Composition for inhibiting VEGF-induced angiogenesis, comprising CCL8-binding peptide

Abstract

The present invention relates to a composition for inhibiting VEGF-induced angiogenesis, the composition comprising a CCL8-binding peptide. The composition according to the present invention can contribute to preventing cancer progression and metastasis by inhibiting cancer cell proliferation and inhibiting cancer cell migration and invasion. On the basis thereof, the present invention can be effectively used in various medicines and foods for preventing, alleviating, or treating cancer, as well as in the feed industry.

Description

Composition for inhibiting VEGF-induced angiogenesis comprising a CCL8 binding peptide

[0001] The present invention relates to a composition for inhibiting VEGF-induced angiogenesis comprising a CCL8 binding peptide.

[0002] Cancer is emerging as the greatest threat to public health, and health threats and deaths due to cancer are expected to increase due to various environmental changes, including an aging population. Cancer is classified as benign and malignant. Benign tumors grow relatively slowly and do not metastasize from their primary site to other tissues. On the other hand, malignant tumors are characterized by rapid growth, infiltrating other tissues beyond their primary site, posing a significant threat to life and leading to death.

[0003] Furthermore, cancer can easily recur even after treatment through processes such as metastasis, making treatment difficult. Metastasis is a phenomenon that accompanies the progression of malignant tumors. As malignant tumor cells proliferate and the cancer progresses, they acquire new genetic traits necessary for metastasis. These cells then infiltrate blood vessels and lymph nodes, circulate through the blood and lymph, and settle in other tissues, where they proliferate. Active research is underway to elucidate the mechanisms of metastasis and to suppress it.

[0004] Meanwhile, CCL8 (CC Motif Chemokine Ligand 8), also known as monocyte chemoattractant protein 2 (MCP2), belongs to the CC chemokine subfamily and is known to play a role in neoplastic and inflammatory host responses as a chemotactic factor that attracts monocytes, lymphocytes, basophils, and eosinophils. Regulation of this chemokine activity influences tumor histology, which promotes the stages of the metastatic process. Recent studies have reported combined treatment of colorectal cancer with CCL8 and CCR5 (P Yang, 2021) and attenuation of cancer metastasis to the lungs by CCL8 inhibition (C Yan, 2018). Therefore, there is a greater need to deeply understand the correlation between CCL8 and cancer and to develop novel substances that can effectively inhibit cancer progression induced by CCL8.

[0005] Under the above-described technical background, the present invention aims to provide a composition for inhibiting angiogenesis, comprising a peptide represented by sequence number 1, which effectively inhibits the growth or proliferation and metastasis of cancer cells.

[0006] In addition, the present invention aims to provide a method for inhibiting angiogenesis, comprising the step of administering a composition comprising a peptide represented by the above sequence number 1 to a subject other than a human.

[0007] To solve the above problem, the present invention provides a composition for inhibiting angiogenesis, comprising a peptide represented by sequence number 1.

[0008] The above peptide may inhibit the proliferation, migration and invasion of cancer cells.

[0009] The above peptide may inhibit the expression of vascular endothelial growth factor A in cancer cells.

[0010] The above cancer may include metastatic cancer.

[0011] The above cancer may be at least one selected from the group consisting of breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer, ovarian cancer, cervical cancer, gallbladder cancer, bile duct cancer, and osteosarcoma.

[0012] The above peptide may bind to CCL8 (CC Motif Chemokine Ligand 8) and inhibit the function of CCL8.

[0013] The present invention also provides a method for inhibiting angiogenesis, comprising the step of administering to a non-human subject a composition comprising a peptide represented by sequence number 1.

[0014] The composition according to the present invention can suppress cancer cell proliferation by inhibiting angiogenesis, and contribute to the prevention of cancer progression and metastasis by inhibiting cancer cell migration and invasion. Based on this, it can be usefully utilized in various pharmaceutical, food, and feed industries for the prevention, improvement, and treatment of cancer.

[0015] Figure 1 is a diagram showing the sequence of the peptide of the present invention, which is a CCL8-binding peptide and its derivative.

[0016] Figure 2 is a diagram comparing the changes in cell mobility (left) and invasiveness (right) in the CCL8 treatment group and the CCL8 and peptide treatment group of the present invention in MDA-MB-231 cells.

[0017] Figure 3 is a diagram comparing the changes in vascular endothelial growth factor in MDA-MB-231 cells treated with CCL8 and in the peptide treatment group of the present invention.

[0018] Figure 4 is a diagram comparing the changes in VEGF-A promoter binding of c-Jun and Sp-1 transcription factors by the peptide of the present invention.

[0019] Figure 5 is a diagram showing the effect of the peptide of the present invention (DSP-1) on inhibiting angiogenesis of endothelial colony forming cells (ECFCs) induced by CCL8.

[0020] Figure 6 is a diagram comparing the cell growth changes of the CCL8 treatment group and the CCL8 and peptide treatment group of the present invention in MDA-MB-231 cells.

[0021] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0022] Terms defined in commonly used dictionaries should be interpreted as having meanings consistent with their meanings in the context of the relevant technology, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. Furthermore, when describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to unnecessarily obscure the gist of the present invention.

[0023] Additionally, when a part of the present invention is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0024]

[0025] Peptide represented by sequence number 1

[0026] The peptide according to the present invention is characterized by including an amino acid sequence represented by sequence number 1, and has an excellent effect of inhibiting the proliferation, migration, and invasion of cancer cells increased under the reaction of CCL8 (CC Motif Chemokine Ligand 8).

[0027] The term "peptide" is used interchangeably with "protein" and includes reference to a polymer of amino acid residues. In the present invention, the peptide may be synthesized using genetic recombination and a protein expression system, and preferably, may be synthesized in vitro using a peptide synthesizer or the like.

[0028] The peptide according to the present invention is one of the derivatives of a peptide synthesized based on the property of evasin P672, a protein present in ticks, to bind to CCL8. Specifically, it includes all homologs, analogs, fragments, or derivatives of a CCL8-binding peptide produced after confirming the sequence through sequence analysis of the N-terminal region of the evasin P672 protein that binds to CCL8.

[0029] In one specific example, the peptide of the present invention isolated from the CCL8-binding peptide has an amino acid sequence represented by SEQ ID NO: 1 as shown in FIG. 1.

[0030] Sequence number 1: YEDEDYEDFFKPVTCYF

[0031]

[0032] The above "CCL8 (CC Motif Chemokine Ligand 8)" belongs to the CC chemokine subfamily and is known to play a role in neoplastic and inflammatory host responses as a chemotactic factor that attracts monocytes, lymphocytes, basophils, and eosinophils. Modulation of this chemokine activity influences the histology of tumors, promoting stages of the metastatic process.

[0033] CCL8 production in peritumoral fibroblasts can be upregulated in response to signals induced by breast cancer cells, particularly triple-negative breast cancer cells. CCL8 expressed in fibroblasts is secreted extracellularly, forming a CCL8 gradient. This gradient acts as a chemoattractant for breast cancer cells, inducing their migration toward fibroblasts. Cancer cells activate surrounding fibroblasts to promote increased CCL8 expression, a process that promotes cancer cell metastasis.

[0034] The present inventors, considering that both genetic inhibition and antibody-mediated inhibition of CCL8 activity interfere with the motility of cancer cells and their invasiveness / metastasis, conceived that anti-CCL8 therapy should be considered for the management of metastatic breast cancer, and confirmed the ability of the peptide of the present invention, which is a derivative of the CCL8-binding peptide, to inhibit the growth, motility and invasiveness of cancer cells increased by CCL8.

[0035]

[0036] The present invention provides a composition for inhibiting angiogenesis, comprising a peptide represented by the above sequence number 1.

[0037] The peptide of the present invention may inhibit the proliferation, migration and invasion of cancer cells.

[0038] The peptide of the present invention may inhibit the expression of vascular endothelial growth factor A in cancer cells.

[0039] The composition comprising the peptide of the present invention has an excellent effect of inhibiting the growth, mobility, invasiveness, and expression of vascular endothelial growth factor-A (VEGF-A) of cancer cells increased by CCL8, and thus has an effect of preventing or treating cancer.

[0040] According to one embodiment of the present invention, the peptide represented by the sequence number 1 may be included in an amount of 0.000001 to 30% by weight, preferably 0.0006 to 10% by weight, and more preferably 0.0006 to 1% by weight, based on 100% by weight of the total pharmaceutical composition containing the peptide. When the peptide represented by the sequence number 1 is included within the above range, the side effects of the pharmaceutical composition are minimized, while the effect of inhibiting the growth, mobility, invasiveness, and expression of vascular endothelial growth factor-A (VEGF-A) of cancer cells increased by CCL8 is maximized, thereby further improving the effect of preventing or treating cancer, and in particular, there is an advantage of improving the effect of inhibiting metastasis of cancer cells.

[0041] Therefore, the above cancer may include metastatic cancer.

[0042] The cancer may be at least one selected from the group consisting of breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer, ovarian cancer, cervical cancer, gallbladder cancer, bile duct cancer, and osteosarcoma, and preferably may be breast cancer, more preferably malignant breast cancer.

[0043] The peptide of the present invention may bind to CCL8 (CC Motif Chemokine Ligand 8) and inhibit the function of CCL8.

[0044] The peptide of the present invention can be used in the management of metastatic breast cancer as an anti-CCL8 therapy because it inhibits the motility of cancer cells and their invasiveness / metastasis by inhibiting CCL8 activity.

[0045] The present invention provides a method for inhibiting angiogenesis, comprising the step of administering to a non-human subject a composition comprising a peptide represented by SEQ ID NO: 1.

[0046] The above composition can be administered to a subject in a pharmaceutically effective amount.

[0047] At this time, the above “individual” may mean all animals including rats, mice, and livestock, including humans, and preferably may be mammals including humans.

[0048] The term "administration" as described above refers to introducing a given substance into an individual through an appropriate method, and may include oral administration or parenteral administration. As such, the pharmaceutical composition may be administered via any common route as long as it can reach the target tissue. Examples of parenteral administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0049] The above “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the level of the effective dose can be easily determined by those skilled in the art based on factors including the patient’s sex, age, weight, health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route, and excretion rate, treatment period, drugs used in combination or simultaneously, and various other factors well known in the medical field, and it is also possible to estimate the amount to be used in humans from the effective amount determined through animal testing.

[0050] In this way, the daily dosage of the pharmaceutical composition can be applied in various ways by those skilled in the art, and is not specifically limited thereto in the present invention, but for example, it can be 0.0001 to 100 mg / kg, and preferably 5 to 25 mg / kg.

[0051] Additionally, the above administration may be divided into one or several times a day.

[0052] The above pharmaceutical composition may be in one or more formulations selected from the group consisting of oral, topical, suppository, sterile injectable solution and spray, each according to a conventional method, but is not limited thereto, and may preferably be an topical formulation.

[0053] The above external preparation may be in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or gel-type external preparation for skin, for example, but is not limited thereto, and may refer to any form applied externally to the skin without any particular limitation.

[0054]

[0055] The present invention will be described in more detail through the following examples. These examples are intended merely to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

[0056]

[0057] Peptide of the present invention that binds to CCL8

[0058] The synthesis of this peptide was commissioned to CSBio (Shanghai) Ltd, a peptide synthesis company.

[0059]

[0060] Example 1: Inhibition of cell motility and invasion by the peptide of the present invention

[0061] Using breast cancer cells MDA-MB-231, an experiment was conducted to determine whether the peptide of the present invention represented by SEQ ID NO: 1 affects cell invasiveness and mobility increased by CCL8.

[0062] Cell motility and invasion were measured using 24-well transwells with polycarbonate filters measuring 8.0 μm in diameter and 6.5 mm (Falcon, USA). For motility experiments, the outer part of the filter was coated only with type I collagen at a concentration of 0.5 mg / mL and dried before use. For invasion measurements, the outer part was coated under the same conditions as for motility experiments, and the inner part was coated with reconstituted basement membrane material (Matrigel) at a concentration of 0.5 mg / mL and then air-dried before use before adding cells.

[0063] Add 600 ㎕ of growth medium to the bottom of the well and add 2Х10 4 100 μl of medium containing dispersed cells was placed on the upper part of the transwell, and CCL8 (100 ng / ml) and the peptide of the present invention (20 μM) were treated on both the outside and inside of the transwell. After fixing the cells with methanol, they were stained with a 0.5% crystal violet solution, and the number of cells that migrated to the lower part of the filter was eluted with 30% acetic acid, and the number of migrated and invaded cells was measured using ELISA. Each sample was tested three times, and the results are shown in Fig. 2.

[0064] Referring to Figure 2, when MDA-MB-231 cells were treated with CCL8 (100 ng / ml) for 48 hours, both cell migration and invasion increased, whereas when CCL8 (100 ng / ml) and the peptide of the present invention (20 μM) were treated together, the cell migration and invasion increased by CCL8 decreased. These results confirmed that the peptide of the present invention can inhibit the migration and invasion of cancer cells.

[0065]

[0066] Example 2: Inhibition of VEGF-A expression by the peptide of the present invention

[0067] CCL8 (CC Motif Chemokine Ligand 8) increases the expression of vascular endothelial growth factor A (VEGF-A) in MDA-MB-231 cells. To examine the effect of the peptide of the present invention on the increase in VEGF-A expression induced by CCL8, RT-qPCR and Westren blot analysis were performed in MDA-MB-231 cells.

[0068] To examine the change in VEGF-A expression, MDA-MB-231 cells were treated with CCL8 (100 ng / ml) or CCL8 (100 ng / ml) and the peptide of the present invention (20 μM), and then total RNA was extracted from the cells using Tri-RNA reagent (Tri-RNA reagent). cDNA was obtained using the extracted RNA (1.0 μg), d(T) primer, and reverse transcriptase. The synthesized cDNA and SYBR green dye (Takara, Japan) were used for measurement in an ABI StepOne plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's suggested method. The results were corrected for the relative value of the beta-actin gene.

[0069] For protein analysis, MDA-MB-231 cells were treated and cultured with CCL8 (100 ng / ml) or CCL8 (100 ng / ml) and the peptide of the present invention (20 μM), and then the cells were lysed by adding 0.18 ml of cell lysis buffer (50 mM Tris.Cl pH 6.8, 2% SDS, 1 mM EDTA, 100 mM DTT, protease inhibitor). After quantifying the protein using the Bradford method, an equal amount of protein was heated to 95°C or higher for 5 minutes, cooled on ice for another 5 minutes, and then electrophoresed on a 12% SDS-PAGE gel at 90 V. Afterwards, the separated proteins were transferred to a PVDF membrane and blocked with 5% skim milk. Anti-VEGF-A and anti-actin antibodies were attached, reacted at 4°C for one day, and washed with PBST. After binding the secondary antibody, the reaction was performed for 1 minute using the ECL detection system, and the degree of protein expression was observed using the FluorChem E documentation system. The results are shown in Figure 3.

[0070] Looking at Figure 3, it was observed that the peptide treatment of the present invention significantly reduced the VEGF-A expression increased by CCL8 (left), and the protein level of VEGF-A was also reduced by the peptide of the present invention (right).

[0071]

[0072] Example 3: Inhibition of VEGF-A promoter binding of c-Jun and Sp-1 transcription factors by the peptide of the present invention

[0073] To determine which transcription factors are involved in CCL8-induced VEGF-A transcriptional activation, transcription factor-DNA interactions were observed using a chromatin immunoprecipitation (ChIP) assay. Extracts from CCL8-treated cells were collected and immunoprecipitated using antibodies to transcription factors such as c-Jun, Sp1, c-fos, and C / EBP. Protein-DNA complexes bound to the antibodies were collected and PCR was performed using primers encompassing the VEGF-A promoter region. The results are shown in Figure 4.

[0074] As shown in Figure 4, a significant increase in c-Jun and Sp1 promoter binding was observed when CCL8 was treated (left). These results demonstrate that c-Jun and Sp1 are required for CCL8-induced VEGF-A transcriptional activation.

[0075] In addition, it was observed whether the peptide (DSP-1) of the present invention affects the promoter binding of transcription factors involved in VEGF-A transcriptional activity, and it was confirmed that treatment with the CCL8 binding peptide (DSP-1) significantly reduced the transcription factor-DNA binding, such as c-Jun and Sp1, increased by CCL8 (right).

[0076]

[0077] Example 4: Inhibition of angiogenesis by the peptide of the present invention

[0078] To verify whether the peptide of the present invention (DSP-1) effectively inhibits angiogenesis of endothelial colony forming cells (ECFCs) induced by CCL8, angiogenesis experiments were performed using MDA-MB-436 cells, which are triple-negative breast cancer cells overexpressing CCR5.

[0079] The results of measuring the degree of blood vessel formation by treating breast cancer cells (MDA-MB-436) with CCL8 alone or a mixture of CCL8 and the peptide of the present invention (DSP-1) and then treating the culture medium of the cancer cells with ECFCs using a tube formation assay are shown in Figure 5.

[0080] As shown in Fig. 5, angiogenesis of ECFCs was significantly increased by treatment with culture medium of cancer cells stimulated with CCL8, whereas treatment with the peptide of the present invention (DSP-1) significantly reduced the angiogenesis increased by CCL8. These results suggest that increased CCL8 in the tumor microenvironment can promote angiogenesis of ECFCs by increasing the expression of VEGF-A, a vascular endothelial growth factor, in cancer cells, and it was confirmed that such angiogenesis was reduced by treatment with the peptide of the present invention (DSP-1), which binds to CCL8 and blocks its ability. In other words, it was confirmed that the peptide of the present invention (DSP-1) is a substance that effectively inhibits angiogenesis induced by CCL8 in tumors.

[0081]

[0082] Example 5: Inhibition of cell growth by the peptide of the present invention

[0083] To determine whether the peptide of the present invention affects cell growth regulated by CCL8, experiments were conducted using a 3D spheroid culture method similar to the human body.

[0084] After removing breast cancer cells Hs578T from the culture dish, 10Х10 4After adding to the medium at a density of 100 ng / ml, CCL8 (100 ng / ml) or CCL8 (100 ng / ml) / the peptide of the present invention (20 μM) were mixed. 50 μl of the mixed cell solution was gently transferred to the bottom of a 96-well conical bottom plate (Corning, Corning, NY, USA), and the edges of the plate were filled with sterile PBS to reduce evaporation, maintaining an appropriate concentration of the medium throughout the experiment. The plate was placed in an incubator and cultured for 72 hours. The formation of 3D structures was monitored using an inverted light microscope, and the results are shown in Fig. 6.

[0085] Referring to Figure 6, when Hs578T cells were treated with CCL8 (100 ng / ml) or CCL8 (100 ng / ml) / the peptide of the present invention (20 μM) together, and the growth of cancer cells was observed for 72 hours, it was observed that cancer cell growth increased by CCL8 treatment, whereas cancer cell growth decreased when the peptide of the present invention was treated. These results confirmed that the peptide of the present invention can inhibit the growth or proliferation of cancer cells.

[0086] The composition according to the present invention inhibits cancer cell proliferation by suppressing angiogenesis, and contributes to the prevention of cancer progression and metastasis by inhibiting cancer cell migration and invasion. Based on this, it can be usefully utilized in various pharmaceutical, food, and feed industries for the prevention, improvement, and treatment of cancer, demonstrating its industrial applicability.

Claims

1. A composition for inhibiting angiogenesis, comprising a peptide consisting of an amino acid sequence of sequence number 1.

2. A composition for inhibiting angiogenesis, characterized in that the peptide in claim 1 inhibits angiogenesis induced by CCL8 (CC Motif Chemokine Ligand 8).

3. A composition for inhibiting angiogenesis, characterized in that the peptide in claim 1 inhibits angiogenesis induced by vascular endothelial growth factor A.

4. A method for inhibiting angiogenesis, comprising the step of administering a peptide consisting of an amino acid sequence of sequence number 1 to a subject other than a human.

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