JPH09176002A - Gingerol-containing agent for suppressing synthesis of protein of hsp27 family - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the above suppressing agent containing gingerol or the derivative thereof as an active component, effective for suppressing the expression of a heat shock protein group having a specific molecular weight and useful for the treatment of cancer related with the protein and disseminated sclerosis, etc., relating to the crisis of the cancer. SOLUTION: This agent suppresses the synthesis of heat shock proteins having a molecular weight of 16-40kD and contains a gingerol (derivative) e.g. (S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone of the formula which is a [6]-gingerol produced by extracting a gingerol-containing plant such as SHOUKYOU (rhizome of Zingiber officinale) with a solvent such as ethyl alcohol, a mixture of ethyl alcohol and water, etc.}. The suppressing agent is administered at a daily rate of 1mg to 10g for adult in terms of [6]-gingerol in 1-4 divided doses by oral or parenteral administration.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a molecular weight of 16 kilodaltons (kD) to 40 containing gingerol or a derivative thereof, particularly [6] -gingerol as an active ingredient.
Heat shock protein group up to kD (hereinafter HSP
27 family)). The inhibitor for the synthesis of a protein belonging to the HSP27 family according to the present invention is particularly one wherein the protein belonging to the HSP27 family is involved in the onset, malignancy, or disorder of treatment by inhibiting the in-tissue synthesis of the protein belonging to the HSP27 family. It is possible to effectively improve a patient's physiological condition such as a disease considered to be effective, for example, cancer or multiple sclerosis, and to effectively treat the disease.

[0002]

2. Description of the Related Art Despite recent advances in chemotherapy, surgery, radiation therapy, and immunotherapy, cancer malignant transformation is still directly or indirectly involved in the cause of cancer death. Overcoming malignancy has become one of the major challenges in cancer treatment in the future. The malignancy of a cancer is determined by the proliferative, invasive, or metastatic nature of the cancer. Metastasis, which is one of the phenomena of malignant transformation, depends on the type of primary cancer and the organs that are apt to undergo metastasis differ. Cancer metastasis is a complex event that begins with the growth of the primary tumor, withdrawal of cancer cells from the primary focus and invasion / proliferation of surrounding tissues, tumor angiogenesis, invasion of cancer cells into nearby blood vessels, blood New tumor blood vessels are born, which is visible after the movement to the distant site by flow and adhesion / implantation to vascular endothelial cells, infiltration outside the blood vessel, and the initiation of proliferation at the distant site (metastasis). It consists of a complex reaction cascade leading to the formation of a metastatic focus. In general, cancers are classified into those with a high grade and those with a relatively low grade. However, no radical treatment has been established for highly aggressive cancers, and patients are most likely to eventually die.

[0003] Hyperthermia for cancer (hyperthermia;
Hyperthermia) is a method of selectively killing tumor cells by heating a cancer tissue to treat cancer, and has recently attracted attention. Cancer treatment with hyperthermia
Looking at the biological effects of heat, a cell-killing effect can be obtained with relatively mild heating of 41 to 45 ° C, and a synergistic effect can be obtained when used in combination with radiation or an anticancer agent. There are many advantages. Treatment of cancer by hyperthermia has been tried in almost all departments in the clinic. However, one of the problems of hyperthermia is thermal tolerance that is transiently induced after heating. That is, since the cancer cells become temporarily resistant to heat by the first heating, the cell killing effect by the next heating is reduced. The thermotolerance means that once a cell (or tissue) is heated sub-lethal once, the cell (or tissue) becomes transiently heat resistant to the next heating. Due to heat resistance, the current practice is that thermal treatment is performed in most facilities only once or twice a week.

[0004] In cancer chemotherapy, there are still solid cancers such as lung cancer and colon cancer which hardly respond to chemotherapy. Is a problem. 1
According to United States statistics of 988, 49% of cancers diagnosed in one year are intrinsically resistant to chemotherapy initially, and 47% were initially chemotherapy effective and the tumor regressed It is said to have acquired relapse resistance after relapse.
These facts indicate that one of the most important problems that hinders the effects of chemotherapy on cancer is resistance to cytotoxic drugs.

[0005] Also, multiple sclerosi
s, MS) is an inflammatory demyelinating disease that specifically impairs the central nervous white matter, and its pathogenesis has been demonstrated by the immune system to attack the myelin sheath that surrounds nerve fibers. Immune disease. Multiple sclerosis often repeats acute hatred / remission in the early stages, but then gradually progresses. Adrenocorticotropic hormone (ACTH) and corticosteroids are used to improve symptoms in the acute phase, and azathioprine and cyclophosphamide are used to prevent recurrence during remission and to prevent the development of chronic progressive symptoms. Immunosuppressive agents such as e.g. However, many of the drugs currently being administered to multiple sclerosis patients are not as effective as expected,
At present, it is difficult to say that it is not enough, as many side effects are seen with non-specific therapies. The development of more specific treatments for multiple sclerosis is expected.

On the other hand, heat shock proteins (heat shock proteins)
protein; also called HSP, stress protein)
A group of proteins expressed in cells by stimulating the cells with some stress, such as heat, heavy metals, drugs, amino acid analogs, or hypoxia (low oxygen concentration). Heat shock proteins are ubiquitous in nature and are produced by higher animals, including bacteria, yeast, plants, insects, and humans.

[0007] HSPs are of various types,
Due to the molecular weight, the HSP90 family (for example, 9
0 kD or 110 kD HSP, etc.), HSP70 family (eg, 70-73 kD HSP, etc.), HS
P60 family (eg, 57-68 kD HSP etc.), small molecule HSP family (eg, 20 kD, 2
5-28 kD or 47 kD HSP). In the present specification, an HSP having a specific molecular weight is represented by HSP and a number described immediately after it, and for example, an HSP having a molecular weight of 27 kD is referred to as “HSP27”. As described above, there are many types of HSPs, which differ not only in molecular weight but also in structure, function, or property. In addition to the response to stress, some of these proteins are constitutively synthesized and, under normal circumstances, regulate protein folding, unfolding, protein subunit assembly, and protein membrane trafficking. It has been shown to play an essential physiological role. These functions as heat shock proteins are called molecular chaperones.

[0008] The expression of proteins belonging to the HSP27 family is significantly correlated with lymph node metastasis, lymph and blood vessel invasion, and shorter survival in human breast cancer ("J. Natl. Cancer Inst."). 83 : 170-178, 1991). It has been reported that a protein belonging to the HSP27 family is also a negative prognostic factor in gastric cancer ("Br.J.
Surg. ", 78 : 334-336, 1991). It has been reported that the expression level of a protein belonging to the HSP27 family in primary cancer cells is positively correlated with cancer malignancy, particularly the recurrence rate of cancer (" Breast Cancer "). Res. Treat. ", 12 : 130,
1988; "Proc. Am. Assoc. Cancer Res.", 30 : 252, 19
89) By suppressing the expression of proteins belonging to the HSP27 family, malignant transformation of cancer can be prevented.

[0009] The hyperthermia, which is a problem in hyperthermia for cancer,
There are reports that proteins belonging to the P27 family are involved. When the human HSP27 gene was introduced into mouse or hamster cells and expressed, the number of heat-resistant cells surviving after heat shock was induced and increased depending on the amount of HSP27 protein ("J. Cell. Biol.", 109
: 7-15, 1989). Also, in Chinese hamster cells, a mutant strain that has become capable of constantly expressing HSP27 can acquire thermotolerance ("J. Cell. Physio").
l. ", 137 : 157, 1988) α-B crystallin is a protein which is induced by heat shock treatment and has a high homology in amino acid sequence with HSP27, and is one of the proteins belonging to the HSP27 family. Cells overexpressing -B crystallin also acquire resistance to heat stress ("J. Cell. Biol.", 125 : 1385-1393, 1994), which suppresses the expression of proteins belonging to the HSP27 family. This has shown the possibility of suppressing hyperthermia and enhancing the effect of hyperthermia on cancer, and has also been reported to correlate the expression of proteins belonging to the HSP27 family with drug resistance ("Breast Ca
ncer Res. Treat. ", 23 : 178, 1992;" Cancer Res. ", 5
1 : 5245-5252, 1991), by suppressing the expression of proteins belonging to the HSP27 family, it is also possible to suppress drug resistance and enhance the effect of chemotherapy.

The immunodominant antigen in multiple sclerosis is αα, a protein belonging to the HSP27 family.
-B crystallin ("N
Nature ", 375 : 739-740, 1995). The expression of α-B crystallin in nervous tissue of multiple sclerosis patients is stronger than that in non-diseased tissues and is very immunogenic. ("Nature", 375 : 798-801, 1995).
HSP27 is the autoantigen in multiple sclerosis
Α-B crystallin, which is one of the proteins belonging to the family, and shows that suppressing the expression of α-B crystallin in the myelin sheath leads to fundamental treatment of multiple sclerosis.

[0011]

SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors effectively improve the physiological condition of a patient suffering from a disease such as cancer or multiple sclerosis, and effectively treat the disease. In order to develop a method capable of performing the above-mentioned methods, various studies have been made on compounds that exhibit a synthesis inhibitory effect on proteins belonging to the HSP27 family. As a result, the present inventors have surprisingly found that gingerol, which is a component of ginger, or a derivative thereof, particularly [6] -gingerol, specifically synthesizes a protein belonging to the HSP27 family in cells of tissues showing pathological conditions. It was found to suppress to. That is, it was found that the administration of zingerol or its derivative suppresses the intracellular synthesis of proteins belonging to the HSP27 family, and thus can treat diseases such as cancer and multiple sclerosis. is there. The present invention is based on such findings, and an object of the present invention is to provide a synthesis inhibitor of a protein belonging to the HSP27 family, which can effectively treat diseases such as cancer and multiple sclerosis.

[0012]

Therefore, the present invention is characterized by containing gingerol or a derivative thereof, particularly [6] -gingerol as an active ingredient.
It relates to a synthetic inhibitor of heat shock proteins (ie proteins belonging to the HSP27 family) between the kilodaltons and 40 kilodaltons. In the present specification, the “HSP27 family” means a heat shock protein group having a molecular weight of 16 kD to 40 kD, as described above. Examples of proteins belonging to the HSP27 family include mammalian HSP27 (that is, heat shock protein having a molecular weight of 27 kD) [or HS
P28 (ie, a heat shock protein having a molecular weight of 28 kD)], HSP25 of a bird (ie, a molecular weight of 25 kD)
D heat shock protein), or yeast HSP26
(That is, a heat shock protein having a molecular weight of 26 kD)
And the like. In general, the molecular weights of proteins differ somewhat depending on, for example, differences in molecular weight measurement methods or experimental conditions. Therefore, among proteins belonging to the HSP27 family, for example, HSP27 and HSP28 in mammals are different from each other. As described above, even if the molecular weight notation is different, it is not clear at present whether they are different proteins having different amino acid sequences, or whether they are the same proteins that differ only in molecular weight notation. include. HSP2
The proteins belonging to the 7 family are
It is the most major heat shock protein in mammals among the heat shock proteins belonging to the P family,
It exhibits well-conserved characteristics across species. But H
Unlike other heat shock proteins, the proteins belonging to the SP27 family have different molecular weights depending on the species, and are extremely diverse proteins with a molecular weight of 16 kD to 40 kD. Α-B crystallin having a high amino acid sequence homology with HSP27 is also induced by heat shock treatment and is one of the proteins belonging to the HSP27 family.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The synthesis inhibitor of the present invention contains gingerol or its derivative as an active ingredient. In the present specification, gingerol refers to, for example, [3] -gingerol ([3]
-Gingerol), [4] -gingerol ([4]
-Gingerol), [5] -gingerol ([5]
-Gingerol), [6] -gingerol ([6]
-Gingerol), [8] -gingerol ([8]
-Gingerol), [10] -gingerol ([1
0] -gingerol) or [12] -gingerol ([12] -gingerol). In the present invention, those gingerol homologues may be used alone or in combination of a plurality of different gingerols at the same time. As the gingerol used in the synthesis inhibitor of the present invention, particularly [6] -gingerol [(S) -5-hydroxy-1- (4-hydroxy-3-methoxyphenyl)]
_{3-decanone; C 17 H 26 O 4 =} 294.38 ] are most preferred. [6] -gingerol has the formula

Embedded image And is contained in crude drugs such as ginger.

The gingerol or its derivative contained in the synthetic inhibitor of the present invention can be prepared by chemical synthesis or by extraction from a natural product and purification. Alternatively, a commercially available product may be used. Further, zingerol used as an active ingredient in the synthetic inhibitor of the present invention, when extracted from natural products, whole or part of a plant containing zingerol (for example, whole grass, leaves, roots,
Extraction is performed using the rhizome, stem, root bark, or flower) as it is, or by using a processed material (eg, dried, cut, or powdered) (eg, crude drug). The extraction conditions are not particularly limited as long as they are generally used for plant extraction.

When the gingerol in the present invention is extracted from a crude drug, it is not limited to this, but it is preferably extracted from ginger, for example. Ginger: Zingiberis Rh
is ginger (Zingiber of)
(ficinale Roscoe) rhizome raw or dried, and the parts thereof may be used alone or in any combination.

The ginger extract used as an active ingredient in the synthetic inhibitor according to the present invention may contain the above-mentioned gingerol, particularly [6] -gingerol,
Therefore, a crude extract of ginger can be used. As a method for producing a ginger extract used in the present invention, ginger is extracted with, for example, water (for example, warm water, preferably hot water) or an organic solvent. Examples of the organic solvent include methyl alcohol, ethyl alcohol, n-propyl alcohol,
Isopropyl alcohol, butyl alcohol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, acetone, methyl isobutyl ketone, petroleum ether, cyclohexane, carbon tetrachloride, toluene, benzene, dichloromethane, chloroform, ether, pyridine, polyethylene glycol, propylene glycol , Butylene glycol, acetonitrile or the like can be used, and these organic solvents can be used alone or in appropriate combination, mixed at a fixed ratio, and further used in an anhydrous or hydrous state. Preferably, ethyl alcohol-water mixed solvent, ethyl alcohol, ether, chloroform, benzene,
Acetone, hot petroleum ether or the like is preferable.

As the method of water extraction or organic solvent extraction,
It is possible to use the method used for usual herbal medicine extraction,
For example, it is desirable to use 5 to 300 parts by weight of water or an organic solvent with respect to 1 part by weight of (dry) ginger, and heat and reflux at a temperature below the boiling point thereof while stirring. The extraction step is usually carried out for 5 minutes to 7 days, preferably 10 minutes to 24 hours, and the extraction time can be shortened by adding an auxiliary means such as stirring if necessary.

After the completion of the extraction step, a crude extract can be obtained by separating insolubles from the water or organic solvent extract by a suitable method such as filtration or centrifugation. In the synthesis inhibitor of the present invention, when using gingerol extracted and fractionated from a natural product, particularly [6] -gingerol,
The crude extract may be used as it is without any particular purification. In addition to a hot water extract or an organic solvent extract by a conventional method, a purified extract obtained by further treating the crude extract with various organic solvents or adsorbents is also used as an active ingredient of the synthesis inhibitor of the present invention. be able to. The Ginger extract containing these crude extracts and refined extracts that have undergone various purification treatments may be used as the solution as it is extracted, or may be an extract obtained by concentrating the solvent, or distilling off the solvent. Then, a dried powder, a crystallized and purified product, or a viscous substance may be used, or a diluted solution thereof may be used. The ginger extract thus obtained is a gingerol contained in ginger (for example,
[3] -gingerol, [4] -gingerol, [5]
-Zingerol, [6] -Zingerol, [8] -Zingerol, [10] -Zingerol, and / or [1]
2] -gingerol and the like) as a mixture, and at the same time, it also contains impurities derived from the raw material Ginger.

The gingerol derivative is, for example, a gingerol derivative originally contained in a natural product, a zingerol derivative converted by a chemical treatment during extraction or fractionation, and a chemically modified zingerol derivative. Is included. Examples of the gingerol derivative originally contained in a natural product include, for example, [6] -gingerdiol ([6] -gingerdiol),
[8] -gingerdiol ([8] -gingerd
iol), [10] -gingerdiol ([10]-
gingerdiol), methyl [6] -gingerdiol (methyl [6] -gingerdio
l), methyl [6] -gingerdiol diacetate (methyl [6] -gingerdiol di
acetate) or dehydrozingerone (dehy)
drozingerone) and the like.
Examples of the gingerol derivative converted by a chemical treatment during extraction or fractionation include, for example, [6] -shogaol ([6] -shogaol), [8] -shogaol ([8] -shogaol), and [10]. ] -Shogaol ([10] -shogaol), zingerone (zingerone), etc. can be mentioned. Examples of the chemically modified zingerol derivative include, for example, methylgingerol.
l), dimethyl gingerol
gerol) or dinitrophenyl zingerol (d
and the like. It should be noted that the above-mentioned gingerol or its derivative has stereoisomers, and in the present invention, any pure stereoisomers thereof or a mixture thereof can be used.

The synthesis inhibitor of the present invention is a gingerol or a derivative thereof, or a gingerol-containing plant, for example,
A gingerol-containing extract extracted and fractionated from a gingerol-containing crude drug (particularly ginger) is used alone,
Alternatively, it can be administered to an animal, preferably a mammal (particularly a human), preferably with a conventional carrier that is pharmaceutically or veterinarily acceptable. The dosage form is not particularly limited, and examples thereof include powder, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, pills, and other oral preparations, or injections. And parenteral preparations such as external preparations, external preparations, ointments, suppositories, creams for topical administration, and eye drops. These oral agents include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose,
Excipients such as mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc, magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate. , Binders, disintegrants, surfactants, lubricants, flow promoters, diluents, preservatives, colorants, flavors, flavoring agents, stabilizers, humectants, preservatives, antioxidants, etc. make use of,
It can be manufactured according to a conventional method. For example, [6]-
For example, capsules and the like filled by mixing 1 part by weight of gingerol and 99 parts by weight of lactose.

Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) and rectal administration. Of these, injections are most preferably used. For example, in the preparation of injectables, zingerol or a derivative thereof (particularly [6] -gingerol) as an active ingredient, or a gingerol-containing plant extract, for example, a zingerol-containing herbal medicine extract (particularly ginger extract) In addition, for example, physiological saline, water-soluble solvent such as Ringer's solution, non-water-soluble solvent such as vegetable oil or fatty acid ester, isotonic agent such as glucose or sodium chloride, solubilizing agent, stabilizer, preservative Agents, suspending agents, emulsifiers and the like can be optionally used. Further, the synthetic inhibitor of the present invention,
You may administer using the method of the sustained release formulation using a sustained release polymer etc. For example, the synthetic inhibitors of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated.

The synthetic inhibitor of the present invention includes, but is not limited to, gingerol or its derivative in an amount of 0.
It can be contained in an amount of 01 to 99% by weight, preferably 0.1 to 80% by weight. In addition, the synthesis inhibitor of the present invention containing a zingerol-containing plant extract, for example, a zingerol-containing herbal medicine extract (particularly, a ginger extract) is a gingerol (particularly [6 ] -Gingerol) can be prepared by appropriately adjusting the amount range to the above range. In addition,
When a gingerol-containing plant extract, for example, a gingerol-containing crude drug extract (particularly a ginger extract) is used as an active ingredient, a synthetic inhibitor should be pharmaceutically acceptable. It is preferable to formulate using a carrier that can be used.

The dose of the synthetic inhibitor of the present invention varies depending on the type of disease, age, sex, body weight of patient, degree of symptom, administration method, etc. and is not particularly limited, but is [6]- The amount of gingerol is usually 1 per adult
About 10 mg to 10 g is orally or parenterally administered in about 1 to 4 times a day. Furthermore, the use is not limited to pharmaceuticals, and various uses, for example, functional foods and health foods can be given in the form of food and drink.

[0024]

As described above, the gingerol or its derivative, particularly [6] -gingerol contained in the synthetic inhibitor of the present invention has an action of specifically suppressing the synthesis of intracellular proteins belonging to the HSP27 family. Therefore, when the above-mentioned gingerol or its derivative is administered, HS in cells is increased.
Biosynthesis of proteins belonging to the P27 family is specifically reduced. Therefore, the above-mentioned gingerol or its derivative is used, for example, for the prevention and treatment of cancer in which a protein belonging to the HSP27 family is associated with malignant transformation, HSP27.
Enhancement of the effects of cancer hyperthermia associated with hyperthermia, where proteins belonging to the family interfere with the therapy, or H
A protein belonging to the SP27 family can be used for prevention and treatment of autoimmune diseases such as multiple sclerosis associated with its onset. In addition, there is also a report that the expression of proteins belonging to the HSP27 family is correlated with drug resistance ("Breast Cancer Res. Treat.", 2
3 : 178, 1992; "Cancer Res.", 51 : 5245-5252, 199
1) By suppressing the expression of proteins belonging to the HSP27 family, it is possible to suppress drug resistance and enhance the effects of chemotherapy.

[0025]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1: Measurement of HSP expression level of human cultured cancer cells (1) Culture of human cultured cancer cells The following human cultured cancer cells were cultured at 37 ° C under 5% carbon dioxide conditions except for heat shock treatment. Cultured. Lung cancer cell line H69 (ATCC HTB 119) is 10%
The cells were cultured in RPMI1640 medium containing inactivated fetal bovine serum (hereinafter abbreviated as FBS). Uterine cancer cell line He
La S3 (ATCC CCL 2.2) was cultured in MEM medium containing 10% inactivated FBS. Neuronal tumor cell line (neuroblastoma) SK-N-MC (ATCC HT
B 10) is a non-essential amino acid (L-alanine 8.9 m
g / l, L-asparagine.H ₂ O 15.0 mg / l,
L-aspartic acid 13.3 mg / l, L-glutamic acid 14.7 mg / l, glycine 7.5 mg / l, L-proline 11.5 mg / l and L-serine 10.5 mg / l.
1) and 10% inactivated FBS in MEM medium.

(2) [6] -Zingerol treatment and heat shock treatment [6] -Zingerol (Matsuura Yakugyo Co., Ltd.) was added to the medium of the various cultured human cancer cells 2 days after seeding so that the final concentration was 100 μM. And cultured for 24 hours. After that, 45 ℃
After heat shock treatment for 15 minutes at 37 ° C., it was cultured at 37 ° C. overnight. The control test was carried out as above except that [6] -gingerol was not added.

(3) Measurement of HSP expression level in cultured human cancer cells Each cell treated in the above section (2) was homogenized by the following method, and the HSP expression level was measured by Western blotting. That is, the cells treated in the above (2) were treated with phosphate buffered saline [composition: KCl = 0.2 g / l,
KH ₂ PO ₄ = 0.2 g / l, NaCl = 8 g / l, N
a ₂ HPO ₄ (anhydrous) = 1.15 g / l; hereinafter, PBS
(-), And then lyse buffer [1.0% NP-40,
0.15 M sodium chloride, 50 mM Tris-HCl
(PH 8.0), 5 mM-EDTA, 2 mM-N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 2 μg / ml leupeptin and 2 μg / ml pepstatin] 1 ml were added, and the mixture was allowed to stand on ice for 20 minutes. Thereafter, centrifugation was performed at 12,000 rpm at 4 ° C. for 20 minutes. 10 μl of the supernatant after centrifugation was added to PBS (−) 790 μ
In addition to l, protein assay stain (Dye Reagen
t Concentrate: Bio-Rad, Catalog No. 500-000
6) 200 μl was added. After standing at room temperature for 5 minutes, the protein was quantified by measuring the absorbance at 595 nm.

Using the sample for which protein was quantified, L
aemmli buffer system (Laemmli, NK, "Natur
e ", 283 : pp. 249-256, 1970), lysates containing equal amounts of protein were subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, blotting and subsequent blocking were performed. Using a transfer device (Trans-Blot Electrophoretic Transfer Cell: Bio-Rad, Catalog No. 170-3946), adhere the gel to a 0.45 μm nitrocellulose membrane (Schleicher & Schuell, Catalog No. 401196) at room temperature and 100 V at room temperature. A tris glycine buffer (Tris Gly Running and Bloom) containing 0.025 M Tris and 0.192 M glycine and adjusted to pH 8.5 was used as a blotting buffer.
tting Buffer; Enprotech, Massachusetts, USA
A buffer prepared by adding methyl alcohol to catalog number SA100034) to 20% was used. After blotting, the nitrocellulose membrane was added to a 10% skim milk (Snow Brand Milk Products) -PBS (-) solution at room temperature for 30 minutes.
Incubate to block non-specific binding.

After blocking, an anti-human HSP27 mouse monoclonal antibody (Stre
ssGen, Victoria, BC, Canada, Cat.No. SPA-8
00), a primary antibody reaction was performed. This anti-human HSP
The 27 mouse monoclonal antibody is a human breast cancer cell line MC
HSP2 isolated from F7 (ATCC HTB 22)
4 was used as an immunogen ("Cancer Res.",
42 , 4256-4258, 1982), HSP27 (human HSP2)
7, Chimpanzee HSP27 and sheep HSP27)
("Cancer Res.", 42 , 4256-4258, 1
982; "Cancer Res.", 43 , 4297-4301, 1983), HSP
Specifically reacts with HSP24 and HSP28. After the primary antibody reaction, the solution was exchanged with PBS (-) for 5 minutes each for 2 minutes.
Washing was performed by a slow rocking shaker, and further, washing was performed four times with a PBS (−)-0.1% Tween 20 (Bio-Rad, Cat. No. 170-6531) solution for 15 minutes, and the solution was replaced. Was. Finally,
Washing was performed twice for 5 minutes each with PBS (-).

After the washing was completed, a peroxidase-labeled goat anti-mouse IgG antibody (CAPPEL, Catalog No. 55550) was
% For 2 hours using 5 ml of an antibody solution prepared by diluting 5000-fold with a PBS (-) solution containing 5% skim milk.
The next antibody reaction was performed. After the completion of the reaction, the nitrocellulose membrane was washed twice with a PBS (-) solution for 5 minutes while changing the solution twice, and further with a PBS (-)-0.1% Tween20 solution for 15 minutes for 5 times. Performed by slow rocking shaker. Finally PBS (-)
The solution was washed twice for 5 minutes each. Extra PBS
(-) After removing the solution, the western blotting detection reagent (ACL Western blotting detection reagent; Ame
rsham, catalog number RPN2106), sprinkle onto the nitrocellulose membrane, incubate for 1 minute, remove excess detection reagent, wrap the nitrocellulose membrane in wrap, and cover the reaction surface with X-ray film (Kodak X-OMAT, AR, (Catalog No. 165 1454), exposed, developed and HSP2
7 was examined. The results are shown in Table 1. In the table,
"↓" means that the expression level of HSP27 was decreased as compared with the control.

[0031]

[Table 1] Cancer type Cancer cell Uterus HeLa S3 ↓ Lung H69 ↓ Nerve SK-N-MC ↓

In the control test, that is, cells to which [6] -gingerol was not added, one band having a molecular weight of about 27 kD was detected. The molecular weight is the same as the anti-human HS.
Binding was determined by P27 mouse monoclonal antibody and molecular weight markers (soybean trypsin inhibitor and bovine carbonic anhydrase). As shown in Table 1, [6] -gingerol is a uterine cancer cell line HeLa.
S3, lung cancer cell line H69, nerve tumor cell line SK-N-
The expression of HSP27 was suppressed in MC. That is,
It is concluded that [6] -gingerol has the activity of a synthetic inhibitor that suppresses the expression of HSP27.

[0033]

INDUSTRIAL APPLICABILITY As described above in detail, zingerol or a derivative thereof, particularly [6] -gingerol, is an intracellular HS.
It has the activity of a synthetic inhibitor that suppresses the expression of proteins belonging to the P27 family. Therefore, by administering zingerol or a derivative thereof, particularly [6] -gingerol, for example, a cancer in which a protein belonging to the HSP27 family is associated with its malignant transformation or a decrease in the effect of hyperthermia, multiple sclerosis associated with its development. It can effectively improve the physiological condition of patients with autoimmune diseases such as illness and effectively treat the diseases. In addition, it has been reported that the expression of proteins belonging to the HSP27 family is correlated with drug resistance ("Breast Cancer Res. Treat.", 2
3 : 178, 1992; "Cancer Res.", 51 : 5245-5252, 199
1) By suppressing the expression of proteins belonging to the HSP27 family, it is possible to suppress drug resistance and enhance the effects of chemotherapy.

Front page continuation (72) Inventor Yosuke Seisuke 3-26-1-303 Hyakunin-cho, Shinjuku-ku, Tokyo (72) Inventor Makoto Yoshimura 1154-91 Shimoasao, Aso-ku, Kawasaki-shi, Kanagawa (72) Inventor Chikushi Yoshikumi 2-19-46 Higashi, Kunitachi, Tokyo (72) Inventor Nagahiro Saijo 2-5-28 Higashigaoka, Meguro-ku, Tokyo

Claims (4)

[Claims] 1. A heat shock protein synthesis inhibitor having a molecular weight of from 16 kilodaltons to 40 kilodaltons, which contains gingerol or a derivative thereof as an active ingredient. 2. The molecular weight of 16 kilodaltons to 4 according to claim 1, wherein the gingerol is [6] -gingerol.
Inhibitor of heat shock protein synthesis up to 0 kilodalton. 3. A heat shock protein synthesis inhibitor having a molecular weight of from 16 to 40 kilodaltons, which contains an extract of a gingerol-containing plant as an active ingredient. 4. A heat-shock protein synthesis inhibitor having a molecular weight of from 16 kilodaltons to 40 kilodaltons, which contains an extract of ginger as an active ingredient.