KR101350868B1 - A pharmaceutical composition for inhibiting angiogenesis comprising HSP27 fragment and a method of screening an active material for inhibiting angiogenesis - Google Patents

Abstract

The present invention provides an HSP27 fragment (Heat Shock Protein 27 fragment) having a N-terminal amino acid sequence of HSP27 deleted or a nucleic acid encoding a fragment thereof. To provide.
The present invention also provides a method of inhibiting growth of vascular endothelial cells in vitro using the HSP27 fragment.
In addition, the present invention provides a method for screening an angiogenesis inhibitor or an active substance for cancer treatment using the HSP27 fragment.

Description

A pharmaceutical composition for inhibiting angiogenesis comprising HSP27 fragment and a method of screening an active material for inhibiting angiogenesis

The present invention relates to a method for screening an angiogenesis-inhibiting pharmaceutical composition and an angiogenesis-inhibiting active substance, and more specifically, angiogenesis, including a heat shock protein fragment found to inhibit vascular endothelial cell growth. A pharmaceutical composition for inhibiting cancer, a pharmaceutical composition for treating cancer, a method for screening an angiogenesis inhibitor by selecting a substance that increases the action of the heat shock protein fragment, and a cancer treatment by selecting a substance for increasing the content of the heat shock protein. The present invention relates to a method for screening an active substance for use.

Angiogenesis is a phenomenon in which endothelial cells of existing blood vessels decompose, migrate, divide, and differentiate extracellular matrix to form new capillaries, except in special cases such as growth, reproduction, and wound healing. I never do that. However, the growth and metastasis of malignant tumors, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis, and chronic inflammation Excessive angiogenesis has been reported in such diseases (Non Patent Literature 1), and for this reason, studies have been conducted to treat angiogenesis-related diseases, particularly malignant tumors, using angiogenesis inhibitors (Patent Document 1). .

Angiogenesis requires a complex series of processes such as growth, migration, differentiation, and capillary formation of vascular endothelial cells, and many angiogenesis and angiogenesis inhibitors have been found to be involved in this process. Angiogenesis inhibitors are activated against the activity of angiogenesis promoters required for angiogenesis. Angiogenesis inhibitors that are naturally present in the body are less toxic and can be used to inhibit pathological angiogenesis.

Vascular endothelial growth factor (VEGF) is a representative protein that regulates angiogenesis. VEGF regulates new angiogenesis from differentiation of endothelial precursors (angiocytes) in situ and is expressed in embryonic tissues, macrophages, and proliferating epithelial keratinocytes during wound healing and may be responsible for tissue edema associated with inflammation. Can be. In situ hybridization studies have demonstrated that VEGF is highly expressed in many human tumor lines, including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasia, and HIV-associated Kaposi's sarcoma (Patent Document 1).

VEGF binds to VEGF receptors 1, 2, or 3 (VEGFR1, VEGFR2, VEGFR3) present in vascular endothelial cells to induce tyrosine phosphorylation of VEGF receptors (VEGFR) resulting in activation of vascular endothelial cells. This will have a significant impact on. Phosphorylation of VEGFR2 is a receptor that plays the most important role in neovascular signaling. VEGF is upregulated in malignant tumor cells involved in angiogenesis and its receptor is upregulated in tumor infiltrating vascular endothelial cells, but expression of VEGF and its receptor is low in normal cells not associated with angiogenesis. Thus, these normal cells block the interaction between VEGF and its receptors, thereby inhibiting angiogenesis and thus no tumor growth.

High levels of VEGFR2 are expressed by endothelial cells infiltrating glioma and are specifically upregulated by VEGF produced by human glioblastomas. Since VEGFR2 transcripts are rarely detected in normal brain endothelial cells, expression of high levels of VEGFR2 in glioblastoma-associated endothelial cells (GAEC) indicates that receptor activity is induced during tumor formation.

Therefore, studies are being actively conducted to suppress tumor growth by inhibiting angiogenesis by inhibiting the activity of VEGF expressed in tumor growth sites. Among the drugs targeting VEGF, the most representative clinically used drug is Avastin, which corresponds to VEGF neutralizing antibody and received FDA approval as an anti-tumor agent.

Meanwhile, the low molecular heat shock protein 27 (hereinafter referred to as "HSP27") forms clusters by itself for external environmental factors such as free radicals, heat, and toxins. It is a low molecular weight protein with Chaperon (Charperon) function that protects against stress (NCBI Gene Bank Accession Number: NP_001531.1). Such HSP27 is known to bind to a protein that induces death of cancer cells, thereby increasing the cancer cell's resistance to radiation and anticancer agents, and the like (Non-Patent Document 2). However, there is no known correlation between HSP27 and HSP27 fragments in inhibiting VEGF function, angiogenesis, or cancer treatment.

1. Korean Patent Publication 2008-0109417

Cameliet and Jain, Nature, 407: 249, 2000 2.Oncogene, 2005 26; 24 (23): 3715-25

Therefore, the present inventors studied to develop an effective substance for inhibiting angiogenesis, HSP27 inhibits the activation of VEGFR2 by VEGF, which inhibits the growth of vascular endothelial cells, and this effect is the N-terminus of HSP27. It was first discovered that the HSP27 fragment of the deletion amino acid sequence was further increased, and based on this, a method for screening an angiogenesis inhibiting composition and an angiogenesis inhibiting active material was developed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for inhibiting angiogenesis.

Another object of the present invention to provide a pharmaceutical composition for treating cancer.

Another object of the present invention to provide a method for inhibiting the growth of vascular endothelial cells in vitro.

Still another object of the present invention is to provide a method for screening an angiogenesis inhibitor.

Still another object of the present invention is to provide a method for screening an active substance for cancer treatment.

In order to achieve the above object, an aspect of the present invention provides a pharmaceutical composition for inhibiting angiogenesis comprising an HSP27 fragment in which N-terminal amino acid sequences of Nos. 1 to 58 of HSP27 (Heat Shock Protein 27) are deleted.

In another aspect, the present invention provides a pharmaceutical composition for treating cancer comprising an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

Another aspect of the present invention provides a pharmaceutical composition for inhibiting angiogenesis comprising a nucleic acid encoding an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising a nucleic acid encoding an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

Another aspect of the present invention provides a method of inhibiting growth of vascular endothelial cells in vitro using an HSP27 fragment from which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

Another aspect of the present invention is

Treating the sample with a mixture of HSP27 or a mixture of VEGF with the HSP27 fragment from which N-terminal amino acid sequences of Nos. 1 to 58 of HSP27 are deleted;

Measuring the degree of binding of the HSP27 or HSP27 fragment with VEGF in the mixture; And

It provides a method for screening an angiogenesis-inhibiting active substance comprising the step of selecting a sample in which the degree of binding is increased compared to the control.

Another aspect of the present invention is

Treating the sample with a mixture of HSP27 or a mixture of VEGF with the HSP27 fragment from which N-terminal amino acid sequences of Nos. 1 to 58 of HSP27 are deleted;

Measuring the degree of binding of the HSP27 or HSP27 fragment with VEGF in the mixture; And

It provides a method for screening an active substance for cancer treatment comprising the step of selecting a sample in which the degree of binding is increased compared to the control.

Another aspect of the present invention is

Processing a sample on HSP27;

Measuring a degree of deletion of the N-terminal amino acid sequences of the first to the 58th HSP27; And

It provides a method for screening an angiogenesis-inhibiting active substance comprising the step of selecting a sample in which the degree of deletion is increased compared to the control.

Another aspect of the present invention is

Processing a sample on HSP27;

Measuring a degree of deletion of the N-terminal amino acid sequences of the first to the 58th HSP27; And

It provides a method for screening an active substance for cancer treatment comprising the step of selecting a sample in which the degree of deletion is increased compared to the control.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to in this specification are incorporated herein by reference in their entirety.

As used herein, "HSP27 N-terminal" refers to the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27.

"HSP27 fragment" refers to an amino acid sequence in which the N-terminal amino acid sequences of HSP27 1-58 are deleted.

Since HSP27 is an HSP found in humans and corresponds to HSP25 in mice, the term HSP27 should be interpreted to apply to HSP25 in mice. Therefore, in the present invention, it should be understood that the HSP27 can be applied to the HSP25 as it is when targeting the mouse.

The pharmaceutical composition for inhibiting angiogenesis or the pharmaceutical composition for cancer treatment provided by the present invention is characterized in that it comprises an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted. Phosphorylation of VEGFR2 is decreased, and phosphorylation of VEGFR2 is further reduced by HSP27 fragment from which the N-terminus of HSP27 (amino acids 1 to 58) is removed. It is based on the first finding that the growth of the anti-cancer effect is inhibited and the cancer metastasis is inhibited when expressed in cancer cells, and this effect is further enhanced by the HSP27 fragment.

The present inventors specifically express DNA sequences expressing HSP27 and the N-terminus of HSP27 (amino acid sequence of Nos. 1 to 58), and the HSP27 fragment (amino acid sequence of Nos. 59 to 203) from which the N-terminus of HSP27 is removed. Was prepared (FIG. 1). The vector was transfected into a COS-7 cell line to stably screen for cell lines expressing each of the above proteins. HSP27-expressing cell lines inducing HSP27 secretion were measured by intracellular HSP27 expression and extracellular HSP27 secretion. It was confirmed that the secretion of increased (Fig. 2).

When HSP27 tagged with FLAG was reacted with VEGF and immunoprecipitated with antibody against FLAG tag, the binding of HSP27 and VEGF was measured. As a result, the N-terminus of HSP27 (amino acid sequence Nos. 1 to 58) did not bind to VEGF. The binding of the HSP27 fragment (amino acid sequence of Nos. 59 to 203) with the N-terminus of HSP27 to VEGF was found to be higher than that of native HSP27 (wild type HSP27) and VEGF (Example 2, FIG. 3A). In addition, human vascular endothelial (HUVEC) cultures were treated with HSP27 or HSP27 fragments (amino acids 59 to 203) from which the N-terminus of HSP27 was removed, followed by VEGF treatment, followed by phsopho-VEGFR2 and VEGFR2 contents. As a result, the phospho-VEGFR2 content of the natural HSP27 treatment group was decreased compared to the control group (PPTLS administration), and the phospho-VEGFR2 content of the HSP27 fragment treatment group was significantly lower than that of the natural HSP27 treatment group. The content of appeared to be almost unchanged (Fig. 3b).

When VEGF was treated in human vascular endothelial cell (HUVEC) cultures alone or after treatment with native HSP27 or HSP27 fragments, VEGF alone increased vascular endothelial cell growth rate compared to the control group. The VEGF combination treatment group showed decreased vascular endothelial cell growth rate compared to the VEGF alone treatment group, and the HSP27 fragment and VEGF combination treatment group significantly reduced vascular endothelial cell growth rate compared to the combination treatment group of HSP27 and VEGF. (FIG. 4).

When the B16F10 melanoma cell line was administered to mice to metastasize to the lung, the B16F10 melanoma cell line that was stably expressed HSP27 was found to inhibit metastasis to the lung compared to the control group, and stably express the HSP27 fragment in the B16F10 melanoma cell line. It was confirmed that the B16F10 melanoma cell line significantly reduced metastasis to the lung compared to the HSP27 expressing B16F10 melanoma cell line-administered group (FIG. 5).

Therefore, HSP27 has a cancer therapeutic effect such as inhibiting the growth of vascular endothelial cells and inhibiting cancer metastasis, and this effect is further increased in the case of HSP27 fragments without the N-terminus, and the inhibitory effect is HSP27. And it can be seen that the HSP27 fragment is due to the action of inhibiting the phosphorylation of VEGFR2 by binding to VEGF. Therefore, the substance that enhances the binding of HSP27 or HSP27 fragment to VEGF or the N-terminus of HSP27 is effective in inhibiting angiogenesis and in treating malignant tumors and inhibiting cancer metastasis.

Accordingly, in one aspect, the present invention provides a pharmaceutical composition for inhibiting angiogenesis comprising an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

The angiogenesis may be accompanied by tumor growth and metastasis, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis, or chronic inflammation, but is not limited thereto, and angiogenesis causes or progresses the disease. May be accompanied by any disease.

In another aspect, the present invention provides a pharmaceutical composition for treating cancer comprising an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

The cancer may be colorectal cancer, pancreatic cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, bone metastasis cancer of the prostate cancer, ovarian cancer, or hematologic cancer, but is not limited thereto, and any cancer in which angiogenesis is involved. It includes. In addition, the cancer treatment is also effective in suppressing metastasis of cancer.

The HSP27 fragment contained in the pharmaceutical composition for inhibiting angiogenesis and the pharmaceutical composition for cancer treatment may be obtained from HSP27, or only the fragment may be synthesized using a chemical synthesis method using any method known in the art, or the fragment may be synthesized. A recombinant expression vector encoding may be introduced into a suitable host to culture the transfectant and then isolated from it.

The amino acid sequence and nucleic acid sequence of HSP27 are listed in NCBI Gene Bank Accession Numbers: NP_001531.1 and NM_001540.3. The HSP27 is to include HSP27 of natural origin, HSP27 of recombinant origin, or variants of these proteins. The HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted may be expressed in a vector with the N-terminal amino acid sequence deleted, or the intact HSP27 may be cleaved by enzyme treatment or the like. It can be prepared by any method known in the art. In addition, the HSP27 fragment is one comprising the HSP27 fragment obtained from the HSP27, HSP27 fragment of recombinant origin, or variants of these proteins.

The HSP27 is isolated from a source of natural origin using methods commonly known in the art, synthesized using a chemical synthesis method, or introduced into a suitable host by introducing a recombinant expression vector encoding HSP27 into a suitable host, followed by culturing the transfectant. Can be separated. In addition, the HSP27 fragment may be obtained from the HSP27, synthesized using a chemical synthesis method, or introduced into a suitable host by introducing a recombinant expression vector encoding the HSP27 fragment into a suitable host and then cultured from the transfectant.

Isolation and purification of HSP27 containing culture or cell extracts can be carried out according to several known methods. Examples of such separation and purification methods include solubility such as salt precipitation and solvent precipitation, methods using molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, ion-exchange columns The method using the difference of charge, such as chromatography, the method using the difference of hydrophilicity, such as reverse phase high performance liquid chromatography, the method using the difference of isoelectric points, such as isoelectric focusing electrophoresis, etc. can be illustrated.

One or more amino acids in the substitutional variant of the amino acid sequence variant may be substituted with another amino acid sequence of similar polarity. Substitutions of amino acids may be substituted with other amino acids of the class to which they belong, which class of non-polar (hydrophobic) amino acids (e.g. alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, etc.). ), Polar neutral amino acids (e.g. glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc.), positively charged (basic) amino acids (e.g. arginine, lysine, histidine, etc.), negatively charged (acidic) amino acids (e.g. aspartic acid) , Glutamic acid, and the like).

Still other amino acid sequence variants can be exemplified by deletions, insertions, or combinations of one or more amino acids.

Amino acid sequence variants induce DNA site coding mutations of nucleotides in DNA encoding HSP27 using conventional cassette mutagenesis or other techniques well known in the art to produce DNA encoding the variants followed by recombinant cell culture. It is prepared by expressing through. Such amino acid sequence variants typically exhibit biologically identical biological activities as the native form. Substitutions of amino acids are typically substitutions of a single base, but one or more substitutions are possible as long as they exhibit functionally identical biological activity, and insertions may typically consist of a sequence of about 1 to 20 amino acids, but larger insertions It may be possible. Deletion typically ranges from about 1 to 30 residues, but in some cases larger deletions may also be possible, such as one of the domains may be deleted. Variants are typically functional equivalents that exhibit the same biological activity as the native form, but variants may be selected if desired that modify the properties of their proteins.

In addition, the HSP27 and HSP27 fragments include post-translational modifications including myristylization, phosphorylation, glycosylation, proteolytic cleavage, and the like.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting angiogenesis comprising a nucleic acid encoding an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

The angiogenesis may be accompanied by tumor growth and metastasis, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis, or chronic inflammation, but is not limited thereto, and the angiogenesis may cause disease or It may be accompanied by any disease that progresses.

In another aspect, the present invention provides a pharmaceutical composition for treating cancer comprising a nucleic acid encoding an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted.

The cancer may be colorectal cancer, pancreatic cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, bone metastasis cancer of the prostate cancer, ovarian cancer, or hematologic cancer, but is not limited thereto, and any cancer in which angiogenesis is involved. It includes. In addition, the cancer treatment is also effective in suppressing metastasis of cancer.

The nucleic acid included in the pharmaceutical composition of the present invention is a sequence encoding the HSP27 fragment from which the N-terminal amino acid sequence of HSP27 1st to 58th is deleted, and amplifies a nucleic acid encoding HSP27 isolated from nature by PCR. The product can be prepared by any method known in the art, such as by inserting it into a specific vector as it is or by treating with an enzyme, or by inserting a fragment into a specific vector by treating the nucleic acid itself with an enzyme. In addition, the HSP27 fragment may be artificially synthesized and / or modified.

Such nucleic acid sequences may be single- or double-stranded, and may be DNA molecules (genome, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules corresponding to DNA molecules comprising introns and mRNA molecules not comprising introns. It may also include additional coding sequences or non-coding sequences.

The nucleic acid encoding the HSP27 fragment may be modified by substitution, deletion or insertion of one or more nucleic acid bases, and the protein expressed by such modification should not include a significant change in its biological functionality. Such modifications include modifications to heterologous homologous genes.

The nucleic acid encoding the HSP27 fragment may be provided as a recombinant expression vector operably linked to a vector expressing it. Gene expression vectors of HSP27 fragments include plasmids, phages, cosmids, viral vectors and the like. The vector may be self-replicating or integrated into the host DNA.

HSP27 fragment nucleic acid molecules can be combined with expression control sequences such as promoter / enhancer sequences and other sequences necessary for transcription, translation or processing. Regulatory sequences include tissue-specific regulatory and / or inducible sequences as well as direct constitutive expression of nucleotides. The design of the expression vector can be determined by factors such as the host cell to be transfected, the level of expression desired, and the like.

The expression vector expressing the HSP27 fragment is preferably a viral vector and includes, for example, replication-defective retroviruses, adenoviruses, adenovirus-associated viruses, and the like. Viral vectors must meet the following criteria: (1) be able to infect the cells of interest, and thus a viral vector with an appropriate host range must be selected, and (2) the genes delivered will be preserved in the cells for an appropriate period of time. Must be able to be expressed and expressed, and (3) the vector must be safe for the host. Other viral vectors that can be used for gene delivery intracellularly include murine leukemia virus (MLV), JC, SV40. Polyoma, Epstein-Barr virus, papilloma virus, vaccinia, poliovirus, herpes virus, Sindbis virus, lenti virus, other human and animal viruses.

The pharmaceutical composition comprising the HSP27 fragment of the present invention or a nucleic acid encoding the fragment thereof may be formulated and administered as a topical preparation or injection according to a conventional method in the pharmaceutical art.

Such topical formulations or injections may be prepared according to conventional methods for preparing injections known in the art.

Formulations for topical administration include liquid or semi-liquid formulations suitable for penetration through the skin to the area in need of treatment. Examples of liquid formulations include, but are not limited to, topical solutions. Examples of semi-liquid formulations include, but are not limited to, linings, lotions, creams, ointments, or pastes, gels, emugels, and the like. Such pharmaceutical ingredients are those commonly used and are generally recognized by those of ordinary skill in the art of pharmaceutical preparations.

Topical solutions of the present invention may be prepared as aqueous or oily solutions or suspensions. Such formulations may be prepared by dissolving the pharmaceutical compound in a suitable aqueous solution of a bactericidal agent and / or fungicide and / or other suitable preservative, preferably including a surfactant. Suitable solvents for the preparation of oily solutions include glycerol, diluted alcohols, and propylene glycol. Optionally, L-methanol can be added to the topical solution.

Lotions and liniments according to the present invention include those suitable for application to the skin containing a sterile aqueous solution and optionally a bactericide. It may also include agents that promote drying and cooling of the skin, such as alcohol or acetone, and / or humectants, such as glycerol or oils such as castor oil or peanut oil.

Creams, ointments, or pastes are semisolid preparations. Such semi-solid formulations may be prepared by mixing pharmaceutically acceptable salts in finely divided or powdered form, either alone or with an aid of a suitable machine together with an oily or non-fatty base, or in solution in an aqueous or non-aqueous fluid. It can be prepared as a suspension. The base may contain a hydrocarbon. Examples of hydrocarbons include hard, soft, or liquid paraffin, glycerol, beeswax, metal soaps, mucous, natural raw oils (such as almond oil, corn oil, peanut oil, or olive oil), wool fat, or derivatives thereof, and / or Fatty acids such as stearic acid or oleic acid. The formulation may also contain surfactants such as anionic, cationic, or nonionic surfactants. Examples of surfactants are sorbitan esters or polyoxyethylene derivatives thereof (eg polyoxyethylene fatty acid esters), and carboxymethylene derivatives thereof (eg carbopol). Suspending agents such as natural gums, cellulose derivative inorganics such as silicon silica, and other ingredients such as lanolin. In the case of ointments, polyethylene glycol 540, polyethylene glycol 3350, and propyl glycol can also be used in combination with pharmaceutical compounds.

Gel or emugel formulations of the invention include any gel forming reagent commonly used in pharmaceutical formulations. Examples of gel forming agents include cellulose derivatives such as methyl cellulose, hydroxyethyl cellulose, and carboxymethyl cellulose; Vinyl polymers such as polyvinyl alcohol, polyvinyl pyrrolidone; And carboxypoly-methylene derivatives such as carbopol. Still other gel forming agents that can be used in the present invention are pectin, gums such as gum arabic, tragacanta gum, alginate, carrageenate, agar, and gelatin. Preferred gel forming agent is carbopol. In addition, gel or emugel preparations may contain adjuvants commonly used in this type of preparation, such as preservatives, antioxidants, stabilizers, colorants, and flavoring agents.

Excipients and auxiliaries used in the aforementioned formulations and methods for the preparation of the formulations can be selected and prepared according to what is well known in the art (for example methods described in the latest edition of Remington's Pharmaceutical Science).

Dosage and timing of the pharmaceutical composition comprising the HSP27 fragment of the present invention will vary depending on the age, sex, condition, weight, route of administration, frequency of administration, and form of the subject. The daily dosage is about 0.01 ug / kg to 10 g / kg, preferably 0.01 mg / kg to 100 mg / kg.

An effective amount of a pharmaceutical composition comprising a nucleic acid encoding an HSP27 fragment of the present invention is easily taken into account in consideration of factors such as the size and weight of the subject, the extent of disease progression, age, health condition, sex, route of administration and local or systemic administration. You can decide. In general, an effective amount of nucleic acid encoding an HSP27 fragment of the invention comprises an intracellular concentration of about 1 nM to about 100 nM at or near the disease site. If necessary, it may be administered higher or lower than the concentration.

Nucleic acids encoding the HSP27 fragment of the invention can be administered to a subject either directly by injection or in combination with a delivery reagent, as such or as a recombinant plasmid or viral vector expressing them. For viral vectors, the amount of recombinant virus containing the viral vector ranges from 10 ³ to 10 ¹² pfu / kg. Suitable delivery reagents include lipofectin, lipofectamine, celfectin, macrocationic (eg polylysine) or liposomes. The pharmaceutical composition comprising a nucleic acid encoding the HSP27 fragment of the present invention can be delivered into cells using a gene gun, ultrasound or electric shock.

According to the present invention, since HSP27 fragment has an effect of effectively inhibiting the phosphorylation of VEGFR2 by binding to VEGF and inhibiting the growth of vascular endothelial cells, it can be used to inhibit the growth of vascular endothelial cells.

Accordingly, in another aspect, the present invention provides a method of inhibiting growth of vascular endothelial cells in vitro using the HSP27 fragment.

The HSP27 fragment may be an HSP27 fragment of HSP27 origin, a HSP27 fragment of recombinant origin, or a variant of these proteins as described above, and the HSP27 fragment of HSP27 origin, which is intended to inhibit growth inhibition of vascular endothelial cells, may be used. By adding HSP27 of origin, or variants of these proteins.

In addition, according to the present invention, since HSP27 or HSP27 fragment is effective in reducing angiogenesis or cancer suppression activity by binding to VEGF, by selecting a substance that increases the degree of binding of HSP2 or HSP27 fragment and VEGF among unknown substances Active substances for inhibiting angiogenesis or active substances for treating cancer can be selected.

Therefore, in another aspect, the present invention provides a method for screening an angiogenesis-inhibiting active substance based on the finding that the HSP27 or HSP27 fragment inhibits angiogenesis by binding to VEGF.

The method for screening the active material for angiogenesis inhibition

Treating the sample with a mixture of HSP27 or a mixture of VEGF with the HSP27 fragment from which N-terminal amino acid sequences of Nos. 1 to 58 of HSP27 are deleted;

Measuring the degree of binding of the HSP27 or HSP27 fragment with VEGF in the mixture; And

Selecting a sample in which the degree of binding is increased relative to the control.

In still another aspect, the present invention provides a method for screening an active agent for cancer treatment based on the finding that the HSP27 or HSP27 fragment inhibits angiogenesis by binding to VEGF.

The method of screening the active substance for cancer treatment

Treating the sample with a mixture of HSP27 or a mixture of VEGF with the HSP27 fragment from which N-terminal amino acid sequences of Nos. 1 to 58 of HSP27 are deleted;

Measuring the degree of binding of the HSP27 or HSP27 fragment with VEGF in the mixture; And

Selecting a sample in which the degree of binding is increased relative to the control.

The degree of binding of the HSP27 or HSP27 fragment and VEGF is determined by, for example, Western blot and immunoprecipitation, fluorescence resonance transfer (FRET, Fluorescence Resonance Energy Transfer), protein transduction, and reporter gene activity assay. Such a method may be carried out using a method known in the art.

In addition, according to the present invention, since HSP27 fragment has better activity to reduce angiogenesis or cancer suppression than HSP27, angiogenesis inhibition by selecting a substance that enhances deletion of the N-terminus of HSP27 among unknown substances The active substance or active substance for cancer treatment can be selected.

Accordingly, in another aspect, the present invention provides a method for screening an angiogenesis-inhibiting active substance based on the finding that a substance that enhances the N-terminal deletion of HSP27 inhibits angiogenesis.

The method for screening the active material for angiogenesis inhibition

Processing a sample on HSP27;

Measuring a degree of deletion of the N-terminal amino acid sequences of the first to the 58th HSP27; And

Selecting a sample in which the degree of deletion is increased relative to the control.

In still another aspect, the present invention provides a method for screening an active substance for cancer treatment based on the finding that the substance enhancing the N-terminal deletion of HSP27 exhibits cancer inhibitory activity.

The method of screening the active substance for cancer treatment

Processing a sample on HSP27;

Measuring a degree of deletion of the N-terminal amino acid sequences of the first to the 58th HSP27; And

Selecting a sample in which the degree of deletion is increased relative to the control.

The degree to which the N-terminal amino acid sequence of Nos. 1 to 58 of the HSP27 is deleted can be determined by measuring the content of the HSP27 fragment. The content of the HSP27 fragment can be determined, for example, by measuring the amount of mRNA of the HSP27 fragment by the Northern blot method or by measuring the expression level of the HSP27 protein using an antibody against the HSP27 fragment. It can carry out using a well-known method.

As described above, the nucleic acid encoding the HSP27 fragment or HSP27 fragment according to the present invention is not only effective in inhibiting angiogenesis, but also in the treatment of various diseases related to angiogenesis by such angiogenesis inhibitory effect. In addition, the nucleic acid encoding the HSP27 fragment or HSP27 fragment according to the present invention can be effectively used for cancer treatment by the angiogenesis inhibitory effect.

In addition, by measuring whether any sample increases the binding of HSP27 or HSP27 fragment with VEGF, the angiogenesis-promoting and cancer therapeutic actives can be screened. In addition, an angiogenesis-promoting active agent and an active agent for cancer treatment can be screened by measuring an increase in the degree of deletion of the N-terminal amino acid sequence of HSP27 1st to 58th. Thus, the technique according to the present invention is an advanced technique for discovering new drug targets by systematic studies of signal transduction and signal mapping between intracellular proteins.

1 shows HSP27, an N-terminal fragment of HSP27 (amino acid sequence of Nos. 1 to 58), and an HSP27 fragment (amino acid sequence of Nos. 59 to 203) from which the N-terminus of HSP27 is removed, wherein FLAG, Myc It is a schematic diagram of the vector which attached the tag of, His etc., and the amino acid sequence called PPTLS for inducing extracellular secretion of HSP27 or the said HSP27 fragment.
Figure 2 is a photograph showing the results confirmed by Western blotting the amount of intracellular expression and extracellular secretion according to the type of HSP27 vector when cultured COS7 cells to stably express the HSP27 or HSP27 fragment.
Figure 3a shows the reaction between each HSP27 or HSP27 fragment obtained from COS7 cells made to stably express the HSP27 or HSP27 fragment with VEGF and confirmed the degree of binding between the two by Western blotting for VEGF after immunoprecipitation with an antibody against FLAG Photograph taken of the result.
Figure 3b is the result of treating each HSP27 or HSP27 fragment obtained from COS7 cells made to express the HSP27 or HSP27 fragment to vascular endothelial cells (HUVEC) and confirmed the degree of phosphorylation of VEGFR2 by VEGF by Western blotting It is a photograph.
Figure 4 shows the growth rate using the MTT proliferation assay when each HSP27 or HSP27 fragment obtained from COS7 cells made to stably express the HSP27 or HSP27 fragments treated with vascular endothelial cells (HUVEC), HSP alone It is a graph showing the ratio of cell growth to the HSP and VEGF combination treatment group of the treatment group.
FIG. 5 shows the right side of a mouse injecting a B16F10 cell line stably expressing an HSP27 or HSP27 fragment into the mouse, and two weeks later, a mouse was autopsied to separate lungs and two representative lungs corresponding to each treatment group. The graph showing the results of counting and averaging the colony counts of the B16F10 melanoma cell lines generated in the lungs by treatment groups is shown on the left.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Experimental Method

1) Culture of cell line used:

Fibroblasts (COS-7) derived from monkey kidney and melanoma cells (B16F10) from mouse were treated with DMEM medium containing 10% fetal calf serum (FBS, GIBCO) at 37 ° C and 5% CO _2. It was cultured in the incubator of. Human vascular endothelial cells (HUVEC) were purchased from Lonza, Inc. and cultured in an incubator at 37 ° C. and 5% CO ₂ using EGM medium containing various growth factors required for vascular endothelial cells.

2) Protein analysis using electrophoresis and immune response:

In order to analyze the protein in the sample, PAGE (polyacrylamide gel electrophoresis) was first performed, followed by Western blotting. Cells to be tested were first suspended in lysate [120 mM NaCl, 40 mM Tris (pH 8.0), 0.1% NP40] to burst the cells, and then a certain amount of protein was separated by molecular weight through 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and analyzed using immunoblotting. In addition, VEGF protein and HSP27 are reacted with each other to measure the binding degree of the sample, and then immunoprecipitated with an antibody against the FLAG tag attached to the HSP27, followed by electrophoresis by the above method, and then immunoblotting for VEGF. Analyzed.

3) Construction of DNA vector expressing HSP27 or HSP27 fragment and acquisition of transformed cell line:

The cDNA encoding HSP27 was inserted into a vector to express the FLAG tag at the N-terminus and the c-Myc tag at the C-terminus. In addition, to induce secretion, cDNA encoding HSP27 or HSP27 fragment was inserted into the vector to express PPTLS peptide and FLAG tag at the N-terminus, c-Myc tag and His tag at the C-terminus. PPTLS peptide stands for preprotrypsin leader sequence and is known to induce extracellular secretion of protein and the amino acid sequence is 'SALLILALVGAAVA' (Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala).

All vectors designed to express HSP27 or HSP27 fragments carry Neomycin resistance genes. Vectors expressing HSP27 or HSP27 fragments were transfected into a COS-7 cell line or B16F10 melanoma cell line and, after 3 days, appropriately aliquoted cells to grow and form a single colony, containing 500 μg / ml of G418 for 2 weeks. The culture was carried out in a medium. Even after two weeks, the cells proliferating in the medium containing G418 are cells whose vectors are intercalated into the cell's chromosome and continuously express Neomycin resistance genes, and the cells are passaged several times when the genes encoding the HSP27 or HSP27 fragments are inserted together. Even in culture, the protein can be continuously expressed. Therefore, in order to confirm this, the colonies selected after 2 weeks were cultured in different culture vessels, and some of the cells were lysed to perform Western blotting. Among them, cell lines stably expressing HSP27 or HSP27 fragments were selected. The cell line of the comparative group was able to obtain a colony showing resistance to G418 by transfecting the vector without HSP27.

4) Proliferation assay:

Vascular endothelial cells were incubated in a 37 well CO ₂ incubator for 24 hours with 7000 cells per well in a 24 well cell culture dish. The cells were washed twice with PBS and changed to OPTI-MEM medium (GIBCO) with 1% fetal calf serum (FBS, GIBCO), followed by HSP27 (Stressgen, Canada) (1 ug / ml) or the transformation. The HSP27 fragment obtained from the COS-7 cell line was treated (1ug / ml). After 16 hours of treatment, VEGF (30 ng / ml) was added and 3 days later, MTT assay was performed as follows. After removing the medium and washing the cells twice with PBS buffer solution, 100 mg of 5 mg / ml Thiozolyl Blue Tetrazolium Bromide (MTT) solution was added to each well and reacted at 37 ° C for 4 hours. After removing the MTT solution, 200 ul 4 mM HCl solution was added to the cells, followed by shaking for 15 minutes in a shaker to measure absorbance at 590 nm with a control wavelength of 620 nm.

4) Animal model for measuring metastasis inhibition of tumor

We measured the production of HSP27 made to stably expressing mouse melanoma cell line B16F10 2X10 ⁵ gae by the injection into the tail vein of C57BL / 6 mice two weeks after the weight of the lung (g), a colony of the B16F10 cell line proliferation in the lung Numbers were counted to observe the extent to which the melanoma cell line metastasized to the lung.

Example 1 Construction of Transgenic Cell Lines Expressing HSP27 or HSP27 Fragments

As described above, a DNA vector expressing HSP27, an N-terminal protein of HSP27 (amino acid sequence of Nos. 1 to 58), and an HSP27 fragment (amino acid sequence of Nos. 59 to 203) from which the N-terminus of HSP27 was removed, were prepared. (FIG. 1), this vector was transfected into a COS-7 cell line to select cell lines stably expressing HSP27 or HSP27 fragment.

The COS-7 cell line stably expressing the HSP27 or HSP27 fragment thus obtained was seeded in OPTI-MEM (GIBCO) medium containing 1% fetal bovine serum (LONZA) for two days, and then the intracellular expression amount and cells of the HSP27 or HSP27 fragment. The amount of secretion out was measured by Western blotting. The western blot photograph thus obtained is shown in FIG. 2.

According to Figure 2, it was confirmed that the secretion of HSP27 or HSP27 fragment increases when the cell line expressing HSP27 induced by secretion by adding the PPTLS amino acid sequence to the N-terminus of the HSP27 or HSP27 fragment.

Example 2: Binding of HSP27 Fragment with VEGF and Inhibitory Effect of HSP27 Fragment on VEFGR2 Phosphorylation

10 ug / ml of the transformed COS-7 cell line-derived HSP27 or fragment thereof was reacted with 10 ug / ml of human vascular endothelial growth factor (VEGF), and then immunoprecipitated using FLAG antibody, and antibodies against FLAG and VEGF. Western blotting was performed. Thus, the photograph taken the result confirmed by Western blotting is shown in Figure 3a.

In addition, the human vascular endothelial cell line HUVEC was treated with the transformed COS-7 cell line-derived HSP27 fragment (1ug / ml) for 16 hours, and then treated with 30 ng / ml of human vascular endothelial growth factor (VEGF) for 5 minutes. Fractions were subjected to Western blotting using Phospho-VEGFR2 and VEGFR2 antibodies. Thus, the photograph taken the result confirmed by Western blotting is shown in Figure 3b.

Figure 3a is a photograph of the results confirmed by Western blotting the degree of binding of HSP27 or HSP27 fragment and VEGF when the HSP27 or HSP27 fragment reacted with VEGF.

Figure 3b is a photograph showing the results confirmed by Western blotting the degree of phosphorylation of VEGFR2 by VEGF when HSP27 or HSP27 fragment is treated in vascular endothelial cells (HUVEC).

According to Figure 3a, it was confirmed that the binding of VEGF and HSP27 is further increased when the N-terminal (amino acids 1 to 58) of HSP27 is removed. In contrast, the HSP27 fragment including only the N-terminus (amino acids 1 to 58) of HSP27 did not bind VEGF.

According to Figure 3b, it was confirmed that phosphorylation of VEGFR2 by VEGF was significantly reduced when HSP27 treatment, and further decreased when HSP27 fragment (amino acids 59 to 203) from which the N-terminus of HSP27 was removed It was confirmed.

Example 3: Confirmation of the inhibition of vascular endothelial cell growth of HSP27 fragment

Human vascular endothelial cell line HUVEC was cultured in a medium containing recombinant HSP27 or HSP27 or HSP27 fragments derived from the transformed COS-7 cell line. After 16 hours of incubation, 30 ng / ml of human vascular endothelial growth factor (VEGF) was added, and after 3 days, the degree of cell growth was measured by MTT assay. Cell growth for PPTLS treatment group, S-HSP27 (WT) treatment group, S-HSP27 (1-58) treatment group, S-HSP27 (59-203) treatment group, recombinant HSP27 treatment group alone and VEGF combination treatment group The degree was measured, and the result is shown graphically in FIG. 5 as the ratio of vascular endothelial growth change of the VEGF combination treatment group to the treatment group alone. The Y axis of the graph of FIG. 5 shows [+ VEGF treatment group cell growth degree] / [-VEGF treatment group cell growth degree].

According to Figure 4, the cells of the S-HSP27 (WT) and VEGF combination treatment group or HSP25 and VEGF combination treatment group was less than the vascular endothelial cell growth rate in the VEGF alone treatment group, and the S-HSP27 (59- 203) and the cells of the VEGF combination treatment group were less than the vascular endothelial cell growth rate in the S-HSP27 (WT) and VEGF combination treatment group or the HSP25 and VEGF combination treatment group. On the other hand, cells of the N-terminal S-HSP27 (1-58) and VEGF combination treatment group had little effect on the growth rate of VEGF-induced vascular endothelial cells.

Example 4: Confirmation of Inhibition of Cancer Metastasis

The DNA vector expressing the prepared HSP27 or HSP27 fragment (in amino acid sequence of Nos. 59 to 203) from which the N-terminus of HSP27 and HSP27 were secreted was transfected into a B16F10 melanoma cell line. Colonies were obtained by culturing for 2 weeks in a medium containing G418 to obtain colonies. After blotting some cells by colony, Western blotting was performed, and cell lines stably expressing HSP27 or HSP27 fragments were selected and used in animal experiments to observe cancer metastasis.

Experiments in which the B16F10 melanoma cell line is administered intravenously in mice have been used as an animal model to observe metastasis to the lung. Therefore, this animal model was used to determine whether secreted-induced HSP27 and HSP27 fragments affect cancer cell metastasis.

To this end, the B16F10 melanoma cell line to express the prepared HSP27 or fragment was administered to the veins of Balb / c mice. Photographed to the right of FIG. 5. In addition, the colony count of all B16F10 melanoma cell lines generated in the lung for each treatment group was counted and averaged, and the results are shown graphically on the left side of FIG. The two bars for each treatment group in the graph show the results of two repeated experiments.

According to Figure 5, B16F10 melanoma cell line was confirmed that lung metastasis is inhibited when the expression of the HSP27 protein. In addition, when the B16F10 melanoma cell line was to express the HSP27 (59-203) protein fragment from the N-terminus, the degree of metastasis to the lung was further reduced.

In summary, the experimental results of Experimental Example 1-5, HSP27 inhibits the growth and cancer metastasis of vascular endothelial cells, it can be seen that this effect is further increased when the N-terminus is removed. In addition, it can be seen that this inhibitory effect is due to the action of HSP27 binds to VEGF and inhibits phosphorylation of VEGFR2. Therefore, a substance that enhances the binding of HSP27 or HSP27 fragment and VEGF, or a substance that removes the N-terminus of HSP27, may be effective in inhibiting angiogenesis and in treating malignant tumors.

Claims (15)

NCBI Gene Bank Accession Number: An pharmaceutical composition for inhibiting angiogenesis comprising an HSP27 fragment (Heat Shock Protein 27 fragment) from which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 of NP_001531.1 is deleted. NCBI Gene Bank Accession Number: A pharmaceutical composition for treating cancer, comprising an HSP27 fragment deleted from N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 of NP_001531.1. The pharmaceutical composition according to claim 2, which is for inhibiting cancer metastasis. The pharmaceutical composition according to any one of claims 1 to 3, wherein the HSP27 fragment is a recombinant protein. NCBI Gene Bank Accession Number: An pharmaceutical composition for inhibiting angiogenesis comprising a nucleic acid encoding an HSP27 fragment in which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 of NP_001531.1 is deleted. NCBI Gene Bank Accession Number: A pharmaceutical composition for treating cancer comprising a nucleic acid encoding an HSP27 fragment from which the N-terminal amino acid sequence of HSP27 of HSP27 of NP_001531.1 is deleted. The pharmaceutical composition according to claim 6, which is for inhibiting cancer metastasis. The pharmaceutical composition according to any one of claims 5 to 7, wherein the nucleic acid encoding the HSP27 fragment is a recombinant vector. The method of claim 1 or 5, wherein the angiogenesis includes tumor growth and metastasis, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, and psoriasis. (psoriasis), or a pharmaceutical composition characterized by accompanying chronic inflammation. The cancer according to any one of claims 2, 3, 6, and 7, wherein the cancer is colorectal cancer, pancreatic cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, bone metastasis cancer of prostate cancer, Pharmaceutical composition, characterized in that the ovarian cancer, or blood cancer. NCBI Gene Bank Accession Number: Method of inhibiting growth of vascular endothelial cells in vitro using HSP27 fragments deleted from N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 of NP_001531.1. Treating the sample with a mixture of HSP27 of NCBI Gene Bank Accession Number: NP_001531.1 or the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27, and a mixture of VEGF;
Measuring the degree of binding of the HSP27 or the HSP27 fragment with VEGF in the mixture; And
The method of screening an angiogenesis inhibitor active material comprising the step of selecting a sample of the binding degree is increased compared to the control. Treating the sample with a mixture of HSP27 of NCBI Gene Bank Accession Number: NP_001531.1 or the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27, and a mixture of VEGF;
Measuring the degree of binding of the HSP27 or the HSP27 fragment with VEGF in the mixture; And
The method of screening the active material for cancer treatment comprising the step of selecting a sample with the degree of binding compared to the control. Processing a sample on HSP27 of NCBI Gene Bank Accession Number: NP_001531.1;
Measuring the degree to which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted; And
The method of screening an angiogenesis inhibitor active material comprising the step of selecting a sample with the degree of deletion increased compared to the control. Processing a sample on HSP27 of NCBI Gene Bank Accession Number: NP_001531.1;
Measuring the degree to which the N-terminal amino acid sequence of Nos. 1 to 58 of HSP27 is deleted; And
The method of screening for the active material for cancer treatment comprising the step of selecting a sample of which the degree of deletion is increased compared to the control.

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