WO2014129590A1 - Cyclin D1 GENE EXPRESSION INHIBITOR AND ANTICANCER DRUG - Google Patents

Abstract

Provided are a Cyclin D1 gene expression inhibitor and an anticancer drug that gently suppress growth of cancer cells without having a cell-destroying action, and are capable of preventing the recurrence of cancer, as a novel cancer-inhibiting drug. The present invention pertains to a Cyclin D1 gene expression inhibitor including, as effective components thereof: a peptide (A) having an amino acid sequence indicated by SEQ ID NO. 1; or a peptide (B) produced by deleting, replacing, or adding at least one amino acid residue, in an amino acid sequence indicated by SEQ ID NO. 1, said peptide having a Cyclin D1 gene expression suppressing action.

Description

Cyclin D1 gene expression inhibitor and antitumor agent

The present invention relates to a Cyclin D1 gene expression inhibitor and an antitumor agent comprising a peptide having a specific amino acid sequence.

The number of cancer patients in the world is approximately 12.7 million / year, and the number of cancer patients in Japan is approximately 670,000 / year. The leading cause of death among Japanese is “cancer” (approximately 340,000 people / year). For cancer, multidisciplinary treatment using surgical operation, chemotherapy, radiation therapy, etc. is performed, but the current situation is that cancer has not yet been suppressed.

Many of the anticancer agents currently used are organic compounds having a molecular weight of 200 to 400 that directly act on nucleic acid synthesis and have a cell-killing effect. Therefore, when such an anticancer agent is administered, normal cells, particularly normal cells that are actively proliferating, are affected, and it is difficult to avoid strong side effects.

On the other hand, Patent Document 1 proposes an Akt activity-inhibiting polypeptide. Patent Document 2 discloses a therapeutic cancer peptide vaccine. Since the mechanism of action of such peptides with anticancer activity is similar to that of molecular targeted drugs, it is expected as a new anticancer agent. It is rare.

Japanese Patent No. 4819321 Japan Special Table 2012-501185

An object of the present invention is to provide a Cyclin D1 gene expression inhibitor and an antitumor agent capable of slowly suppressing the proliferation of cancer cells without having a cytocidal action and preventing recurrence of cancer as a new anticancer agent. That is.

The present inventor has previously promoted research focusing on embryonic liver hematopoiesis, which is an organ in which hematopoietic stem cells actively proliferate and differentiate, and as a result, the amino acid sequence of SEQ ID NO: 1 suppresses proliferation and differentiation of hematopoietic stem cells. Invented a novel physiologically active peptide having a molecular weight (molecular weight: 1348.5; number of amino acids: 13) (International Publication No. 2011/040500). In this added culture, KS-13 is taken up by endocytosis in the added culture and suppresses the proliferation and differentiation of mouse / human hematopoietic cells (hereinafter, the peptide is also referred to as “KS-13”).

As a result of intensive research on KS-13, the present inventor has further found that KS-13 and its derivatives can be added to cells of various cancer cell lines such as leukemia cell lines, prostate cancer cell lines, breast cancer cell lines. It was found that by suppressing the expression of the Cyclin D1 gene, which controls the cycle, to 1/100 or less, the growth of cell lines was suppressed by about 20%, that is, KS-13 and its derivatives had anticancer activity. The invention has been completed.

That is, the Cyclin D1 gene expression inhibitor of the present invention is an active ingredient,
(A) a peptide having the amino acid sequence shown in SEQ ID NO: 1; or (B) a peptide obtained by deleting, substituting, or adding at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, A cyclin D1 gene expression inhibitor comprising a peptide having a D1 gene expression inhibitory action.

Moreover, a preferred embodiment of the present invention is the above Cyclin D1 gene expression inhibitor,
The peptide of (B) is a Cyclin D1 gene expression inhibitor, which is a peptide having the amino acid sequence shown in SEQ ID NO: 2.

Furthermore, a preferred embodiment of the present invention is the above Cyclin D1 gene expression inhibitor,
The peptide of (B) is a Cyclin D1 gene expression inhibitor, which is a peptide having any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 10.

The antitumor agent of the present invention is an active ingredient,
(A) a peptide having the amino acid sequence shown in SEQ ID NO: 1; or (B) a peptide obtained by deleting, substituting, or adding at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, An antitumor agent comprising a peptide having a D1 gene expression inhibitory action.

Moreover, a preferred embodiment of the present invention is the above antitumor agent,
The peptide of (B) is an antitumor agent which is a peptide having the amino acid sequence shown in SEQ ID NO: 2.

Furthermore, the suitable form of this invention is in the said antitumor agent,
The peptide (B) is an antitumor agent which is a peptide having any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 10.

According to the present invention, as an active ingredient, (A) a peptide having the amino acid sequence shown in SEQ ID NO: 1 or (B) at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is deleted or substituted In addition, by including a peptide that is added and has a cyclin D1 gene expression inhibitory action, it slowly suppresses the growth of cancer cells without having a cytocidal action, and also prevents recurrence of cancer as a new anticancer agent. Cyclin D1 gene expression inhibitor and antitumor agent that can be prevented can be provided.

FIG. 1 is a graph showing the results of Experimental Example 1. FIG. 2 is a photomicrograph showing the results of Experimental Example 2-1. FIG. 3 is a photomicrograph showing the results of Experimental Example 2-2. FIG. 4 is a graph showing the results of Experimental Example 3-1. FIG. 5 is a graph showing the results of Experimental Example 3-2. FIG. 6 is a graph showing the results of Experimental Example 3-3. FIG. 7 is a graph showing the results of Experimental Example 4 (Example 1). FIG. 8 is a photomicrograph showing the results of Experimental Example 5-1. FIG. 9 is a diagram showing the results of Experimental Example 5-2. FIG. 10 is a diagram illustrating the results of Experimental Example 6. FIG. 11 is a diagram showing the results of Experimental Example 7-1. FIG. 12 is a diagram showing the method and results of Experimental Example 7-2. FIG. 13 is a diagram showing the method and results of Experimental Example 7-2. FIG. 14 is a graph showing the results of Experimental Example 8-1. FIG. 15 is a graph showing the results of Experimental Example 8-2. FIG. 16 is a graph showing the results of Experimental Example 9-1. FIG. 17 is a graph showing the results of Experimental Example 9-2. FIG. 18 is a graph showing the results of Experimental Example 9-3. FIG. 19 is a graph showing the results of Experimental Example 9-4. FIG. 20 is a graph showing the results of Experimental Example 9-5. FIG. 21 is a graph showing the results of Experimental Example 9-6. FIG. 22 is a graph showing the results of Experimental Example 10 (Example 2).

(I) Definition of terms used in the present invention The abbreviations such as amino acid sequences in the present specification are defined by IUPAC-IUB [IUPAc-IUB communication on Biological Nomenclature, Eur. J. et al. Biochem. 138; 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and conventional symbols in the field.

“Cyclin D1 gene” is a gene encoding a cell cycle regulator and is also known as a so-called “cancer-related gene”. When expressed in normal cells, the gene is capable of tumorigenic or cancerous, and Cyclin D1 gene overexpression is caused by parathyroid adenoma, breast cancer, prostate cancer, colon cancer, lymphoma, melanoma And involved in the development of lung cancer (Morgan, D.O., (2008), Cell, 135,764-794, Lung Cancer, (2007), 55, 1-14).

A “gene expression inhibitor” is a drug that generally suppresses the expression of a target gene at the mRNA level or protein level. Although the mechanism of action of Cyclin D1 gene expression suppression in the gene expression inhibitor of the present invention is still unclear, it cannot be suppressed by binding a peptide having Cyclin D1 gene expression inhibitory action directly to the promoter region. The results of Experimental Examples 5-2 to 7-2 below are that they are suppressed by the target molecules such as ATP-dependent RNA helicase (YTHDC2) and Solute carrier family12 member3 (S12A3). Suggested by

"Antineoplastic agent" is a chemotherapeutic agent that is generally administered for the purpose of treating tumors in a broad sense. The antitumor agent of the present invention slowly suppresses the growth of cancer cells by suppressing the expression of the cancer-related gene Cyclin D1 gene that causes normal cells to become tumorous or cancerous, and is not due to cell killing action. Therefore, it has a feature that strong side effects can be avoided.

(II) Peptide according to the present invention The peptide targeted by the present invention is a peptide having a Cyclin D1 gene expression inhibitory action.
One embodiment of such a peptide is a peptide having an amino acid sequence consisting of the amino acid residues shown below:

The peptide consists of a partial sequence of the extracellular domain located at positions 24 to 303 of human Dlk1 (delta-like 1 homolog) protein (SEQ ID NO: 11) consisting of 383 amino acid residues in total length. As mentioned above, this peptide is also referred to herein as “KS-13”.

The peptide targeted by the present invention is a peptide obtained by deleting, substituting or adding at least 1, preferably about 1 to 6 amino acids in the amino acid sequence of KS-13 (SEQ ID NO: 1). And a peptide having an inhibitory effect on the expression of Cyclin D1 gene.

The peptide targeted by the present invention is as described above, but it is 60% or more, preferably 75% or more, more preferably 80% or more, and still more preferably 85% to 90% or more. It preferably has the same or similar amino acid sequence as shown in. Here, the ratio of “identity” or “similarity” can be calculated from the ratio of identical or similar amino acids overlapping with the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 1. . Here, “similar amino acids” mean amino acids similar in physicochemical properties. For example, similar amino acids include aromatic amino acid groups (Phe, Trp, Tyr), aliphatic amino acid groups (Ala, Leu, Ile). Val), polar amino acid group (Gln, Asn), basic amino acid group (Lys, Arg, His), acidic amino acid group (Glu, Asp), amino acid group having a hydroxyl group (Ser, Thr), and small side chain It can be classified into amino acid groups (Gly, Ala, Ser, Thr, Met). Such substitution between similar amino acids is unlikely to affect the properties of the peptide, and in this sense can be referred to as conservative amino acid substitution (for example, Bowie et al., Science, 247: 1306-1310 ( 1990)).

Thus, when the amino acid is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 1, or when some amino acids are deleted or added, the position of the substitution, deletion or addition is The resulting peptide has a Cyclin D1 gene expression inhibitory action.

The fact that the peptide obtained by substitution, deletion or addition has a cyclin D1 gene expression inhibitory effect is evaluated by evaluating the inhibitory effect on the proliferation of human cancer cells as shown in Experimental Examples 1 to 3 described later. It can be confirmed indirectly, and as shown in Experimental Example 4 (Example 1), it can be confirmed directly by analyzing human Cyclin D1 (CCND1) gene expression using real-time PCR. it can.

Examples of the peptide obtained by substitution, deletion or addition include a peptide represented by the following amino acid sequence (SEQ ID NO: 2) and having the above action.

Specific examples of such peptides include peptides corresponding to KS-13 described above, consisting of a partial sequence of the extracellular domain region of a protein corresponding to an ortholog of human Dlk1 protein. For example, mouse Dlk1 protein corresponding to an ortholog of human Dlk1 protein has an amino acid sequence consisting of a total length of 385 amino acid residues (SEQ ID NO: 12). In the amino acid sequence of the mouse Dlk1 protein, the region at positions 24 to 305 is the extracellular domain, and the partial sequence (sequence consisting of 13 amino acid residues at positions 124 to 136: SEQ ID NO: 3) corresponds to KS-13. It is a peptide.

In addition to mice, rodents such as rats and rats, and mammals such as pigs, horses, goats, cattle, wallabies, and giant kangaroos; It has Dlk1 protein corresponding to protein homologue or orthologue. The partial sequences corresponding to the above KS-13 located in the extracellular domain of these Dlk1 proteins are shown in Table 1 together with the amino acid sequences of peptides derived from human-derived KS-13 and mouse KS-13. Amino acid residues conserved in common with human-derived KS-13 are indicated by underlining.

For example, when comparing the sequences of peptides of rodents such as human and mouse or rat, the third amino acid from the N-terminus (corresponding to “XXb” in the amino acid sequence of SEQ ID NO: 2) may be either Lys or His. It can be seen that the fifth amino acid (corresponding to “XXd” in the amino acid sequence of SEQ ID NO: 2) may be either Asp or Ala. Further, comparing the sequences of human and porcine peptides, it can be seen that the 10th amino acid from the N-terminal (corresponding to “XXf” in the amino acid sequence of SEQ ID NO: 2) may be either Ile or Met. Further, when comparing the sequences of human and giant kangaroo peptides, the third amino acid from the N-terminus may be deleted, and the fifth amino acid from the N-terminus (“XXd” in the amino acid sequence of SEQ ID NO: 2). It is understood that either Asp or Glu may be used. Further, when comparing the sequences of human and chicken peptides, the 5th amino acid from the N-terminus may be deleted, and the 9th amino acid from the N-terminus (in the amino acid sequence of SEQ ID NO: 2, “XXe”). It can be seen that any of Val and Ile can be used.

In the peptide according to the present invention, KS-13 is the most preferred peptide, but as shown in SEQ ID NO: 2, any one of amino acids 1 to 6 (XXa to XXf) in the amino acid sequence is another amino acid. It may be substituted, may be a peptide consisting of 12 amino acid residues with one less N-terminal or C-terminal amino acid, or a peptide consisting of 11 amino acid residues with one less amino acid Good. The N-terminal amino acid may be deleted up to 5 or 4 to 3 amino acids.

In the amino acid sequence shown in SEQ ID NO: 1, the peptide formed by adding at least one amino acid has five amino acids derived from human Dlk1 protein at the N-terminus and / or C-terminus of the amino acid sequence, Preferred are those with 4, 3, 2 or 1 added.

In addition, a method for substituting, adding or deleting at least one amino acid in the amino acid sequence of KS-13 (SEQ ID NO: 1) has already been conventionally used in the art. For example, DNA encoding the peptide In the case of performing via the Internet, for example, site-specific mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); 100, 468 (1983); Nucleic Acids Res. , 12, 9441 (1984); secondary biochemistry experiment course 1 "Gene Research Method II", edited by Japanese Biochemical Society, p105 (1986)], etc., phosphate triester method, phosphate amidite method, etc. Chemical synthesis means [J. Am. Chem. Soc. 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett. , 22, 1859 (1981); 24, 245 (1983)] and combinations thereof. More specifically, DNA synthesis can be performed by chemical synthesis by the phosphoramidite method or triester method, and can also be performed on a commercially available automatic oligonucleotide synthesizer. Double-stranded fragments are chemically synthesized single strands produced by synthesizing complementary strands and annealing the strands together under appropriate conditions, or adding complementary strands using DNA polymerase with appropriate primer sequences. It can also be obtained from things. Furthermore, the peptide according to the present invention can also be synthesized by a solid phase synthesis method using a peptide synthesizer, and amino acid substitution, addition or deletion changes the type of protected amino acid when using a peptide synthesizer. This can be done easily. In addition, special amino acids such as D-amino acid and sarcosine (N-methylglycine) can be introduced.

The peptide according to the present invention may be in a free state, in the form of a salt, or in the form of a solvate containing a hydrate. Examples of the salt include physiologically acceptable, that is, pharmaceutically acceptable acid addition salts and base salts. Such acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, nitrate and sulfate; or sulfonic acid salts such as methanesulfonic acid and toluenesulfonic acid; organic acids such as trifluoroacetic acid and succinic acid. Examples include salt. Examples of the base salt include alkali metal salts such as sodium, potassium and lithium; or alkaline earth metal salts such as calcium and magnesium.

The peptides according to the present invention include peptides whose C-terminus is a carboxyl group (—COOH), carboxylate (—COO—), amide (—CONH ₂ ), or ester (—COOR). Here, as R in the ester, for example, an alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and n-butyl; a cycloalkyl group having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl; phenyl, aryl groups having 6 to 12 carbon atoms such as α-naphthyl; phenyl-C1-2 alkyl groups such as benzyl and phenethyl; C7-14 aralkyl groups such as α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl; A pivaloyloxymethyl group can be exemplified. When the peptide according to the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the peptide according to the present invention includes those in which the carboxyl group is amidated or esterified. In the peptide according to the present invention, the amino group of the N-terminal amino acid residue is protected with a protecting group (for example, a C1-6 acyl group such as C1-6 alkanoyl such as formyl group and acetyl group). Or modified with fatty acid (saturated fatty acid of C8-18), N-terminal glutamine residue which can be cleaved in vivo, pyroglutamine oxidized, substituent on side chain of amino acid in molecule (For example, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, C1-6 acyl groups such as C1-6 alkanoyl groups such as formyl group, acetyl group, etc.) Or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound. Furthermore, the peptides according to the present invention include those in which an imidazolyl group or SH group is alkylated (for example, methylated), aralkylated (for example, benzylated), or acylated (for example, acetylated or benzoylated). The fatty acid-modified products include myristoylated peptides in which the amino group of the N-terminal amino acid residue is modified with myristic acid.

Note that the content of International Publication No. 2011/040500 is incorporated in its entirety into this specification. Therefore, as a peptide according to the present invention, any peptide described in International Publication No. 2011/040500 can be used as long as it has an effect of suppressing the expression of the Cyclin D1 gene. Moreover, the peptide which concerns on this invention can be manufactured with reference to description of international publication 2011/040500.

(III) Cyclin D1 gene expression inhibitor The peptide according to the present invention detailed above or a pharmaceutically acceptable salt or solvate thereof as it is or with a carrier that is physiologically acceptable as necessary After mixing to make a composition, it is used as the Cyclin D1 gene expression inhibitor of the present invention. In addition, even if the peptide based on this invention consists of 1 type individually, 2 or more types can also be used in arbitrary combinations. Preferably, human-derived KS-13 (SEQ ID NO: 1), rodent-derived peptide (SEQ ID NO: 3), porcine-derived peptide (SEQ ID NO: 4), chicken-derived peptide (SEQ ID NO: 10), and more Preferred are human-derived KS-13 (SEQ ID NO: 1) and rodent-derived peptides (SEQ ID NO: 3).

Cyclin D1 gene expression inhibitor, for example, dissolves the peptide of the present invention in water or an appropriate buffer solution (eg, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.) to an appropriate concentration. Can be prepared. Moreover, you may mix | blend the preservative, stabilizer, reducing agent, tonicity agent, etc. which are used normally as needed.

(IV) Antitumor Agent The peptide according to the present invention detailed above or a pharmaceutically acceptable salt or solvate thereof is mixed as it is or with a physiologically acceptable carrier as necessary. After preparing the composition, it is used as the antitumor agent of the present invention. In addition, even if the peptide based on this invention consists of 1 type individually, 2 or more types can also be used in arbitrary combinations. Preferably, human-derived KS-13 (SEQ ID NO: 1), rodent-derived peptide (SEQ ID NO: 3), porcine-derived peptide (SEQ ID NO: 4), chicken-derived peptide (SEQ ID NO: 10), and more Preferred are human-derived KS-13 (SEQ ID NO: 1) and rodent-derived peptides (SEQ ID NO: 3).

The antitumor agent is prepared, for example, by dissolving the peptide according to the present invention in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.) to an appropriate concentration. can do. Moreover, you may mix | blend the preservative, stabilizer, reducing agent, tonicity agent, etc. which are used normally as needed.

Hereinafter, the present invention will be described in more detail using examples and experimental examples, including the background to the present invention. However, the present invention is not limited by these experimental examples.

1. Effect of KS-13 on proliferation of human cancer cells (in vitro)
<Experimental example 1>
(1) Materials and Methods Human leukemia cell line (K562) and human prostate cancer cell line (PC-3) were cultured using RPMI 1640 medium containing 10% fetal bovine serum (FBS).
Since K562 cells are suspension cells, the cells are collected by gentle pipetting and 2 × 10 ⁵ cells / ml using RPMI 1640 + 10% fetal bovine serum (FBS) supplemented with 10 μg / ml KS-13. The suspension was cultured in a 48-well tissue culture plate.
Since PC-3 cells are adherent cells, the cells are collected by adding 0.2% trypsin and then with RPMI 1640 + 10% fetal bovine serum (FBS) supplemented with 10 μg / ml KS-13. A cell suspension of × 10 ⁵ cells / ml was cultured in a 48-well tissue culture plate.
Cultures without KS-13 were used as controls.
One day, two days, and five days later, cells stained with trypan blue and unstained live cells were counted under a microscope. After 5 days, a photograph of the cell line morphology was taken under a microscope.

(2) Results As shown in FIG. 1, the number of both K562 and PC-3 cells increased over time, but in the presence of KS-13, both K562 and PC-3 cells proliferated compared to the control. And the number of viable cells decreased at all observations. (The total number of viable cells of K562 cells in the presence of KS-13 after 2 days was 3 times lower than the control. The total number of viable cells after 5 days was KS-13 compared to 1 day and 2 days later. (In the presence of KS-13, both K562 cells and PC-3 cells were reduced by 20-30% of the control.)
This confirmed that KS-13 inhibited the proliferation of leukemia cells and prostate cancer cells in vitro.

<Experimental example 2-1>
As a result of observing the cells cultured in Experimental Example 1 using an inverted microscope, the number of K562 cells in the presence of KS-13 was smaller than that of the control, and the cell morphology was also changed (see FIG. 2). Furthermore, large cells (large cells) were observed only in the presence of KS-13 (arrows). Thereby, it is considered that differentiation is induced by KS-13 and cancer cell proliferation is suppressed.

<Experimental example 2-2>
As in Experiment 1, Kasumi cells (human myeloid leukemia cell line) cultured for 2 days with 10 μg / ml KS-13 and Kasumi cells (control) cultured for 2 days without KS-13 were added. After May-Giemsa staining, observation was performed using an upright microscope (see FIG. 3). As a result, in the presence of KS-13, cells of various sizes and various cell nuclei were observed, and many large cells and multinucleated cells were observed (arrows).
In addition, compared with the control, in the presence of KS-13, the cytoplasm of large cells tended to contain vesicles (arrowhead).

<Experimental example 3-1>
KS-13 was added to the culture medium of the K562 cell line and monitored with a horoscope microscope for 30 minutes.
The results are shown in FIG. White dots indicate the KS-13 added group, and black dots indicate the control (KS-13 non-added group). That is, when KS-13 was added, differentiation of the K562 cell line was induced, the thickness changed, and it became clear that it became an irregular form. Thus, it is considered that cancer cell proliferation is suppressed by KS-13.

<Experimental Example 3-2>
With respect to the K562 cell line that had been cultured for 12 hours with or without KS-13 (control), live cells were observed by the MS3 Cell Track method using a horoscope microscope. FIG. 5 shows the results of representing irregularity on the vertical axis and roughness on the horizontal axis.
As confirmed from the graph of FIG. 5, for irregularity, cells treated with KS-13 (irregularity average = 0.024) were treated with control (irregularity average = 0.006). 4 times the value. On the other hand, regarding the roughness, no difference was observed between the cells treated with KS-13 (roughness average value = 5.57) and the control (roughness average value = 5.86). From these results, it was confirmed that KS-13 changes the cell morphology of K562 cells.

<Experimental Example 3-3>
Using the Kasumi cell line, live cells were observed by the MS3 Cell Track method using a horoscope microscope in the same manner as in Experimental Example 3-2. FIG. 6 shows the results of representing irregularity on the vertical axis and roughness on the horizontal axis.
As confirmed from the graph of FIG. 6, for irregularity, cells treated with KS-13 (irregularity average value = 0.062) were controlled (average irregularity value = 0.002). 30 times the value. On the other hand, with respect to the roughness, a slight decrease was observed in the cells treated with KS-13 (roughness average value = 4.59) compared to the control (roughness average value = 6.24). . From these results, it was confirmed that KS-13 also changed cell morphology in Kasumi cells.

2. Inhibition of Cyclin D1 Expression in Cancer Cell Lines by KS-13 <Experimental Example 4 (Example 1)> Analysis of Human Cyclin D1 (CCND1) Gene Expression by Real-Time PCR According to Experimental Examples 1-3, KS-13 was expressed in vitro. Therefore, the following experiment was conducted focusing on the CyclinD1 gene, which is a gene that controls the cell cycle.

(1) Total RNA extraction and cDNA synthesis RNA was extracted using RNAqueous-4PCR (trademark) (manufactured by Life Technologies) according to the product protocol.
The mRNA was reverse transcribed using High Capacity RNA-to-cDNA Kit (manufactured by Life Technologies).
The quality of cDNA synthesis was checked by PCR amplification of β-actin (ACTB), a human housekeeping gene.

(2) Real-time PCR
The level of human Cyclin D1 (CCND1) gene was evaluated using TaqMan Gene Expression Mat Mix and StepOnePlus ™ RT-PCR machine (Life Technologies). The ACTB gene was used as an endogenous control for real-time PCR.
All probes were from TaqMan Gene Expression Assay (Life Technologies). Samples were analyzed in triplicate. The mRNA level was normalized with β-actin, and the relative amount of expression was compared with a sample added with KS-13 peptide as 1.
(3) Results The results are shown in FIG. Thereby, KS-13 increased the expression of Cyclin D1 (CCND1) gene in human leukemia cell line (Kasumi), human prostate cancer cell line (PC-3), and human breast cancer cell line (T47D) by 141 times, It was found to be suppressed 122 times and 152 times.

3. Examination of cellular uptake of KS-13 and binding of KS-13 to the promoter region of human Cyclin D1 <Experimental example 5-1>
In Kasumi cells, a confirmation test was performed to determine whether KS-13 acts extracellularly or intracellularly.
(1) Materials and Methods Kasumi cells were cultured for 2 days in the presence of Biotin-modified KS-13 (10 μg / mL). Subsequently, 10000 cells were brought into close contact with the slide glass and fixed by cytospin. SA-Alexa488-streptavidin was subjected to secondary reaction with intracellular KS-13-biotin and observed using a focused laser microscope (magnification: 60 times). (See FIG. 8). As a negative control, Kasumi cells cultured in the absence of KS-13 were used and treated with SA-Alexa488-streptavidin as described above.

(2) Results As a result of microscopic observation, it was confirmed that KS-13-biotin was taken up into the cells. In particular, a large amount was taken into the cytoplasm, and the amount taken into the nucleus was relatively small. (The nuclei were stained with TOTO-3 iodide. The scale bar in the micrograph shows 40 μm.)

<Experimental example 5-2>
It was tested whether KS-13 binds directly to the promoter region of human Cyclin D1 in K562 cells.
(1) Materials and methods Chromatin immunoprecipitation (ChIP)
A 2 × 10 ⁵ cell / ml K562 cell suspension was cultured for 2 days using RPMI 1640 + 10% fetal bovine serum (FBS) supplemented with 10 μg / ml biotin-conjugated KS-13. The number of cells used for ChIP is 1 × 10 ⁶ cells.
For protein / peptide binding to chromosomal DNA, it was cross-linked by adding formaldehyde at a final concentration of 1%. After washing with cold PBS, the cells were lysed using a ChIP lysis buffer containing SDS. Cell debris was removed by centrifugation at 12000 rpm for 10 minutes (4 ° C.). Chromosomal DNA was fragmented by sonication (250 W, 5 min / 30 sec on / off cycle) to obtain chromosomal DNA of a small size of 100-1000 bp.
ChIP was performed according to the product instructions (Chromatin immunoprecipitation assay kit, MILLIPORE). That is, the fragmented DNA was diluted with a ChIP binding buffer. Genomic DNA diluted to 1% was used as a control “before ChIP”. Rabbit anti-biotin antibody was used to precipitate KS-13 and genomic DNA bound to KS-13. The precipitated chromosomal DNA was used as a “post-ChIP” template for PCR.

(2) PCR
A 127 bp PCR product was obtained using a primer that binds to the promoter region of human Cyclin D1.
K562 genomic DNA was used as a positive control for PCR, and no template was used as a negative control.
The PCR product was electrophoresed on a 2% agarose gel simultaneously with a DNA ladder marker for 100 bp and stained with SYBER Green.

(3) Results As shown in FIG. 9, PCR products were observed in the “before ChIP” sample, but not in the “after ChIP” sample. This indicates that KS-13 did not bind to the promoter region of Cyclin D1. This suggests that KS-13 does not directly bind to the promoter and suppress the expression of Cyclin D1, but suppress it via other molecules.

4). The target molecule of KS-13 in the K562 leukemia cell line was tested to see if KS-13 binds to other proteins.
<Experimental example 6>
(1) Materials and methods Chromatin immunoprecipitation (ChIP)
2 × 10 ⁵ cells / ml of K562 cells were cultured for 2 days using RPMI 1640 + 10% fetal bovine serum (FBS) supplemented with 10 μg / ml biotin-conjugated KS-13. The number of cells used for ChIP is 1 × 10 ⁶ cells.
After washing with cold PBS, the cells were lysed using an IP lysis buffer (Pierce Classic IP Kit, Thermo Scientific). Cell debris was removed by centrifugation at 12000 rpm for 10 minutes (4 ° C.).
IP was performed according to the product instructions (Pierce Classic IP Kit, Thermo Scientific). Rabbit anti-biotin antibody was used to precipitate KS-13 and KS-13 binding proteins.
The biotin-KS-13 protein complex was separated from unbound protein by adding protein G. After three washes, the protein was separated from the immune complex using low PH buffer.
The eluted protein comprises heavy chain (50 kDa) and light chain (25 kDa) rabbit IgG. The eluted protein was electrophoresed on SDS-PAGE and stained with Coomassie Brilliant blue G-250 (Bio-Rad).

(2) Results The results are shown in FIG. Rabbit IgG heavy and light chains were identified in both control KS-13 (-) and KS-13 (+). This indicates that immunoprecipitation is successful.
Compared with control KS-13 (-), KS-13 (+) showed bands at 50 to 250 kDa and 25 to 50 kDa.
In addition, liquid chromatography mass spectrometry (LC-MS) was used to identify proteins.

<Experimental Example 7-1>
Identification of K-13 binding protein in K562 cells was performed using liquid chromatography mass spectrometry (LC-MS).
As a result, ATP-dependent RNA helicase (YTHDC2) and single carrier family12 member3 (S12A3) seemed to bind to K-13 in K562 cells compared to the control without addition of K-13. (See FIG. 11).
Thus, two target molecules of KS-13 in the K562 leukemia cell line were revealed.

<Experimental Example 7-2>
Liquid chromatograph mass spectrometry (LC-MS) was used to identify K-13 binding proteins in K562 and Kasumi cells. The method and results are shown in FIG. 12 (K562 cells) and FIG. 13 (Kasumi cells).
(1) Materials and Methods 3 × 10 ⁶ K562 cells and Kasumi cells were cultured in the presence of KS-13-biotin. Immunoprecipitation was performed using a rabbit anti-biotin polyclonal antibody, and the immunoprecipitated protein was identified using LC-MS. As a background control, a protein immunoprecipitated from cells cultured in the absence of KS-13-biotin was used. Proteins excluding proteins confirmed by background control were identified as follows.

(2) Identified K-13 binding protein Dermcidin (DCD) and U2 small nuclear ribonucleoproteins A and B (RU2A, RU2B) were identified as K-13 binding proteins of K562 cells.
High mobility group protein 2 (HMGN2) and SUB1 homologue (S. cerevisiae) (SUB1) binding to nucleosomes were identified as K-13 binding proteins of Kasumi cells.

5. Analysis of representative phosphorylated proteins The molecular mechanism of KS-13 in protein phosphorylation of K562 cells was examined.
<Experimental Example 8-1>
(1) Materials and Methods A suspension of 2 × 10 ⁵ cells / ml of K562 cells was cultured for 2 days using RPMI + 10% fetal bovine serum (FBS) supplemented with 10 μg / ml of KS-13. A phosphorylated proteome array (phosphorylation-kinase array kit, R & D Systems) was performed according to the product instructions.
(2) Results There was no difference in protein phosphorylation of K562 cells treated with KS-13 (see FIG. 14).

<Experimental Example 8-2>
(1) Materials and Methods A suspension of 2 × 10 ⁵ cells / ml of K562 cells was cultured for 2 days using RPMI + 10% fetal bovine serum (FBS) supplemented with 10 μg / ml of KS-13. A phosphorylated proteome array (phosphorylation-kinase array kit, R & D Systems) was performed according to the product instructions.
(2) Results There was no difference in protein phosphorylation of K562 cells treated with KS-13 (see FIG. 15).

6). Confirmation of toxicity of KS-13 in vivo <Experimental example 9-1>
To confirm the in vivo toxicity of KS-13, a single dose experiment of KS-13 was performed on ICR mice. Using peripheral blood one day after administration, analysis was performed with a Sysmex multi-item automatic blood cell counter KX-21.
Compared with the control group (PBS administration), in the KS-13 Low (low concentration 0.36 μg / g body weight) administration group and the KS-13 High (high concentration 3.6 μg / g body weight) administration group, leukocytes ( The number of WBC), the number of red blood cells (RBC), the number of platelets (PLT), and the hemoglobin (HGB) values were within normal ranges, and it was revealed that there was no hepatotoxicity (see FIG. 16).

<Experimental Example 9-2>
To confirm the in vivo toxicity of KS-13, a single dose experiment of KS-13 was performed on ICR mice. Using peripheral blood 7 days after administration, analysis was performed with Sysmex's multi-item automatic blood cell counter KX-21.
Compared with the control group (PBS administration), in the KS-13 Low (low concentration 0.36 μg / g body weight) administration group and the KS-13 High (high concentration 3.6 μg / g body weight) administration group, leukocytes ( The number of WBC), the number of red blood cells (RBC), the number of platelets (PLT), and the hemoglobin (HGB) were within normal ranges, and it was revealed that there was no hematologic toxicity (see FIG. 17).

<Experimental Example 9-3>
To confirm the in vivo toxicity of KS-13, a single dose experiment of KS-13 was performed on ICR mice. Using peripheral blood serum 7 days after administration, commissioned analysis was performed by an animal biochemical test (Hitachi 7180 type automatic analyzer) of Oriental Yeast Co., Ltd.
Compared with the control group (PBS administration), in the KS-13 Low (low concentration 0.36 μg / g body weight) administration group and KS-13 High (high concentration 3.6 μg / g body weight) administration group, GOT ( Glutamate oxaloacetate transaminase (= AST) and GPT (glutamate pyruvate transaminase) (= ALT) values were within the normal range, and it was revealed that there was no hepatotoxicity. It was also revealed that urea nitrogen (BUN) value and creatinine (CRE) value were within normal ranges and there was no nephrotoxicity (see FIG. 18).

<Experimental Example 9-4>
To confirm the in vivo toxicity of KS-13, a single dose experiment of KS-13 was performed on ICR mice. Using peripheral blood 14 days after administration, analysis was performed with Sysmex's multi-item automatic blood cell counter KX-21.
Compared with the control group (PBS administration), in the KS-13 (concentration 3.6 μg / g body weight) administration group, the number of white blood cells (WBC), the number of red blood cells (RBC), the number of platelets (PLT), hemoglobin (HGB) The value was within the normal range, and it was revealed that there was no hematological toxicity (see FIG. 19).

<Experimental Example 9-5>
To confirm the in vivo toxicity of KS-13, a single dose experiment of KS-13 was performed on ICR mice. Using peripheral blood 28 days after administration, analysis was performed using a Sysmex multi-item automatic blood cell counter KX-21.
Compared with the control group (PBS administration), in the KS-13 administration group (3.6 μg / g body weight), the white blood cell (WBC) count, red blood cell (RBC) count, platelet (PLT) count, hemoglobin (HGB) level Was within the normal range and was found to have no hematologic toxicity (see FIG. 20).

<Experimental Example 9-6>
To confirm the in vivo toxicity of KS-13, a single dose experiment of KS-13 was performed on ICR mice. Using peripheral blood serum 28 days after administration, commissioned analysis was performed by an animal biochemical test (Hitachi 7180 type automatic analyzer) of Oriental Yeast Co., Ltd.
Compared with the control group (PBS administration), in the KS-13 administration group, GOT (glutamate oxaloacetate transaminase) (= AST) value, GPT (glutamate pyruvate transaminase) (= ALT) value is within the normal range, It became clear that there was no liver toxicity. In addition, urea nitrogen (BUN) value and creatinine (CRE) value were within the normal range, and it was revealed that there was no nephrotoxicity (see FIG. 21).

From the above Experimental Examples 1 to 9, KS-13 (a peptide having the amino acid sequence shown in SEQ ID NO: 1) suppresses the growth of leukemia cell line / prostate cancer cell line, leukemia cell line / breast cancer cell line / prostate It was confirmed that the CyclinD1 gene expression of the cell line was suppressed, that it did not act directly on the CyclinD1 gene expression region, that there were two molecular targets in the K562 leukemia cell line, and that no toxicity was observed after a single administration.

7). Effect of KS-13 in treatment of human blood cancer model <Experimental Example 10 (Example 2)>
The effect of KS-13 (a peptide having the amino acid sequence shown in SEQ ID NO: 1) in the treatment of a mouse human blood cancer model was confirmed as follows.
(1) Materials and Methods Non-obese diabetic mice (NOD mice) are transferred intravenously with 2 × 10 ⁷ human myeloid leukemia cell line (Kasumi). One day after the cells were transferred, (1) PBS, (2) 5FU (anticancer agent), (3) 5FU + 72 μg KS-13, and (4) 72 μg KS-13 were intravenously administered. The survival of the mice was observed every week. The survival rate for each week was plotted on the graph (see FIG. 22). Dead mice were fixed with 4% formaldehyde for further analysis.

(2) Results As shown in FIG. 22, at the 38th week, all mice administered with PBS died. On the other hand, mice administered KS-13 were 100% alive until the 41st week. Thus, when a tumor-bearing mouse using the Kasumi leukemia cell line was prepared and the anticancer effect of a single administration of KS-13 was evaluated, a clear life-prolonging effect was observed.

Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. In addition, this application is based on the US provisional application (61 / 767,322) for which it applied on February 21, 2013, The whole is used by reference.

DETAILED DESCRIPTION OF THE INVENTION The present invention has high technical significance as 1) can contribute to the survival of cancer patients as a new drug; 2) can contribute to target molecule identification of a new drug; 3) can contribute to life science as a research reagent.

Claims (6)

1. A Cyclin D1 gene expression inhibitor comprising the peptide (A) or (B) as an active ingredient.
   (A) A peptide having the amino acid sequence shown in SEQ ID NO: 1 (B) A peptide obtained by deleting, substituting, or adding at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, wherein the Cyclin D1 gene Peptide having expression inhibitory action.
   

   
2. The Cyclin D1 gene expression inhibitor according to claim 1, wherein the peptide of (B) is a peptide having the amino acid sequence shown in SEQ ID NO: 2.
   

   
3. The Cyclin D1 gene expression inhibitor according to claim 1, wherein the peptide of (B) is a peptide having any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 10.
   

   
4. An antitumor agent comprising the peptide (A) or (B) as an active ingredient.
   (A) A peptide having the amino acid sequence shown in SEQ ID NO: 1 (B) A peptide obtained by deleting, substituting, or adding at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, wherein the Cyclin D1 gene Peptide having expression inhibitory action.
   

   
5. The antitumor agent according to claim 4, wherein the peptide of (B) is a peptide having the amino acid sequence shown in SEQ ID NO: 2.
   

   
6. The antitumor agent according to claim 4, wherein the peptide of (B) is a peptide having any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 10.
   

   

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