WO2006054773A1

WO2006054773A1 - Activation of caspase in the cell division stage of cancer cells and utilization of caspase inhibitor in anticancer agent and so on

Info

Publication number: WO2006054773A1
Application number: PCT/JP2005/021470
Authority: WO
Inventors: Shinji Kamada; Ushio Kikkawa; Toshiaki Hashimoto
Original assignee: Shinji Kamada; Ushio Kikkawa; Toshiaki Hashimoto
Priority date: 2004-11-22
Filing date: 2005-11-22
Publication date: 2006-05-26

Abstract

It is newly found out that caspase is activated in the cell division stage of cancer cells. By administering a caspase inhibitor, the progress of cell division is retarded in cancer cells and normal chromosome partition is inhibited in the division stage. Moreover, it is observed that the administration of a caspase inhibitor brings about an effect of inhibiting the proliferation of liver cancer origin-cells, cervical cancer-origin cells, etc. An anticancer agent, which comprises as the active ingredient a caspase inhibitor having the effect of inhibiting cancer cell proliferation as described above, is usable an agent against solid cancer such as liver cancer and cervical cancer.

Description

Specification

Activation of caspases during cancer cell division and use of caspase inhibitors as anticancer agents

Technical field

[0001] The present invention relates to the use of a caspase inhibitor as an anticancer agent. More specifically, the present invention has found a cancer cell growth inhibitory effect as a new effect of a caspase inhibitor. It is used as an agent, functional food with anti-cancer effect, or supplement.

Background art

[0002] Apoptosis is a major mechanism for eliminating cells that are no longer necessary for a living body. Apoptosis dysregulation, i.e., excessive apoptosis, or failure to apoptose, is associated with many diseases such as acute inflammation, autoimmune diseases, ischemic diseases, and neurodegenerative diseases (see below). Non-patent literature 1 · 2).

[0003] Caspase has been identified as a major molecule that executes apoptosis!

Caspase is a cysteine protease having a cysteine residue at the active center and has the activity of cleaving the C-terminal side of aspartate. Caspase is synthesized as an inactive proenzyme (pr oform) and is composed of three regions. Prodomain, large subunit, and small subunit are called prodomain, large subunit, and small subunit from the N-terminal side, and proteolytic processing occurs between the domains, and the large and small subunits form heterodimers and become tetramers. (Active form) (non-patent documents 1 and 3 described later).

[0004] So far, 14 types of caspases have been identified from mammals, and they are roughly classified into those involved in the execution of apoptosis and those involved in the inflammatory response. Further, those involved in apoptosis are classified into an initiator-caspase that functions upstream and an effector-caspase that functions downstream. The initiator-caspase has a long prodomain, and is activated by binding to an adapter molecule in this region, and includes caspases 2, 8, 9, and 10. The effector caspase has a short prodomain, and is cleaved at the C-terminal side of aspartic acid by the cytheter caspase. It is divided into groups and activated. Effector caspases include caspases 3, 6, and 7. An activated effector caspase performs apoptosis by cleaving various substrates (Non-patent Documents 1, 3, 4 described later).

[0005] In recent years, it is thought that by suppressing caspase activity, it is possible to treat diseases caused by abnormal apoptosis, and various caspase inhibitors have been developed and identified. Substances that inhibit caspase enzyme activity include naturally occurring inhibitory proteins and artificially chemically synthesized peptide inhibitors. Naturally occurring inhibitory proteins include IAP family proteins (for example, cIAPl, cIAP2, XIAP, survivin, etc.), p35 protein derived from baculovirus, and crmA protein derived from cowpox virus. Utilizing the property of cleaving the C-terminal side of the peptide, it is an inhibitor containing aspartic acid in its peptide, and reversibly or irreversibly binds to caspase to inhibit its activity. For example, the peptide inhibitor rz-Asp-CH2-DCBj described later is described in Patent Document 14 described later.

[0006] To date, the usefulness of caspase inhibitors to treat various mammalian disease states associated with increased apoptosis has been shown. For example, in preclinical studies using animal models, caspase inhibitors suppress liver-myocardium, kidney, intestine, and brain ischemia-induced apoptosis caused by reperfusion, and preserve the function of these organs. (Non-patent document 5-9 described later). In addition, caspase inhibitors suppress neuronal apoptosis due to traumatic brain injury, atrophic lateral sclerosis, Parkinson's disease, etc. in preclinical studies using animal models (Non-patent Documents 10-12 below) .

[0007] As described above, caspase is considered to be a major factor in the execution of apoptosis, and research and development have been conducted for the treatment of various diseases caused by abnormal apoptosis by negatively controlling its activity. In recent years, new functions of caspases have been reported. For example, cell proliferation, cell motility, cell cycle control, cell differentiation, etc., but the detailed mechanism remains unclear (Non-patent Documents 13-17 below). By elucidating the new functions of these caspases and their points of action, new uses of caspase inhibitors for the treatment of diseases may be found.

[0008] Recently, some types of cancer cells (prostate cancer, breast cancer, colon cancer, etc.) An increase in caspase activity not related to potosis has been reported (see the following non-patent literature)

18). In these cancer cells, an increase in the expression of IAP family proteins, which are caspase inhibitory proteins, was observed at the same time, and the risk of falling into apoptosis due to increased caspase activity was avoided by increasing the expression of caspase inhibitory proteins. (Non-Patent Documents 19-25 below). In addition, attempts have been made to treat cancer by inducing apoptosis specifically in cancer cells by inhibiting the function of these inhibitory proteins (Non-patent Documents 26-28 below).

Patent Document 1: US Patent No. 5985838

Patent Document 2: US Patent No. 6576614

Patent Document 3: Japanese Patent Application Laid-Open No. 7-025865

Patent Document 4: Japanese Patent Laid-Open No. 7-069894

Non-patent literature l: Thornberry et al., Science, 281, 1312-1316 (1998)

Non-Patent Document 2: Nicholson et al., Nature, 407, 810-816 (2000)

Non-Patent Document 3: Earnshaw et al., Annu. Rev. Biochem., 68, 383-424 (1999) Non-Patent Document 4: Fischer et al., Cell Death Differ., 10, 76-100 (2003)

Non-Patent Document 5: Cursio et al "FASEB J., 13, 253-261 (1999)

Non-Patent Document 6: Mocanu et al., Br. J. Pharmacol, 130, 197-200 (2000)

Non-Patent Document 7: Farber et al., J. Vase. Surg., 30, 752-760 (1999)

Non-Patent Document 8: Daemen et al., J. Clin. Invest., 104, 541-549 (1999)

Non-patent document 9: Endres et al., J. Cereb. Blood Flow Metab., 18, 238-247 (1998) Non-patent document 10: Yakovlev et al., J. Neurosci., 17, 7415-7424 (1997)

Non-patent literature ll: Li et al "Science, 288, 335-339 (2000)

Non-Patent Document 12: Schierle et al., Nature Med., 5, 97-100 (1999)

Non-Patent Document 13: Los et al., Trends Immunol. 22, 31-34 (2001)

Non-Patent Document 14: Algeciras- Schimnich et al., Curr. Opin. Cell Biol. 14, 721-726 (200

2)

Non-patent document 15: Schwerk et al., Biochem. Pharmacol. 66, 1453-1458 (2003) Non-patent document 16: Newton et al., Genes Dev. 17, 819-825 (2003) Non-Patent Document 17: Woo et al, Nature Immunol. 4, 1016-1022 (2003)

Non-Patent Document 18: Yang et al., Cancer Res., 63, 6815-6824 (2003)

Non-Patent Document 19: Satoh et al., Cancer, 92, 271-278 (2001)

Non-Patent Document 20: Ambrosini et al., Nat. Med. 3, 917-921 (1997)

Non-Patent Document 21: Serela et al., Ann. Surg. Oncol, 8, 305-310 (2001)

Non-Patent Document 22: Tanaka et al., Clin. Cancer Res., 6, 127-134 (2000)

Non-Patent Document 23: Ferreira et al., Ann. Oncol, 12, 799-805 (2001)

Non-Patent Document 24: Li et al., Endocrinology, 142, 370-380 (2001)

Non-Patent Document 25: Sasaki et al., Cancer Res., 60, 5659-5666 (2000)

Non-Patent Document 26: Liston et al., Nat. Cell Biol, 3, 128-133 (2001)

Non-Patent Document 27: Mesri et al "J. Clin. Investig., 108, 981-990 (2001)

Non-Patent Document 28: Schimmer et al., Cancer Cell, 5, 25-35 (2004)

Disclosure of the invention

Problems to be solved by the invention

[0010] As described above, an increase in caspase activity that is not associated with apoptosis in cancer cells has been reported. However, the reason for the increase in cancer cell-specific caspase activity is unknown, and if the function of cancer cell-specific caspase can be clarified, a new cancer treatment method by inhibiting caspase activity can be found. Can be presented.

[0011] The present invention has been made in view of the above-described circumstances, and the object thereof is the physiological role of caspase in cancer cells, and the availability of caspase inhibitors as anticancer agents. The aim is to provide new anti-cancer drugs targeting caspases based on the findings.

Means for solving the problem

[0012] As a result of diligent research in view of the above problems, the present inventor has (1) that an active form of caspase is detected specifically in the cell division stage of cancer cells, and (2) a caspase inhibitor. Administration of this drug causes delay in cell division in cancer cells, inhibits normal chromosomal segregation during mitosis, and (3) administers a caspase inhibitor. For cancer cells derived from liver cancer, cervical cancer, and acute T-cell leukemia As a result, the inventors have found that a growth inhibitory effect has been recognized, and have completed the present invention.

That is, the present invention includes the following inventions as medically and industrially useful inventions.

A) A pile cancer agent containing a caspase inhibitor as an active ingredient. Caspase inhibitors include (1) substances that have a substrate-like structure, etc., that bind to the active site of caspase, thereby inhibiting the activity of force spurase, and (2) interact with sites other than the active site. , A substance that inhibits caspase activity, (3) a substance that inhibits caspase activity and inhibits force spase, and (4) a substance that inhibits and inhibits caspase expression. Substances that directly or indirectly inhibit caspases, such as substances, are widely included.

B) A pile cancer agent containing as an active ingredient an inhibitor of caspase activated during the cell division phase of cancer cells.

C) An anticancer agent comprising as an active ingredient any one or more of caspases 1, 3, 4, 7, 8, and 9.

D) The anticancer agent according to any one of the above A) to C), which is applied to solid cancers such as liver cancer and cervical cancer.

E) The anticancer agent according to any one of A) to C) above, wherein the caspase inhibitor is a peptidic compound, a non-peptidic compound, or a protein derived from a living organism.

F) The anticancer agent according to E) above, wherein the caspase inhibitor is any one of the following compounds (1) to (8).

(1) Z- Asp- CH2- DCB

(2) Boc-Asp (OMe) -FMK

(3) Boc- Asp (OBzl)-CMK

(4) Ac-AAVALLPAVLLALLAP-YVAD-CHO

(5) Ac- AAVALLPAVLLALLAP- DEVD- CHO

(6) Ac- AAVALLPAVLLALLAP- LEVD- CHO

(7) Ac— AAVALLPAVLLALLAP— IETD— CHO

(8) Ac- AAVALLPAVLLALLAP- LEHD- CHO G) An edible composition having an anticancer effect by containing a caspase inhibitor.

H) A method for suppressing the growth of cancer cells by introducing RNA that specifically suppresses the expression of caspase protein into the cancer cells.

The RNA (RNAi) may be a siRNA (short interference RNA: also referred to as “short interfering RNA”, “small interfering RNA”, etc.) or an RNAi expression vector (“siRNA expression vector”). ). siRNA and RNAi expression vectors can be designed according to known methods based on the gene sequence of the caspase to be suppressed (for example, Ambion TechNotes 9 (1): 3-5 (2002), Proc. Natl. Acad. Sci. USA 99 (8): 5515-5520 (2002), Proc. Natl. Acad. Sci. USA 99 (9): 6047-6052 (2 002), Nature Biotechnology 20: 505-508 (2002) )). The RNAi expression vector (1) is designed to express dsRNA having a hairpin structure of an appropriate length with a single RNA in the target cell, (2) sense strand, antisense strand Any of those designed to express and associate with each other in the target cell may be used.

RNA can be introduced into cancer cells according to a conventional method (see, for example, Nature 411: 494-498 (2001), Science 296: 550-553 (2002)). It is also possible to use the method developed in (1).

I) Stake cancer agent consisting of RNA that specifically suppresses caspase protein expression, or RNAi expression vector constructed to express the RNA in target cancer cells.

As the “RNAi expression vector”, a viral vector, plasmid, phage, cosmid or the like can be used, and a promoter that functions in the target cancer cell (for example, an RNA polymerase III promoter such as U6 or H1 promoter, or RNA polymerase II promoters etc.) may be used that are incorporated upstream of the siRNA sequence that expresses it.

Here, “specifically suppress caspase protein expression” is sufficient if it substantially reduces the expression level of caspase protein in the target cancer cell. Caspase protein expression is completely suppressed. It does not have to be.

The invention's effect [0014] The present invention utilizes a caspase inhibitor as an anticancer agent or a functional food having an anticancer effect. Conventionally, caspase inhibitors have been researched and developed from the viewpoint of suppressing / preventing apoptosis (for example, treatment of neurodegenerative diseases caused by abnormal apoptosis). The present invention has been found that caspases are activated during the cell division phase of cancer cells and are involved in the control thereof. Further, based on this finding, a new use of suppressing the growth of cancer cells by caspase inhibitors is provided. It is to provide.

[0015] Thus, according to the present invention, the growth of cancer cells can be suppressed by inhibiting caspases that are thought to play an important role in the execution of apoptosis. Despite being non-apoptotic in many cancer cells, caspase activity is increased, and caspase inhibitors are likely to act specifically on cancer cells. Conventional acupuncture, radiation therapy, etc. used for cancer treatment are aimed at inducing apoptosis in cells, and have a great influence on normal cells other than cancer cells. The effect is a big problem. By using the anticancer agent of the present invention that targets a caspase that specifically activates cancer cells, the establishment of a new type of cancer treatment method that has extremely low side effects and is unprecedented can be expected.

Brief Description of Drawings

[0016] FIG. 1 is a diagram showing the activity of caspase 3 specific to cell division phase by immunofluorescence staining. (a) is the result of using an antibody that recognizes the large subunit of caspase 3 (pi 7), (b) is the result of using an antibody that recognizes the small subunit of caspase 3 (pi 2)

FIG. 2 is a diagram showing the results of examining caspase activity during cell division by treating hepatoma-derived HepG2 cells with nocodazole. (A) shows the results of cell cycle analysis by flow cytometry, and (b) shows the results of examining the activity of caspase by Western blotting and the presence or absence of substrate cleavage by caspase.

FIG. 3 shows the results of examining caspase activity during cell division by treating cervical cancer-derived HeLa cells with thymidine. (A) shows the results of cell cycle analysis by flow cytometry. (B) shows the activity of caspase and caspase by Western blotting. The result of having investigated the presence or absence of the substrate cleavage by a case is shown.

FIG. 4 is a graph showing the delay in cell cycle progression by caspase inhibitors.

FIG. 5 is a graph showing an increase in the number of cells having condensed nuclei by caspase inhibitors.

FIG. 6 is a diagram showing inhibition of progression of chromosome segregation during cell division by caspase inhibitors.

FIG. 7 is a graph showing the effect of suppressing proliferation of cancer cells by a general-purpose caspase inhibitor.

FIG. 8 is a graph showing the growth inhibitory effect of each caspase-specific inhibitor on HepG2 cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Hereinafter, preferred embodiments of the present invention will be described. In the present specification and drawings, when a compound such as an amino acid is represented by an abbreviation, the notation is based on an abbreviation by IUPAC-IUB Commission on Biochemical Nomenclature or an abbreviation commonly used in the field.

[0018] [1] Anticancer agent of the present invention

As described above, the anticancer agent of the present invention comprises a caspase inhibitor, that is, a caspase inhibitor as an active ingredient. As shown in the examples below, administration of several caspase inhibitors actually suppressed and inhibited the growth of hepatoma-derived HepG2 cells and cervical cancer-derived HeLa cells. (Figures 7 and 8).

Traditionally, caspases are active during apoptosis and positively control apoptosis, so inhibitors have been used to suppress apoptosis. In contrast to this, the present inventor newly discovered that caspases are activated specifically in the cell division phase in cancer cells such as HepG2 cells and HeLa cells in the process of elucidating the various physiological roles of caspases. (Figure 1-3). In addition, administration of caspase inhibitors slowed cell cycle progression in cancer cells and inhibited normal chromosome segregation during mitosis (Figure 46). Details will be described later.

[0019] Based on the above new findings, the present invention confirms the growth inhibitory effect of caspase inhibitors against cancer cells derived from liver cancer and cervical cancer, and provides anticancer drugs against caspase inhibitors. It provides a novel method of use as an agent. [0020] Since the caspase inhibitor also suppressed the proliferation of urkat cells due to acute T cell leukemia (FIG. 7), the anticancer agent of the present invention can also be used for the treatment of cancer such as leukemia. In addition, caspase activity in a non-apoptotic state in cancer cells has been reported in prostate cancer, breast cancer, colon cancer, etc., and therefore caspase inhibitors can also be used against these cancer cells. May suppress proliferation. Furthermore, if caspase activity in cancer cells in a non-apoptotic state is widely recognized, it becomes possible to use caspase inhibitors as anticancer agents by inhibiting growth for all cancers. .

In other words, the anticancer agent of the present invention can be applied to cancer cells in which caspase activation is observed in the cell division phase. Examples include use in various solid cancers such as cervical cancer and squamous cell carcinomas such as cervical cancer.

[0022] The type of caspase to be inhibited by the caspase inhibitor is not particularly limited, but the caspase 1, 3, 4, 7, 8, and 9 inhibitors were actually observed to have a growth inhibitory effect. Therefore (FIG. 8), it is preferable to use a caspase inhibitor that inhibits one or more of caspases 1, 3, 4, 7, 8, and 9. In particular, activity in the mitotic phase has been confirmed in liver cancer, cervical cancer, etc., where the use of inhibitors of caspases 1, 3, 4, and 7 that have a high growth inhibitory effect is preferred. Particularly preferred is the use of inhibitors against caspases 3,7. In addition, a general-purpose caspase inhibitor that inhibits multiple types of caspases may be used, or a caspase inhibitor that controls upstream activity of caspase 3 and the like may be used.

[0023] The caspase inhibitor, that is, the caspase inhibitor, may be any of a peptide compound, a non-peptide compound, or a biological protein. Examples of peptidic compounds include artificially chemically synthesized peptidic compounds (1) to (8) below.

(1) Z- Asp- CH2- DCB (Molecular weight 454. 26)

(2) Boc- Asp (OMe)-FMK (Molecular weight 263.3)

(3) Boc- Asp (OBzl)-CMK (Molecular weight 355.8)

(4) Ac-AAVALLPAVLLALLAP-YVAD-CHO (Molecular weight 1990.5) (5) Ac- AAVALLPAVLLALLAP- DEVD- CHO (Molecular weight 2000. 4)

(6) Ac- AAVALLPAVLLALLAP- LEVD- CHO (Molecular weight 1998.5)

(7) Ac— AAVALLPAVLLALLAP— IETD— CHO (Molecular weight 2000. 5)

(8) Ac- AAVALLPAVLLALLAP- LEHD- CHO (Molecular weight 2036.5)

[0024] The compounds (1) to (3) are!, All of which are cell membrane permeable and are general-purpose caspase inhibitors that inhibit multiple types of caspases. Z-Asp-CH2-DCB in (1) is the official name Benzyloxycarbo-Lu L-Spartau 1-Lu [(2, 6-Dichloro-benzo Noreno ^3- r)] methane (Benzyloxycarbonyl— L— Aspart— 1— y ([2,6—Dichlorobenzoyl) oxy ”me thane). (2) Boc-Asp (OMe) -FMK is the official name N— (tert-butoxycarbo-nore) aspanoretinore (O-methinore)- Fnorolelomethinoleketone (N- (tert-butoxycarbonyl) aspartyl (0-methyl) -fluoromethylketone). (3) Boc- Asp (OBzl) -CMK is the official name N ― (tert-butoxycarbo -L) Aspartyl (O-benzyl) Chloromethylketone (N- (tert-Dutoxycarbonyl) aspartyl (0-benzyl)-chioromethylketone) & Z- Asp- CH2- DCB ゝ Boc- Asp (OMe)-FMKゝ Boc- Asp (OBzl) -CMK inhibited the growth of HepG2 cells, HeLa cells, and Jurkat cells in a concentration-dependent manner (Fig. 7). CMK, Z-Asp-CH2-DCB, and Boc-Asp (OMe) -FMK were favorable in this order.

[0025] The above (4) is a caspase 1 inhibitor, and cell membrane permeability is present on the N-terminal side of the 4 amino acids of YVAD (ie, tyrosine monoparin / alanine / aspartate) involved in caspase 1 inhibition. In order to enhance, a hydrophobic region of force-positive fibroblast growth factor (Kaposi fibroblast growth factor) having the following amino acid sequence ability is added.

Amino acid 酉己歹 U: AAVALLPAVLLALLAP (ie, Alanine, Alanine, Parin, Lanin, Leucine, Leucine, Proline, Alanine, and Parin, Leucine, Leucine, Alanine, Leucine, Leucine, Alanine, Proline)

[0026] The above (5) is a caspase 3 and 7 inhibitor, and permeates through the cell membrane on the N-terminal side of the 4 amino acids of DEVD (ie, aspartate glutamate monoparin aspartate) involved in the inhibition of caspase 3 and 7. In order to enhance the properties, a hydrophobic region of the force positive fibroblast growth factor is added. [0027] The above (6) is a caspase 4 inhibitor. Cell membrane permeability is increased on the N-terminal side of the 4 amino acids of LEVD (ie, leucine monoglutamate monoparin monoaspartate) involved in the inhibition of caspase 4. To enhance, a hydrophobic region of the force positive fibroblast growth factor is given.

[0028] The above (7) is a caspase 8 inhibitor, and it has cell membrane permeability on the N-terminal side of 4 amino acids of IETD (ie isoleucine glutamate 1 threonine 1 aspartate) involved in caspase 8 inhibition. In order to enhance, a hydrophobic region of the force positive fibroblast growth factor is added.

[0029] The above (8) is a caspase 9 inhibitor, in order to enhance cell membrane permeability on the N-terminal side of the 4 amino acids of LEHD (ie, leucine glutamate histidine aspartate) involved in caspase 9 inhibition. The hydrophobic region of the force positive fibroblast growth factor is given.

[0030] The compounds of (4) to (8) above have an acetyl group (Ac) on the N-terminal side and an aldehyde group (CHO) on the C-terminal side of the oligopeptide having a total of 20 amino acids. Have Each of these caspase specific inhibitors showed growth inhibitory activity against HepG2 cells, although there was a difference in their inhibitory effects (Fig. 8).

[0031] The compounds (1) to (8) can be easily produced by using known chemical synthesis methods such as various existing peptide synthesis methods. In addition, peptide compounds are not limited to these compounds, and other peptide compounds that can suppress / inhibit caspase activity may be used in the anticancer agent of the present invention. Oh ,.

[0032] For example, as a caspase inhibitor of a peptide compound, (l) VX-740-Vertex Pharmeuticals (Leung-Toung et al "Curr. Med. Chem. 9, 979-1002 (2002), (2) HMR -3480-Aventis Pharma AG (Randle et al., Expert Opin. Investig. Drugs 10, 1207-1209 (2001).

Non-peptide compound caspase inhibitors include (1) anlinoqu inazolines (AQZs) -AstraZeneca Pharmaceuticals (Scott et al "J. Pnarmacol. Exp. Ther. 304, 433-440 ( 2003)), (2) M826-Merck Frosst (Han et al., J. Biol. Chem. 277, 30128-30136 (2002)), (3) M867-Merck Frosst (Methot et al., J. Exp. Med. 199, 199-207 (2004)), (4) Nicotinyl aspartyl ketones-Merck Frosst (Isabel et al., Bioorg. Med. Chem. Lett. 13, 2137-2140 (2003) )), Etc.

Further, as caspase inhibitors of other non-peptidic compounds, (l) IDN-6556-Id un Pharmaceuticals (Hoglen et al., J. Pharmacol. Exp. Ther. 309, 634-640 (2004), (2) MF-286 and MF-867-Merck Frosst (Los et al., Drug Discov. Today 8, 67-77 (2003), (3) IDN— 5370-Idun Pharmaceuticals (Deckwerth et al., Drug Dev. Res. 52 ,

579-586 (2001), (4) IDN— 1965-Idun Pharmaceuticals (Hoglen et al "J. Pharmaco 1. Exp. Ther. 297, 811—818 (2001), (5) VX-799-Vertex Pharmaceuticals (Los et a 1., Drug Discov. Today 8, 67-77 (2003)), etc. In addition, M-920 and M—791-Merck Frosst (Hotchkiss et al., Nat. Immunol. 1, 496-501 (2000) and the like can also be mentioned as caspase inhibitors.

Biologically derived proteins include IAP family proteins (eg cIAPl, cIAP2, XIAP, survivin, etc.) that are caspase activity-inhibiting proteins, p35 protein derived from vaccinia virus, crmA protein derived from cowpox virus, etc. Can be illustrated. By expressing these proteins specifically for cancer cells, it is possible to suppress the growth of cancer cells. Examples of a method for expressing a protein in cancer cells include use of a gene expression vector encoding the protein. In the case of this method, the modified protein may be expressed artificially by substituting, deleting, inserting, and Z or adding one or several bases in the base sequence of the gene expression vector according to a conventional method. Good.

Still other caspase inhibitors include RNA that specifically suppresses caspase protein expression, and RNAi expression vectors constructed to express the RNA in target cancer cells, as described above. In fact, when RNAi method was used to reduce the amount of multiple caspase proteins in cancer cells, the results showed that cancer cell growth was suppressed. Therefore, siRNA that suppresses the expression of caspase protein can be used for cancer treatment. As a method of introducing siRNA into cancer cells, vectors and nucleic acids can be used to transfer specific vectors or nucleic acids in order to efficiently and selectively introduce siRNA into target cancer cells. Known carriers and drug delivery systems proposed to be transported to specific cells can be used.

The siRNA and RNAi expression vectors can be designed based on the caspase gene sequence as described above. For example, the cDNA sequence and amino acid sequence of human caspase 3 are disclosed in DDBJ / EMBL / GenBank databases, such as accession numbers “NM_004346” and “NM_032991,” and the target sequence is determined based on these sequence information. It is possible to design and prepare siRNA and RNAi expression vectors that can be determined and suppress caspase protein expression.

[0034] As described above, the anticancer agent of the present invention is a caspase inhibitor, that is, a caspase inhibitory substance as an active ingredient. Not only a known caspase inhibitor but also a caspase inhibitor found in the future. May be used. Caspase inhibitors include (1) substances that have a substrate-like structure, etc., that bind to the active site of caspase, thereby inhibiting the activity of force spurase, and (2) interact with sites other than the active site. , A substance that inhibits caspase activity, (3) a substance that inhibits caspase activity and inhibits force spase, and (4) a substance that inhibits and inhibits caspase expression. Substances that directly or indirectly inhibit caspases, such as substances, are widely included. For example, in an experimental system in which apoptosis is induced in cells by the activity of caspase, a substance that suppresses apoptosis can also be said to be a substance that directly or indirectly inhibits caspase, and as a caspase inhibitor of the present invention. Can be used.

[0035] When a substance that inhibits the enzyme activity of caspase is used, the degree of inhibition is not particularly limited, but in a normal enzyme activity assay performed in vivo or in vitro, a 50% inhibitory concentration is used. It is preferable to have a high inhibitory activity of several picomoles (pM) to several tens of micromoles M), in vivo several micromoles M) or less, and in vitro, several hundred nanomoles (nM) or less. More preferred,

[0036] [2] Examples of use of the anticancer agent of the present invention

The above is the case where a caspase inhibitor is used as an anticancer agent (anticancer agent). However, the present invention is not limited to this. A caspase inhibitor is a food such as a functional food or a supplement (edible composition). Food with anti-cancer and cancer-preventing effects It can also be used for product development. Alternatively, it can be used as a raw material for cosmetics, and can be used to develop cosmetics that have anticancer effects and cancer prevention effects.

Hereinafter, an example of using a caspase inhibitor as an anticancer agent will be described. The caspase inhibitor can be administered to humans (or animals) as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary. For example, oral preparations such as tablets, capsules, granules, fine granules, powders, injections, etc. Parenterals such as suppositories, suppositories, and coating agents.

[0038] Oral preparations such as tablets, capsules, granules, fine granules, powders and the like are commonly used, for example, starch, lactose, sucrose, trenorose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, etc. Manufactured according to. The amount of caspase inhibitor in these preparations is not particularly limited and can be set as appropriate. In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, flavors, and the like can be appropriately used.

[0039] In the case of parenteral agents, the dosage is adjusted according to the patient's age, weight, disease severity, etc., for example, intravenous injection, intravenous infusion, subcutaneous injection, intraperitoneal injection, intramuscular injection, intratumoral injection For example, systemically or locally. This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent. Further, if necessary, bactericides, preservatives and stabilizers may be added. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling in a vial or the like, water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use. Furthermore, an isotonic agent, stabilizer, preservative, and soothing agent may be added as necessary. The compounding amount of the caspase inhibitor in these preparations is not particularly limited and can be arbitrarily set. Examples of other parenteral agents include liquid preparations for external use, coating agents such as ointments, suppositories for rectal administration, etc., and these are also produced according to conventional methods.

[0040] It should be noted that, using a known DDS (drug 'delivery' system), for example, a caspase inhibitor or a gene expression vector encoding a protein acting as an inhibitor is enclosed in a carrier such as a ribosome. It may be administered. At this time, the target site (cancer cell) is specific If a carrier that recognizes the target is used, the caspase inhibitor can be efficiently transported to the target site.

[0041] As described above, the caspase inhibitor can be used in foods (edible compositions) such as supplements and functional foods. In other words, it is added to caspase inhibitors as raw materials for various beverages and various processed foods, and if necessary, it can be added to pellets, tablets, granules, etc. together with excipients such as dextrin, lactose, starch, flavorings, pigments, etc. It can be processed, or coated with gelatin and molded into capsules for use as health food or health food. Example

Hereinafter, examples of the present invention will be described with reference to the drawings, but the present invention is not limited to these examples.

[Example 1: Activation of caspase during cell division of cancer cells]

First, in order to investigate the activity of caspase 3 in a non-apoptotic state of cancer cells, the following experiment was conducted.

[0043] HepG2 cells derived from hepatoma cells were seeded at 2 x 10 ⁵ cells per well in a 6-well dish with a cover glass in the bottom and cultured for 24 hours. 3. After fixing with phosphate buffer containing 7% formaldehyde for 10 minutes, wash twice with phosphate buffer, treat with phosphate buffer containing 0.5% Triton X-100 for 10 minutes, Washed twice with phosphate buffer.

[0044] The cells treated in this manner were subjected to 4 ° overnight in a phosphate buffer (containing 1% ushi serum albumin) containing anti-active caspase 3 antibody and anti-tubulin antibody as primary antibodies. Incubation with C. After washing twice with phosphate buffer, it was incubated for 10 minutes in a phosphate buffer containing a secondary antibody labeled with TXRD or FITC, and then washed twice with phosphate buffer. Nuclei were stained with 10 _i u M Hoechst 33342 (Calbiochem) and then examined with a fluorescence microscope (product name Laborlux, Leitz).

[0045] Fig. 1 (a) shows the result of using an antibody that recognizes the C terminus of the large subunit (pl7) of active caspase 3 as the primary antibody. The result of using an antibody that recognizes the N-terminus of the unit (p 12). In the figure, cells other than the interphase show tubulin polymerization and chromosome condensation, and are considered to be in the cell division phase. It can be seen that caspase 3 is active in the early, early, middle, late, and late stages of cell division. The

[0046] Furthermore, in order to confirm the activity of caspase specific to cell division phase, the following experiment was performed using HepG2 cells derived from liver cancer and HeLa cells derived from cervical cancer.

[0047] First, HepG2 cells (1 X 10 ⁶ / 6cm dish) were cultured in nocodazole (0. 8 μ g / ml) in the presence, over time the cells were harvested and subjected to Western blotting, activity I spoon We detected caspase 3 (and caspase 8 · 9) active fragments as a result of cleavage by the above method. The accumulation of mitotic cells by nocodazole treatment was confirmed by flow cytometry c

[0048] As shown in Fig. 2 (a), the number of cells (= cells with DNA content) increased with time, and the number of cells (cell number) increased with nocodazole treatment. After time, over 80% of the cells were in mitotic phase. As shown in FIG. 2 (b), the active form fragment of caspase 3 was detected 12 hours after nocodazole treatment and increased with time. In addition, caspase 8 and caspase 9 active form fragments were detected in the same time course, and the caspase 3 substrates poly ADP ribose polymerase (PARP), lamin Bl (LaminBl), protein kinase C δ (PKC δ) cleavage (cleav age fragment) was also detected. Lane A in the figure is a cell in which apoptosis was induced with an anti-Fas antibody as a control.

[0049] From these results, it was confirmed that caspase 3 (and 8.9) was activated in the cell division phase of HepG2 cells derived from liver cancer.

[0050] Next, cell-phase specific caspase activation in cervical cancer-derived HeLa cells was examined. HeLa cells (4 × 10 ⁵ Z6 cm dishes) were cultured for 24 hours and then cultured for 18 hours in the presence of 2.5 mM thymidine. After washing 3 times with phosphate buffer, a new medium was added and the cells were cultured for 10 hours, and again cultured in the presence of 2.5 mM thymidine for 14 hours. After washing 3 times with phosphate buffer, the cells are cultured in a new medium from which thymidine has been removed. The cells are collected over time, and fragments of caspases 3/8, 9 cleaved by active protein by Western blotting are collected. Detected. Progression of the cell cycle at each recovery was confirmed by flow cytometry.

[0051] By performing thymidine treatment twice, the cells can be stopped at the boundary between almost 100% G1 phase and S phase. As shown in Figure 3 (a), when thymidine is removed from the culture (release), the cells Begins around the cell cycle again, and after thymidine removal, the cells are in the G2ZM phase approximately 8-12 hours and reenter the G1 phase approximately 14 hours later.

[0052] As shown in Fig. 3 (b), caspase 3, caspase 8 and caspase 9 active form fragments were detected 10-14 hours after thymidine removal. In addition, cleavage of the caspase 3 substrates poly ADP ribose polymerase (PARP), lamin Bl (LaminBl), and protein kinase C δ (PKC δ) was also detected at the same time. In the figure, lane N is the result of HeLa cells cultured under normal conditions, and lane A is the result of cells in which apoptosis was induced with anti-Fas antibody.

[0053] From the above results, caspase activity specific to cell division was also confirmed in cervical cancer-derived HeLa cells.

[Example 2: Delayed progression of cell division by caspase inhibitor]

As described above, caspase activation was confirmed specifically in the cell division phase of cancer cells. Next, its role was examined.

[0055] In the same manner as in Example 1, the cell cycle of HeLa cells was stopped at the boundary between the G1 phase and the S phase using thymidine. Then, a new medium was added to resume cell cycle progression, and after 7 hours, Z-Asp-CH2-DCB (final concentration in culture medium: 200 μΜ), a cell membrane-permeable general-purpose caspase inhibitor, was added. The cells were dissolved in dimethyl sulfoxide (DMSO) and added, and the progression of the cell cycle was examined by flow cytometry.

The results are shown in FIG. The control medium was supplemented with 0.5% DMSO in the same manner as when the inhibitor was added. Cells with inhibitor and control cells were both in G2ZM phase after 8 hours.

[0057] Thereafter, most cells in control cells were in M phase after 10 hours, some cells progressed to G1 phase, and most cells entered G1 phase after 14 hours. In contrast, a cell cycle delay of about 2 hours was observed in cells to which a caspase inhibitor was added, compared to control cells. From these results, it was shown that caspase inhibitors delay the progression of the cell cycle of cancer cells by inhibiting the activity of caspases activated during the cell division stage of HeLa cells.

[Example 3: Prevention of normal chromosome segregation during cell division by caspase inhibitor] Furthermore, in order to examine the effect of caspase inhibitors on the cell division phase, HepG2 cells and HaLa cells were cultured in the presence of the caspase inhibitors to examine changes in the morphology of the nucleus. HepG2 cells or HeLa cells were seeded at 2 × 10 ⁵ cells per lwell in a 6-well dish with a cover glass at the bottom. The next day, Z-Asp-CH2-DCB (final concentration 200 μΜ) or DMSO as a control was covered, and each day, the nuclei were stained with 10 ΜHoechst 33342. The proportion was examined. As a result, apoptosis was suppressed by the caspase inhibitor, and cells having fragmented nuclei were not observed, but cell nuclei corresponding to the early to middle cell division were observed. In addition, as shown in FIG. 5, an increase in the proportion of cells with condensed nucleus (nuclear condensation) different from nuclear degeneration due to apoptosis was observed by treatment with caspase inhibitors for both HepG2 cells and HeLa cells.

[0059] Next, in order to clarify the action point of the caspase inhibitor in the cell cycle, the morphological change of the nucleus over time was analyzed using a confocal laser microscope. In order to visualize the chromosome, HeLa cells (HeLa-GFP-H2B) in which histone H2B was fused with GFP and highly expressed were used. Seed these HeLa-GFP-H2B cells (2 X 10 ⁵ cells Z3.5 cm glass bottom dish), and add Z-Asp-CH2-DCB (final concentration 300 μΜ) or DMSO as a control the next day. After 1 day, the morphological changes of the nucleus were observed for 1 to 2 hours at 1 minute intervals using a confocal laser microscope.

[0060] Figure 6 shows typical nuclear morphology of cells treated with control and caspase inhibitors. In control cells with DMSO, cell division progressed normally, and chromosome condensing power progressed to separation in about 90 to 120 minutes (Figs. 6a and b).

[0061] In contrast, cells in which caspase inhibitor was added to the culture solution to a final concentration of 300 μΜ showed obvious abnormalities in the progression of the cell division phase, and cells with chromosomes dividing into three (Fig. C and d in 6), cells in which condensation of chromosomes was not completed in 120 minutes (e and f in Fig. 6), cells that remained in the middle stage and chromosome division did not proceed (g in Fig. 6), etc. were observed . These results indicate that caspase inhibitors inhibit chromosome condensation and division during cell division.

[Example 4: Inhibition of cancer cell proliferation by caspase inhibitor]

From the above, caspase was activated during the cell division stage of cancer cells, and caspase inhibitor was It has been shown to inhibit cancer cell division, especially chromosome segregation. In some cancer cells, caspase 3 activity increases despite being in a non-apoptotic state, suggesting that some functions specific to cancer cells are occurring. The possibility of functioning as a cell-specific cell growth inhibitor was considered. Therefore, in order to investigate this possibility, the following experiment was conducted.

[0063] WST-1 reagent (Roche) was used for the cell proliferation assay. HepG2 cells derived from liver cancer, HeLa cells derived from cervical cancer, or urkat cells derived from acute T cell leukemia were seeded in 96 well dishes at 4 × 10 ³ cells per well and cultured for 24 hours. After each caspase inhibitor is prepared at each concentration, WST-1 reagent is prepared every day, and the dehydrogenase activity localized in mitochondria is measured by measuring the absorbance at 450 nm and 690 nm. And used as an index of cell proliferation.

[0064] As caspase inhibitors, the following compounds (1) to (8) were used.

(1) Z- Asp- CH2- DCB

(2) Boc-Asp (OMe) -FMK

(3) Boc- Asp (OBzl)-CMK

(4) Ac-AAVALLPAVLLALLAP-YVAD-CHO (Caspase 1 inhibitor)

(5) Ac- AAVALLPAVLLALLAP- DEVD- CHO (Caspase 3 and 7 inhibitor)

(6) Ac- AAVALLPAVLLALLAP- LEVD- CHO (Caspase 4 inhibitor)

(7) Ac- AAVALLPAVLLALLAP- IETD-CHO (caspase 8 inhibitor)

(8) Ac- AAVALLPAVLLALLAP- LEHD-CHO (Caspase 9 inhibitor)

[0065] The above (1) to (3) are cell membrane permeation-type versatile inhibitors. A hydrophobic region of force-positive fibroblast growth factor is added to the N-terminal side of each of the caspase-specific inhibitors (4) to (8) above in order to enhance cell membrane permeability. (1) is an inhibitor made by Peptide Institute, and (2) to (8) are inhibitors made by Calbiochem. All inhibitors were dissolved in DMSO, and a portion of it was added to the culture medium, so the control burned DMSO at the same concentration as the inhibitor was added.

[0066] The experimental results are shown in FIG. 7 and FIG. The inhibitor concentration shown in each graph is the final concentration in the culture medium. Z-Asp-CH2-DC is a cell membrane permeation inhibitor B, Boc-Asp (OMe) -FMK and Boc-Asp (OBzl) -CMK all inhibited the growth of HepG2, HeLa, and Jurkat cells in a concentration-dependent manner (FIG. 7). Each caspase-specific inhibitor showed cytostatic activity against HepG2 cells, although there was a difference in the inhibitory effect (Fig. 8).

Industrial applicability

As described above, the anticancer agent of the present invention comprises a caspase inhibitor that has been shown to be effective in inhibiting the growth of cancer cells as an active ingredient, and solids such as liver cancer and cervical cancer It can be used as an anticancer agent against cancer.

The present invention can also be used as functional foods, supplements and the like having an anticancer effect and a cancer prevention effect.

Claims

Claims [1] A pile cancer agent comprising a caspase inhibitor as an active ingredient. [2] An anticancer agent comprising as an active ingredient an inhibitor of caspase activated during the cell division phase of cancer cells. [3] An anticancer agent comprising as an active ingredient any one or more of caspases 1, 3, 4, 7, 8, and 9. [4] The anticancer agent according to any one of claims 1 to 3, which is applied to solid cancers such as liver cancer and cervical cancer. [5] The anticancer agent according to any one of claims 1 to 3, wherein the caspase inhibitor is a peptide compound, a non-peptide compound, or a protein derived from a living organism. [6] The anticancer agent according to claim 5, wherein the caspase inhibitor is any one of the following compounds (1) to (8).

(1) Z- Asp- CH2- DCB

(2) Boc-Asp (OMe) -FMK

(3) Boc- Asp (OBzl)-CMK

(4) Ac-AAVALLPAVLLALLAP-YVAD-CHO

(5) Ac- AAVALLPAVLLALLAP- DEVD- CHO

(6) Ac- AAVALLPAVLLALLAP- LEVD- CHO

(7) Ac— AAVALLPAVLLALLAP— IETD— CHO

(8) Ac- AAVALLPAVLLALLAP- LEHD- CHO

[7] An edible composition having an anticancer effect by containing a caspase inhibitor.

[8] A method for suppressing the growth of cancer cells by introducing RNA that specifically suppresses the expression of caspase protein into the cancer cells.

[9] RNA that specifically suppresses the expression of caspase protein, or a stake cancer agent such as an RNAi expression vector constructed to express the RNA in target cancer cells.