KR102039367B1 - Anticancer composition comprising prostate cancer cell line-derived autocrine motility factor as effective component - Google Patents

Abstract

The present invention relates to a composition for anticancer containing a prostate cancer cell line-derived AMF as an active component. More specifically, the prostate cancer cell line-derived AMF of the present invention has an excellent effect of inhibiting the proliferation of pancreatic cancer, leukemic cancer, and brain cancer, and causing cell death. Therefore, the composition containing the prostate cancer cell line-derived AMF of the present invention can be used as an anticancer therapeutic agent.

Description

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

The present invention relates to an anticancer composition containing an AMF-derived prostate cancer cell line as an active ingredient, and more particularly for an anticancer containing an AMF-derived prostate cancer cell line-derived AMF as an active ingredient against pancreatic cancer, leukemia and brain 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 the prostate 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 containing AMF derived from prostate cancer cell line as an active ingredient, wherein the composition inhibits proliferation of pancreatic cancer, leukemia and brain 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 autocrine motility factor (AMF) derived from prostate cancer cell line 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 AMF-derived prostate cancer cell line as an active ingredient. The prostate cancer cell line-derived AMF of the present invention inhibits proliferation of pancreatic cancer, leukemia and brain cancer cells, and has excellent effects of causing cell death. Therefore, the composition of the present invention containing the prostate 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 other cancer cell lines conditioned medium (CM). AsPC-1 is pancreatic cancer cell line, Hep 3B is liver cancer cell line, HL60 is leukemia cell line, MCF-7 is breast cancer cell line, PC3 is prostate cancer cell line, SNU 484 is gastric cancer cell line, U-87 MG is brain cancer cell line.
Figure 2 is a result confirming that the proliferation inhibitory effect of prostate cancer cell-mediated medium (CM) on HL60 leukemia cells by AMF. 5% (v / v) CM is treated with 5% (v / v) prostate cancer cell-mediated medium compared to culture medium, and E4P is treated with erythrose 4-phosphate (E4P), an inhibitor of AMF activity, by concentration. 5% (v / v) IP-CM is a group treated with 5% (v / v) of AMF-depleted prostate cancer cell media using AMF antibody.
Figure 3 is a result of comparing the amino acid sequence of prostate cancer-derived recombinant AMF and GPI homologous protein (Genbank accession number: BC004982.1). A is the amino acid sequence of the GPI isoprotein and B is the amino acid sequence of recombinant AMF derived from prostate cancer. Indicates that the sequences of A and B are identical.
4 shows the results of confirming that the purified AMF-derived recombinant AMF causes the death of cancer cells by microscopy (A) and cell count (B). rAMF is prostate cancer-derived recombinant AMF, AsPC-1 is pancreatic cancer cell, HL60 is leukemia cell, U-87 MG is brain cancer cell.

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

The prostate cancer cell line is preferably DU145, which is a human-derived prostate cancer cell line, but is not limited thereto.

The prostate cancer cell line-derived AMF consists of the amino acid sequence of SEQ ID NO: 1. The range of AMFs derived from prostate 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 prostate 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 brain 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, or the like, oral preparations, 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 such solid preparations may contain 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). Pancreatic cancer cells AsPC-1, liver cancer cells Hep 3B, leukemia cells HL60, prostate cancer cells DU145 and PC3, breast cancer cells MCF-7, gastric cancer cells SNU 484, brain cancer cells U-87 MG 10% (v / v) Fetal bovine serum (FBS), 100U / ㎖ penicillin G and 100μg / ㎖ of streptomycin containing streptomycin was incubated under 37 ° C, 5% CO ₂ conditions.

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

When the DU145 prostate cancer cell line was grown at approximately 80% in a 75 cm 2 culture flask, it was 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, DU145 prostate cancer cell lines were prepared by culturing in a 175 cm 2 culture flask, washed three times with PBS when grown to approximately 80% ratio, 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), and then obtained for a final 72 hours at 24 hour intervals to obtain a total of 4 L of DU145 CM. DU145 CM was filtered through 0.22㎛ 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 DU145 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 DU145 CM was dissolved in Tris buffer (50 mM Tris-HCl, pH 7.5) and dialyzed against the same buffer at 4 ° C. After dialysis DU145 CM samples were 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 HL60 cell growth using MTT assay, and after confirming the size of the bioactive sample on a 12% SDS-PAGE gel, MALDI-TOF mass spectrometry was used for protein identification. Was carried out.

5. AMF blocking assay

Survival cell counts after 3 days of cell culture under treatment or no treatment conditions of DU145 CM, DU145 CM with 0.5 mM E4P (AMF activity inhibitor) or IP-DU145 CM with AMF depleted by immunoprecipitation (IP) Measured. 1 ml of DU145 CM was incubated with 1 μg of anti-GPI / AMF antibody for 1 hour to prepare IP-DU145 CM, 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 DU145 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 pDU145-AMF. Escherichia coli BL21 containing pDU145-AMF was incubated for 16 hours at 200 rpm and 37 ° C. in LB medium containing 100 μg / ml Ampicillin, and then diluted 1: 100 in fresh medium, and 200 rpm and 15 ° 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, 1 nM DTT and 0.5 mM PMSF), and then French pressure cell press (Model FA-078A, Thermo IEC, Milford, MA, USA) was used to disrupt cells 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. DU145 recombinant AMF (DU145 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. DU1 45 CM's  Cancer cell proliferation inhibitory effect

In order to confirm the cancer cell proliferation inhibitory effect of DU145 CM, various cancer cells were treated with DU145 CM, and MTT analysis was performed. As a result, as shown in Figure 1 DU145 CM inhibited the proliferation of pancreatic cancer (AsPC-1), leukemia (HL60) and brain cancer (U-87 MG) cells, liver cancer (Hep 3B), breast cancer (MCF-7 ), Proliferation of prostate cancer (PC3) and gastric cancer (SNU 484) cells had no effect or rather promoted proliferation.

Through this, DU145 CM was confirmed to specifically inhibit the proliferation of pancreatic cancer (AsPC-1), leukemia (HL60) and brain cancer (U-87 MG) cells.

Example  2. Analysis of DU145 Secreted Proteins

Proteins were purified and identified to analyze proteins that exhibit cancer cell proliferation inhibitory effect in DU145 CM. The fractions showing 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 re-fractions were obtained. Confirmed. Physiological activity was confirmed by inhibiting the proliferation of cancer cells by treating the fractions to HL60 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 DU145 CM inhibited proliferation of cells. As a result, as shown in FIG. 2, when DU145 CM was treated with E4P, an AMF active inhibitor, or AMF antibody was previously removed using AMF antibody (IP-CM), leukemia caused by DU145 CM. It was confirmed that the antiproliferative effect of (HL60) cells was restored. This confirmed that AMF in DU145 CM has the effect of inhibiting the proliferation of certain cancer cells.

Example  4. Origin of DU145 AMF  Gene structure analysis

DU145 AMF cDNA was amplified by reverse transcription and PCR using DU145 RNA and cloned into pCold DNA1 vector to construct pDU145-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. DU145-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, as shown in FIG. 4A, brain cancer (U-87 MG) cells in recombinant AMF-treated cells were transformed into small particles in comparison with the untreated group, and their death was observed under a microscope. When treated with (2, 10, 50 and 100 ng / ml) of recombinant AMF for 3 days, pancreatic cancer (AsPC-1), leukemia (HL60) and brain cancer (U-87 MG) cells were concentration-dependently compared to the untreated control group. It was confirmed that the proliferation of was suppressed and killed.

<110> DAEGU CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Anticancer composition comprising prostate cancer cell          line-derived autocrine motility factor as effective component <130> PN18184 <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 Ser 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 Glu Thr 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 Val Lys Gln Leu Ala Lys Lys Ile Glu Pro Glu         515 520 525 Leu Asp Gly Ser Ala Gln Val Thr Ser Gln Gln Ala Ser Ser Lys Gly     530 535 540 Leu Leu Asn Tyr Met 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 agcaatggtc ccgagagcac 60 cgctcagagc 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 ctacccagga gaccatcacg 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 ggtaaagcag 1560 ctggctaaga aaatagagcc tgagcttgat ggcagtgctc aagtgacctc tcaacaagct 1620 tcttcgaaag gactcttgaa ttacatgaag cagcagcgcg aggccagagt ccaataa 1677

Claims (8)

A pharmaceutical composition for anticancer cancer against at least one carcinoma selected from the group consisting of pancreatic cancer, leukemia and brain cancer, which contains AMF (autocrine motility factor) derived from prostate cancer cell line DU145 consisting of the amino acid sequence of SEQ ID NO: 1. delete delete delete According to claim 1, wherein the prostate 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 brain cancer, characterized in that to inhibit the proliferation of cancer cells. According to claim 1, wherein the prostate 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 brain cancer, characterized in that inducing apoptosis of cancer cells. According to claim 1, wherein the pharmaceutical composition is pancreatic cancer, leukemia and brain 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. Claims 1 to 5 for at least one carcinoma selected from the group consisting of pancreatic cancer, leukemia and brain cancer comprising the step of administering the pharmaceutical composition for cancer according to any one of claims 5 to 7 in mammals other than humans Proliferation Inhibition or Apoptosis Enhancement of Cancer Cells.

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