WO2011108699A1 - Gene related to the action of isoflavones - Google Patents

Abstract

Disclosed is a gene (or product thereof), "Pap associated domain containing 5" (Papd5), identified as a gene necessary for equol to exhibit anticancer activity, for use as a biomarker for diagnosing the effectiveness, in a subject, of an anticancer substance selected from a group comprising equol, daidzein, and analogs thereof. Also disclosed is a product for detecting a biomarker using PCR, a DNA array, ELISA, a protein array, mass spectrometry, or immunochromatography. Further disclosed is a method for determining the effectiveness, in a subject, of an anticancer substance selected from a group comprising equol, daidzein, and analogs thereof. Said method includes a step for detecting the transcription or expression of Papd5 in a sample taken from the subject.

Description

Genes related to the action of isoflavones

The present invention relates to a gene on which isoflavones, particularly equol, depend upon acting on a subject. More particularly, the present invention relates to Papd5. According to the present invention, Papd5 or a product thereof can be used as a biomarker related to cancer. The present invention is useful in the fields of medicine, alternative medicine, and research and development of pharmaceuticals, foods and cosmetics.

Cancer has been the number one cause of death in Japan since 1981, and the number of cancers has been increasing year by year. It can be said that an effective treatment for cancer has not been established yet. Under these circumstances, we are focusing on food ingredients as substances useful for the prevention and treatment of cancer. There have been many reports on foods effective for cancer. Anti-cancer activity has been reported to Conjugated Linoleic Acid (CLA) contained in meat and eggs and Epigallocatechin-3-O-gallate (EGCG), which is a major component in green tea leaves (Non-patent Documents 1 and 2). In addition, anticancer activity has been reported to isoflavones such as genistein and daidzein (Non-patent Document 3).

Isoflavones are a kind of flavonoid contained in soybean, mainly soybean germ, and there are three kinds of genistein, daidzein and glycitein. In soybean fermented foods such as miso and natto, these are contained in large amounts as aglycones, but in most foods, they are contained in the state of glycosides. Glycosides are hydrolyzed by digestive enzymes and β-glucosidase of enteric bacteria to produce aglycones. Daidzein is also metabolized to equol by certain intestinal bacteria. About half of the Japanese and about 20% of Westerners are said to have enteric bacteria that convert daidzein to equol.

Isoflavones have a variety of physiological functions and have been reported to improve menopause and osteoporosis (non-patent document 4) in postmenopausal women. For cancer, it is widely known that it exhibits anticancer activity against hormone-dependent cancers such as breast cancer (Non-patent document 5) and prostate cancer (Non-patent document 6). It has been. Isoflavones are similar in structure to estrogen, which is a type of steroid hormone, and therefore, when they bind to estrogen-receptor (ER) in vivo, they exhibit estrogen-like action or antagonize estrogen and exert antiestrogenic action. It is considered.

On the other hand, the existence of ER-independent action of isoflavones has been attracting attention. For example, genistein exhibits an anticancer action against hormone-independent cancers such as gastric cancer, melanoma, and pancreatic cancer by inhibiting tyrosine kinase and topoisomerase (Non-patent Document 7). Moreover, although genistein contracts the thymus, it is reported that a part of its action is an ER-independent action (Non-patent Document 8). On the other hand, it has been clarified that an antiallergic function (Non-Patent Document 9) and a vascular relaxation function (Non-Patent Document 10) are ER-independent actions as equol functions. However, the mechanism of such ER-independent action is unclear.

Attempts have been made to use food factors that exhibit anticancer effects as alternative medicines such as cancer prevention, treatment, and recurrence prevention, but adverse effects such as the possibility of cancer progression have been pointed out. Yes. As an in vitro report on isoflavones, it has been reported to induce apoptosis and inhibit growth of human cancer cells (Non-Patent Document 5), while suggesting an effect of promoting carcinogenesis (Non-Patent Document 11). ). In in vivo animal experiments, some organs have been reported to have a carcinogenic promoting effect (Non-patent Documents 12 and 13).

On the other hand, for the gene Pap associated domain containing 5 (Papd5), the effect of camptothecin, an anticancer agent targeting topoisomerase, has only been reported to be reduced by the presence of Papd5 (Non-patent literature). 14).

In order to use isoflavones safely and effectively as cancer preventive / therapeutic agents, it is necessary to individually identify cancers for which isoflavones exhibit anticancer effects. On the other hand, the sensitivity to the physiological effects of functional food factors is being recognized as being different between individuals and organs, as is the case with pharmaceuticals, but in many cases it is unclear what causes these differences. One of the causes is that almost no gene gene (food factor-sensing gene) on the living body responsible for the functional expression of food factors has been clarified.

In the case of green tea catechin EGCG, the present inventors have identified 67 kDa laminin receptor (67 LR) as a gene (EGCG sensing gene) expressed on the side of cancer cells indispensable for its anticancer activity. (Tachibana H, Koga K, Fujimura Y, Yamada K. A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol. 11, 380-381 (2004)). Recently, intensive research was conducted to identify genes essential for equolequ to exert its ER independent anticancer activity. First, the present inventor found that equol, a kind of isoflavones, suppresses the growth of the prostate cancer cell line PC-3 and the human cervical cancer cell line HeLa. Furthermore, even in the presence of ICI 182,780, an estrogen receptor (ER) antagonist, the cell growth inhibitory effect of this equol was not inhibited, so it was clarified that this effect is ER-independent (Example and FIG. 1) . Furthermore, we attempted to identify genes responsible for the expression of equol's cancer cell growth-inhibiting activity and cell-killing activity using the genetic-suppressor-elements- (GSE) method. As a result, Pap associated domain containing 5 (Papd5) was identified as a gene essential for equol to exert its cancer cell proliferation inhibitory activity, and the present invention was completed.

The present invention provides the following.
[1] A biomarker for diagnosing the effectiveness of an anticancer substance selected from the group consisting of equol, daidzein and their analogs in a subject, from Papd5 or a product thereof A biomarker.
[2] A method for determining the effectiveness of an anticancer substance selected from the group consisting of equol, daidzein, and analogs thereof, comprising a step of detecting the transcription or expression of Papd5 in a sample collected from the subject.
[3] For detecting the biomarker according to [1], including oligonucleotide, peptide, antibody, for PCR, DNA array, ELISA, protein array, mass spectrometry, or immunochromatography , Product.
[4] The method according to [2], wherein the detection of transcription or expression of Papd5 is performed by PCR, DNA array, ELISA, protein array, mass spectrometry, or immunochromatography.
[5] Drugs, foods or cosmetics, or their application plans for the treatment of diseases or conditions ameliorated by equol or daidzein using the presence or absence of Papd5 mutation in the subject, or detection of transcription or expression as an index, Design method.
[6] Apply a test product that is a natural product, food, cosmetic, pharmaceutical product or compound to a subject, a sample collected from the subject, or a cell that has Papd5 and is capable of transcription or expression of Papd5. A method for screening for an effective treatment for cancer, comprising a step of detecting whether the amount of transcription or expression of Papd5 changes.
[7] The method according to 6, wherein an object suitable for application to a subject in combination with equol, daidzein or an analog thereof is screened.

FIG. 1 is a graph showing the cell growth inhibitory activity and ER dependence of equol against various cancer cells. Human cervical cancer cell line HeLa or human prostate cancer cell line PC-3 was seeded in a 24-well culture plate at 2 × 10 ⁴ cells / mL and pre-cultured for 24 hours. After treatment with a medium supplemented with 1 mM of ICI182,780, which is an ER antagonist, for 30 minutes, equol was added and cultured for 72 hours, and the number of cells was counted. FIG. 2 is a graph showing the cytostatic activity of equol against mouse melanoma cell line B16. The mouse melanoma cell line B16 was seeded at 2 × 10 ⁴ cells / mL in a 24-well culture plate, cultured for 72 hours in a medium supplemented with equol, and the number of cells was counted. FIG. 3 is a graph showing the effect of ICI on the equol sensitivity of B16 cells. B16 cells were seeded in a 24-well plate at 2 × 10 ⁴ cells / mL, pretreated for 30 minutes with a medium supplemented with ICI182,780 at a final concentration of 1 mM, and equol at each concentration was then added. After culturing for 72 hours, the number of cells was measured. ICI: ER inhibitor ICI182,780 FIG. 4 is a graph showing the involvement of Papd5 expression on the equol sensitivity of HeLa cells. a) Scramble-shRNA expression vector introduced HeLa cells or Papd5-shRNA expression vector introduced RNA was collected from HeLa cells, and after cDNA synthesis, the expression level of Papd5 mRNA was examined by Real time RT-PCR. b) HeLa cells transfected with Scramble-shRNA or Papd5-shRNA expression vector were seeded in a 24-well culture plate at 2 x 10 ⁴ cells / mL, and cultured for 72 hours in a medium supplemented with each concentration of equol. It was measured. scramble: HeLa cells introduced with scramble-shRNA expression vector, siPapd5: HeLa cells introduced with Papd5-shRNA expression vector FIG. 5 is a graph showing the involvement of Papd5 expression on the equol sensitivity of B16 cells. a) RNA was collected from Scramble-shRNA expression vector-introduced B16 cells or Papd5-shRNA expression vector-introduced B16 cells, and after cDNA synthesis, the expression level of Papd5 mRNA was examined by RT-PCR. b) B16 cells transfected with Scramble-shRNA or Papd5-shRNA expression vector were seeded at 2 x 10 ⁴ cells / mL in a 24-well culture plate, cultured in medium supplemented with equol for 72 hours, and the number of cells was measured. FIG. 6 is a graph showing the involvement of Papd5 expression in the cancer cell growth inhibitory action of functional food factors. Each concentration of equol, genistein, zedaidzein, and EGCG was added to HeLa cells into which Scramble-shRNA or Papd5-shRNA expression vector was introduced, and the number of cells was measured after culturing for 72 hours. FIG. 7 is a graph showing the effect of oral intake of equol on tumors knocked down by Papd5 expression. Cancer was transplanted by subcutaneously injecting cells into which the Papd5-shRNA5- expression vector had been introduced into the back of the mouse, and then equal was orally administered, and the change in tumor volume over time was measured. FIG. 8 is a graph showing the cell growth promoting effect of equol on cancer cells knocked down by Papd5pd expression. Each concentration of equal was added to B16 cells (siPapd5) introduced with Papd5-shRNA expression vector or B16 cells (scramble) introduced with Scramble-shRNA, and the number of cells was counted. FIG. 9A is a view showing nucleotide sequences and amino acid sequences related to the present invention. FIG. 9B shows the nucleotide sequence and amino acid sequence related to the present invention. FIG. 9C is a view showing a nucleotide sequence and an amino acid sequence related to the present invention.

The present invention is a biomarker for diagnosing the effectiveness of Papd5 or a product thereof in a subject of an anticancer substance selected from the group consisting of equol, daidzein and analogs thereof. Regarding use.

[Papd5 or its product, biomarker]
In the present invention, “Papd5” is used in the meaning of the Papd5 gene unless otherwise specified. Specifically, the polynucleotide comprising the nucleotide sequence represented as SEQ ID NO: 1, 3 or 5 in the sequence listing, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4 or 6, or any of them Refers to a homolog. More specifically, it is one of the following (a), (b), (c), (d), (e) or (f).
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 5 (more specifically, DNA);
(B) a polynucleotide that hybridizes with a polynucleotide comprising a sequence complementary to the polynucleotide of (a) under stringent conditions and can exhibit cytostatic activity in the presence of equol or daidzein (more specific Then DNA);
(C) a polynucleotide (more specifically, DNA) having high sequence identity with the polynucleotide of (a) and capable of exhibiting cytostatic activity in the presence of equol or daidzein;
(D) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, or 6 (more specifically, DNA);
(E) The amino acid sequence of SEQ ID NO: 2, 4 or 6 encodes an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and inhibits cell proliferation in the presence of equol or daidzein A polynucleotide (more specifically, DNA) that can exhibit:
(F) a polynucleotide that encodes an amino acid sequence having high sequence identity with the amino acid sequence of SEQ ID NO: 2, 4, or 6 and that can exhibit cytostatic activity in the presence of equol or daidzein (more specifically, DNA).

In the sequence listing of the present specification, the nucleotide sequence of human Papd5 is shown as SEQ ID NO: 1, and the amino acid sequence of human Papd5 protein is shown as SEQ ID NO: 2. Further, as SEQ ID NO: 3, nucleotide sequence of mouse Papd5, as SEQ ID NO: 4, amino acid sequence of mouse Papd5 protein, as SEQ ID NO: 5, as nucleotide sequence of rat Papd5, as SEQ ID NO: 6, as rat The amino acid sequence of Papd5 protein is shown.

Papd5 is preferably derived from a mammal, more preferably from a human.

In the present invention, the term “product” of Papd5 refers to an antibody that specifically recognizes the above-mentioned Papd5 transcription product (mRNA), expression product (protein), or any one of them unless otherwise specified. .

An example of the product is mRNA consisting of the nucleotide sequence represented as SEQ ID NO: 1, 3 or 5 or a homologue thereof. However, in SEQ ID NO: 1, 3, or 5, t is replaced with u. Hereinafter, the same applies to SEQ ID NO: 1, 3, or 5 as the mRNA sequence. Specifically, mRNA or a homologue thereof is any of the following (a ′), (b ′), (c ′), (d ′), (e ′), or (f ′).
(A ′) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, 3, or 5 (more specifically, RNA);
(B ′) a polynucleotide that hybridizes with a polynucleotide comprising a sequence complementary to the polynucleotide of (a ′) under stringent conditions and can exhibit cell growth inhibitory activity in the presence of equol or daidzein ( More specifically, RNA);
(C ′) a polynucleotide (more specifically, RNA) having high sequence identity with the polynucleotide of (a ′) and capable of exhibiting cytostatic activity in the presence of equol or daidzein;
(D ′) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, or 6 (more specifically, RNA);
(E ′) encoding an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4 or 6, and cell growth inhibition in the presence of equol or daidzein A polynucleotide (more specifically, RNA) capable of exerting activity;
(F ′) a polynucleotide that encodes an amino acid sequence having high sequence identity with the amino acid sequence of SEQ ID NO: 2, 4, or 6 and that can exhibit cytostatic activity in the presence of equol or daidzein (more specifically, , RNA).

Another example of a product is a protein consisting of 2, 4 or 6 amino acid sequences or a homologue thereof. Specifically, it is one of the following (d ″), (e ″) or (f ″).
(D ″) a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 6;
(E ″) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, 4 or 6, and cell growth inhibitory activity in the presence of equol or daidzein A protein that can exert
(F ″) a protein comprising an amino acid sequence having high identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6, and capable of exhibiting cell growth inhibitory activity in the presence of equol or daidzein.

The term “stringent conditions” as used in the present invention refers to the conditions of 6M urea, 0.4% SDS, 0.5 × SSC or a hybridization condition equivalent thereto, unless otherwise specified. May be applied under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC or equivalent hybridization conditions. Under each condition, the temperature can be about 40 ° C. or higher, and if higher stringency conditions are required, for example, about 50 ° C. or about 65 ° C. may be used.

In the present invention, the number of amino acids to be substituted when “one or more amino acids are substituted, deleted, inserted, and / or added” is the number of amino acids to be replaced or the protein encoding the protein. The nucleotide is not particularly limited as long as it has a desired function, but it is about 1 to 9 or 1 to 4. Means for preparing a polynucleotide according to such an amino acid sequence include, for example, the site-directed mutagenesis method (Kramer W & Fritz H-J: Methods Enzymol 154: 350, 1987).

In the present invention, regarding nucleotides, when “identity” is “high”, it means sequence identity of at least 80% or more, preferably 90% or more, more preferably 95% or more. In the present invention, when referring to amino acid sequences, identity refers to sequence identity of at least 80% or more, preferably 90% or more, more preferably 95% or more. Nucleotide or amino acid sequence identity is determined using the algorithm BLAST by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) it can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, parameters are set, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov/).

In the present invention, “polynucleotide” refers to DNA or RNA unless otherwise specified. Which one is indicated will be clear to the skilled person from the context. DNA can be single-stranded or double-stranded.

In the present invention, the term “Biomarker” is objectively measured as an index of a pharmacological response to a normal biological process, pathological process, or therapeutic intervention, unless otherwise specified. It is used to refer to a substance (tangible) that can be evaluated.

In the present invention, “a biomarker comprising Papd5 or a product thereof” includes not only the whole (full length) of Papd5 or a product thereof but also a part thereof, unless otherwise specified. This “part” is a part that is long enough that the presence of Papd5 or its product can be inferred. In the present invention, Papd5 or a product thereof is referred to as “use as a biomarker”, not only when all of Papd5 or a product thereof is used, but also a part of Papd5 or a product thereof, and Papd5 or a product thereof. It also includes the case where a part having such a length that the existence of the above can be estimated is used. Also, use as a biomarker includes use for detection of SNP, use for detection of gene mutation, use for detection of localization change.

[Anti-cancer substances, equal, daidzein]
Compounds used in or related to the present invention include the following:

According to the study by the present inventors, among functional food factors that exhibit anticancer effects, equol, genistein, daidzein, and epigallocatechin gallate (EGCG), genistein and EGCG inhibit the growth of HeLa cells in the same way as equol. However, the effect was not affected by the decrease in Papd5 expression. On the other hand, the cell growth inhibitory action of daidzein was inhibited in the cells in which the expression of Papd5 was reduced, like equol (Example and FIG. 6).

In the present invention, the term “anticancer substance” means a general component effective for the treatment of cancer, unless otherwise specified. The anticancer substance may be a food component or a medicine (anticancer agent). In the present invention, an anticancer substance selected from the group consisting of equol, daidzein and analogs thereof is used. “These analogs” have anti-cancer activity (more specifically, cancer cell growth-inhibitory activity) similar to that of daidzein glycosides, equol or daidzein, but the effect is due to decreased expression of Papd5. Substances that are inhibited are included. Such substances may act in an ER-independent manner.

According to another study by the inventors, the human cervical cancer cell line HeLa and the human prostate cancer cell line PC-3 were inhibited by equol and treated with ER antagonist ICI182,780 (ICI). Even in the case of the action, the action was not inhibited. From this, it can be said that equol exhibits cytostatic activity against these cancer cell lines by an action mechanism independent of ER (FIG. 1).

In this specification, “estrogen receptor (ER)” has the ability to bind to estrogen (estrone (E1), estradiol (E2) and estriol (E3)), unless otherwise specified, and accepts steroids. One of the molecules belonging to the body superfamily, and refers to a receptor protein also called follicular hormone receptor. The estrogen receptor has two isoforms, ERα (NR3A1, 595 amino acid residues) and ERβ (NR3A2, 530 residues). Estrogens have the function of promoting the formation of reproductive functions and cell proliferation.

In the present invention, “ER-independent” or “ER-independent” means acting on a target without depending on ER, unless otherwise specified. Whether or not the action of a substance is “ER-independent” can be appropriately determined by those skilled in the art. For example, due to the presence of an antagonist of ER, the effect to be exerted is unaffected or can be neglected (eg, effects within ± 30% compared to the effect in the absence) Can be judged using the index of For more detailed conditions, reference can be made to the description of the examples in the present specification.

[Diagnosis of effectiveness of anticancer substances in subjects]
In the present invention, a disease or condition that is improved by the action of equol or daidzein, unless otherwise specified, cancer, particularly cervical cancer, endometrial cancer, prostate cancer; stomach cancer, melanoma, Pancreatic cancer (Non-patent document 7); Thymic hypertrophy or atrophy (Non-patent document 8); Allergy (Non-patent document 9), vascular stenosis or relaxation (Non-patent document 10). Preferably it is cancer. There are two types of pathogenesis of body cancer, estrogen-dependent and independent, with the former being representative of endometrioid adenocarcinoma and the latter being serous adenocarcinomas.

In the present invention, when “treatment” is concerned with cancer, unless otherwise specified, it prevents cancer, reduces the risk of developing cancer, treats cancer, and suppresses progression of cancer. Including doing.

According to the present invention, for detection of the biomarker of the present invention, oligonucleotides, peptides, antibodies, PCR (RT-PCR, real-time PCR), DNA array, ELISA, protein array, or immunochromatography Products (kits, etc.) for graphy are provided. Such products specifically amplify all or part of the appropriate length of the oligonucleotide probe, Papd5 or its transcript, which can be detected by specifically hybridizing with Papd5 or its transcript. An antibody (which can be expressed in Escherichia coli) and may contain an oligonucleotide primer of an appropriate length that can specifically recognize Papd5, its transcription product or its expression product. From the amino acid sequence information of the recombinant protein or Papd5, a Papd5-specific antibody can be obtained by immunization with a highly antigenic peptide derived from Papd5), and may contain a SELEX product or the like. Those skilled in the art should appropriately design and manufacture such primers and the like based on the sequence information shown in SEQ ID NOs: 1 to 6 in the sequence listing with reference to the common general technical knowledge and the description of the present specification. Can do.

Specifically, the product including the above-described primers can be a PCR kit, a DNA chip (microarray), or a protein array.

Further, according to the present invention, a group consisting of equol or daidzein and analogs thereof, comprising a step of detecting transcription or expression of Papd5 in a subject or a sample collected from the subject (usually not assumed to be returned to the subject). Methods for determining the effectiveness of a more selected anticancer substance in a subject are provided. In this determination method, the method used is preferably a PCR method, a DNA array method, an ELISA method, a protein array method, or an immunochromatography method.

In the present invention, when the transcription or expression of Papd5 is suppressed, the effectiveness of the anticancer substance selected from the group consisting of equol or daidzein and their analogs is low or ineffective It can be judged. In general, anything assumed as a cause of suppression of gene transcription or expression is considered to affect the transcription or expression of Papd5. For example, due to the presence of disease risk factors such as congenital factors, oxidative stress, genetic mutations due to carcinogenic factors, diet, lifestyle (smoking, alcohol consumption, exercise, etc.), obesity, hypertension, hyperglycemia and hyperlipidemia, Papd5 Transcription or expression can be suppressed.

Regarding the application of an anticancer substance selected from the group consisting of equol or daidzein and their analogs in the present invention, the term “subject” refers to cancer animals, healthy animals, cancers, unless otherwise specified. Includes patients and healthy individuals. In the present invention, when it is `` applied '' with respect to anticancer substances and natural products that are candidates for anticancer substances, in addition to testing on samples, administration of pharmaceuticals to individuals, intake of food, This includes applying cosmetics, including when it is done for others. The “medicine” referred to in the present invention includes animal drugs in addition to drugs for humans, unless otherwise specified, and the “food” referred to in the present invention includes not only solid foods, unless otherwise specified. Including beverages and for humans as well as for animals, such as pet food, feed. The “cosmetics” as used in the present invention refers to those used for the purpose of cosmetics, which are not intended for treatment of illness, except when specifically described.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for screening a substance effective for cancer treatment from existing natural products, foods, cosmetics, pharmaceuticals or compounds. More specifically, the screening method of the present invention may comprise the following steps:
1) A step of applying a test product which is a natural product, food, cosmetic, pharmaceutical product or compound to a subject, a sample collected from the subject or a cell having Papd5 and capable of transcription or expression of Papd5;
2) detecting whether or not the amount of transcription or expression of Papd5 in the subject or sample changes; and 3) if the amount of transcription or expression increases, the test substance is effective for treating cancer. The process of selecting as a simple thing.

The screening method of the present invention is particularly suitable for screening an object suitable for application to a subject in combination with equol, daidzein or an analog thereof.

According to the present invention, the presence or absence of mutation of Papd5, or the amount of transcription or expression, as an index, according to the individuality of the subject, a pharmaceutical, food or cosmetic for treatment of a disease or condition improved by the action of equol or daidzein, Or the application plan can be designed. For example, in a subject with high expression of Papd5, the effect of the anticancer drug camptothecin is reduced (Non-patent Document 14), but it seems that the action of equol or daidzein can be expected.

Implementation of the present invention including actions such as “treatment”, “application”, and “design” includes medical actions by doctors and actions that do not rely on doctors. *

Among the following experiments, experiments using human-derived cells 1, 5 and 6 are genes and sequences that cause transcription of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 in the sequence listing (mRNA of human Papd5). The experiment using a mouse-derived cell of 2-4 and 7-9, corresponding to the implementation of a protein consisting of the amino acid sequence of No. 2 (human Papd5 protein), This corresponds to the execution of a protein (mouse Papd5 protein) consisting of the amino acid sequence of SEQ ID NO: 4, a gene that generates nucleotides (mRNA of mouse Papd5) by transcription.

<Experimental materials and experimental methods>
1． The human cervical cancer cell line HeLa used for the measurement of equol's cytostatic activity and ER-dependent cell count on various human cancer cells is DMEM medium supplemented with 10% fetal bovine serum (FCS) (BIOLOGICAL). The human prostate cancer cell line PC-3 was subcultured and maintained in RPMI1640 medium supplemented with 10% FCS at 37 ° C. under 5% CO ₂ with water vapor saturation. Cells were maintained in culture in the logarithmic growth phase. The DMEM medium used for the culture was Dulbecco's MEM medium (Cosmo Bio Co., Ltd.) 13.38 g per 1 L of dH ₂ O, 5.958 g of HEPES (Wako Pure Chemical Industries, Ltd.), 200,000 units of penicillin G for injection (Meiji Seika Co., Ltd.) Company) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (nacalai tesque) 3.7 g were suspended and then sterilized with a 0.22 μm filter. RPMI-1640 medium is 10.4 g of RPMI-1640 medium (Nissui Pharmaceutical) per 1 L of dH ₂ O, 2.38 g of 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane sulfonic acid (HEPES), 100 U / penicillin. mL, streptomycin 100 mg / L, and NaHCO ₃ (Wako Pure Chemical Industries, Ltd.) 2.0 g were suspended, and the pH was adjusted to 6.8 to 7.4 by blowing carbon dioxide, and then sterilized by filtration through a 0.22 μm filter. Then, 10% of fetal calf serum (FCS) was added and used for cell culture. When the cells were passaged, the cells were washed with PBS and then detached with a trypsin solution. PBS suspends 8.0 g of NaCl (nacalai tesque) per 1 L of dH ₂ O, 0.2 g of KCl (nacalai tesque), 1.15 g of Na ₂ HPO ₄ (Wako Pure Chemical Industries, Ltd.), 0.2 g of KH ₂ PO ₄ (nacalai tesque). The trypsin solution was prepared by suspending 0.05 g of EDTA · 2Na (Wako Pure Chemical Industries, Ltd.) and 0.02 g of trypsin (nacalai tesque) per 100 mL of PBS, and sterilizing the filter. , 5 mL or 10 mL adhesive dish (nunc ^™ ) was used Equol (Funakoshi), Daidzein (Funakoshi), Genistein (Funakoshi) were dissolved in DMSO (nacalai tesque) to 100 mM and stored at 4 ° C. ICI182,780 (invitrogen) was dissolved in DMSO to a concentration of 10 mM and stored at −80 ° C.

For the measurement of the number of cells, each cell was adjusted to 2 × 10 ⁴ cells / mL, seeded in a 24-well plate (nunc ^™ ), and precultured in a medium containing 10% FCS for 24 hours. Thereafter, the medium was replaced with 2% FCS-containing medium supplemented with ICI182,780 to 1 μM, treated for 30 minutes, and equol (LC Labolatories) was added to each concentration. After culturing for 72 hours, the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.

2． The mouse melanoma cell line B16 (American Type Culture Collection) used for the measurement of the cell proliferation inhibitory activity cell number and cell viability of equol against the mouse melanoma cell line B16 was 5% FCS-added DMEM medium at 37 ° C with water vapor saturation. Passaged and maintained under% CO ₂ conditions. Cells were maintained in culture in the logarithmic growth phase. For the measurement of cell number and cell viability, mouse melanoma cell line B16 was adjusted to 2 × 10 ⁴ cells / mL, seeded in a 24-well plate, and pre-cultured in DMEM medium containing 5% FCS for 24 hours. Thereafter, the cells were counted for 72 hours or 24, 48, 72, 96 hours in a 2% FCS-containing DMEM medium containing equol having a final concentration of 0, 1, 5, 10, 25, 50 μM, and the number of cells was counted using a cell counter. Student's t test was used for statistical processing of the experimental results.

3． Effect of ICI on equol sensitivity of B16 cells To measure the number of cells, B16 cells were adjusted to 2 × 10 ⁴ cells / mL, seeded in 24-well plates, and pre-cultured in DMEM medium containing 5% FCS for 24 hours. After treatment with 2% FCS-containing DMEM medium containing ICI 182,780 at a final concentration of 1 μM for 30 minutes, equol was added to a final concentration of 0, 1, 5, 10, 25 μM. After culturing for 72 hours, the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.

Four. Identification of equol-sensing gene EcoPack2-293 and AmphoPack-293 packaging cells (Clontech) were adjusted to 2 × 10 ⁵ cells / mL, seeded in a 5 mL dish, and cultured in DMEM medium containing 10% FCS. The next day, Mouse Embryo cDNA library ML8000BB (Clontech, Mountain View, CA) was fragmented with restriction enzymes EcoRI and Sph I, and introduced into pLPCX-modified retroviral vector FGS library (MFL-ESP) (1.0 μg / μL) 3 μL, pVSV-G vector (Takara Bio Inc.) (1.0 μg / μL) 3 μL and FuGENE 6 Transfection Reagent (Roche) 6 μL were mixed and introduced into each packaging cell. The next day, the packaging cell culture supernatant is passed through a filter (0.22 μm), and polybrene (Hexadimethrine bromide) (SIGMA-ALDRICH) is added to a final concentration of 8 μg / mL, and then adjusted to 1 × 10 ⁴ cells / mL the previous day. Then, the cells were seeded in a 5 mL dish and sprinkled on B16 cells that had been cultured in a DMEM medium containing 5% FCS to infect the virus in the culture supernatant. To the packaging cells, a fresh 10% FCS-containing DMEM medium was added, and the culture was continued. This operation was performed four times every 12 hours. The infected B16 cells were adjusted to 1 × 10 ⁴ cells / mL, seeded in a 96-well plate (nunc ^™ ), and recovered for 24 hours in 2% FCS-containing DMEM medium. After recovery culture, the cells were cultured in a DMEM medium containing 1% FCS in a medium containing 80 μM equol to obtain equol-resistant B16 cells.

To Equol resistant cells, 1 mL of TRIzol (registered trademark) Reagent (invitogen) was added and allowed to stand at room temperature for 5 minutes. Next, 200 μL of chloroform (nacalai tesque) was added and stirred, allowed to stand at room temperature for 3 minutes, and then centrifuged (12000 × g) at 4 ° C. for 15 minutes. Thereafter, the upper layer was taken out, 500 μL of 2-propanol (nacalai tesque) was added and stirred, allowed to stand at room temperature for 10 minutes, and then centrifuged (12000 × g) at 4 ° C. for 10 minutes. Thereafter, the supernatant was removed, and 1 mL of 75% EtOH (nacalai tesque) in DEPE water (1 mL of DEPC (SIGMA-ALDRICH) dissolved in 1 L of dH ₂ O and autoclaved) was added and stirred, then 5 ° C at 4 ° C. Centrifuge for 1 minute (12000 xg). The supernatant was completely removed, and 10 to 20 μL of DEPC water was added and suspended. In addition, (dT) 20 primer (5'-TTT TTT TTT TTT TTT TTT TT-3 ', which was commissioned to Genenet Co., Ltd.) was adjusted to 0.5 μg / μL with DEPC water. All primers were also commissioned to Genenet Co., Ltd.) 1.0 μL was added, allowed to stand at 70 ° C. for 10 minutes, and then allowed to stand on ice for 10 minutes. Then, add dNTP 2.0 μL, M-MLV Reverse Transcriptase buffer 4.0 μL, M-MLV Reverse Transcriptase (Promega) 0.1 μL, RNase inhibitor (Takara Bio Inc.) 0.1 μL and let stand at 37 ° C. for 1 hour, then 5 minutes Boiled. The prepared cDNA was stored at -20 ° C. Using the cDNA as a template, pLPCX-S (5'-GAT CCG CTA GCG CTA CCG GAC TCA GAT-3 ', SEQ ID NO: 7) and pLPCX-A (5'-CTT TCA TTC CCC CCT TTT TCT GGA GAC-3 ', And the primer set of SEQ ID NO: 8) was used by adjusting to 20 μM with TE buffer (10 mM Tris (nacalai tesque) -HCl, 1 mM EDTA, pH 8.0). The same applies to the primers used in the following PCR.

The introduced cDNA fragment was amplified by PCR using TE buffer. As a PCR device, TGRADIENT (Biometra (registered trademark ⁾ , Germany) or PC320 (ASTEC, Fukuoka) was used. PCR is 0.5 μL of each primer (sense primer and antisense primer) per sample, dNTP mix 1 μL, 10 x buffer 1 μL, Ex taq 0.1 μL and dH ₂ O 5.9 μL purchased from Takara Bio Inc. (Shiga) 1 μL of the template was suspended, initial denaturation at 94 ° C. for 2 minutes, denaturation reaction at 94 ° C. for 30 seconds, annealing at 67.1 ° C. for 30 seconds, extension reaction at 72 ° C. for 1 minute, and denaturation reaction, annealing and extension reaction were performed 40 cycles. . After PCR, 1 μL of BPB solution and 0.5 μL of SYBR (registered trademark) Green I nucleic acid gel stein (Molecular probes (invitrogen)) were added per sample. The BPB solution was dissolved in dH ₂ O to be 0.25% Bromophenol blue (Wako Pure Chemical Industries, Ltd.) and 30% glycerin (nacalai tesque). SYBR (registered trademark) Green I nucleic acid gel stein was used after diluting 1000 times with dH ₂ O. The sample was used for electrophoresis on a 1.5% agarose gel. The agarose gel was prepared by dissolving Agarose S (Wako Pure Chemical Industries, Ltd.) at a concentration of 1 to 1.5% in TAE buffer. As a TAE buffer, 242 g of Tris (nacalaitesque) -HCl per 1 L, 37.2 g of EDTA 2Na, 57.1 mL of glacial acetic acid (nacalai tesque) are dissolved in dH ₂ O, adjusted to pH 8.5, and then filled up to 1 L with dH ₂ O. The mixture was sterilized by autoclaving to prepare a 50 × TAE buffer, which was diluted 50 times with dH ₂ O and used. GelMate (TOYOBO) was used for the electrophoresis tank. After electrophoresis, the cells were immersed in 500 mL TAE buffer supplemented with 250 μL EtBr for 15 minutes. Thereafter, DNA was detected using ChemiImager ^™ 5500 (Alpha Innotech). Then, the amount of DNA was digitized from the photoshop histogram. As a result, a pLPCX-specific band confirmed was collected from an agarose gel using GENECLEAN II kit (Funakoshi). The DNA fragment was TA cloned into pTargeT ^™ Mammalian Expression Vector System (Promega) using DNA Ligation Kit Ver.2.1 (Takara Bio Inc.). This vector was transformed into E. coli as follows. Escherichiacoli JM109 (Takara Bio Inc.) was used for E. coli. Add the glycerol stock of Escherichia coli JM109 stored at -80 ° C to SOB, shake at 16 ° C until OD ₆₀₀ = 0.6, centrifuge, discard the supernatant, and suspend in TB (Transfer Buffer). It became cloudy. It was centrifuged and the supernatant was discarded. The supernatant was suspended in DMSO-added TB and stored in liquid nitrogen. SOB is 3.0 g of Bacto Tryptone (Wako Pure Chemical Industries, Ltd.), 0.75 g of Bacto Yeast Extract (Wako Pure Chemical Industries, Ltd.), 0.078 g of NaCl, 0.027 g of KCl, and dH ₂ O per 150 ml. Added to 148.5 ml. After cooling the solution to room temperature autoclaved, which 2M Mg ²⁺ solution separately autoclaved and (12.324g MgSO ₄ · 7H ₂ O of (Wako Pure Chemical Industries, Ltd.) + 10.165g of MgCl ₂ · 6H ₂ 1.5 ml of O (nacalai tesque) was mixed with water to make 50 ml and sterilized by autoclaving). TB is mixed with 0.3 g of PIPES (Wako Pure Chemical Industries, Ltd.), 0.22 g of CaCl ₂ (Wako Pure Chemical Industries, Ltd.) and 1.86 g of KCl in 100 ml of water per 100 ml, and then the pH is adjusted to KOH (nacalai tesque) adjusted to 6.7. After adding 1.09 g of MnCl ₂ .4H ₂ O (Wako Pure Chemical Industries, Ltd.), the total amount was adjusted to 100 ml, filter sterilized (0.22 μm), and stored at 4 ° C. E. coli was cultured using ampicillin-containing liquid LB medium or ampicillin-containing LB plates. Ampicillin-containing liquid LB medium was filled with 1 g of Bacto Tryptone 10 g, Bacto Yeast Extract 5 g, and NaCl 10 g to 1 L with dH ₂ O to a pH of 7.0, and autoclaved. Before use, 150 mg / ml ampicillin was added at a 1000-fold dilution. The LB plate was added with 2 g of Bacto Tryptone, 1 g of Bacto Yeast Extract, 2 g of NaCl, and 5 g of Bacto Agar (Wako Pure Chemical Industries, Ltd.) per 200 ml, filled up to 200 ml with dH2O and sterilized by autoclave. After autoclaving and cooling to 60 ° C., 200 μl of 150 mg / ml ampicillin was added, and 10 ml each was dispensed into a 10 ml dish (Falcon 1029) (Nippon Becton Dickinson Co., Ltd.). Ampicillin used was ampicillin sodium (Wako Pure Chemical Industries, Ltd.) dissolved in dH ₂ O to 150 mg / ml, filter sterilized (0.22 μm) and stored at −20 ° C. For transformation, the vector was mixed with 100 μL of Escherichiacoli JM109 suspension and left on ice for 30 minutes. Thereafter, it was left at 42 ° C. for 30 seconds, then returned to ice and cooled for 1 to 3 minutes. 0.9 mL of SOC was added thereto, and recovery culture was performed at 37 ° C. for 1 hour. The SOC was 100 mL SOB per 100 mL, 1 mL of 2M glucose solution (18.016 g mixed in water and autoclaved to 50 mL), dispensed, and stored at room temperature or 4 ° C. It was centrifuged (1000 × g) for 5 minutes at room temperature to remove 900 μL of supernatant.

The Escherichia coli was applied to an ampicillin-containing LB plate and cultured at 37 ° C. overnight, and the colonies that had risen were applied to an ampicillin-containing LB plate coated with x-gal and IPTG for blue-white determination. The plate was used by applying 25 μL of 20 mg / ml X-gal and 25 μL of 0.1M IPTG to an LB plate containing ampicillin. For 20 mg / ml X-gal, 100 mg of X-gal was dissolved in 5 ml of NN-dimethyl-formamide (Wako Pure Chemical Industries, Ltd.) and stored at -20 ° C. For 0.1M IPTG, 1 g of IPTG (Wako Pure Chemical Industries, Ltd.) was dissolved in 4.2 ml of dH ₂ O and stored at −20 ° C. After overnight culture at 37 ° C, a white colony that has risen as a template, 0.5 μL each of primer set of pLPCX-S and pLPCX-A, 2.5 mM dNTP mix 1 μL, 25 mM MgCl ₂ purchased from Fermentas UAB 1 × L of 10 × buffer with (NH ₄ ) ₂ SO ₄ , 0.1 μL of Taq DNA Polymerase (Recombinant) and 4.9 μL of dH ₂ O were suspended in appropriate amounts. PCR was performed at an initial denaturation of 94 ° C. for 2 minutes, a denaturation reaction at 94 ° C. for 30 seconds, an annealing at 67.1 ° C. for 30 seconds, an extension reaction at 72 ° C. for 2 minutes, and a denaturation reaction, annealing, and extension reaction were performed for 30 cycles. After PCR, 1 μL of BPB was added to each sample, and electrophoresis was performed on a 1.5% agarose gel.

Plasmid DNA was recovered from the clones in which the insert was confirmed using LaboPass ^™ Mini (LaboPass) and suspended in TE buffer. Using the recovered plasmid DNA as a template, an extension reaction for sequencing was performed. The reaction was performed using the BigDye Terminator v3.1Cycle Sequencing Kit according to the Big Dye Terminator cycle sequencing protocol. The reaction solution was mixed with 3.5 μL of buffer, 3.2 pmol of primer, appropriate amount of template, 1.0 μL of BigDye Terminator v3.1Cycle Sequencing Kit (premix), and adjusted to 20 μL with dH ₂ O. The reaction was performed at initial denaturation 96 ° C. for 1 minute, denaturation reaction 96 ° C. for 10 seconds, annealing 50 ° C. for 5 seconds, extension reaction 60 ° C. for 4 minutes, and denaturation reaction, annealing, and extension reaction were performed 25 cycles. T7 (5'-CTA ATA CGA CTC ACT ATA GGG C-3 ', SEQ ID NO: 9), LacZ (5'-AGA TAT GAC CAT GAT TAC GCC-3', SEQ ID NO: 10) are used as primers. The buffer was adjusted to 1.0 pmol / μL before use. Thereafter, the base sequence was determined with an ABI3130xl DNA sequencer. The equol resistance causative gene was identified from the nucleotide sequence information.

Five. Construction of HeLa cells in which expression of Papd5 was knocked down Construction of human cervical cancer cell line HeLa in which Papd5 was knocked down was performed as follows. As a vector (shPapd5-RNA) for knocking down the expression of Papd5, a pLKO.1-puro vector (SIGMA-ALDRICH) having 5′-GCCACATATAGAGATTGGATA-3 (SEQ ID NO: 11) as a target sequence was used. In the gene transfer method, HeLa cells were seeded at 2 × 10 ⁴ cells / mL, and 24 hours later, 2 μg of vector and 6 μL of Fugene 6 were mixed with DMEM to a total volume of 200 μL, and added to the cells. As a method for selecting a cell into which a gene was introduced, a vector was added to the cell, and the cell was collected 48 hours later, seeded in a 96-well plate, and selected with puromycin (invivogen). Using the surviving and proliferating cells, knockdown of Papd5 was confirmed as follows. The reagents used were those prepared at the time of identification of equol's anticancer activity-related gene. 1 mL of TRIzol® Reagent was added to the cells and left at room temperature for 5 minutes. Next, 200 μL of chloroform was added and stirred, allowed to stand at room temperature for 3 minutes, and then centrifuged (12000 × g) at 4 ° C. for 15 minutes. Thereafter, the upper layer was taken out, 500 μL of 2-propanol was added and stirred, allowed to stand at room temperature for 10 minutes, and then centrifuged (12000 × g) at 4 ° C. for 10 minutes. Thereafter, the supernatant was removed, 1 mL of 75% EtOH in DEPE water was added and stirred, and then centrifuged (12000 × g) at 4 ° C. for 5 minutes. The supernatant was completely removed, and 10 to 20 μL of DEPC water was added and suspended. (DT) 20 0.5 μL and Ramdom 6 mers (5′-NNN NNN-3 ′, adjusted to 0.5 μg / μL with DEPC water) 0.5 μL, 5 x PrimeScript Buffer (for Real time) 2 μL, PrimesScript RT Enzyme Mix I 0.5 μL (both Takara Bio) were added, and a reverse transcription reaction was performed at 37 ° C. for 15 minutes. After treating at 85 ° C for 5 seconds, it was stored at -4 ° C. In Real Time PCR, a primer for amplifying Papd5, Papd5-F (5'-CACAAAGTCGCAGATGAGGA-3 ', SEQ ID NO: 12), Papd5-R (5'-TGGACTGTGTGGCAGAAGAG-3', SEQ ID NO: 13) becomes 10 mM. As described above, a solution diluted with TE buffer was used. 2 μL of the prepared cDNA, 1 μL of each primer, and 12.5 μL of SYBR Premix Ex Taq II (2 ×, SYBR PrimerScript RT-PCR Kit II) were mixed and set in the Thermal Cycler Dice Real Time System for reaction. PCR conditions were initial denaturation at 95 ° C. for 10 seconds, then 95 ° C. for 5 seconds and 60 ° C. for 20 seconds. Similarly, the expression level of GAPDH as an internal standard was examined in the same manner as before.

On the other hand, HeLa cells into which a control vector (scramble-shRNA) was constantly introduced were also prepared.

6． Papd5-shRNA-introduced HeLa cells and scramble-shRNA-introduced HeLa cells created in 5 of Papd5 on equol sensitivity of HeLa cells are subcultured and maintained in the same manner as WildType HeLa cells in DMEM medium containing 10% FCS. did.

For the measurement of the number of cells, HeLa cells were seeded in a 24-well plate at 2 × 10 ⁴ cells / mL and pre-cultured in DMEM medium containing 10% FCS for 24 hours. Thereafter, the cells were replaced with 10% FCS-containing DMEM medium containing equol having a final concentration of 1,5,10,25 μM, cultured for 72 hours, and the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.

7． Construction of B16 cells in which expression of Papd5 was knocked down Construction of B16 cells in which Papd5 was knocked down was performed as follows. As a vector (shPapd5-RNA) for knocking down the expression of Papd5, a pLKO.1-puro vector (SIGMA-ALDRICH) having 5′-CTGACGAGGATTCCGTGAAAG-3 ′ (SEQ ID NO: 14) as a target sequence was used. In the gene transfer method, B16 cells were seeded at 2 × 10 ⁴ cells / mL, 24 hours later, 2 μg of vector and 6 μL of Fugene 6 were mixed with DMEM to a total volume of 200 μL and added to the cells. As a method for selecting a cell into which a gene was introduced, a vector was added to the cell, and the cell was collected 48 hours later, seeded in a 96-well plate, and selected with puromycin (invivogen). Using the surviving and proliferating cells, knockdown of Papd5 was confirmed as follows. The reagents used were those prepared at the time of identification of equol's anticancer activity-related gene. 1 mL of TRIzol (Registered Trademark) Reagent was added to the cells and left at room temperature for 5 minutes. Next, 200 μL of chloroform was added and stirred, allowed to stand at room temperature for 3 minutes, and then centrifuged (12000 × g) at 4 ° C. for 15 minutes. Thereafter, the upper layer was taken out, 500 μL of 2-propanol was added and stirred, allowed to stand at room temperature for 10 minutes, and then centrifuged (12000 × g) at 4 ° C. for 10 minutes. Thereafter, the supernatant was removed, 1 mL of 75% EtOH in DEPE water was added and stirred, and then centrifuged (12000 × g) at 4 ° C. for 5 minutes. The supernatant was completely removed, and 10 to 20 μL of DEPC water was added and suspended. There, 0.5 μL of (dT) 20 and 0.5 μL of Ramdom 6 mers (5′-NNN NNN-3 ′, adjusted to 0.5 μg / μL with DEPC water), 5 × PrimeScript Buffer (for Real time ) 2 μL and PrimesScript RT Enzyme Mix I 0.5 μL (both Takara Bio) were added, and a reverse transcription reaction was performed at 37 ° C. for 15 minutes. After treating at 85 ° C for 5 seconds, it was stored at -4 ° C. RT-PCR is 10 mM of primers that amplify Papd5, Papd5-F (5′-GGAGGTAGTGAGCAGGATCG-3 ′, SEQ ID NO: 15), Papd5-R (5′-ATCCTCGTCAGCGACTTTGT-3 ′, SEQ ID NO: 16). As described above, a solution diluted with TE buffer was used. Using 1 μL of prepared cDNA, 0.5 μL of each primer, 2.5 μm dNTP mix 1 μL, 25 mM MgCl ₂ 1 μL, 10 x buffer with (NH ₄ ) ₂ SO ₄ 1 μL, Taq DNA Polymerase (Fermentas) 0.1 μL, dH ₂ O 4.9 μL And subjected to PCR. PCR was performed at an initial denaturation of 94 ° C. for 2 minutes, a denaturation reaction at 94 ° C. for 1 minute, an annealing at 60 ° C. for 1 minute, an extension reaction at 72 ° C. for 1 minute, and a denaturation reaction, annealing, and extension reaction were performed for 35 cycles. Similarly, the expression level of β-Actin as an internal standard was examined in the same manner as before. After PCR, 1 μL of BPB was added, and electrophoresis was performed on a 1.5% agarose gel to which EtBr Solution (Wako Pure Chemical Industries, Ltd.) was added at a 10,000-fold dilution.

On the other hand, B16 cells into which a control vector (scramble-shRNA) was constantly introduced were also prepared.

8． Papd5-shRNA-introduced B16 cells created in 7 involved in Papd5 on equol sensitivity of B16 cells, and B16 cells with cramble-shRNA were subcultured in the same manner as Wild Type B16 cells in DMEM medium containing 5% FCS. Maintained.

For the measurement of the number of cells, B16 cells were seeded on a 24-well plate at 2 × 10 ⁴ cells / mL and pre-cultured in DMEM medium containing 5% FCS for 24 hours. Thereafter, the medium was replaced with 2% FCS-containing DMEM medium containing equol having a final concentration of 10 μM and cultured for 72 hours, and the number of cells was counted with a cell counter. Student's t test was used for statistical processing of experimental results.

9． Functional food factors (equol, genistein (Tokyo Kasei Kogyo), daidzein (Tokyo Kasei Kogyo), EGCG) using Papd5-shRNA-introduced HeLa cells created in Papd5's involvement 5 on the cell growth inhibitory action of functional food factors The effect of was examined. Equol, genistein and daidzein were dissolved in DMSO to 100 mM and stored at -30 ° C. EGCG (Teavigo) was dissolved in ultrapure water to 5 mM and stored at 4 ° C.

For the measurement of the number of cells, HeLa cells were seeded on a 24-well plate at 2 × 10 ⁴ cells / mL and pre-cultured in DMEM medium containing 10% FCS for 24 hours. Thereafter, the cells were replaced with 2% FCS-containing DMEM medium containing each sample at final concentrations of 1, 5, 10, and 20 μM, cultured for 72 hours, and the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.

<Result>
1． Equol cell growth-inhibitory activity against various human cancer cells and its ER-dependent human cervical cancer cell line HeLa and human prostate cancer cell line PC-3 at 2 x 10 ⁴ cells / mL in 24-well plates After seeding and treatment with ER antagonist ICI182,780 (ICI), equol was added and cultured for 72 hours to count the number of cells (FIG. 1). As a result, the growth of these cancer cell lines was suppressed by equol, and even when treated with ICI, the action was not inhibited. This suggests that equol exhibits cytostatic activity against these cancer cell lines by an ER-independent mechanism of action (FIG. 1).

2． In order to identify a gene essential for the anticancer activity of equol's cytostatic activity Equol against mouse melanoma cell line B16, it becomes equol resistant by inhibiting the function of the gene by introducing a gene fragment. We decided to use a technique (genetic suppressor elements (GSE) method) to screen cancer cell lines and obtain the gene fragment that caused the disease from resistant cells. In order to use the GSE method, a cancer cell line in which growth suppression and lethal action are induced by equol is necessary. The mouse melanoma cell line B16 was seeded on a 24-well culture plate at 2 × 10 ⁴ cells / mL and cultured for 72 hours in a medium supplemented with equol or the number of cells was measured every 24 hours (FIG. 2). As a result, this cell line was found to be a cancer cell line to which the GSE method can be applied because its growth was remarkably suppressed by equol (FIG. 2).

3． On the other hand, equol's cytostatic activity on B16 cells was not inhibited by treatment with ICI. This showed that ER was not involved in the cell growth inhibitory action of equol in B16 cells (FIG. 3).

Four. Mouse embryo-derived cDNA fragments detected from Equol-resistant B16 cells B16 cells into which a mouse embryo-derived cDNA library was introduced were cultured in a medium containing equol (80 μM), and cells that became equol-resistant were screened. As a result of comprehensive collection of gene fragments from these equol-resistant cells and analysis of the gene sequence, the Pap associated domain containing 5 (Papd5) gene (polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 (mouse Papd5) mRNA) was found to be a fragment derived from a gene that generates by transcription and a gene that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 4.

Five. Effect of Papd5 knockdown on equol sensitivity of HeLa cells In order to specifically knockdown Papd5 expression using RNA interference method, a Papd5-specific shRNA expression vector (Papd5-shRNA) and its control vector (scramble-shRNA) are used. A human cervical cancer cell line HeLa into which expression was constantly introduced was prepared. As a result, HeLa cells with reduced Papd5 expression could be obtained (FIG. 4a).

HeLa cells introduced with Papd5-shRNA or scramble-shRNA were seeded in a 24-well culture plate at 2 x 10 ⁴ cells / mL, cultured for 72 hours in a medium supplemented with equol of each concentration, the number of cells was measured, and equol The sensitivity to was investigated. As a result, HeLa cells into which scramble-shRNA had been introduced were inhibited by equol. On the other hand, the sensitivity to equol completely disappeared in HeLa cells in which Papd5-shRNA was introduced to reduce the amount of Papd5 expression (FIG. 4b).

6． Effect of Papd5 knockdown on equol sensitivity of B16 cells To elucidate the involvement of Papd5 in the equol sensitivity of mouse melanoma cell line B16, we used Papd5-specific shRNA expression vector (Papd5-shRNA) and its control vector (scramble-shRNA). A mouse melanoma cell line B16 into which expression was constantly introduced was prepared. As a result, B16 cells with reduced Papd5 expression could be obtained (FIG. 5a).

B16 cells transfected with Papd5-shRNA or scramble-shRNA were seeded in a 24-well culture plate at 2 x 10 ⁴ cells / mL, cultured for 72 hours in a medium supplemented with equol of each concentration, the number of cells was measured, and equol The sensitivity to was investigated. As a result, B16 cells into which scramble-shRNA had been introduced were inhibited by equol. On the other hand, in B16 cells in which Papd5-shRNA was introduced and the expression level of Papd5 was reduced, the sensitivity to equol completely disappeared (FIG. 5b).

7． Involvement of Papd5 in the cell growth inhibitory action of functional food factors The involvement of Papd5 in the cancer cell growth inhibitory action of functional food factors (equol, genistein, daidzein, EGCG), which show anticancer action, was examined. HeLa cells into which Papd5-shRNA or Scramble-shRNA had been introduced were cultured for 72 hours in a medium supplemented with various concentrations of food factors, and the number of cells was measured. Genistein and EGCG, like equol, inhibited the growth of HeLa cells, but their effects were not affected by the decrease in Papd5 expression. On the other hand, the cell growth inhibitory action of daidzein was inhibited in cells with decreased expression of Papd5, as in equol (FIG. 6).

<Discussion>
Equol was found to suppress cell growth of human cervical cancer cell line HeLa, human prostate cancer cell line PC-3, and mouse melanoma cell line B16. Furthermore, it was revealed that ER is not involved in these cell growth inhibitory activities. Papd5 was identified as a gene candidate responsible for equol's cytostatic activity from B16 cells, where Equol exhibits ER-independent growth-suppressing activity. Papd5 is a gene involved in functional expression of ER-independent equol. It was thought that. When the expression of Papd5 in B16 cells and HeLa cells was specifically reduced using RNA interference, the sensitivity of equol to cytostatic activity disappeared from both cancer cell lines. From the above results, it was revealed that Papd5 is an essential gene for functional expression of ER independent of equol. It was also revealed that Papd5 is an essential gene in the anticancer activity of daidzein, the precursor of equol.

* There is only one paper on Papd5, and detailed functions are unknown. This discovery enables the development of anticancer drugs based on a completely new concept that targets Papd5, and diagnoses the effectiveness and adaptability of anticancer drugs using the expression level of Papd5 as an index. Use as a biomarker (diagnostic agent) can be expected. In addition, development of tailor-made foods and alternative medicines using Papd5 as a biomarker to determine how equol works (adapters) can be expected.

1． Effects of oral intake of equol on tumors knocked down in Papd5 expression
In the same manner as in the previous example, a mouse melanoma cell line B16 cell in which expression of Papd5 was knocked down was obtained by introducing the Papd5-shRNA expression vector, and 5 x 10 ⁵ cells / 100 mL PBS per mouse was added to C57BL. Cancer was transplanted by subcutaneous injection into the back of / 6J mice (male, 6 weeks old). Thereafter, equol 0.8 mg dissolved in 400 mL of 1% DMSO-H ₂ O per mouse was forcibly orally administered once every two days, and changes in tumor volume over time were measured. On day 24 after cancer transplantation, the mice were sacrificed and the tumor weight was measured. The control group received 1% DMSO-H ₂ O as a solvent.

The results are shown in FIG. Tumors knocked down in the expression of Papd5 increased in volume and weight as a result of oral intake of equal compared to the case of ingesting only the solvent. It was suggested that the expression of Papd5 is important for the equal anti-cancer effect, and that even if Papd5 expression is knocked down, equal can promote cancer.

2． Equal cell growth-promoting action on cancer cells knocked down in Papd5 expression In the same manner as in the previous example, a mouse melanoma cell line B16 cell or Scramble in which Papd5-shRNA expression vector was introduced and Papd5 expression was knocked down was introduced. -Equal concentrations of each concentration were added to shRNA-introduced B16 cells and cultured for 72 hours, and the number of cells was counted. The test group to which only the equal solvent (DMSO) was added was used as a control, and the relative fine number (%) was calculated with the number of control cells as 100%.

The results are shown in FIG. In B16 cells in which Papd5 expression was knocked down, cell proliferation was significantly promoted by addition of equal. It is suggested that Papd5 expression is important for equal cancer cell growth inhibitory activity, and that equal can promote cell proliferation in cancer cells in which Papd5 expression is knocked down. It was.

Claims (7)

1. A biomarker for diagnosing the effectiveness of an anticancer substance selected from the group consisting of equol, daidzein, and their analogs in a subject, and comprising Papd5 or a product thereof.
2. A method for determining the effectiveness of an anticancer substance selected from the group consisting of equol, daidzein, and analogs thereof, comprising a step of detecting the transcription or expression of Papd5 in a sample collected from the subject.
3. A product for detecting the biomarker according to claim 1, comprising an oligonucleotide, a peptide, or an antibody, for PCR, for DNA array, for ELISA, for protein array, for mass spectrometry, or for immunochromatography.
4. 3. The method according to claim 2, wherein the detection of transcription or expression of Papd5 is carried out by PCR method, DNA array method, ELISA method, protein array method, mass spectrometry method, or immunochromatography method.
5. A method for designing a pharmaceutical, food, or cosmetic product, or an application plan thereof, for the treatment of a disease or condition ameliorated by equol or daidzein using the presence or absence of Papd5 mutation in a subject, or detection of transcription or expression as an index.
6. Apply a test product that is a natural product, food, cosmetic, pharmaceutical product or compound to a subject, a sample collected from the subject, or a cell that has Papd5 and is capable of transcription or expression of Papd5;
   A method for screening for an effective treatment for cancer, comprising a step of detecting whether the amount of transcription or expression of Papd5 changes.
7. 7. The method according to claim 6, wherein an object suitable for application to a subject in combination with equol, daidzein or an analog thereof is screened.

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