KR101746143B1 - A pharmaceutical composition containing fibulin-3 for the prevention and treatment of pancreatic cancers - Google Patents

A pharmaceutical composition containing fibulin-3 for the prevention and treatment of pancreatic cancers Download PDF

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KR101746143B1
KR101746143B1 KR1020150096986A KR20150096986A KR101746143B1 KR 101746143 B1 KR101746143 B1 KR 101746143B1 KR 1020150096986 A KR1020150096986 A KR 1020150096986A KR 20150096986 A KR20150096986 A KR 20150096986A KR 101746143 B1 KR101746143 B1 KR 101746143B1
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pancreatic cancer
protein
pibulin
pharmaceutical composition
cells
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KR20170006415A (en
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김인규
이재하
김서연
김정열
신병철
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한국원자력연구원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1738—Calcium binding proteins, e.g. calmodulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of pancreatic cancer, which comprises fibulin-3 protein as an active ingredient. The pibulin-3 protein inhibits c-Met and ALDH1 in pancreatic cancer cells, 3 can be used as a radiation sensitivity enhancer for pancreatic cancer treatment and pancreatic cancer because it decreases the growth, penetration and transfer ability and promotes radiation sensitivity.

Description

Technical Field [0001] The present invention relates to a pharmaceutical composition for preventing and treating pancreatic cancer containing an FBLN-3 protein as an active ingredient,

The present invention relates to a composition for prevention and treatment of pancreatic cancer, which comprises fibulin-3 protein, a pibuline-3 over-expressing cell line or a culture solution thereof as an active ingredient, and a radiation sensitivity enhancer.

The fibulin (FBLN) protein family is a protein associated with basement membrane and elastic extracellular matrix (ECM), which is a protein that contributes to cell-cell interactions or cell-substrate interactions. Type 1 6 types are known. The prophylactic protein group has been proposed as a new gene target that inhibits various tumors according to the species. Recently, it has been reported that the Pf-5 and Pf-3 protein act on cell growth regulation in some tumors. Recently, Pfalin-5 has been reported to be involved in tumor suppression by participating in cell proliferation, penetration, vascular endothelial growth factor and tumor angiogenesis [1-4]. However, it has been reported that pibuline-3 is a factor promoting cell growth in brain tumor cells (glioma) and, in contrast, some tumors including hepatocellular carcinoma inhibit tumor cell growth such as tumor angiogenesis inhibition [ 5-7]. Recently, it has been shown that the PB-3 protein strongly inhibits tumorigenesis through the IGF1R / PI3K / AKT / GSK3b pathway in cancer stem cells isolated based on ALDH1 activity, as well as inhibiting the penetration ability of non-small cell lung cancer cells , And inhibits c-Met gene expression in pancreatic cancer by controlling the expression of ALDH 1 [8, 9].

 Cancer stem cells are caused by specific cell populations that have the potential to differentiate into various cell populations and exhibit self renewal [10]. The presence of cancer stem cells has been implicated in the pathogenesis of acute myelogenous leukemia ), And in recent years, the presence of stem cells has also been confirmed in solid tumors by demonstrating cancer stem cells in general solid tumors including breast cancer [11, 12]. Among them, aldehydrogeanse 1 (ALDH 1), a marker of promising cancer stem cells, is a detoxifying enzyme that oxidizes intracellular aldehydes, which causes significant resistance to alkylating agents and oxidative stress [13, 14 ]. In addition, ALDH converts retinol (Vitamine A) into retinoic acid, the most active form of retinoids important for the treatment and prevention of cancer, and affects cell growth and proliferation. ALDH is generally known to have 19 genes and is classified into ALDH 1, II, and III classes. Among them, only ALDH 1 has retinal dehydrogenase activity, which converts retinal to retinoic acid, and ALDH1A1 and retinaldehyde dehydrogenase 3 (ALDH1A3) have the most efficient activity [ 15]. Therefore, the activity of ALDH 1 is important for isolating subpopulations of cancer stem cells in cancer cells such as lung cancer cells [16].

 c-Met is a ligand for hepatocyte growth factor (HGF) and is an activating receptor tyrosine kinase that regulates various cell signaling pathways involved in cell proliferation, motility and metastasis. In normal cells, it is important to control the homeostasis of the tissue, but it is known that it is activated specifically by mutation, amplification or protein overexpression in cancer.

Increased expression of c-Met gene and protein has been reported in various cancer types. [17] This increase in c-Met gene may occur during tumor progression, and interestingly, in recent years, (Epidermal Growth Factor Receptor; EGFR) inhibitor and c-Met. This suggests that combination therapy, which inhibits EGFR and c-Met simultaneously, is required based on drug resistance [18].

Thus, the present inventors have shown that pancreatic cancer cell lines can inhibit the growth and metastasis of pancreatic cancer cells by pibolin-3 methylation and overexpression, and this results in decreased expression of c-Met and ALDH 1 And thus can be used as a protein therapeutic drug for controlling pancreatic cancer cells. Thus, the present invention has been completed.

1. W.P. Schiemann, G.C. Blobe, D.E. Kalume, A. Pandey, H.F. Lodish, J. Biol. Chem. 277 (2002): 27367-27377 2. A.R. Albig, W.P. Schimann, DNA. Cell Biol. 23 (2004): 367-379 3. A.R. Albig, W.P. Schimann, Future Oncology 1 (2005): 23-35 4. W. Yue, Q. Sun, R. Landreneau, C. Wu, J.M. Siegfried, J. Yu, L. Zhang, Cancer Res. 69 (2009): 6339-6346 5. A.R. Albig, J.R. Neil, W.P. Schimann, Fibulin-3 and -5 antagonize tumor angiogenesis in vivo, Cancer Res. 66 (2006) 2621-2629 6. B. Hu, K. K. Thirtamara-Rajamani, H. Sim, M.S. Viapiano. Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion. Mol. Cancer Res. 7 (2009) 1756-1770 7. R. Luo, M. Zhang et al, Decrease of fibulin-3 in hepatocellular carcinoma indicates poor prognosis, PLoS One 8 (8) (2013) e70511 8. I.G. Kim et al, Fibulin-3-mediated inhibition of epithelial-to-mesenchymal transition and self renewal of ALDH + lung cancer stem cells through IGF1R signaling, ONCOGENE 33 (30) (2014) 3908-3917 9. E.J. Kim et al, MMP-7 and MMP-2 regulation of the invasive behavior of non-small cell lung cancer cells via methylation of the Fibulin-3 promoter 10. B.M. Boman, M.S. Wicha, Cancer stem cells: a step toward the cure, J. Clin. Oncol. 26 (2008) 2795-2799 11. D. Bonnet, J.E. Dick, Human acute myeloid leukemia is organized as hierachy that originates from a primitive hematopoietic cell. Nat. Med 3 (1997) 730-737 12. M. Al-Hajj, M.F. Clarke, Self-renewal and tumor stem cells. Oncogene 23 (2003) 7274-7284 13. M. Magni, S. Shammah, R. Schiro. et al, Induction of cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer. Blood 87 (1996) 1097-1103 14. N.A. Sophos, V. Vasiliou, Aldehyde dehydrogenase gene superfamily: the 2002 update, Chem. Biol. Interact. 143-144 (2003) 5-22 15. A. Sima, M. Parisotto, S. Mader, P.V. Bhat, kinetic characterization of recombinant mouse retinal dehydrogenase type 3 and 4 for retinal substrates. Biochim. Biophys. Acta 1790 (2009) 1660-1664 16. J. Feng, Q. Qiu, K. Abha et al., Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cacer, Mol. Cancer. Res. 7 (2009) 330-338 17. Tong, C. Y., Hui, A. B., Yin, X. L., Pang, J. C., Zhu, X. L., Poon, W.S. et al. Detection of oncogene amplifications in medulloblastomas by comparative genomic hybridization and array-based comparative genomic hybridization. J Neurosurg 100 (2004): 187-193 18. Bean, J., Brennan, C., Shih, J. Y., Riely, G., Viale, A., Wang, L. et al. Metamplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci 104: 20932-20937

It is an object of the present invention to provide a composition for the prevention and treatment of pancreatic cancer containing fibulin-3 protein, a pibulin-3 over-expressing cell line or a culture solution thereof as an active ingredient, and a radiation sensitivity enhancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition and a health functional food for the prevention and treatment of pancreatic cancer containing fibulin-3 protein as an active ingredient.

The present invention also relates to a pharmaceutical composition for the prevention and treatment of pancreatic cancer comprising as an active ingredient a pibolin-3 over-expressing cell line transformed with the vector comprising the polynucleotide encoding the pibulin-3 protein, or a culture thereof .

Further, the present invention provides a pharmaceutical composition for inhibiting pancreatic cancer metastasis and a health functional food containing a pibulin-3 protein as an active ingredient.

In addition, the present invention provides a radiation sensitivity enhancer for pancreatic cancer, which contains a pibulin-3 protein as an active ingredient.

The present invention relates to a composition for preventing and treating pancreatic cancer, a composition for inhibiting pancreatic cancer metastasis, and a radiation sensitizer for pancreatic cancer, which comprises a fibulin-3 protein as an active ingredient, 3 can be used as a radiation sensitivity enhancer for pancreatic cancer treatment and pancreatic cancer because it inhibits c-Met and ALDH1 and reduces cell growth, penetration and metastatic ability and promotes radiation sensitivity.

Figure 1 shows the expression of fibulin-3 gene and protein in pancreatic cancer cells:
AsPC-1: pancreatic cancer cell line;
BxPC-3: pancreatic cancer cell line;
Mia PaCa-2: pancreatic cancer cell line; And
FBLN3: Phillipine -3.
2A is a nucleotide sequence of fibrosine-3 and methylation sites of fibulin-3 in pancreatic cancer cell lines (Aspc1, Bxpc3, MIA PaCa2)
Underlined nucleotide sequence: pyrosequencing site; And
Y: methylation site (Position 1-4).
2b shows the degree of methylation of the CpG island in the promoter region present in pibuline-3 in pancreatic cancer cells:
AsPC-1: pancreatic cancer cell line;
BxPC-3: pancreatic cancer cell line;
Mia PaCa-2: pancreatic cancer cell line;
Position 1-4: methylation site; And
mean: mean.
FIG. 3 is an analysis of gene and protein changes in a pancreatic cancer cell line overexpressing or inhibiting the pibulin-3 gene using a sequential polymerase reaction and Western blotting:
AsPC-1: pancreatic cancer cell line;
BxPC-3: pancreatic cancer cell line;
Mia PaCa-2: pancreatic cancer cell line;
FBLN3: Pbull-3;
VC and siCtl: control group;
FBLN3 O / E: Pibulin-3 overexpressing group;
siFBLN3: PbuLin-3 inhibition group;
c-Met: MET proto-oncogene, receptor tyrosine kinase;
ALDH1A1: retinaldehyde dehydrogenase 1; And
ALDH1A3: retinaldehyde dehydrogenase 3.
Figure 4 shows the radiation sensitivity of pancreatic cancer cell lines overexpressing or inhibiting the pibulin-3 gene:
AsPC-1: pancreatic cancer cell line;
BxPC-3: pancreatic cancer cell line;
Mia PaCa-2: pancreatic cancer cell line;
pcDNA and con: control;
pcDNA-FBLN3: Pibulin-3 overexpressing group; And
siFBLN3: PfuBrin-3 inhibitor.
FIG. 5 shows penetration and metastatic ability of cells of a pancreatic cancer cell line overexpressing or inhibiting the pibulin-3 gene and epithelial-to-mesenchymal transition (EMT) marker changes.
AsPC-1: pancreatic cancer cell line;
BxPC-3: pancreatic cancer cell line;
Mia PaCa-2: pancreatic cancer cell line;
pcDNA and con: control;
pcDNA-FBLN3 (+): Pibulin-3 overexpressing group;
siFBLN3: PbuLin-3 inhibition group;
Migration: Transition; And
Invasion.
Figure 6 shows sphere formation and stemness marker analysis of the pibuline cancer cell line overexpressing the pibulin-3 gene:
AsPC-1: pancreatic cancer cell line;
Mia PaCa-2: pancreatic cancer cell line;
pcDNA and con: control;
pcDNA-FBLN3 (+): Pibulin-3 overexpressing group;

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of pancreatic cancer containing fibulin-3 protein as an active ingredient.

The pibulin-3 protein preferably has an amino acid sequence selected from the amino acid sequences of the following 1) to 3), but is not limited thereto:

1) an amino acid sequence represented by SEQ ID NO: 1;

2) an amino acid sequence represented by a part of SEQ ID NO: 1; And

3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.

The present invention also relates to a pharmaceutical composition for the prevention and treatment of pancreatic cancer comprising as an active ingredient a pibolin-3 over-expressing cell line transformed with the vector comprising the polynucleotide encoding the pibulin-3 protein, or a culture thereof .

The pibulin-3 protein preferably has an amino acid sequence selected from the amino acid sequences of the following 1) to 3), but is not limited thereto:

1) an amino acid sequence represented by SEQ ID NO: 1;

2) an amino acid sequence represented by a part of SEQ ID NO: 1; And

3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.

The vector comprising the pibulin-3 polynucleotide may be a vector containing a linear DNA, a plasmid vector, a viral expression vector or a recombinant retrovirus vector, a recombinant adenovirus vector, It is preferably a recombinant viral vector comprising a recombinant adeno-associated virus (AAV) vector, a recombinant herpes simplex virus vector or a recombinant lentivirus vector, and is preferably a pcDNA3.1 vector But is not limited thereto.

The cells containing the pibulone-3 polynucleotide may be isolated from a group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells and established tumor cells But it is not limited thereto.

In a specific example of the present invention, in order to compare the expression level of pibilin-3 in the pancreatic cancer cell lines AsPC-1, BxPC-3 and Mia PaCa-2 cells used in the experiments of the present invention, PCR) and Western blotting. As a result, it was confirmed that the pbuLIN-3 gene and protein were not expressed in AsPC-1 and MiaPaCa-2 cells, and the pbuLIN-3 protein was expressed in BxPC-3 cells 1).

In order to biochemically confirm the methylation state of CpG islands in the promoter region present in the pibulin-3, the inventors of the present invention used pibolin-3 of each of the pancreatic cancer cell lines AsPC-1, BxPC-3 and MiaPaCa-2 The gene promoter was subjected to pyrosequencing. As a result, as shown in FIG. 2A, the underlined nucleotide sequence indicated the pyrosequencing site of FBLN3, and the four regions marked with Y were methylated at the cancer cell line (See Fig. 2a). It was also confirmed that about 95% or more methylation occurred in AsPC1 and MiaPaCa2 cell lines and about 35% in BxPC-3 cell line (see FIG. 2B).

In order to artificially overexpress the pibulin-3 gene in pancreatic cancer cells, the present inventors prepared a pcDNA3.1 clone for overexpressing FBLN-3 using pibulin-3 gene in pancreatic cancer cell line BxPC-3.

In order to confirm the expression of c-Met and ALDH1 expression by overexpression of the pibulin-3 protein, the inventors of the present invention examined whether the above-described vector was overexpressed in AsPC-1 and MiaPaCa-2 cells without pibulin- 3 was overexpressed in AsPC-1 and MiaPaCa-2 pancreatic cancer cells by the pibulin-3 gene overexpression clone, and c- Met and ALDH1 expression was decreased (see Fig. 3).

In order to confirm the change of c-MET and ALDH1 expression through inhibition of the pibulin-3 protein, the present inventors inhibited the expression of pibulin-3 by introducing siRNA into BxPC-3 cells expressing pibulin- , RT-PCR and Western blot analysis. As a result, pbuLin-3 was inhibited by pbuLin-3 siRNA in BxPC-3 cells, and expression of c-Met and ALDH1 was increased (See Fig. 3).

Therefore, in the present invention, it has been confirmed that the expression of piburin-3 is inhibited by the expression of the pibulin-3 protein in the pancreatic cancer cell line and the expression of c-Met and ALDH1 is inhibited. It can be used as a composition.

The therapeutically effective amount of the composition of the present invention may vary depending on a variety of factors, such as the method of administration, the site of administration, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington ' s Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Sodium chloride, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, antioxidants, buffers, And other conventional additives may be added.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the protein, based on the total weight of the composition.

The composition of the present invention may be administered orally or non-orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered in accordance with a desired method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, more preferably administered once to several times a day.

In the case of a vector comprising a polynucleotide encoding the fibrin-3 protein of the present invention, the vector preferably contains 0.05 to 500 mg, more preferably 0.1 to 300 mg, and the fibrin- In the case of a recombinant virus comprising a polynucleotide, it is preferable that it contains 10 3 to 10 12 IU (10 to 10 10 PFU), more preferably 10 5 to 10 10 IU, but is not limited thereto.

In addition, in the case of a cell containing a polynucleotide encoding the fibrin-3 protein of the present invention, it is preferable that the cell contains 10 3 to 10 8, more preferably 10 4 to 10 7 , It is not limited.

The effective dose of a vector containing a polynucleotide encoding the fibrin-3 protein of the present invention or a composition containing cells as an active ingredient is 0.05 to 12.5 mg / kg in the case of a vector per kg of body weight, case 10 7 to 10 11 viral particles (10 5 to 10 9 IU) / ㎏, in the case of the cells 10 3 to 10 6 cells / ㎏, and for preferably a vector is from 0.1 to 10 ㎎ / ㎏, the recombinant virus in the case of 10 8 to 10 10 particles, the case of (10 6 to 10 8 IU) / ㎏, cells, and 10 2 to 10 5 cells / ㎏, it may be administered twice or three times a day. Such composition is not necessarily limited to this, but may vary depending on the condition of the patient and the degree of neurological disease.

The present invention also provides a health functional food for prevention and improvement of pancreatic cancer, which contains pibulin-3 protein as an active ingredient.

The pibulin-3 protein preferably has an amino acid sequence selected from the amino acid sequences of the following 1) to 3), but is not limited thereto:

1) an amino acid sequence represented by SEQ ID NO: 1;

2) an amino acid sequence represented by a part of SEQ ID NO: 1; And

3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.

In the present invention, it was confirmed that pibulin-3 inhibited the expression of c-Met and ALDH1 by the expression of pibulin-3 protein in pancreatic cancer cell line. Thus, the pibulin-3 was found to be a health functional food for prevention and improvement of pancreatic cancer Can be used.

The present invention also provides a pharmaceutical composition for inhibiting pancreatic cancer metastasis comprising a pibuline-3 protein as an active ingredient.

The pibulin-3 protein preferably inhibits pancreatic cancer stem cell growth, but is not limited thereto.

The pibulin-3 protein preferably has an amino acid sequence selected from the amino acid sequences of the following 1) to 3), but is not limited thereto:

1) an amino acid sequence represented by SEQ ID NO: 1;

2) an amino acid sequence represented by a part of SEQ ID NO: 1; And

3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.

The pancreatic cancer stem cell is preferably selected from any one marker selected from the group consisting of CD133 (prominin-1; AC133), hyaluronate receptor (P-glycoprotein 1) and ALDH1, which are markers of cancer stem cells, and ALDH1 , But it is not limited thereto.

The composition preferably has an epithelial-to-mesenchymal transition (EMT) inhibitory activity, but is not limited thereto.

In a specific example of the present invention, the present inventors used Migration and Invasion (Invasion) using 0.8 um pore size Transwell (Falcon, USA) for pancreatic cancer cell mediated transformation of pibulin- ) And an epithelial-to-mesenchymal transition (EMT) protein marker. As a result, AsPC-1 and MiaPaCa-2 pancreatic cancer (Fig. 5). It was confirmed that BxPC-3 cells inhibited Pbu1-3 by the PbuRn-3 siRNA, and metastasis and infiltration ability were increased in the cells. In addition, the expression level of E-cadherin, an epithelial cell marker, was increased in AsPC-1 and MiaPaCa-2 cells overexpressing pibuline-3 and N-cadherin, Snail, Twist expression was decreased, and reverse results were confirmed in PB-PC-3 cells inhibited with pibulin-3 (see FIG. 5).

The present inventors also confirmed sphere formation assay and stem cell protein marker changes in order to confirm the sphere formation ability of the above-produced pibuline-3 overexpressed pancreatic cancer cells. As a result, In the AsPC-1 and MiaPaCa-2 cells overexpressed, the size of the sphere, which is a major characteristic of malignant transformation, was relatively small, did not grow, and the stem protein marker was also decreased. It was confirmed that the expression of pibulin-3 suppresses cell growth and stemming ability (see FIG. 6).

Thus, in the present invention, pibulin-3 has been shown to decrease the cell metastatic potential by expression of the pibulin-3 protein in pancreatic cancer cell lines, and thus the pibulin-3 may be used as a pharmaceutical composition for inhibiting pancreatic cancer metastasis Respectively.

In addition, the present invention provides a health functional food for inhibiting pancreatic cancer metastasis which contains a pibuline-3 protein as an active ingredient.

In the present invention, pibulin-3 has been shown to decrease the cell metastatic potential by expression of the pibulin-3 protein in pancreatic cancer cell lines, thereby confirming that pibulin-3 can be used as a health functional food for inhibiting pancreatic cancer metastasis Respectively.

In addition, the present invention provides a radiation sensitivity enhancer for pancreatic cancer containing the pibulin-3 protein as an active ingredient.

The radiation can be gamma radiation, X-rays or electron beams, and more specifically gamma radiation, more specifically, but not exclusively, can be used 60 Co gamma-rays.

The radiation dose may be, but is not limited to, 0.2-20 Gy, more specifically 0.5-10 Gy, and more specifically 2 Gy. The radiation dose rate may be 0.02-2 Gy / min, more specifically 0.05-1 Gy / min, more specifically 0.2 Gy / min, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors have found that AsPC-1 and MiaPaCa-2 cells into which a pibulin-3 vector has been introduced, radiation by modulation of the expression of the pegylated gene through PbuLin- In order to confirm the sensitivity, a colony formation assay was carried out using a gamma ray irradiation facility (source: 60-Co) of the Korea Atomic Energy Research Institute and irradiated with 6 Gy of gamma rays. As a result, In the overexpressed AsPC-1 and MiaPaCa-2 pancreatic cancer cells, the number of colony formation by gamma irradiation was decreased, and the number of colony formation by gamma irradiation was increased in BxPC-3 cells inhibited by pylon-3 by siRNA (Fig. 4).

Thus, in the present invention, pibulin-3 has been shown to increase radiation sensitivity by expression of the pibulin-3 protein in pancreatic cancer cell lines, and thus the pibulin-3 can be used as a radiation sensitizer for pancreatic cancer Respectively.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> pancreatic cancer cell line Blood ( FBLN ) -3 Confirmation of gene and protein expression level

Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed to compare the expression levels of pibilin-3 in pancreatic cancer cell lines AsPC-1, BxPC-3 and Mia PaCa-2 cells.

Specifically, total RNA was extracted from pancreatic cancer cell lines (AsPC-1, BxPC-3, and Mia PaCa-2) using High Pure RNA Isolation Kit (Roche, Germany), and then transcriptor first strand cDNA synthesis kit (Roche, Germany) (Reverse transcription polymerase chain reaction, RT-PCR) was performed on 1 μg of total cDNA. The PCR was carried out under the conditions of denaturation at 94 ° C for 5 minutes using the primer pair shown in Table 1, followed by 1 minute denaturation at 94 ° C, 1 minute at 56 ° C, and extension at 72 ° C for 1 minute and 30 seconds After repeating 30 times, a post-extension was performed at 72 캜 for 5 minutes. By carrying out PCR under such conditions, the amount of pibulin-3 gene expression was confirmed.

In order to confirm the expression of the pibulin-3 protein, 50 μl of the lysis solution was added to each cell of the pancreatic cancer, followed by reaction at 4 ° C. for 30 minutes, followed by centrifugation using a 4 ° C. centrifuge to obtain supernatant, 40 μg of each protein was loaded onto SDS-gel using the following protein quantitation kit (sigma). Then, the protein loaded on the SDS gel was transferred to a nitrocellulose membrane and reacted with BSA buffer at room temperature for 30 minutes to prevent other antibodies from binding. The primary antibody, anti-fibrin-3 antibody (santa cruz) And anti-β-actin antibody (Sigma) in a 1: 1000 dilution of PBS buffer for 4 hours. The secondary antibody, anti-mouse signaling, was then added to PBS buffer solution diluted 1: 10000 for 1 hour Lt; / RTI &gt; The nitrocellulose membrane was then washed 5 times with PBS and then sensitized to the film with the detection solution.

As a result, as shown in Fig. 1, it was confirmed that the pbuLIN-3 gene and protein were not expressed in AsPC-1 and MiaPaCa-2 cells and the pbuLIN-3 protein was expressed in BxPC-3 cells ).

FBLN-3-forward 5'-ATGTTGAAAGCCCTTTTCC-3 ' SEQ ID NO: 2 FBLN-3-reverse 5'-CTAAAATGAAAATGGCCCC-3 ' SEQ ID NO: 3 GAPDH-forward 5'-ATGGGGAAGG TGAAGG-3 ' SEQ ID NO: 4 GAPDH-reverse 5'-TTACTCCTTGGAGGCC-3 ' SEQ ID NO: 5

< Example  2> Pyrosequencing ( pyrosequencing ) Blood -3 methylation ( 메틸레이션 )

In order to confirm biochemically the methylation state of CpG islands in the promoter region present in Pibulin-3, the pibuline-3 gene promoter of each of the pancreatic cancer cell lines AsPC-1, BxPC-3 and MiaPaCa-2 Pyrosequencing was performed.

Specifically, whole genomic DNA of each of AsPC-1, BxPC-3 and MiaPaCa-2 was isolated and 200 ng of genomic DNA was treated with bisulfite using EZ DNA methylation-Gold kit (ZymoResearch, USA) Respectively. When DNA is treated with bisulfite, unmethylated cytosine is transformed into uracil and methylated cytosine is retained without change. The bisulfite-treated DNA was eluted with 20 μl of sterilized distilled water to perform pyrosequencing. 20 ng of bisulfite-treated genomic DNA was amplified by PCR. The primers of the PCR are shown in Table 2 below. The PCR was carried out by denaturing at 95 ° C for 10 minutes, followed by 30 seconds at 95 ° C, 30 seconds at 58 ° C, and 30 seconds at 72 ° C. Lt; RTI ID = 0.0 &gt; 72 C &lt; / RTI &gt; for 5 minutes. Subsequently, the template DNA labeled with the biotin was purified and the base sequence was analyzed using PyroMark ID (Biotage, USA). Sequencing primers used were 5'-GGTGGTGATGGTTTTTA-3 'and the PCR and sequencing primers were designed using the PSQ assay design program (Biotage, USA).

As a result, as shown in FIG. 2A, the underlined nucleotide sequence indicates the pyrosequencing region of FBLN3, and the four regions marked with Y indicate the methylation site of the cancer cell line. As shown in FIG. 2B, about 95% or more methylation was observed in the AsPC1 and MiaPaCa2 cell lines and about 35% in the BxPC-3 cell line (FIG. 2).

Me-FBLN3-F 5'-TGGATTTTATAGGAGTTGGTTAGAAGT-3 ' SEQ ID NO: 6 Me-FBLN3-R 5'-ACCRCAACCCAAAATACCAAT-3 ' SEQ ID NO: 7 Sequencing primer 5'-GGTGGTGATGGTTTTTA-3 ' SEQ ID NO: 8 FBLN3 sequencing primer 5'-TTTATAGGAGTTGGTTAGAAG-3 ' SEQ ID NO: 9 FBLN3 sequence for analysis ttgggaygttgagtagttttaggggatygtygygtta SEQ ID NO: 10

< Example  3> Blood -3 Overexpression clone production

The present inventors produced an overexpressed clone of the pibulin-3 gene to artificially overexpress the pibulin-3 gene in pancreatic cancer cells.

Specifically, total RNA was extracted from the pancreatic cancer cell line BxPC-3 using High Pure RNA Isolation Kit (Roche, Germany), and then reverse transcription polymerase chain reaction (PCR) was performed on 1 μg of total RNA using Transcriptor First Strand cDNA Synthesis Kit The reverse transcription polymerase chain reaction (RT-PCR) was performed. The PCR conditions were as follows: 55 ° C for 30 minutes and 85 ° C for 5 minutes. ATATAAGCTTATGTTGAAAGCCC-3 'and reverse primer 5'-ATATCTCGAGCTAAAATGAAAATGGCCCC-3 (SEQ ID NO: 3) prepared so as to include the HindIII restriction enzyme recognition sequence at the 5'-end and the XhoI restriction enzyme recognition sequence at the 3'- , Followed by 30 cycles of denaturation at 94 ° C for 1 minute, extension at 56 ° C for 1 minute, and extension at 72 ° C for 1 minute and 30 seconds. Lt; 0 &gt; C for 5 minutes. By performing PCR under such conditions, a pibulin-3 gene of 1,482 bp was obtained. The obtained PCR product and pcDNA3.1 (Invitrogen, USA) vector were treated with restriction enzymes, respectively, and ligated with ligase to prepare pcDNA3.1 / fibulin-3 vector for overexpression of pibulin-3. And ligated with ligase to prepare pcDNA3.1 clone for overexpressing FBLN-3.

5Hind-FBLN3 5'-ATATAAGCTTATGTTGAAAGCCC-3 ' SEQ ID NO: 11 3Xho-FBLN3 5'-ATATCTCGAGCTAAAATGAAAATGGCCCC-3 ' SEQ ID NO: 12

< Example  4> in pancreatic cancer cells Blood -3 overexpression and inhibition of c- Met  And ALDH1  Identification of expression changes

<4-1> Blood -3 overexpression of c- Met  And ALDH1  Identification of expression changes

The present inventors overexpressed the vector prepared in Example 3 to confirm the change in c-Met and ALDH1 expression through overexpression of the pibulin-3 protein, followed by reverse transcription-polymerase chain reaction (RT-PCR) Respectively.

Specifically, AsPC-1 and MiaPaCa-2 cells without pibolin-3 expression in pancreatic cancer cells were each divided into 5 × 10 3 cells, and then 4 μg of pibilin-3 vector and lipofectamine 2000 were used to express penicillin-streptomycin -streptomycin solution, Hyclone) was not added. After 12 hours, the medium was replaced with medium supplemented with 100 units / ml of penicillin-streptomycin and cultured for 48 hours.

AsPC-1 and MiaPaCa-2 cells into which the pFB-3 vector was introduced were collected and RNA was extracted by the same method as in Example 1 to synthesize cDNA to confirm the expression of the pFB-3 gene. Thereafter, primers of c-Met and ALDH1 genes shown in Table 4 were used for denaturation at 94 DEG C for 5 minutes with I-taq polymerase (Intron), followed by denaturation at 94 DEG C for 30 seconds; 56 ° C for 30 seconds; The elongation at 72 ° C for 30 seconds was repeated 30 times, followed by extension at 72 ° C for 10 minutes, and the obtained product was loaded on 1% agarose gel. AsPC-1 and MiaPaCa-2 cells into which the pibulin-3 vector was introduced were collected and Western blotting experiments were carried out in the same manner as in Example 1, in order to confirm the change in the expression of the pibulin-3 protein. The cells were washed twice with PBS buffer (1: 1000) diluted with primary antibody anti-pibulin-3 (santa cruz), anti-ALDH1A1, anti-ALDH1A3 (abcam), anti- For 4 hours, and then reacted with secondary antibody anti-rabbit or mouse signaling in PBS buffer solution diluted 1: 10000 for 1 hour. The nitrocellulose membrane was then washed 5 times with PBS buffer, and then sensitized to the film with the detection solution.

As a result, p-boron-3 was overexpressed by the pibulin-3 gene overexpressing clone in AsPC-1 and MiaPaCa-2 pancreatic cancer cells, and the expression of c-Met and ALDH1 was decreased 3).

<4-2> Blood Lt; RTI ID = 0.0 &gt; c- Met  And ALDH1  Identification of expression changes

The present inventors conducted the following experiments to confirm the change of c-MET and ALDH1 expression through inhibition of the pibulin-3 protein.

Specifically, as shown in the following Table 4, in order to suppress the expression of pibilin-3 in BxPC-3 cells with a high expression of pibuline-3, the forward primer 5'-UAGAAUGUAGGGAUCUUGACAAGG-3 ' Fibulin-3 stealth-RNAi, a 25 mer each of primers 5'-CCUUGUCAAGAUCCCUACAUUCUAA-3 ', was ordered from Invitrogen. Intracellular introduction of the siRNA was performed by taking 100 nM of each RNAi per 2 x 10 5 of BxPC-3 cells using Lipofectamine RNAi MAX (invitrogen), and cells transfected with a Scrambled Stealth TM RNA molecule (negative control ; siControl) was used to transduce cells in a medium without penicillin-streptomycin using Lipofectamine RNAi MAX (Invitrogen) taking 100 nM of Scrambled Stealth TM RNA molecules. After 4 to 6 hours of transfection, the medium was replaced with medium supplemented with 100 units / ml penicillin-streptomycin and cultured for 72 hours. Further, BxPC-3 cells in which pbuLin-3 was inhibited were collected and tested for expression of PfuBin-3 gene and protein in the same manner as in Example <4-1>.

As a result, as shown in FIG. 3, pbuLin-3 was inhibited by the pibuline-3 siRNA in BxPC-3 cells, and the expression of c-Met and ALDH1 was increased (FIG. 3).

Therefore, it was confirmed that the expression of pibulin-3 in the pancreatic cancer cell line inhibited the expression of c-Met and ALDH1.

Fibulin-3 siRNA forward 5'-UAGAAUGUAGGGAUCUUGACAAGG-3 ' SEQ ID NO: 13 Fibulin-3 siRNA Reverse 5'-CCUUGUCAAGAUCCCUACAUUCUAA-3 ' SEQ ID NO: 14 c-Met Forward 5'-GCCCGTTCCTTAGATCCTAT-3 ' SEQ ID NO: 15 c-Met Reverse 5'-ATGGTCGAATTGTCCCAATG-3 ' SEQ ID NO: 16 ALDH1A1 Forward 5'-ATGTCATCCTCAGGCACGCC-3 ' SEQ ID NO: 17 ALDH1A1 reverse direction 5'-TTATGACTGTGACTGTTTTGACC-3 ' SEQ ID NO: 18 ALDH1A3 Forward 5'-GCCCTGGAGACGATGGATAC-3 ' SEQ ID NO: 19 ALDH1A3 reverse direction 5'-TCCACTGCCAAGTCCAAGTC-3 ' SEQ ID NO: 20

< Example  5> in pancreatic cancer cells Blood -3 gene overexpression or inhibition

The present inventors investigated the radiation sensitivity by controlling the expression of the fused gene through the AsPC-1 and MiaPaCa-2 cells and the PfuRin-3-inhibited BxPC-3 cells into which the pibulin-3 vector was introduced. (Source: 60-Co) was used to conduct a colony formation assay after gamma irradiation.

Specifically, pancreatic cancer cells were each fractionated in the same manner as in Example 4, and then penicillin-streptomycin solution (Hyclone) was used by using 4 μg of pibulin-3 vector or 100 nM of RNAi as lipofectamine 2000, Lt; / RTI &gt; was added in the absence of the culture medium. After 12 hours, the medium was replaced with a medium supplemented with 100 units / ml of penicillin-streptomycin, cultured for 48 hours, irradiated with 6 Gy, and cultured in a 5% CO 2 incubator for 8 days. The colonies were then washed twice with PBS buffer, stained with a crystal violet solution for 10 minutes, washed five times with PBS buffer, and colonies were observed.

As a result, as shown in Fig. 4, the number of colonies formed by gamma irradiation was decreased in the AsPC-1 and MiaPaCa-2 pancreatic cancer cells overexpressed with the PfuLin-3 vector, It was confirmed that the number of colonies formed by gamma irradiation was increased in BxPC-3 cells (Fig. 4). Therefore, it was confirmed that the radiation sensitivity was increased by pibulin-3 in the pancreatic cancer cell line.

< Example  6> in pancreatic cancer cells Blood -3 transgene overexpression or inhibition

Migration and invasion analysis and epithelial-to-mesenchymal transition were performed in pancreatic cancer cells using 0.8 μm pore size Transwell (Falcon, USA) -mesenchymal transition (EMT) protein markers.

Specifically, pancreatic cancer cells were each fractionated in the same manner as in Example 4, and then penicillin-streptomycin solution (Hyclone) was used by using 4 μg of pibulin-3 vector or 100 nM of RNAi as lipofectamine 2000 Lt; / RTI &gt; was added in the absence of the culture medium. After 12 hours, the medium was replaced with a medium supplemented with 100 units / ml of penicillin-streptomycin, cultured for 48 hours, irradiated with 6 Gy, and cultured in a 5% CO 2 incubator for 8 days. Then, each cell of a transition (Migration) well contains 1 × 10 kiwotgo 72 hours for five to incubator 37 ℃ inoculated, infiltration (Invasion) well is 5 × 10 then inoculated with 5 to 48 hours to 37 ℃ incubator After incubation for 10 minutes with 0.5% of crystal violet reagent, the cells were washed several times with PBS buffer and observed with a microscope. Using the primary antibodies N-cadherin, E-cadherin, Snail, Twist (BD Bioscience) and β-actin (cell signaling) used as markers for epithelial to mesenchymal transition (EMT) To confirm protein changes.

As a result, as shown in Fig. 5, both the metastasis and infiltration capacity were decreased in the AsPC-1 and MiaPaCa-2 pancreatic cancer cells overexpressed with the pig-borne-3 vector, and the pibulin- Gt; BxPC-3 &lt; / RTI &gt; cells (FIG. 5).

In addition, the expression level of E-cadherin, an epithelial cell marker, was increased in AsPC-1 and MiaPaCa-2 cells overexpressing pibuline-3 and N-cadherin, Snail, Twist expression was decreased, and reverse results were confirmed in PB-PC-3 cells inhibited with pibulin-3 (FIG. 5). Therefore, it was confirmed that the expression of pibuline-3 in the pancreatic cancer cell line decreased cell metastasis ability.

< Example  7> Blood -3 overexpression of pancreatic cancer cells sphere formation  formation

In order to confirm the analysis of the sphere formation ability of piboulin-3 overexpressed pancreatic cancer cells prepared in Example 4, sphere formation assay and stem cell protein marker changes were confirmed.

Specifically, 2 × 10 4 pancreatic cancer cells were suspended in DMEM (Invitrogen) containing stem cell-permissive medium, and 20 ng / ml EGF and 20 ng / ml basic fibroblast growth facotr were added to DMEM-F12 medium (invitrogen) (bFGF) and B27 Serum-free Supplement (50 x; Invitrogen) were mixed and cultured on a 60 mm plate pretreated with 0.8% agar, and the vector was used as a control. Cells were maintained in a 5% CO 2 incubator at 37 ° C and Sphere formation was observed after 7 days of incubation. In addition, protein changes were confirmed by the same method as in Example 1 using the primary antibodies Sox2, Oct4, and β-actin (cell signaling) used as stymeness markers.

As a result, as shown in Fig. 6, the sphere of the major characteristic of malignant transformation in AsPC-1 and MiaPaCa-2 cells overexpressing pibulin-3 was relatively small and did not grow, and the stem protein marker . It was confirmed that the expression of pibulin-3 suppressed cell growth and stemming ability (FIG. 6).

<110> Korea Atomic Energy Research Institute <120> A pharmaceutical composition containing fibulin-3 for the          prevention and treatment of pancreatic cancers <130> 2015P-06-053 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 493 <212> PRT <213> Homo sapiens <400> 1 Met Leu Lys Ala Leu Phe Leu Thr Met Leu Thr Leu Ala Leu Val Lys   1 5 10 15 Ser Gln Asp Thr Glu Glu Thr Ile Thr Tyr Thr Gln Cys Thr Asp Gly              20 25 30 Tyr Glu Trp Asp Pro Val Arg Gln Gln Cys Lys Asp Ile Asp Glu Cys          35 40 45 Asp Ile Val Pro Asp Ala Cys Lys Gly Gly Met Lys Cys Val Asn His      50 55 60 Tyr Gly Gly Tyr Leu Cys Leu Pro Lys Thr Ala Gln Ile Ile Val Asn  65 70 75 80 Asn Glu Gln Pro Gln Gln Glu Thr Gln Pro Ala Glu Gly Thr Ser Gly                  85 90 95 Ala Thr Thr Gly Val Ala Ala Ser Ser Ala Thr Ser Gly Val             100 105 110 Leu Pro Gly Gly Gly Ply Val Ala Ser Ala Ala Ala Val Ala Gly Pro         115 120 125 Glu Met Gln Thr Gly Arg Asn Asn Phe Val Ile Arg Arg Asn Pro Ala     130 135 140 Asp Pro Gln Arg Ile Pro Ser Asn Pro Ser His Arg Ile Gln Cys Ala 145 150 155 160 Ala Gly Tyr Glu Gln Ser Glu His Asn Val Cys Gln Asp Ile Asp Glu                 165 170 175 Cys Thr Ala Gly Thr His Asn Cys Arg Ala Asp Gln Val Cys Ile Asn             180 185 190 Leu Arg Gly Ser Phe Ala Cys Gln Cys Pro Pro Gly Tyr Gln Lys Arg         195 200 205 Gly Glu Gln Cys Val Asp Ile Asp Glu Cys Thr Ile Pro Pro Tyr Cys     210 215 220 His Gln Arg Cys Val Asn Thr Pro Gly Ser Phe Tyr Cys Gln Cys Ser 225 230 235 240 Pro Gly Phe Gln Leu Ala Ala Asn Asn Tyr Thr Cys Val Asp Ile Asn                 245 250 255 Glu Cys Asp Ala Ser Asn Gln Cys Ala Gln Gln Cys Tyr Asn Ile Leu             260 265 270 Gly Ser Phe Ile Cys Gln Cys Asn Gln Gly Tyr Glu Leu Ser Ser Asp         275 280 285 Arg Leu Asn Cys Glu Asp Ile Asp Glu Cys Arg Thr Ser Ser Tyr Leu     290 295 300 Cys Gln Tyr Gln Cys Val Asn Glu Pro Gly Lys Phe Ser Cys Met Cys 305 310 315 320 Pro Gln Gly Tyr Gln Val Val Arg Ser Ser Thr Cys Gln Asp Ile Asn                 325 330 335 Glu Cys Glu Thr Thr Asn Glu Cys Arg Glu Asp Glu Met Cys Trp Asn             340 345 350 Tyr His Gly Gly Phe Arg Cys Tyr Pro Arg Asn Pro Cys Gln Asp Pro         355 360 365 Tyr Ile Leu Thr Pro Glu Asn Arg Cys Val Cys Pro Val Ser Asn Ala     370 375 380 Met Cys Arg Glu Leu Pro Gln Ser Ile Val Tyr Lys Tyr Met Ser Ile 385 390 395 400 Arg Ser Asp Arg Ser Val Pro Ser Asp Ile Phe Gln Ile Gln Ala Thr                 405 410 415 Thr Ile Tyr Ala Asn Thr Ile Asn Thr Phe Arg Ile Lys Ser Gly Asn             420 425 430 Glu Asn Gly Glu Phe Tyr Leu Arg Gln Thr Ser Pro Val Ser Ala Met         435 440 445 Leu Val Leu Val Lys Ser Leu Ser Gly Pro Arg Glu His Ile Val Asp     450 455 460 Leu Glu Met Leu Thr Val Ser Ser Ile Gly Thr Phe Arg Thr Ser Ser 465 470 475 480 Val Leu Arg Leu Thr Ile Val Gly Pro Phe Ser Phe                 485 490 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> FBLN-3-forward <400> 2 atgttgaaag cccttttcc 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> FBLN-3-reverse <400> 3 ctaaaatgaa aatggcccc 19 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-forward <400> 4 atggggaagg tgaagg 16 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-reverse <400> 5 ttactccttg gaggcc 16 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Me-FBLN3-F <400> 6 tggattttat aggagttggt tagaagt 27 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Me-FBLN3-R <400> 7 accrcaaccc aaaataccaa t 21 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> sequencing <400> 8 ggtggtgatg gttttta 17 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 9 tttataggag ttggttagaa g 21 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> analysis primer <400> 10 ttgggaygtt gagtagtttt aggggatygt ygygtta 37 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 5Hind-FBLN3 <400> 11 atataagctt atgttgaaag ccc 23 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 3Xho-FBLN3 <400> 12 atatctcgag ctaaaatgaa aatggcccc 29 <210> 13 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> siRNA_F <400> 13 uagaauguag ggaucuugac aagg 24 <210> 14 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA_R <400> 14 ccuugucaag aucccuacau ucuaa 25 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Met_F <400> 15 gcccgttcct tagatcctat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Met_R <400> 16 atggtcgaat tgtcccaatg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A1_F <400> 17 atgtcatcct caggcacgcc 20 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A1_R <400> 18 ttatgactgt gactgttttg acc 23 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A3_F <400> 19 gccctggaga cgatggatac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A3_R <400> 20 tccactgcca agtccaagtc 20

Claims (12)

A pharmaceutical composition for preventing and treating pancreatic cancer containing fibulin-3 protein as an active ingredient.
The pharmaceutical composition for the prevention and treatment of pancreatic cancer according to claim 1, wherein the pibulin-3 protein has the amino acid sequence of SEQ ID NO: 1.
3. A pharmaceutical composition for the prevention and treatment of pancreatic cancer, comprising as an active ingredient, a vector comprising a polynucleotide encoding a pibulin-3 protein or a pibuline-3 over-expressing cell line transformed with said vector, or a culture thereof.
4. The pharmaceutical composition for the prevention and treatment of pancreatic cancer according to claim 3, wherein the vector is any one selected from the group consisting of linear DNA, plasmid DNA and recombinant viral vectors.
The pharmaceutical composition for preventing and treating pancreatic cancer according to claim 4, wherein the recombinant virus is any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and lentivirus.
4. The method of claim 3, wherein the cell is any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells. A pharmaceutical composition for therapeutic use.
A health functional food for prevention and improvement of pancreatic cancer which contains PfuBulin-3 protein as an active ingredient.
A pharmaceutical composition for inhibiting pancreatic cancer metastasis comprising a pibulin-3 protein as an active ingredient.
9. The pharmaceutical composition for inhibiting pancreatic cancer metastasis according to claim 8, wherein said pibulin-3 protein inhibits pancreatic cancer stem cell growth.
9. The pharmaceutical composition according to claim 8, wherein the composition has an epithelial-to-mesenchymal transition (EMT) inhibitory activity.
delete Radiation sensitivity enhancer for pancreatic cancer containing pibulin-3 protein as an active ingredient.
KR1020150096986A 2015-07-08 2015-07-08 A pharmaceutical composition containing fibulin-3 for the prevention and treatment of pancreatic cancers KR101746143B1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014113406A1 (en) 2013-01-15 2014-07-24 Fred Hutchinson Cancer Research Center Compositions and methods for treating pancreatic cancer
KR101440487B1 (en) 2011-08-09 2014-09-18 한국원자력연구원 Pharmaceutical composition containing a fibulin-3 protein for inhibiting the growth of cancer stem cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101440487B1 (en) 2011-08-09 2014-09-18 한국원자력연구원 Pharmaceutical composition containing a fibulin-3 protein for inhibiting the growth of cancer stem cells
WO2014113406A1 (en) 2013-01-15 2014-07-24 Fred Hutchinson Cancer Research Center Compositions and methods for treating pancreatic cancer

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