 μmol or less against mononuclear cells isolated and cultured from human blood. The IC ₅₀ value of thiostrepton for cancer cell lines identified by the inventors is approximately 1 μmol to 5 μmol.

The anticancer composition of thiostrepton according to the present invention or the anticancer composition containing thiostrepton as an active ingredient may be parenterally or orally administered as desired. Thiostrepton may be prepared as an anticancer composition alone or in admixture with a pharmaceutically acceptable carrier. The thiostrepton anticancer composition of the present invention may include a conventionally used excipient, disintegrant, glidant, and the like in a conventional manner.

In addition, the thiostrepton anticancer agent according to the present invention is a unit dosage form or a multi-dose preparation such as tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, liquids or parenteral preparations by well-known conventional methods. It can be formulated as.

The effective dose of the thiostrepton anticancer agent according to the present invention is about 1.7 to 8.3 mg per day per patient weight, preferably 5-8 mg / KG per day.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

Figure 1 is a structural formula of thiostrepton, Figures 2a to 2k are normal cells (MNC: Figure 2a), cancer cell lines AGS (Figure 2b), MKN-74 (Figure 2c), SNU-638 (Figure 2d), Hep G2 (FIG. 2E), SK-Hep1 (FIG. 2F), Hep 3B (FIG. 2G), Lovo (FIG. 2H), KM 1214 (FIG. 2i), SK-BR-3 (FIG. 2J), and Jurkat (FIG. 2K). Figure 3 is a graph showing the effect on the growth, Figure 3 is a photograph showing the apoptosis of gastric cancer cell line (AGS) caused by thiostrepton.

Example 1 Extraction of Thiostrepton

Streptomyces laurentii (ATCC NO. 31,255) was incubated for 65 hours by a standardized known method, the water layer and the culture were separated by centrifugation, and the mycelium was harvested. Next, the mycelium was extracted with chloroform to obtain an extract. The chloroform extract was concentrated in vacuo at 40 ° C., then impurities were removed with methanol, dissolved in chloroform, and finally crystallized by adding methanol. Tone crystals were harvested.

Example 2: Effect of Thiostrepton on Growth of Cancer Cell Lines

Mononuclear cells (MNC), gastric cancer cell lines (AGS, MKN-74, SNU638) isolated from healthy human blood, liver cancer cell lines (Hep G2, SK-Hep1, Hep 3B), colon cancer cell lines (Lovo, KM 1214), breast cancer Cell line (SK-BR-3) and leukemia cell line (Jurkat) were treated with thiostrepton dissolved in DMSO (dimethyl sulfoxide) at various concentrations (10 nmol to 10 μmol) as shown in FIG. 2 and cultured for 72 hours. By measuring the growth rate of cancer cells, the effect of thiostrepton on the growth of the cancer cell lines was evaluated.

Specifically, first, the normal cell MNC and each cancer cell line were liquid cultured in DMED (Hep G2, SK-Hep1) and RPMI (MNC and all other cancer cell lines) containing 10% fetal bovine serum. After spreading on a 6-well plate in the amount of ⁴ -10 ⁵ , it was incubated for 24 hours at 37 ℃ under 5% CO ₂ conditions.

After then cultured in under a number of processes in various concentrations at 37 ℃ for 72 hours in 5% CO ₂ conditions, as shown in the graph of Fig. 2 a thio streptavidin Tondo on each culture medium thereof, irradiation of cell proliferation by MTT assay It was.

MTT analysis Each cancer cell was treated with 5 mg / ml MTT to 1/10 of the culture volume, and left at 37 ° C. for 4 hours to remove the medium. After dissolving the purple MTT pigment staining only living cells using 0.05 M HCl isopropanol solution, the absorbance of the pigment was read at 570 nm. At this time, the absorbance at 630 nm, which is a background wavelength, was subtracted from the value of 570 nm, and then data was processed.

The results of the above test are shown in FIG. 2, and as shown, the blood concentrations of healthy humans are shown in proportion to the specific proportional specific toxicity in the range of concentrations of thiostrepton administered to various cancer cell lines (FIGS. 2B to 2K). It can be seen that there is no effect on the mononuclear cells (MNC: Figure 2a) isolated from.

Example 3: Mechanism of Thiostrepton on Growth of Cancer Cell Lines

The mechanism by which thiostrepton inhibits the growth of cancer cells was observed by an apoptosis assay. The apoptosis assay was performed according to the manufacturer ( Promega's Apoptosis Detection System, Fluorescein protocol).

Specifically, the cancer cell line AGS was first liquid cultured in RPMI medium containing 10% fetal calf serum and spread on a Lab-Tek chamber slide, followed by incubation at 37 ° C. under 5% CO ₂ conditions for 24 hours.

Next, 0.5 nmol thiostrepton was treated in the cancer cell line, incubated for 6 hours, washed twice with PBS (phosphate-buffered saline), and then the cancer cell line was 4 ° C. in 4% methanol-free formaldehyde PBS solution. Treated for 30 minutes at and fixed and washed three times with PBS.

Next, the cancer cell line was treated with 0.2% Triton X-100 PBS solution at room temperature for 15 minutes to permeabilization, and then washed three times with PBS and treated with 100 μl equilibration buffer and left at room temperature for 10 minutes. .

Next, TdT incubation buffer (45 μl equilibration buffer, 5 μl nucleotide mix, and 1 μl TdT enzyme) was treated with the cancer cell line, and left in a humidified chamber at 37 ° C. for 1 hour, and 2 × SSC was placed on the slide. Treated and left at room temperature for 15 minutes.

Finally, washed 3 times with PBS, treated with 1 ㎍ / ml propidium iodide (left) in the dark for 15 minutes, dyed, and washed with deionized water for 5 minutes three times Afterwards, the green fluorescence of apoptotic cells was observed under a fluorescence microscope, and the results are shown in FIG. 3.

As shown in FIG. 3, it can be clearly seen that thiostrepton induces apoptosis in cancer cell lines.

Anti-cancer agent using thiostrepton according to the present invention described above, while showing no toxicity to various cancer cells without showing toxicity to normal cells induces the selective killing of cancer cells only when applied to various cancer patients Therefore, it is useful as cancer treatment.

Claims (2)

An anticancer agent comprising thiostrepton as an active ingredient. [Claim 2] The anticancer agent according to claim 1, wherein the effective amount of thiostrepton is 1.7 mg to 8.3 mg per 1 kg of patient weight per day as an active ingredient.