JP2006169242A - Activation of caspase in cell division stage of cancer cell and utilization of caspase inhibitor as anticancer agent or the like - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new anticancer agent capable of being aimed at caspase as a target, by investigating a physiological function of the caspase in a cancer cell and a possibility of utilization of a caspase inhibitor as the anticancer agent and then developing the agent based on information obtained thereby. <P>SOLUTION: This anticancer agent contains the caspase inhibitor as an active ingredient, wherein the agent is developed based on a new discovery that the caspase is activated in a cell division stage of the cancer cell, further advance of cell division in the cancer cell is retarded and normal chromosome disjunction in the division stage is inhibited, when the caspase inhibitor is administered, and furthermore an antiproliferative effect on a cell derived from lever cancer, a cell derived from cervical cancer, etc., is recognized, when the caspase inhibitor is administered. Thus, the agent contains the caspase inhibitor which is recognized to have the antiproliferative effect on the cancer cell, so that the agent is useful as an anticancer agent against solid cancer, including the liver cancer and the uterine cervical cancer. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the use of a caspase inhibitor as an anticancer agent, and more specifically, as a new effect of a caspase inhibitor, a cancer cell growth inhibitory effect is found, and this is used as an anticancer agent, It is used as a functional food or supplement with anticancer effects.

  Apoptosis is a major mechanism for eliminating cells that are no longer needed by the body. Poor control of apoptosis, i.e., excessive apoptosis or failure of apoptosis, has been implicated in a number of diseases such as acute inflammation, autoimmune diseases, ischemic diseases, and neurodegenerative diseases (non-patents below) References 1 and 2).

  Caspase has been identified as the main molecule that performs apoptosis. Caspase is a cysteine protease having a cysteine residue at the active center and has an activity of cleaving the C-terminal side of aspartic acid. Caspases are synthesized as inactive proenzymes (proforms) and are composed of three regions. From the N-terminal side, it is called a prodomain, large subunit, or small subunit, and proteolytic processing occurs between the domains, and the large and small subunits form heterodimers and become tetramers to form active enzymes ( active form) (non-patent documents 1 and 3 described later).

  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 reaction. Those involved in apoptosis are further classified into initiator caspases that function upstream and effector caspases that function downstream. Initiator caspases have a long prodomain and are activated by binding to adapter molecules in this region, and include 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 initiator caspase, and is divided into large and small subunits and activated. Effector caspases include caspases 3, 6, and 7. The activated effector caspase performs apoptosis by cleaving various substrates (Non-patent Documents 1, 3 and 4 described later).

  In recent years, it has been 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 the enzyme activity of caspases include naturally occurring inhibitory proteins and artificially chemically synthesized peptide inhibitors. Naturally occurring inhibitory proteins include IAP family proteins (eg, cIAP1, 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, it is an inhibitor containing aspartic acid in its peptide, and reversibly or irreversibly binds to caspase to inhibit its activity. For example, the below-mentioned peptide inhibitor “Z-Asp-CH2-DCB” is described in Patent Documents 1-4 described later.

  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 apoptosis due to damage caused by ischemia-reperfusion of the liver, myocardium, kidney, intestine, and brain, and preserve the function of these organs. (Non-Patent Documents 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 to 12 described later). .

  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. A new function of Nari caspase is being reported. For example, cell proliferation, cell motility, cell cycle control, cell differentiation, etc., but the detailed mechanism remains unclear (the following non-patent documents 13-17). By clarifying the new functions of these caspases and their points of action, new uses for caspase inhibitors for the treatment of diseases may be found.

  Recently, an increase in caspase activity not related to apoptosis has been reported in certain types of cancer cells (prostate cancer, breast cancer, colon cancer, etc.) (Non-patent Document 18 below). In these cancer cells, an increase in the expression of IAP family proteins, which are caspase inhibitory proteins, has been observed at the same time, and the risk of falling into apoptosis due to increased caspase activity is avoided by increasing the expression of caspase inhibitory proteins. (Non-Patent Documents 19-25 below). In addition, attempts have been made to induce cancer treatment by inhibiting the function of these inhibitory proteins to specifically induce cancer cells (the following non-patent documents 26 to 28).

US Pat. No. 5,985,838 US Pat. No. 6,766,614 Japanese Unexamined Patent Publication No. 7-025865 Japanese Unexamined Patent Publication No. 7-069894 Thornberry et al., Science, 281, 1312-1316 (1998) Nicholson et al., Nature, 407, 810-816 (2000) Earnshaw et al., Annu. Rev. Biochem., 68, 383-424 (1999) Fischer et al., Cell Death Differ., 10, 76-100 (2003) Cursio et al., FASEB J., 13, 253-261 (1999) Mocanu et al., Br. J. Pharmacol., 130, 197-200 (2000) Farber et al., J. Vasc. Surg., 30, 752-760 (1999) Daemen et al., J. Clin. Invest., 104, 541-549 (1999) Endres et al., J. Cereb. Blood Flow Metab., 18, 238-247 (1998) Yakovlev et al., J. Neurosci., 17, 7415-7424 (1997) Li et al., Science, 288, 335-339 (2000) Schierle et al., Nature Med., 5, 97-100 (1999) Los et al., Trends Immunol. 22, 31-34 (2001) Algeciras-Schimnich et al., Curr. Opin. Cell Biol. 14, 721-726 (2002) Schwerk et al., Biochem. Pharmacol. 66, 1453-1458 (2003) Newton et al., Genes Dev. 17, 819-825 (2003) Woo et al., Nature Immunol. 4, 1016-1022 (2003) Yang et al., Cancer Res., 63, 6815-6824 (2003) Satoh et al., Cancer, 92, 271-278 (2001) Ambrosini et al., Nat. Med. 3, 917-921 (1997) Serela et al., Ann. Surg. Oncol., 8, 305-310 (2001) Tanaka et al., Clin. Cancer Res., 6, 127-134 (2000) Ferreira et al., Ann. Oncol., 12, 799-805 (2001) Li et al., Endocrinology, 142, 370-380 (2001) Sasaki et al., Cancer Res., 60, 5659-5666 (2000) Liston et al., Nat. Cell Biol., 3, 128-133 (2001) Mesri et al., J. Clin. Investig., 108, 981-990 (2001) Schimmer et al., Cancer Cell, 5, 25-35 (2004)

  As described above, an increase in caspase activity 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 could be developed. There is a possibility that it can be presented.

  The present invention has been made in view of the above circumstances, and its purpose is to investigate the physiological role of caspases in cancer cells and the applicability of caspase inhibitors as anticancer agents, and the findings To provide a new anticancer agent targeting caspase based on the above.

  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 phase of cancer cells, and (2) administration of a caspase inhibitor. Causes delay of cell division in cancer cells, inhibits normal chromosome segregation in the division phase, and (3) by administering a caspase inhibitor, The present inventors have found that a growth inhibitory effect was observed for each cancer cell derived from cervical cancer and acute T-cell leukemia, and completed the present invention.

That is, the present invention includes the following inventions as medically and industrially useful inventions.
A) An anticancer agent containing a caspase inhibitor as an active ingredient. The caspase inhibitor (1) has a structure similar to a substrate and binds to the active site of caspase to bind to the caspase activity, (2) interacts with a site other than the active site, Substances that inhibit caspase activity, (3) substances that inhibit caspase activation and inhibit caspase, (4) substances that inhibit caspase expression by inhibiting and inhibiting caspase expression, etc. Substances that directly or indirectly inhibit are widely included.
B) An anticancer agent comprising as an active ingredient an inhibitor of caspase activated in 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 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 biological protein.
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 of 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 siRNA (short interference RNA: also referred to as “short interfering RNA”, “small interfering RNA” or the like), or an RNAi expression vector (also referred to as “siRNA expression vector” or the like). There may be. 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 (2002), Nature Biotechnology 20: 505-508 (2002) etc.) . The RNAi expression vector is (1) a single RNA designed to express dsRNA having a hairpin structure of an appropriate length in the target cell, and (2) a sense strand and an antisense strand, respectively. Any of those designed to be expressed and associated in the target cell may be used.
RNA can be introduced into cancer cells according to a conventional method (for example, see Nature 411: 494-498 (2001), Science 296: 550-553 (2002)). You may use the method developed.
I) An anticancer agent comprising RNA that specifically suppresses the expression of caspase protein or an 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 type promoter such as U6 or H1 promoter, or RNA What is necessary is just to use what was incorporated in the upstream of the sequence | arrangement of siRNA which expresses a polymerase II type | system | group promoter etc.).
Here, “specifically suppress caspase protein expression” may be anything that substantially reduces the expression level of caspase protein in the target cancer cell, and completely suppresses caspase protein expression. It does not have to be a thing.

  The present invention utilizes a caspase inhibitor as an anticancer agent or a functional food having an anticancer effect. Conventionally, caspase inhibitors have been used for research and development from the viewpoint of suppression / prevention of apoptosis (for example, treatment of neurodegenerative diseases caused by abnormal apoptosis). The present invention finds that caspase is activated during the cell division phase of cancer cells and participates in the control thereof, and further provides a new use of suppressing the growth of cancer cells by caspase inhibitors based on this finding. Is.

  Thus, according to the present invention, 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. Chemotherapy and radiation therapy that have been used for cancer treatment so far have the purpose of inducing apoptosis in the cells, and have a great influence on normal cells other than cancer cells, and their side effects are large. It is a problem. By using the anticancer agent of the present invention that targets a caspase that is activated specifically for cancer cells, it is expected to establish an unprecedented new type of cancer treatment method with extremely low side effects.

  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.

[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 described later, by administering several caspase inhibitors, it was possible to actually suppress / inhibit the growth of liver cancer-derived HepG2 cells and cervical cancer-derived HeLa cells ( (Figs. 7 and 8).

  Traditionally, caspases are activated during apoptosis and positively control apoptosis, so inhibitors thereof have been used to suppress apoptosis. On the other hand, the present inventor newly found that caspases are specifically activated in the cell division phase in cancer cells such as HepG2 cells and HeLa cells in the process of elucidating various physiological roles of caspases. (FIG. 1-3). In addition, administration of a caspase inhibitor delayed the cell cycle progression in cancer cells and inhibited normal chromosome segregation during the mitotic phase (FIGS. 4-6). Details will be described later.

  Based on the above new findings, the present invention has confirmed the effect of inhibiting the growth of caspase inhibitors against cancer cells derived from liver cancer and cervical cancer, and is a novel caspase inhibitor as an anticancer agent. It provides usage.

  Since the caspase inhibitor also suppressed the growth of acute T-cell leukemia-derived Jurkat cells (FIG. 7), the anticancer agent of the present invention can be used for the treatment of cancer such as leukemia. In addition, caspase activation in cancer cells in a non-apoptotic state has been reported in prostate cancer, breast cancer, colorectal cancer, and the like, and the growth of these cancer cells can be suppressed by a caspase inhibitor. there is a possibility. Furthermore, if activation of caspase in a non-apoptotic state in cancer cells is widely recognized, it becomes possible to use a caspase inhibitor as an anticancer agent 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, specifically, digestive system cancer such as liver cancer. Examples thereof include use for various solid cancers such as squamous cell carcinoma such as cervical cancer.

  The type of caspase to be inhibited by the caspase inhibitor is not particularly limited, but since the growth inhibitory effect was actually observed for each inhibitor of caspase 1, 3, 4, 7, 8, 9 (Fig. 8) The use of a caspase inhibitor that inhibits any one or more of caspases 1, 3, 4, 7, 8, and 9 is preferred. In particular, it is preferable to use inhibitors of caspases 1, 3, 4, and 7 that have a high growth inhibitory effect, and inhibit caspases 3 and 7 that have been confirmed to be activated in the mitotic phase in liver cancer, cervical cancer, and the like. The use of agents is particularly preferred. A general-purpose caspase inhibitor that inhibits multiple types of caspases may be used, or a caspase inhibitor that controls the activation of caspase 3 and the like upstream may be used.

The caspase inhibitor, that is, the caspase inhibitor, may be a peptide compound, a non-peptide compound, or a protein derived from an organism. Examples of the peptide compound include the following peptide compounds (1) to (8) which are artificially chemically synthesized.
(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 19988.5)
(7) Ac-AAVALLPAVLLALLAP-IETD-CHO (Molecular weight 2000.5)
(8) Ac-AAVALLPAVLLALLAP-LEHD-CHO (Molecular weight 2036.5)

  All of the compounds (1) to (3) are cell membrane permeable and are general-purpose caspase inhibitors that inhibit multiple types of caspases. Z-Asp-CH2-DCB of (1) has the official name benzyloxycarbonyl-L-aspart-1-yl-[(2,6-dichlorobenzoyl) oxy] methane (Benzyloxycarbonyl-L-Aspart-1-yl- [(2,6-Dichlorobenzoyl) oxy] methane). (2) Boc-Asp (OMe) -FMK is the official name N- (tert-butoxycarbonyl) aspartyl (O-methyl) -fluoromethylketone (N- (tert-butoxycarbonyl) aspartyl (O-methyl) -fluoromethylketone) ). (3) Boc-Asp (OBzl) -CMK is the official name N- (tert-butoxycarbonyl) aspartyl (O-benzyl) -chloromethylketone (N- (tert-butoxycarbonyl) aspartyl (O-benzyl) -chloromethylketone) ). Z-Asp-CH2-DCB, Boc-Asp (OMe) -FMK, and Boc-Asp (OBzl) -CMK all inhibited the growth of HepG2 cells, HeLa cells, and Jurkat cells in a concentration-dependent manner (FIG. 7). The degree of the growth inhibitory effect was favorable in the order of Boc-Asp (OBzl) -CMK, Z-Asp-CH2-DCB, and Boc-Asp (OMe) -FMK.

The above (4) is a caspase 1 inhibitor. In order to enhance the cell membrane permeability on the N-terminal side of 4 amino acids of YVAD (ie, tyrosine-valine-alanine-aspartic acid) involved in caspase 1 inhibition, A hydrophobic region of Kaposi fibroblast growth factor consisting of an amino acid sequence is added.
Amino acid sequence: AAVALLPAVLLALLAP (ie alanine-alanine-valine-alanine-leucine-leucine-proline-alanine-valine-leucine-leucine-alanine-leucine-leucine-alanine-proline)

  The above (5) is an inhibitor of caspase 3 and 7, and cell membrane permeability is increased on the N-terminal side of the 4 amino acids of DEVD (ie, aspartic acid-glutamic acid-valine-aspartic acid) involved in the inhibition of caspase 3 and 7. In order to enhance, the hydrophobic region of the Kaposi fibroblast growth factor is added.

  The above (6) is a caspase 4 inhibitor, in order to increase the cell membrane permeability on the N-terminal side of 4 amino acids of LEVD (that is, leucine-glutamic acid-valine-aspartic acid) involved in the inhibition of caspase 4, A hydrophobic region of Kaposi fibroblast growth factor is added.

  The above (7) is a caspase 8 inhibitor. In order to enhance cell membrane permeability on the N-terminal side of 4 amino acids of IETD (ie, isoleucine-glutamic acid-threonine-aspartic acid) involved in the inhibition of caspase 8, A hydrophobic region of Kaposi fibroblast growth factor is added.

  The above (8) is a caspase 9 inhibitor. In order to increase cell membrane permeability on the N-terminal side of 4 amino acids of LEHD (ie, leucine-glutamic acid-histidine-aspartic acid) involved in the inhibition of caspase 9, A hydrophobic region of Kaposi fibroblast growth factor is added.

  The compounds (4) to (8) have an acetyl group (Ac) on the N-terminal side and an acetaldehyde group (CHO) on the C-terminal side of the oligopeptide consisting of a total of 20 amino acids. Although these inhibitors specific to each caspase showed a difference in their suppressive effects, they all showed growth inhibitory activity against HepG2 cells (FIG. 8).

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

For example, as caspase inhibitors of peptide compounds, (1) VX-740-Vertex Pharmaceuticals (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)).
Examples of caspase inhibitors of non-peptide compounds include (1) anilinoquinazolines (AQZs) -AstraZeneca Pharmaceuticals (Scott et al., J. Pharmacol. 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. Can be illustrated.
Further, as caspase inhibitors of other non-peptidic compounds, (1) IDN-6556-Idun 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. Pharmacol. Exp. Ther. 297, 811-818 (2001)), (5) VX-799- Vertex Pharmaceuticals (Los et al., Drug Discov. Today 8, 67-77 (2003)). In addition, M-920 and M-791-Merck Frosst (Hotchkiss et al., Nat. Immunol. 1, 496-501 (2000)) can also be mentioned as caspase inhibitors.

Examples of biological proteins include IAP family proteins (for example, cIAP1, cIAP2, XIAP, survivin, etc.) that are caspase activity-inhibiting proteins, baculovirus-derived p35 proteins, cowpox virus-derived crmA proteins, etc. can do. By expressing these proteins specifically for cancer cells, it is possible to suppress the growth of cancer cells. Examples of the method for expressing a protein in cancer cells include use of a gene expression vector encoding the protein. In this method, the modified protein may be expressed artificially by substituting, deleting, inserting and / or adding one or several bases in the base sequence of the gene expression vector according to a conventional method. .
Still other caspase inhibitors include RNA that specifically suppresses caspase protein expression as described above, and RNAi expression vectors constructed to express the RNA in target cancer cells. When the amount of a plurality of caspase proteins in cancer cells was actually reduced using the RNAi method, the result of suppressing the growth of cancer cells was obtained. Therefore, siRNA that suppresses the expression of caspase protein can be used for cancer treatment. As a method for introducing siRNA into cancer cells, known carriers and drugs that have been proposed to transport vectors and nucleic acids to specific parts of living organisms and specific cells in order to efficiently and selectively introduce siRNA into target cancer cells A delivery system can be used.
The siRNA and RNAi expression vector can be designed based on the gene sequence of caspase as described above. For example, the cDNA sequence and amino acid sequence of human-derived caspase 3 are disclosed in accession numbers “NM_004346” and “NM_032991” of DDBJ / EMBL / GenBank databases, and the target sequence is determined based on these sequence information. In addition, siRNA and RNAi expression vectors that can suppress the expression of caspase protein can be designed and prepared.

  As described above, the anticancer agent of the present invention comprises a caspase inhibitor, that is, a caspase inhibitor, as an active ingredient, but not only a known caspase inhibitor but also a caspase inhibitor found in the future is used. You may do. The caspase inhibitor includes (1) a substance having a structure similar to a substrate and the like, which binds to the active site of caspase, thereby inhibiting the activity of caspase, (2) interacts with a site other than the active site, Substances that inhibit caspase activity, (3) substances that inhibit caspase activation and inhibit caspase, (4) substances that inhibit caspase expression by inhibiting and inhibiting caspase expression, etc. Substances that directly or indirectly inhibit are widely included. For example, in an experimental system that induces apoptosis in cells by caspase activation, a substance that suppresses apoptosis can also be said to be a substance that directly or indirectly inhibits caspase and can be used as the caspase inhibitor of the present invention.

  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 several picomoles (pM ) To several tens of micromolar (μM), preferably having a high inhibitory activity, more preferably several micromolar (μM) or less in vivo and several hundred nanomolar (nM) or less in vitro.

[2] Examples of use of anticancer agent and the like of the present invention The above is a case where a caspase inhibitor is used as an anticancer agent (anticancer agent), but the present invention is not limited thereto, and caspase inhibition. The agent can be used as a raw material for foods (edible compositions) such as functional foods and supplements, and can be used for the development of foods having anticancer effects and cancer prevention effects. Alternatively, it can be used as a raw material for cosmetics and can be used for the development of cosmetics having an anticancer effect and a cancer prevention effect.

  Hereinafter, an example in the case 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, Non-oral agents such as suppositories and coating agents can be mentioned.

  Oral preparations such as tablets, capsules, granules, fine granules, powders and the like are produced according to a conventional method using, for example, starch, lactose, sucrose, trehalose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like. The amount of the 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, fragrances and the like can be appropriately used.

  In the case of a parenteral preparation, the dose is adjusted according to the age, weight, degree of disease, etc. of the patient, for example, intravenous injection, intravenous infusion, subcutaneous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, etc. Administer 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. Furthermore, you may add a disinfectant, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the 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, you may add an isotonic agent, a stabilizer, an antiseptic | preservative, and a soothing agent as needed. The 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, and the like, and these are also produced according to conventional methods.

  In addition, using a known DDS (drug delivery system), for example, a caspase inhibitor or a gene expression vector encoding a protein that acts as an inhibitor may be enclosed in a carrier such as a liposome and administered in vivo. . At this time, if a carrier that specifically recognizes the target site (cancer cell) is used, the caspase inhibitor can be efficiently carried to the target site, which is effective.

  As described above, the caspase inhibitor can be used for foods (edible compositions) such as supplements and functional foods. In other words, caspase inhibitors are added to foods and drinks as raw materials for various beverages and various processed foods, and processed into pellets, tablets, granules, etc. together with excipients such as dextrin, lactose, starch, fragrances, and pigments as necessary Or coated with gelatin or the like and molded into capsules for use as health food or health food.

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 activation of caspase 3 in a non-apoptotic state of cancer cells, the following experiment was performed.

Hepatoma cell-derived HepG2 cells were seeded at 2 × 10 ⁵ cells per well in a 6-well dish with a cover glass in the bottom and cultured for 24 hours. After fixing with a phosphate buffer containing 3.7% formaldehyde for 10 minutes, washing with a phosphate buffer twice and treating with a phosphate buffer containing 0.5% Triton X-100 for 10 minutes, Washed twice with phosphate buffer.

  The cells thus treated were incubated overnight at 4 ° C. in a phosphate buffer solution (containing 1% bovine serum albumin) containing anti-active caspase 3 antibody and anti-tubulin antibody as primary antibodies. After washing twice with a phosphate buffer, it was incubated in a phosphate buffer containing a secondary antibody labeled with TXRD or FITC for 10 minutes, and then washed twice with a phosphate buffer. Nuclei were stained with 10 μM Hoechst 33342 (Calbiochem) and then examined with a fluorescence microscope (product name: Laborlux, Leitz).

  FIG. 1 (a) shows the result of using an antibody that recognizes the C-terminal of the large subunit (p17) of active caspase 3 as the primary antibody, and FIG. 1 (b) shows the small subunit (p12 of active caspase 3). The results of using an antibody that recognizes the N-terminus of In the figure, cells other than interphase showed tubulin polymerization and chromosome condensation, and are considered to be in the cell division phase. It can be seen that caspase 3 is activated in the early, middle, middle, late, and final stages of cell division.

  Furthermore, in order to confirm the activation of caspase specific to the cell division phase, the following experiment was performed using hepatoma-derived HepG2 cells and cervical cancer-derived HeLa cells.

First, HepG2 cells (1 × 10 ⁶ / 6cm Deisshu) cultured in the presence nocodazole (0.8 [mu] g / ml), over time the cells were harvested and subjected to Western blotting, caused a result of cleavage by activated caspase 3 (and caspase 8.9) were detected. The accumulation of mitotic cells by nocodazole treatment was confirmed by flow cytometry.

  As shown in FIG. 2 (a), the number of cells (cell number) increased with the passage of time (= cells with 4N DNA content (DNA content) of 4N), and 18 hours after treatment with Nocodazole. More than 80% of the cells were in the mitotic phase. As shown in FIG. 5B, the active form fragment of caspase 3 was detected 12 hours after nocodazole treatment and increased with time. In addition, active form fragments of caspase 8 and caspase 9 were also detected in the same time course. Furthermore, poly ADP ribose polymerase (PARP), lamin B1 (LaminB1), protein kinase Cδ (PKCδ), which are caspase 3 substrates. ) (Cleavage fragment) was also detected. In the figure, lane A is a cell in which apoptosis was induced with an anti-Fas antibody as a control.

  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.

Next, activation of caspase specific for cell division in cervical cancer-derived HeLa cells was examined. After HeLa cells (4 × 10 ⁵ / 6cm Deisshu) cultured for 24 hours, and 18 hours of culture in the presence 2.5mM thymidine. After washing three times with a phosphate buffer, a new medium was added and 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 caspase 3, 8, 9 cleaved by activation are detected by Western blotting. did. Progression of the cell cycle at each recovery was confirmed by flow cytometry.

  By performing thymidine treatment twice, the cells can be stopped at the boundary between almost 100% G1 phase and S phase. As shown in FIG. 3 (a), when thymidine is removed from the culture medium (release), the cells begin to go around the cell cycle again, and in about 8-12 hours after thymidine removal, the cells are in the G2 / M phase. Enter G1 again in 14 hours.

  As shown in FIG. 3 (b), active form fragments of caspase 3, caspase 8 and caspase 9 were detected 10-14 hours after thymidine removal. In addition, cleavage of the caspase 3 substrates, poly ADP ribose polymerase (PARP), lamin B1 (LaminB1), and protein kinase Cδ (PKCδ) 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 an anti-Fas antibody.

  From the above results, activation of caspase specific for 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, the role and function were examined.

  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. Thereafter, a new medium was added to resume the cell cycle, and after 7 hours, Z-Asp-CH2-DCB (final concentration in the culture medium: 200 μM), a cell membrane-permeable general-purpose caspase inhibitor, was added to dimethyl sulfoxide (DMSO). The cell cycle was examined by flow cytometry.

  The result is shown in FIG. To the control medium, 0.5% DMSO was added in the same manner as when the inhibitor was added. Both the cells to which the inhibitor was added and the control cells were in the G2 / M phase after 8 hours.

  Thereafter, in the control cells, most of the cells were in the M phase after 10 hours, some of the cells progressed to the G1 phase, and most of the cells shifted to the G1 phase after 14 hours. In contrast, a cell cycle delay of about 2 hours was observed in the cells to which the caspase inhibitor was added, compared to the control cells. From this result, it was shown that a caspase inhibitor delays the progression of the cell cycle of cancer cells by inhibiting the activity of caspases activated in the cell division phase 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 inhibitor, and the change in the morphology of the nucleus was examined. HepG2 cells or HeLa cells were seeded at 2 × 10 ⁵ cells per well in a 6-well dish with a cover glass at the bottom. On the next day, Z-Asp-CH2-DCB (final concentration 200 μM) or DMSO as a control was added, and nuclei were stained with 10 μM Hoechst 33342 every day to examine the ratio of condensed nuclei per total cell nucleus. 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 having a condensed nucleus (nuclear condensation) different from the nuclear degeneration due to apoptosis was observed in both HepG2 cells and HeLa cells treated with caspase inhibitors.

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. The HeLa-GFP-H2B cells (2 × 10 ⁵ cells / 3.5 cm glass bottom dish) are seeded, and the next day, Z-Asp-CH2-DCB (final concentration 300 μM) or DMSO as a control is added, and the confocal laser 2 days later. Using a microscope, changes in the morphology of the nucleus were observed at 1 minute intervals for 1 to 2 hours.

  FIG. 6 shows typical nuclear morphology of cells treated with control and caspase inhibitors. In control cells to which DMSO was added, cell division proceeded normally and progressed from chromosome condensation to separation in about 90 to 120 minutes (FIGS. 6a and b).

  In contrast, cells in which a caspase inhibitor was added to the culture solution to a final concentration of 300 μM showed obvious abnormalities in the progression of the cell division phase, and cells whose chromosomes were divided into three (c and d in FIG. 6). Cells that did not complete chromosome condensation in 120 minutes (e and f in FIG. 6), cells that remained in the middle stage and did not proceed with chromosome division (g in FIG. 6), and the like 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, it has been clarified that caspases are activated at the cell division stage of cancer cells, and caspase inhibitors inhibit the cell division of cancer cells, particularly the separation of chromosomes. In some cancer cells, the activity of caspase 3 increases despite being non-apoptotic, suggesting that caspase inhibitors perform some function specific to cancer cells. The possibility of functioning as a specific cell growth inhibitor was considered. Therefore, in order to investigate this possibility, the following experiment was conducted.

For cell proliferation assay, WST-1 reagent (Roche) was used. Liver cancer-derived HepG2 cells, cervical cancer-derived HeLa cells, or acute T-cell leukemia-derived Jurkat cells were seeded at 4 × 10 ³ cells per well in a 96-well dish and cultured for 24 hours. After each caspase inhibitor was added at each concentration, WST-1 reagent was added every day, and the dehydrogenase activity localized in mitochondria was measured by measuring the absorbance at 450 nm and 690 nm. It was used as an index.

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)

  The above (1) to (3) are cell membrane permeation-type versatile inhibitors. A hydrophobic region of Kaposi fibroblast growth factor is added to the N-terminal side of each of the caspase-specific inhibitors (4) to (8) described above in order to enhance cell membrane permeability. (1) is an inhibitor manufactured by Peptide Institute, and (2) to (8) are inhibitors manufactured by Calbiochem. Since all the inhibitors were dissolved in DMSO and a part thereof was added to the culture solution, the control was added with DMSO at the same concentration as when the inhibitor was added.

  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-DCB, Boc-Asp (OMe) -FMK, and Boc-Asp (OBzl) -CMK, which are cell membrane permeation-type general-purpose inhibitors, all proliferate HepG2, HeLa, and Jurkat cells. Was suppressed in a concentration-dependent manner (FIG. 7). Moreover, although each caspase specific inhibitor showed the difference in the suppression effect, all showed the cell growth inhibitory activity with respect to HepG2 cell (FIG. 8).

As described above, the anticancer agent of the present invention comprises a caspase inhibitor that has been shown to have a cancer cell growth inhibitory effect as an active ingredient, such as solid cancers such as liver cancer and cervical cancer. It can be used as an anticancer agent against
The present invention can also be used as functional foods, supplements and the like having an anticancer effect and a cancer prevention effect.

It is a figure which shows activation of caspase 3 specific to a cell division phase by an immunofluorescence staining method. (A) is the result of using an antibody that recognizes the large subunit (p17) of caspase 3, and (b) is the result of using an antibody that recognizes the small subunit (p12) of caspase 3. It is a figure which shows the result of having treated the hepatoma origin HepG2 cell with nocodazole and examined the activation of caspase in a cell division phase. (A) shows the results of cell cycle analysis by flow cytometry, and (b) shows the results of examining the presence or absence of caspase activation and substrate cleavage by caspase by Western blotting. It is a figure which shows the result of having treated the cervical cancer origin HeLa cell with thymidine, and examined the activation of caspase in a cell division phase. (A) shows the results of cell cycle analysis by flow cytometry, and (b) shows the results of examining the presence of caspase activation and substrate cleavage by caspase by Western blotting. It is a graph which shows the delay of cell cycle progress by a caspase inhibitor. It is a graph which shows the increase in the number of cells with the condensed nucleus by a caspase inhibitor. It is a figure which shows the progress inhibition of the chromosome separation in a cell division phase by a caspase inhibitor. It is a graph which shows the growth inhibitory effect of the cancer cell by a versatile caspase inhibitor. It is a graph which shows the growth inhibitory effect of each caspase specific inhibitor with respect to HepG2 cell.

Claims (9)

  An anticancer agent containing a caspase inhibitor as an active ingredient.   An anticancer agent comprising as an active ingredient an inhibitor of caspase activated during the cell division phase of cancer cells.   An anticancer agent comprising as an active ingredient any one or more inhibitors of caspases 1, 3, 4, 7, 8, and 9.   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.   The anticancer agent according to any one of claims 1 to 3, wherein the caspase inhibitor is a peptidic compound, a non-peptidic compound, or a biological protein. 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   An edible composition having an anticancer effect by containing a caspase inhibitor.   A method for suppressing the growth of cancer cells by introducing RNA that specifically suppresses the expression of caspase protein into the cancer cells. An anticancer agent comprising an RNA that specifically suppresses the expression of a caspase protein, or an RNAi expression vector constructed to express the RNA in a target cancer cell.