JP4560612B2 - Decorin-derived peptide - Google Patents

Description

  The present invention relates to a peptide having an action of regulating cell proliferation.

Decorin is a kind of extracellular matrix protein and belongs to the SLRP (small leucine-rich proteoglycan) family. This family consists of 13 members. All SLRP proteins have a molecular weight of about 40 kDa and share a central domain that repeats leucine rich repeats consisting of 20-30 amino acid residues 6-10 times. SLRP family members have cysteine-rich domains at the N-terminus and C-terminus, and are further classified into four classes depending on the characteristic cysteine arrangement at the N-terminus. Decorin is classified into class I together with biglycan and asporin, and the N-terminal cysteine is arranged as CX ₃ CXCX ₆ C.

  Decorin is a protein discovered as the first member of the SLRP family and is 359 amino acids long. The gene sequence and amino acid sequence are shown in SEQ ID NOs: 3 and 4, respectively (GenBank accession numbers AF138300, NM_133507). The structure and function of decorin are being analyzed, such as collagen, epidermal growth factor (EGF) receptor, insulin-like growth factor-I (IGF-I) receptor, transforming growth factor-β (TGF-β), etc. In addition to being involved in the formation and stabilization of collagen fibrils, it is known to regulate cell proliferation, differentiation, and apoptosis. Among these various physiological activities, the cell growth inhibitory ability has attracted attention, and attempts have been made to use decorin gene for cancer gene therapy [Reed, CC, et al. Oncogene 24, 1104-1110]. (2005)]. Recently, the Iozzo group has proposed a mechanism by which decorin promotes EGF receptor degradation [Zhu, J.X., et al. J Biol Chem 280, 32468-32479 (2005)].

However, so far, little is known about the function and role of the N-terminal CX ₃ CXCX ₆ C structure conserved in SLRP family class I, including decorin.

Reed, C.C., et al. Oncogene 24, 1104-1110 (2005) Zhu, J.X., et al. J Biol Chem 280, 32468-32479 (2005) Scott, P.G., et al. Proc Natl Acad Sci 101, 15633-15638 (2004) Schonherr, E., et al. J Biol Chem 280, 15767-15772 (2005)

  The present inventors synthesized a partial peptide of a region conserved in SLRP family class I containing decorin and examined its effect on cell proliferation. Surprisingly, the proliferation was suppressed depending on the cell type. It has been found that there are cases where it has an effect of promoting proliferation and an effect of promoting proliferation.

The present invention has the following formula:
Cys Pro X1 Arg Cys Gln Cys His Leu Arg Val Val Gln Cys (SEQ ID NO: 1)
[In the formula, X1 is Phe or Tyr, and the first Cys and the seventh Cys, and the fifth Cys and the 14th Cys are disulfide bonded, respectively]
Or the following formula:
Cys X2 X3 Pro Gly His X4 X5 X6 Lys Ala Ser Tyr Ser Gly Val Cys (SEQ ID NO: 2)
[Wherein X ₂ is Pro or Arg, X3 is Pro or Ala, X4 is Asn or Pro, X5 is Thr or Ser, X6 is Lys or Arg, and the first Cys and The 16th Cys is disulfide bonded]
A human cancer cell growth inhibitor and a fibroblast growth promoter containing a peptide represented by the formula:

Particularly preferably, the human cancer cell proliferation inhibitor and the fibroblast proliferation promoter of the present invention are:
Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val Val Gln Cys (SEQ ID NO: 5),
Cys Pro Pro Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Cys (SEQ ID NO: 6)
It has any amino acid sequence.

  The human cancer cell proliferation inhibitor and fibroblast proliferation promoter of the present invention suppresses the proliferation of cancer cells such as T.Tn, TE2 derived from esophageal cancer and MKN74 derived from gastric cancer, while fibroblasts Rather, it has the effect of promoting the growth of NIH3T3. Therefore, the peptide according to the present invention is predicted to suppress cancer cell growth with selectivity, and is considered useful for the treatment of cancer and neurodegenerative diseases. It is also considered useful for regenerative medicine because of the low risk of cancer cell growth.

The peptide according to the present invention has the following formula:
Cys Pro X1 Arg Cys Gln Cys His Leu Arg Val Val Gln Cys (SEQ ID NO: 1)
[In the formula, X1 is Phe or Tyr, and the first Cys and the seventh Cys, and the fifth Cys and the 14th Cys are disulfide bonded, respectively]
Or the following formula:
Cys X2 X3 Pro Gly His X4 X5 X6 Lys Ala Ser Tyr Ser Gly Val Cys (SEQ ID NO: 2)
It has the amino acid sequence of

  In the present invention, each amino acid residue of these peptides may be conservatively substituted except for a cysteine residue involved in a disulfide bond. A conservative substitution is one in which an amino acid is replaced with another amino acid having similar properties, so that one skilled in the art of peptide chemistry would expect that the secondary structure and hydropathic properties of the polypeptide will not change substantially. Refers to substitution. The following groups of amino acids that are conservative substitutions for each other are generally known: (1) glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine; (2) alanine, valine, leucine, isoleucine, proline. , Phenylalanine, methionine and tryptophan; (3) glycine, alanine, serine, threonine and methionine; (4) leucine, isoleucine and valine; (5) glutamine and asparagine; (6) glutamic acid and aspartic acid; (7) arginine, lysine And (8) phenylalanine, tryptophan and tyrosine.

  Furthermore, the peptide according to the present invention may further contain an additional sequence in addition to the above amino acid sequence. A person skilled in the art can add any amount of sequence to the N-terminal side and C-terminal side in order to achieve the desired effect as a human cancer cell growth inhibitor or fibroblast growth promoter. Of course you can understand. Therefore, for example, on the N-terminal side and / or C-terminal side of the peptide according to the present invention, an amino acid sequence that facilitates formulation, an amino acid sequence that is convenient for expressing a peptide as a fusion protein, An amino acid sequence that is convenient for purification may be added. The peptide according to the present invention may be a chemically modified derivative, or a polymer or sugar chain may be added thereto. Examples of derivatives include, for example, polyethylene glycol derivatives and derivatives in which amino acids are acetylated, guanidylated, carbamylated or biotinylated.

  The peptide according to the present invention is produced by a general chemical synthesis method, an enzymatic degradation method of a protein molecule, a gene recombination technique using a host transformed so as to express a base sequence encoding a target amino acid sequence, and the like. be able to.

  When the target peptide is produced by a chemical synthesis method, it can be produced by a method well known in ordinary peptide chemistry. For example, it can be synthesized by a solid phase synthesis method using a peptide synthesizer. Can do. The crude peptide thus obtained can be purified by purification methods commonly used in protein chemistry, such as salting out, ultrafiltration, reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. Can be purified by.

  When a peptide is produced by gene recombination technology, for example, a synthesized or cloned DNA fragment encoding the target amino acid sequence is incorporated into an appropriate expression vector. Next, a desired peptide can be obtained by transforming microorganisms or animal cells using this expression vector and culturing the obtained transformant. As expression vectors that can be used, plasmids, viral vectors, and the like well known in the art can be used.

  Methods for transforming host cells using expression vectors in this peptide production technique include methods well known in the art, such as calcium chloride method, calcium phosphate coprecipitation method, DEAE dextran method, lipofectin method, electroporation method. Etc. can be used.

  The peptide according to the present invention can be administered, for example, orally or by a transdermal route such as intravenous administration or subcutaneous administration depending on the usage form. Examples of the dosage form include tablets, granules, soft capsules, hard capsules, liquids, oils, and emulsifiers.

  The human cancer cell growth inhibitor and fibroblast growth promoter containing the peptide of the present invention promotes or suppresses the proliferation of cells depending on the type of cells, so that the diseases caused by abnormal cell proliferation such as cancer and neurodegenerative diseases Useful in therapy and regenerative medicine. The human cancer cell growth inhibitor and fibroblast growth promoter of the present invention can be formulated by methods known to those skilled in the art. For example, a pharmaceutically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It can be formulated by mixing in a unit dosage form generally required for pharmaceutical practice, in combination with a binder, etc. as appropriate.

  For oral administration, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries are prepared by mixing the peptides of the present invention or derivatives thereof with pharmaceutically acceptable carriers well known in the art. , And can be formulated as a suspension or the like. As the carrier, those conventionally known in the art can be widely used. For example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like. Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dry starch, sodium alginate, agar powder, laminaran powder, carbonic acid Disintegrating agents such as sodium hydride, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegrating inhibitors such as sucrose, stear cocoa butter, hydrogenated oil; La Absorption promoters such as sodium rilsulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; abundance of purified talc, stearate, boric acid powder, polyethylene glycol, etc. An agent or the like can be used. Furthermore, the tablet can be a tablet with a normal coating, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary.

  For parenteral administration, the extract or compound of the present invention or a salt thereof can be formulated according to conventional pharmaceutical practice using pharmaceutically acceptable vehicles well known in the art.

  Water-soluble vehicles for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. An agent such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactant such as polysorbate 80 (TM), HCO-50 may be used in combination.

  Oily vehicles include sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection is usually filled into a suitable ampoule.

  Suitable administration routes of the human cancer cell growth inhibitor and fibroblast growth promoter of the present invention are not limited, but are oral, rectal, transmucosal, or enteral administration, or intramuscular, subcutaneous, intramedullary, Includes intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection. The administration route and administration method can be appropriately selected depending on the age and symptoms of the patient.

  The human cancer cell growth inhibitor and fibroblast growth promoter of the present invention can be administered, for example, in the range of 0.1 μg to 10 mg per kg of body weight at a time. The dose and interval of administration of the human cancer cell growth inhibitor and fibroblast growth promoter of the present invention to individual patients are determined by a doctor in consideration of the patient's weight, symptoms, administration route, and the like.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

The cells include ras-NIH3T3 (NIH3T3 cells transformed with Ha-ras), decorin / ras-NIH3T3 (cells overexpressing the decorin gene in NIH3T3 cells transformed with Ha-ras), coup / ras-NIH3T3 (Cells overexpressing coup gene in NIH3T3 cells transformed with Ha-ras), T.Tn (esophageal cancer cells), TE-2 (esophageal cancer cells), ECGI-10 (esophageal cancer cells), MKN74 ( Gastric cancer cells), PC6 (lung cancer cells) and A431 (epidermoid carcinoma) were used. Cells were maintained in DMEM with serum at 37 ° C. in a CO ₂ incubator.

  The peptide shown in FIG. 1 was synthesized. In the figure, cysteines cross-linked by S-S bonds are connected by lines. N1 and N2 contain two bridges, and eh, em, E1, and E2 contain one bridge. All peptides were synthesized with the amino terminus biotinylated and the carboxy terminus amidated.

  Each peptide was dissolved in a culture solution (serum-free) and added to cultured cells, and the effect on cell growth was examined by MTT assay. The MTT assay was performed by the method of Hiwasa et al. (Hiwasa et al., FEBS Lett. 552, 177-183, 2003) according to the original method (Mosmann, T. J. Immunol. Methods 65, 55-63, 1983). The procedure is shown below.

The peptide was used after being dissolved in a culture solution (serum-free) at 100 times the maximum concentration. MTT solution (manufactured by Sigma) was dissolved in phosphate buffer (PBS) at 5 mg / ml, sterilized by filtration, and stored at -20 ° C. The STOP solution was 0.04 N HCl in isopropanol.
1. Place 50 μl of serum-free medium in rows 2-11 of a 96-well plate. For 12, add 100 μl.
2. Column 1 contains 98 μl of serum-free medium.
3. Add 2 μl of each drug to column 1.
4. Dilute twice in the 1 → 9 column. Store in a CO ₂ incubator.
5. Peel off the cells with trypsin solution, suspend in a medium containing twice the concentration of serum, and measure with a Coulter counter to calculate the cell concentration (cells / ml).
6. Dilute the cells with medium containing doubled serum to adjust to 1 x 10 ⁵ cells / ml or 5 x 10 ⁴ cells / ml. Prepare at least 6 ml per plate.
7. Add 50 μl of cell suspension to the 11 → 1 well. Reservoir varies from plate to plate.
8. Mix gently.
9. Place in a CO ₂ incubator and incubate for 3-4 days.
10. Add 10 μl of MTT (5 mg / ml in PBS) to column 12 → 1.
11. Incubate for 4 hours in a CO ₂ incubator at 37 ° C.
12. Add 150 μl of STOP solution (0.04 N HCl in isopropanol) and mix well.
13. Wrap in wrap and leave at room temperature for at least 2 hours.
14. Measure with a reader. Measurement wavelength 570 nm, control wavelength 655 nm.
15. Calculate the% of each concentration (average of 2 measurements) with the 10th and 11th columns as 100% control and the 12th column as 0% control.

The results of the MTT assay are shown in Fig. 2-18.
Figure 2: Peptides N1 and N2 promoted proliferation of ras-NIH3T3 cells (mouse fibroblasts). It was promoted to about 1.5 times in a serum concentration of 1% and a culture period of 3 days.

  Figure 3: Peptides N1 and N2 promoted proliferation of ras-NIH3T3 cells (mouse fibroblasts). The serum concentration was promoted to about 3 times in 0.5% and culture period of 4 days.

  Figure 4: Peptides N1 and N2 promoted proliferation of decorin-introduced ras-NIH3T3 cells (mouse fibroblasts). The serum concentration was promoted to about 3 times in 0.5% and culture period of 4 days.

  Figure 5: Peptides N1 and N2 promoted proliferation of coup-TF1-introduced ras-NIH3T3 cells (mouse fibroblasts). The serum concentration was increased to 0.5 times and about 2.6 times in 4 days of culture.

  Figure 6: Peptides N1 and N2 inhibited the growth of T.Tn cells (human esophageal cancer cells). Serum concentration was suppressed to 1% and 40% after 3 days of culture.

  Figure 7: Peptides N1 and N2 inhibited the growth of TE-2 cells (human esophageal cancer cells). Serum concentration was suppressed to 1% and 20% after 3 days of culture.

  Figure 8: Peptides N1 and N2 inhibited the growth of ECGI-10 cells (human esophageal cancer cells). Serum concentration was suppressed to 1% and about 30% after 3 days of culture.

  Figure 9: Peptides N1 and N2 inhibited the growth of MKN74 cells (human gastric cancer cells). Serum concentration was suppressed to 1% and 40% after 3 days of culture.

  Figure 10: Peptides N1 and N2 slightly inhibited the growth of PC6 cells (human lung cancer cells). Serum concentration was suppressed to 1% and about 80% after 3 days of culture.

  Figure 11: Peptides N1 and N2 slightly inhibited the growth of A431 cells (human uterine cancer cells). Serum concentration was suppressed to 1% and about 80% after 3 days of culture.

  Figure 12: Peptides eh and em slightly promoted proliferation of ras-NIH3T3 cells (mouse fibroblasts). E1 was somewhat inhibited.

  Figure 13: Peptides eh and em slightly promoted proliferation of T.Tn cells (human esophageal cancer cells). E1 was somewhat inhibited.

  FIG. 14: Peptide E1 inhibited the proliferation of TE-2 cells (human esophageal cancer cells). eh and em promoted somewhat.

  FIG. 15: Peptide E1 slightly inhibited the growth of ECGI-10 cells (human esophageal cancer cells). em promoted somewhat.

  Figure 16: Peptide E1 slightly inhibited the growth of MKN74 cells (human gastric cancer cells).

  FIG. 17: Peptide em slightly inhibited the growth of PC6 cells (human lung cancer cells). eh and E1 had no effect.

  Figure 18: Peptide E1 inhibited the growth of A431 cells (human uterine cancer cells). eh and em had no effect.

  From the above results, it was revealed that peptides N1 and N2 have the action of suppressing the growth of cancer cells and promoting the growth of fibroblasts. Peptides eh and em had the same effect as N1 and N2, but their actions tended to be weak.

  The human cancer cell proliferation inhibitor and fibroblast proliferation promoter of the present invention are useful in the treatment of diseases (cancer, neurodegenerative diseases, etc.) caused by abnormal cell proliferation, and regenerative medicine.

FIG. 1 shows the structure of the peptide used in the examples. FIG. 2 shows the effect of the peptides N1 and N2 according to the present invention on the proliferation of ras-NIH3T3 cells. FIG. 3 shows the effect of peptides N1 and N2 according to the present invention on the growth of ras-NIH3T3 cells. FIG. 4 shows the effect of peptides N1 and N2 according to the present invention on the proliferation of decorin-introduced ras-NIH3T3 cells. FIG. 5 shows the effects of the peptides N1 and N2 according to the present invention on the proliferation of coup-TF1-introduced ras-NIH3T3 cells. FIG. 6 shows the influence of peptides N1 and N2 according to the present invention on the proliferation of T.Tn cells. FIG. 7 shows the effect of peptides N1 and N2 according to the present invention on the proliferation of TE-2 cells. FIG. 8 shows the influence of peptides N1 and N2 according to the present invention on the proliferation of ECGI-10 cells. FIG. 9 shows the influence of the peptide according to the present invention on the proliferation of MKN74 cells. FIG. 10 shows the effect of peptides N1 and N2 according to the present invention on the proliferation of PC6 cells. FIG. 11 shows the effect of peptides N1 and N2 according to the present invention on the growth of A431 cells. FIG. 12 shows the effect of the peptides eh and em according to the present invention on the growth of ras-NIH3T3 cells. FIG. 13 shows the effect of the peptides eh and em according to the present invention on the proliferation of T.Tn cells. FIG. 14 shows the effect of peptides eh and em according to the present invention on the proliferation of TE-2 cells. FIG. 15 shows the influence of peptides eh and em according to the present invention on the proliferation of ECGI-10 cells. FIG. 16 shows the influence of the peptides eh and em according to the present invention on the proliferation of MKN74 cells. FIG. 17 shows the effect of the peptides eh and em according to the present invention on the proliferation of PC6 cells. FIG. 18 shows the effects of the peptides eh and em according to the present invention on the proliferation of A431 cells.

Claims (2)

The following formula:
Cys Pro X1 Arg Cys Gln Cys His Leu Arg Val Val Gln Cys (SEQ ID NO: 1)
[In the formula, X1 is Phe or Tyr, and the first Cys and the seventh Cys, and the fifth Cys and the 14th Cys are disulfide bonded, respectively]
The human cancer cell growth inhibitor which contains the peptide represented by this as an active ingredient. The following formula:
Cys Pro X1 Arg Cys Gln Cys His Leu Arg Val Val Gln Cys (SEQ ID NO: 1)
[In the formula, X1 is Phe or Tyr, and the first Cys and the seventh Cys, and the fifth Cys and the 14th Cys are disulfide bonded, respectively]
Or the following formula:
Cys X2 X3 Pro Gly His X4 X5 X6 Lys Ala Ser Tyr Ser Gly Val Cys (SEQ ID NO: 2)
[Wherein X2 is Pro or Arg, X3 is Pro or Ala, X4 is Asn or Pro, X5 is Thr or Ser, X6 is Lys or Arg, and the first Cys and 16 The second Cys is disulfide bonded]
A fibroblast growth promoter containing a peptide represented by the formula:

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