KR102028574B1 - Anticancer composition comprising ovary cancer cell line-derived autocrine motility factor as effective component - Google Patents

Abstract

The present invention relates to an anticancer composition containing an ovarian cancer cell line-derived autocrine motility factor (AMF) as an active component. More specifically, the ovarian cancer cell line-derived AMF suppresses the proliferation of pancreatic cancer, leukemia, and breast cancer cells, and has an excellent effect of inducing cell death. Therefore, a composition of the present invention containing the ovarian cancer cell line-derived AMF as the active component can be used as an anticancer agent.

Description

Anticancer composition comprising ovarian cancer cell line-derived AMF as an active ingredient {Anticancer composition comprising ovary cancer cell line-derived autocrine motility factor as effective component}

The present invention relates to an anticancer composition comprising an ovarian cancer cell line-derived AMF as an active ingredient, and more particularly, to an anticancer containing an ovarian cancer cell line-derived AMF as an active ingredient, which exhibits anticancer effects against pancreatic cancer, leukemia and breast cancer. It relates to a pharmaceutical composition.

Cancer is one of the three leading causes of death in the world along with infectious diseases and cardiovascular diseases, and is a major disease that is expected to increase rapidly in the future due to environmental problems, life extension, and westernization of food culture. Despite many studies on cancer for the development of more effective anticancer drugs, the development of new anticancer drugs that can overcome the resistance and overcome resistance due to the diversification of cancer pathology is still urgently needed.

On the other hand, cell competition is a process of comparing the growth and survival between cells, cells that are pushed out of the competition can be killed. Cellular competition is closely related to cancer development, and cells around the inferior growth adaptability compared to cancer cells may disappear. To date, it has been assumed that a winner cell will secrete a death signal early in the cell competition, but it has not been identified.

On the other hand, autocrine motility factor (AMF) is a housekeeping protein, and glucose-6-phosphate and fructose-in the second process of glycolysis in relation to energy metabolism in cells. It is known to play a role in interconversion of 6-phosphate and is also called glucose-6-phosphate isomerase (GPI). . AMF, a cytokine secreted from tumors, is abundant at the tumor site and is known to play an important role in tumor proliferation, differentiation and survival.

On the other hand, Korean Patent Publication No. 2016-0050773 discloses an AMFR-Fc fusion protein and its anticancer use, but an anticancer composition containing AMF derived from ovarian cancer cell line of the present invention as an active ingredient has not been disclosed.

The present invention has been made by the above-described requirements, and provides an anticancer composition comprising an ovarian cancer cell line-derived AMF as an active ingredient, wherein the composition inhibits the proliferation of pancreatic cancer, leukemia and breast cancer cells and prevents cell death. The present invention was completed by confirming that it is caused.

In order to solve the above problems, the present invention provides an anticancer pharmaceutical composition containing an ovarian cancer cell line-derived AMF (autocrine motility factor) as an active ingredient.

The present invention also provides a method for inhibiting proliferation or apoptosis of cancer cells, comprising administering the anticancer pharmaceutical composition in a mammal other than a human.

The present invention relates to an anticancer composition comprising an ovarian cancer cell line-derived AMF as an active ingredient. The ovarian cancer cell line-derived AMF of the present invention is excellent in inhibiting the proliferation of pancreatic cancer, leukemia and breast cancer cells and causing cell death. Therefore, the composition of the present invention containing an ovarian cancer cell line-derived AMF as an active ingredient can be used as an anticancer material.

1 is a result of confirming the proliferation of cancer cells by MTT analysis when treated with ovarian cancer cell conditioned medium (conditioned medium, CM) to other cancer cell lines. A549 is lung cancer cell line, AsPC-1 is pancreatic cancer cell line, DU145 is prostate cancer cell line, HeLa is cervical cancer cell line, Hep G2 is liver cancer cell line, HL60 is white blood cell line, HT-29 is colorectal cancer cell line, MCF-7 is breast cancer cell line to be.
2 is a result confirming that the cell growth inhibition effect of ovarian cancer cell-mediated medium (CM) on MCF-7 breast cancer cells by AMF. Con is negative control group, CM is ovarian cancer cell control medium treatment group, E4P is a group treated with 0.5mM of erythrose 4-phosphate (E4P), an inhibitor of AMF activity, CM + E4P 0.5mM in ovarian cancer cell control medium Is a group treated with, IP-CM is an ovarian cancer cell control medium treatment group in which AMF is removed using AMF antibody.
Figure 3 is a result of comparing the amino acid sequence of ovarian cancer-derived recombinant AMF and GPI homologous protein (Genbank accession number: BC004982.1). A is the amino acid sequence of GPI isoprotein and B is the amino acid sequence of recombinant AMF derived from ovarian cancer. Indicates that the sequences of A and B are identical.
4 is a result of confirming by measuring the change in cell number that the purified ovarian cancer-derived recombinant AMF causes the death of cancer cells. rAMF is ovarian cancer-derived recombinant AMF, AsPC-1 is pancreatic cancer cell, HL60 is leukemia cell, MCF-7 is breast cancer cell.

The present invention relates to a pharmaceutical composition for anticancer containing an ovarian cancer cell line-derived AMF (autocrine motility factor) as an active ingredient.

The ovarian cancer cell line is preferably SKOV3, which is a human-derived ovarian cancer cell line, but is not limited thereto.

The ovarian cancer cell line-derived AMF consists of the amino acid sequence of SEQ ID NO: 1. The range of AMFs derived from ovarian cancer cell lines of the present invention includes functional equivalents of proteins having the amino acid sequence represented by SEQ ID NO: 1. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid; It refers to a protein having 95% or more of sequence homology and exhibiting substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 1. Also included are peptides that exhibit substantially the same physiological activity as AMFs derived from ovarian cancer cell lines. "Substantially homogeneous physiological activity" means activity against cancer prevention or treatment.

In the present invention, the anticancer means inhibiting the proliferation of cancer cells or killing cancer cells. The composition of the present invention may be an anticancer effect in at least one selected from the group consisting of pancreatic cancer, leukemia and breast cancer, but is not limited thereto. .

In the pharmaceutical composition for anticancer according to an embodiment of the present invention, the composition may inhibit the proliferation of cancer and induce cell death, but is not limited thereto.

The pharmaceutical composition according to the present invention may be used in the form of capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, and the like, oral formulations, suppositories, or sterile injectable solutions, respectively, according to a conventional method. Can be.

Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate And various compounds or mixtures including cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the pharmaceutical composition. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include suspensions, solvents, emulsions, syrups, and the like, as well as simple diluents such as water and liquid paraffin, which may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration and the like.

The present invention also provides a method for inhibiting proliferation or apoptosis of cancer cells, comprising administering the anticancer pharmaceutical composition in a mammal other than a human.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Materials and methods

1. Cell Culture

Human cancer cell lines used in this experiment were purchased from Korea Cell Line Bank (Seoul, Korea). 10% (v / v) A549 lung cancer cell line, AsPC-1 pancreatic cancer cell line, DU145 prostate cancer cell line, HeLa cervical cancer cell line, Hep G2 liver cancer cell line, HL60 leukemia cell line, HT-29 colon cancer cell line, MCF-7 breast cancer cell line Fetal bovine serum (FBS), 100 U / ml penicillin G and 100 μg / ml of streptomycin containing streptomycin were incubated under 37 ° C. and 5% CO ₂ conditions.

2. Preparation of serum-free conditioned medium (CM)

When SKOV3 ovarian cancer cell lines were grown to approximately 80% in 75 cm 2 culture flasks, they were washed three times with phosphate-buffered saline (PBS) and then replaced with serum-free medium. Serum-free medium was obtained after 24 hours and concentrated to 1/5 volume in a dialysis tube (7000 MWCO) by dialysis against sucrose, filtered to 0.22 μm and stored at minus 70 ° C.

To obtain a large amount of serum-free media in a similar manner, SKOV3 ovarian cancer cell lines were prepared by culturing in a 175 cm 2 culture flask, washed three times with PBS when grown to approximately 80%, and then fresh radish Serum-free medium was obtained by replacing the cells with serum medium for 24 hours to obtain a serum-free medium (hereinafter, CM), followed by a 24-hour interval, and obtained for a final 72 hours to obtain a SKOV3 CM. SKOV3 CM was filtered through 0.22㎛ and then lyophilized and stored at minus 70 ℃.

3. Cell Growth Assay

Cells were incubated for 24 hours in a 24-well culture dish, and then cultured cells were cultured for 48 to 72 hours by treating SKOV3 CM at a concentration of 5% (v / v) with respect to the culture medium.

Cell growth was observed at a wavelength of 570 nm through MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; thiazolyl blue) analysis.

In addition, trypan blue exclusion assay was performed to determine viable cell number.

4. Protein Purification and Identification

Lyophilized SKOV3 CM was dissolved in Tris buffer (50 mM Tris-HCl, pH 7.5) and dialyzed against the same buffer at 4 ° C. After dialysis, the SKOV3 CM sample was loaded into a column containing 30 ml of DEAE diethylaminoethyl group sepharose pre-equilibrated in Tris buffer. Bound protein was eluted from the column using a linear gradient of 0-1.0 M sodium chloride (NaCl) in Tris buffer at a flow rate of 0.7 ml per minute. Elution fractions containing major peaks with bioactivity were collected and concentrated in dialysis tubes (7000 MWCO) by dialysis against sucrose. The concentrated sample was re-fractionated on a column packed with 0.8 × 30 cm Sephacryl S-200 previously equilibrated in Tris buffer. The bioactive fraction was selected at a flow rate of 0.1 ml per minute and concentrated by dialysis as described above. Bioactivity was assessed by analyzing the inhibitory effect of AsPC-1 cell growth using MTT assay.MalDI-TOF mass for protein identification after confirming the size of bioactive samples on 12% SDS-PAGE gel Analysis was performed.

5. AMF blocking assay

Viable cell counts were obtained after 3 days of cell culture under treatment or no treatment conditions of SKOV3 CM, SKOV3 CM with 0.5 mM E4P (AMF activity inhibitor) or IP-SKOV3 CM depleted of AMF by immunoprecipitation (IP). Measured. To prepare IP-SKOV3 CM, 1 ml of SKOV3 CM was incubated with 1 μg of anti-GPI / AMF antibody for 1 hour, followed by 1 μl of protein A / G agarose beads for 1 hour. Processed. Thereafter, centrifugation was performed at 3000 rpm for 2 minutes to remove the beads to which the antigen-antibody complexes bound.

6. DNA Cloning and Recombinant Protein Expression

RNA was extracted from SKOV3 cells and reverse transcribed and used as a template for amplifying AMF cDNA by PCR (30 times at 95 ° C, 30 seconds at 58 ° C and 100 seconds at 72 ° C for 30 times).

Forward primers containing Nde I restriction enzyme sites and reverse primers containing Xho I restriction enzyme sites used for PCR were constructed based on DNA sequences encoding genbank accession number (AK301103.1).

Forward primer: 5'-TACATATGGCCGCTCTCACCCGGGACCCCCAGTTCCAGAA-3 '(SEQ ID NO: 2)

Reverse primer: 5'-ATCTCGAGTTATTGGACTCTGGCCTCGCGCTGCT-3 '(SEQ ID NO: 3)

The 1.7 kb PCR product confirmed the DNA sequence and was cloned into the pCold DNA1 vector to construct pSKOV3-AMF. Escherichia coli BL21 containing pSKOV3-AMF was incubated in LB medium containing 100 µg / ml of ampicillin for 16 hours under conditions of 200 rpm and 37 ° C, diluted 1: 100 in fresh medium, and 200 rpm, 37 ° C. Recultured for 2 hours under. Thereafter, cells were treated with 0.5 mM IPTG and incubated for 24 hours under conditions of 200 rpm and 15 ° C. The cells harvested after incubation are resuspended in buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl and 0.5 mM PMSF), and then French pressure cell press (Model FA-078A, Thermo IEC, Milford, MA, USA) Cells were disrupted and centrifuged at 12000 rpm for 20 minutes at 4 ° C. to obtain lysed fractions. The obtained fractions passed through a 0.22 μm syringe filter. SKOV3 recombinant AMF (SKOV3 rAMF) was purified by His60 Ni resin affinity chromatography (Promega, Madison, WI, USA) according to the supplier's method, and the amount of purified protein was quantified using Bio-Rad protein assay reagent. It was.

Example  One. SKO V3 CM's  Cancer cell proliferation inhibitory effect

In order to confirm the cancer cell proliferation inhibitory effect of SKOV3 CM, various cancer cells except for ovarian cancer were treated with SKOV3 CM, and MTT analysis was performed. As a result, as shown in FIG. 1, it was confirmed that the proliferation of pancreatic cancer (AsPC-1), leukemia (HL60) and breast cancer (MCF-7) cells treated with SKOV3 CM, an ovarian cancer-derived cell, was inhibited. A549), prostate cancer (DU145), cervical cancer (HeLa), liver cancer (Hep G2) and colon cancer (HT-29) cells did not affect the proliferation.

It was confirmed that SKOV3 CM specifically inhibits the proliferation of pancreatic cancer (AsPC-1), leukemia (HL60) and breast cancer (MCF-7) cells.

Example  2. SKOV3  Analysis of Secretory Proteins

Proteins were purified and identified to analyze proteins that exhibit cancer cell proliferation inhibitory effect in SKOV3 CM. The fractions showing the physiological activity in the DEAE sepharose fraction and the DEAE sepharose fraction were re-fractionated using a column filled with Sephacryl S-200, and the fractions having excellent physiological activity among the refractions were obtained. Confirmed. Physiological activity was confirmed through treatment of fractions with AsPC-1 cells to inhibit proliferation of cancer cells.

As a result of mass spectrometry using a malditope, the fraction showing excellent physiological activity was identified as AMF, a protein related to the mobility and proliferation of cancer cells, and then experimented with AMF.

Example  3. AMF  Activity inhibitory effect

An AMF blocking assay was performed to determine if AMF present in SKOV3 CM inhibits proliferation of cells. As a result, as shown in FIG. 2, SKOV3 CM was treated with E4P, an AMF active inhibitor (CM + E4P), or AMF antibody was used to remove AMF in SKOV3 CM in advance (IP-CM). It was confirmed that the proliferation inhibitory effect of breast cancer (MCF-7) cells by CM is restored. This confirmed that AMF in SKOV3 CM has an effect of inhibiting the proliferation of certain cancer cells.

Example  4. SKO V3 Origin AMF  Gene structure analysis

The SKOV3 AMF cDNA was amplified by reverse transcription and PCR using SKOV3 RNA and cloned into pCold DNA1 vector to construct pSKOV3-AMF. As a result, the DNA sequence was analyzed as shown in SEQ ID NO: 4, and the amino acid sequence of the predicted recombinant AMF showed 99% homology with the GPI homologous protein (Genbank accession number: BC004982) as shown in FIG. Several mutations were observed.

Example  5. SKOV3  Derived recombination To AMF  Cell proliferation inhibitory effect

Purified AMF in Example 4 and then treated with cancer cells to confirm the effect on cell proliferation. As a result, when treated with various concentrations of recombinant AMF (2, 20 and 100 ng / ㎖) as shown in Figure 4 compared to the untreated control group, pancreatic cancer (AsPC-1), leukemia (HL60) and breast cancer ( MCF-7) It was confirmed that the proliferation of cells is inhibited.

<110> DAEGU CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Anticancer composition comprising ovary cancer cell line-derived          autocrine motility factor as effective component <130> PN18183 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 558 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ala Leu Thr Arg Asp Pro Gln Phe Gln Lys Leu Gln Gln Trp   1 5 10 15 Tyr Arg Glu His Arg Ser Glu Leu Asn Leu Arg Arg Leu Phe Asp Ala              20 25 30 Asn Lys Asp Arg Phe Asn His Phe Ser Leu Thr Leu Asn Thr Asn His          35 40 45 Gly His Ile Leu Val Asp Tyr Ser Lys Asn Leu Val Thr Glu Asp Val      50 55 60 Met Arg Met Leu Val Asp Leu Ala Lys Ser Arg Gly Val Glu Ala Ala  65 70 75 80 Arg Glu Arg Met Phe Asn Gly Glu Lys Ile Asn Tyr Thr Glu Gly Arg                  85 90 95 Ala Val Leu His Val Ala Leu Arg Asn Arg Ser Asn Thr Pro Ile Leu             100 105 110 Val Asp Gly Lys Asp Val Met Pro Glu Val Asn Lys Val Leu Asp Lys         115 120 125 Met Lys Ser Phe Cys Gln Arg Val Arg Ser Gly Asp Trp Lys Gly Tyr     130 135 140 Thr Gly Lys Thr Ile Thr Asp Val Ile Asn Ile Gly Ile Gly Gly Ser 145 150 155 160 Asp Leu Gly Pro Leu Met Val Thr Glu Ala Leu Lys Pro Tyr Ser Ser                 165 170 175 Gly Gly Pro Arg Val Trp Tyr Val Ser Asn Ile Asp Gly Thr His Ile             180 185 190 Ala Lys Thr Leu Ala Gln Leu Asn Pro Glu Ser Ser Leu Phe Ile Ile         195 200 205 Ala Ser Lys Thr Phe Thr Thr Gln Val Asp Ile Thr Asn Ala Glu Thr     210 215 220 Ala Lys Glu Trp Phe Leu Gln Ala Ala Lys Asp Pro Ser Ala Val Ala 225 230 235 240 Lys His Phe Val Ala Leu Ser Thr Asn Thr Thr Lys Val Lys Glu Phe                 245 250 255 Gly Ile Asp Pro Gln Asn Met Phe Glu Phe Trp Asp Trp Val Gly Gly             260 265 270 Arg Tyr Ser Leu Trp Ser Ala Ile Gly Leu Ser Ile Ala Leu His Val         275 280 285 Gly Phe Asp Asn Phe Glu Gln Leu Leu Ser Gly Ala His Trp Met Asp     290 295 300 Gln His Phe Arg Thr Thr Pro Leu Glu Lys Asn Ala Pro Val Leu Leu 305 310 315 320 Ala Leu Leu Gly Ile Trp Tyr Ile Asn Cys Phe Gly Cys Glu Thr His                 325 330 335 Ala Met Leu Pro Tyr Asp Gln Tyr Leu His Arg Phe Ala Ala Tyr Phe             340 345 350 Gln Gln Gly Asp Met Glu Ser Asn Gly Lys Tyr Ile Thr Lys Ser Gly         355 360 365 Thr Arg Val Asp His Gln Thr Gly Pro Ile Val Trp Gly Glu Pro Gly     370 375 380 Thr Asn Gly Gln His Ala Phe Tyr Gln Leu Ile His Gln Gly Thr Lys 385 390 395 400 Met Ile Pro Cys Asp Phe Leu Ile Pro Val Gln Thr Gln His Pro Ile                 405 410 415 Arg Lys Gly Leu His His Lys Ile Leu Leu Ala Asn Phe Leu Ala Gln             420 425 430 Thr Glu Ala Leu Met Arg Gly Lys Ser Thr Glu Glu Ala Arg Lys Glu         435 440 445 Leu Gln Ala Ala Gly Lys Ser Pro Glu Asp Leu Glu Arg Leu Leu Pro     450 455 460 His Lys Val Phe Glu Gly Asn Arg Pro Thr Asn Ser Ile Val Phe Thr 465 470 475 480 Lys Leu Thr Pro Phe Met Leu Gly Ala Leu Val Ala Met Tyr Glu His                 485 490 495 Lys Ile Phe Val Gln Gly Ile Ile Trp Asp Ile Asn Ser Phe Asp Gln             500 505 510 Trp Gly Val Glu Leu Gly Lys Gln Leu Ala Lys Lys Ile Glu Pro Glu         515 520 525 Leu Asp Gly Ser Ala Gln Val Thr Ser His Asp Ala Ser Thr Asn Gly     530 535 540 Leu Ile Asn Phe Ile Lys Gln Gln Arg Glu Ala Arg Val Gln 545 550 555 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 tacatatggc cgctctcacc cgggaccccc agttccagaa 40 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 atctcgagtt attggactct ggcctcgcgc tgct 34 <210> 4 <211> 1677 <212> DNA <213> Homo sapiens <400> 4 atggccgctc tcacccggga cccccagttc cagaagctgc agcaatggta ccgcgagcac 60 cgctccgagc tgaacctgcg ccgcctcttc gatgccaaca aggaccgctt caaccacttc 120 agcttgaccc tcaacaccaa ccatgggcat atcctggtgg attactccaa gaacctggtg 180 acggaggacg tgatgcggat gctggtggac ttggccaagt ccaggggcgt ggaggccgcc 240 cgggagcgga tgttcaatgg tgagaagatc aactacaccg agggtcgagc cgtgctgcac 300 gtggctctgc ggaaccggtc aaacacaccc atcctggtag acggcaagga tgtgatgcca 360 gaggtcaaca aggttctgga caagatgaag tctttctgcc agcgtgtccg gagcggtgac 420 tggaaggggt acacaggcaa gaccatcacg gacgtcatca acattggcat tggcggctcc 480 gacctgggac ccctcatggt gactgaagcc cttaagccat actcttcagg aggtccccgc 540 gtctggtatg tctccaacat tgatggaact cacattgcca aaaccctggc ccagctgaac 600 cccgagtcct ccctgttcat cattgcctcc aagaccttta ctacccaggt agacatcacg 660 aatgcagaga cggcgaagga gtggtttctc caggcggcca aggatccttc tgcagtggcg 720 aagcactttg ttgccctgtc tactaacaca accaaagtga aggagtttgg aattgaccct 780 caaaacatgt tcgagttctg ggattgggtg ggaggacgct actcgctgtg gtcggccatc 840 ggactctcca ttgccctgca cgtgggtttt gacaacttcg agcagctgct ctcgggggct 900 cactggatgg accagcactt ccgcacgacg cccctggaga agaacgcccc cgtcttgctg 960 gccctgctgg gtatctggta catcaactgc tttgggtgtg agacacacgc catgctgccc 1020 tatgaccagt acctgcaccg ctttgctgcg tacttccagc agggcgacat ggagtccaat 1080 gggaaataca tcaccaaatc tggaacccgt gtggaccacc agacaggccc cattgtgtgg 1140 ggggagccag ggaccaatgg ccagcatgct ttttaccagc tcatccacca aggcaccaag 1200 atgataccct gtgacttcct catcccggtc cagacccagc accccatacg gaagggtctg 1260 catcacaaga tcctcctggc caacttcttg gcccagacag aggccctgat gaggggaaaa 1320 tcgacggagg aggcccgaaa ggagctccag gctgcgggca agagtccaga ggaccttgag 1380 aggctgctgc cacataaggt ctttgaagga aatcgcccaa ccaactctat tgtgttcacc 1440 aagctcacac cattcatgct tggagccttg gtcgccatgt atgagcacaa gatcttcgtt 1500 cagggcatca tctgggacat caacagcttt gaccagtggg gagtggagct gggaaagcag 1560 ctggctaaga aaatagagcc tgagcttgat ggcagtgctc aagtgacctc tcacgacgct 1620 tctaccaatg ggctcatcaa cttcatcaag cagcagcgcg aggccagagt ccaataa 1677

Claims (8)

An anticancer pharmaceutical composition for at least one carcinoma selected from the group consisting of pancreatic cancer, leukemia, and breast cancer containing an autocrine motility factor (AMF) derived from an ovarian cancer cell line consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. According to claim 1, wherein the ovarian cancer cell line is SKOV3, anticancer pharmaceutical composition for at least one carcinoma selected from the group consisting of pancreatic cancer, leukemia and breast cancer. delete delete According to claim 1, wherein the ovarian cancer cell line-derived AMF is anti-cancer pharmaceutical composition for at least one carcinoma selected from the group consisting of pancreatic cancer, leukemia and breast cancer, characterized in that to inhibit the proliferation of cancer cells. According to claim 1, The ovarian cancer cell line-derived AMF is anti-cancer pharmaceutical composition for at least one carcinoma selected from the group consisting of pancreatic cancer, leukemia and breast cancer, characterized in that induces apoptosis of cancer cells. According to claim 1, wherein the pharmaceutical composition is pancreatic cancer, leukemia and breast cancer, characterized in that the preparation in the form of capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injectable solutions Pharmaceutical composition for anticancer for at least one carcinoma selected from the group consisting of. A method selected from the group consisting of pancreatic cancer, leukemia and breast cancer comprising the step of administering the pharmaceutical composition for cancer according to any one of claims 1, 2 and 5 to 7 in a mammal except for humans. Proliferation inhibitory or apoptosis promoting method of the above cancer cells.

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