KR102304832B1 - Peptide fnin3 inhibiting cancer cell proliferation and uses thereof - Google Patents

Abstract

The present invention relates to a peptide FNIN3 for inhibiting proliferation of cancer cells, and a use thereof. More specifically, the present invention provides a peptide having a cancer cell proliferation inhibitory activity and composed of an amino acid sequence represented by SEQ ID NO: 1; a pharmaceutical composition for enhancing a therapeutic effect of an anticancer agent, wherein the pharmaceutical composition contains a peptide as an active ingredient; and a pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition comprises the peptide and an anticancer agent as active ingredients. The peptide has an excellent cancer cell proliferation inhibitory activity and can enhance an anticancer activity of an anticancer agent. Thus, by co-administering the peptide and an anticancer agent, it is possible to prevent or treat various cancer diseases such as pancreatic cancer, breast cancer, and lung cancer more effectively.

Description

Peptide FNIN3 inhibiting proliferation of cancer cells and uses thereof

The present invention relates to the peptide FNIN3 and its use for inhibiting the proliferation of cancer cells.

Integrins are a family of glycoprotein membrane receptors that mediate cell-matrix and cell-cell interactions. Integrins are heterodimers composed of α and β subunits. To date, at least 24 distinct integrin heterodimers comprising 18 α and 8 β subunits have been reported. Integrins mediate cell adhesion and migration through specific interactions with different extracellular matrix (ECM) proteins.

Several integrins are known to interact with fibronectin via the Arg-Gly-Asp (RGD) motif present in fibronectin (FN). These integrins include five α5 integrins (α5β1, α5β3, α5β5, α5β6, and α5β8) and two β1 integrins (α5β1 and α8β1). Fibronectin is an extracellular matrix high molecular weight (about 440 kDa) glycoprotein that binds to integrins as well as other extracellular matrix proteins. Fibronectin exists as a dimer of two monomers linked by a disulfide bond at the C-terminus. Each fibronectin monomer has a molecular weight of 230-250 kDa containing three types of domains, type I, type II and type III. Type I and II domains are stabilized by intrachain disulfide bonds, whereas type III domains do not contain any disulfide bonds. The absence of disulfide bonds may lead to flexibility in the structure of the FN type III domain.

A distinguishing feature of malignant tumors is a hard physical barrier composed of extracellular matrix, such as collagen, fibronectin, and integrin, which accounts for more than half of the tumor mass. This physical barrier inhibits drug penetration and halves the effectiveness of anticancer drugs. Because of these properties, the binding of fibronectin and integrin is a potential therapeutic target as an important factor in tumor growth. Accordingly, based on the above interaction with integrin and fibronectin, research on various fibronectin-derived peptides that bind to integrin is being actively conducted.

US Registered Patent US 6316412 B1 (Registered on January 13, 2001)

It is an object of the present invention to provide a peptide that inhibits the proliferation of cancer cells.

Another object of the present invention is to provide a composition for enhancing the therapeutic effect of an anticancer agent using the peptide.

Another object of the present invention is to provide a cancer treatment composition in which the above peptide and an anticancer agent are used in combination.

Another object of the present invention is to provide a composition for inhibiting cancer cell proliferation using the above peptide.

Another object of the present invention is to provide a method for inhibiting cancer cell proliferation using the above peptide.

In order to achieve the above object, the present invention provides a peptide having cancer cell proliferation inhibitory activity consisting of the amino acid sequence represented by SEQ ID NO: 1.

The present invention provides a pharmaceutical composition for enhancing the therapeutic effect of an anticancer agent containing the peptide as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the peptide and an anticancer agent as active ingredients.

The present invention provides a reagent composition for inhibiting cancer cell proliferation containing the peptide as an active ingredient.

In addition, the present invention provides a method for inhibiting cancer cell proliferation comprising the step of treating an animal other than a human with the peptide.

The present invention relates to a peptide FNIN3 and a novel use thereof, wherein the peptide has excellent cancer cell proliferation inhibitory activity and can enhance the anticancer activity of an anticancer agent, and can be used to enhance the therapeutic effect of cancer diseases.

The peptide can more effectively prevent or treat various cancer diseases, such as pancreatic cancer, breast cancer, and lung cancer, by being administered in combination with an anticancer agent, and can exhibit excellent therapeutic effects even with a small amount of anticancer agent, thereby reducing the side effects of anticancer agents and treating them more safely can

1 shows a schematic diagram of the design of the FNIN3 peptide focusing on an amino acid site that binds to integrin among a part of human fibronectin (FN) protein. To generate a short mimetic peptide of the FN protein, an amino acid site of 1,267-1,540 (274 amino acids: including RGD, which is an important site for cell adhesion, binding to several integrin receptors) was selected, and the 10-20 amino acid sequence of this site was selected. FNIN3-* peptides were designed by random selection.
Figure 2 shows the results of analyzing the interaction between the FNIN3-* peptide synthesized using the PatchDock and FireDock protocols and the integrins α5β1, α5β3, and αIIbβ3. Through this, the amino acid portion attached to FNIN3-* and integrin was identified. a. FNIN3-* vs. integrin α5β1, b. FNIN3-* vs. αvβ3, c. FNIN3-* vs. αIIbβ3.
Figure 3 is an evaluation of the effect of FNIN3 and / or gemcitabine (Gemcitabine) on the proliferation of pancreatic cancer cells.
Figure 4 is an observation of the effect when FNIN3 and gemcitabine is treated in combination with pancreatic cancer cells.
5 is an evaluation of the effect of FNIN3 and / or doxorubicin (Doxorubicin) on the proliferation of lung cancer cells.
6 is an observation of the effect of FNIN3 and doxorubicin in combination treatment with lung cancer cells.
7 is an evaluation of the effect of FNIN3 and / or doxorubicin on the proliferation of breast cancer cells.
8 is an observation of the effect of FNIN3 and doxorubicin in combination treatment with breast cancer cells.

Hereinafter, the present invention will be described in detail.

The present inventors synthesized a peptide (FNIN3) targeting α5β1-integrin, which binds to human fibronectin and is involved in intercellular binding and cell proliferation, and treated it to pancreatic cancer, breast cancer, and lung cancer cells to model secondary and tertiary culture When the proliferation of cancer cells was observed in the FNIN3 treatment, the proliferation of cancer cells was decreased, and the anticancer effect was enhanced when combined with an anticancer agent, thereby completing the present invention.

The present invention provides a peptide having cancer cell proliferation inhibitory activity.

Preferably, the peptide may consist of the amino acid sequence represented by SEQ ID NO: 1.

In the present specification, the peptide represented by SEQ ID NO: 1 as shown in Table 1 below was named FNIN3.

SEQ ID NO: Peptide name amino acid sequence One FN3 TVYAVTGRGDSPASSKPC

The peptide is produced by amidation at the C-terminus, acetylation at the N-terminus of the peptide, or amidation at the C-terminus and acetylation at the N-terminus of the peptide. ) can be

Preferably, the peptide may be amidated at the C-terminus of the peptide, such as "FNIN3-*" in an embodiment of the present invention, and "*-FNIN3-*, such as the C of the peptide. -Terminal amidation (amidation) and N-terminus may be acetylated (acetylation), but is not limited thereto.

In the peptide according to the present invention, the cancer cells may be one or two or more selected from the group consisting of pancreatic cancer cells, lung cancer cells and breast cancer cells, but is not limited thereto.

The cancer cells may be two-dimensionally cultured or three-dimensionally cultured, preferably three-dimensionally cultured, but is not limited thereto.

According to an experimental example of the present invention, when the peptide was treated with pancreatic cancer cells, lung cancer cells or breast cancer cells, it was confirmed that the proliferation of the cancer cells was effectively inhibited.

The present invention provides a pharmaceutical composition for enhancing the therapeutic effect of an anticancer agent containing a peptide having cancer cell proliferation inhibitory activity as an active ingredient.

Preferably, the peptide may consist of the amino acid sequence represented by SEQ ID NO: 1.

Corresponding features may be substituted for the above-mentioned parts.

In the pharmaceutical composition for enhancing the therapeutic effect of an anticancer agent according to the present invention, the anticancer agent may be selected from the group consisting of a cytotoxic anticancer agent, a targeted anticancer agent, and an immune anticancer agent, and preferably a cytotoxic anticancer agent that attacks cancer cells to show an anticancer effect , specifically gemcitabine, capecitabine, cytarabine, decitabine, enocitabine, doxorubicin, epirubicin, dow It may be one or two or more selected from the group consisting of norubicin (daunorubicin), idarubicin (idarubicin) and mitoxantrone (mitoxantrone), but is not limited thereto.

The targeted anticancer agent is cetuximab, trastuzumab, rituximab, imatinib, nilotinib, erlotinib, gefitinib , sorafenib (sorafenib), axitinib (axitinib), vandetanib (vandetanib), temsirolimus (temsirolimus) and the like may be included, but are not limited thereto.

The immunotherapy may include, but is not limited to, ipilimumab, pembrolizumab, nivolumab, atezolizumab, blinatumomab, and the like.

The pharmaceutical composition for enhancing the anticancer agent therapeutic effect according to the present invention may be administered simultaneously (simultaneous), separately (separate) or sequentially (sequential) with the anticancer agent. Since the composition can enhance the therapeutic effect of the anticancer agent, it can be administered in combination with the anticancer agent, thereby preventing or treating cancer more effectively.

The cancer may be one or two or more selected from the group consisting of pancreatic cancer, lung cancer and breast cancer, but is not limited thereto.

According to an experimental example of the present invention, when the peptide and gemcitabine or doxorubicin were treated in combination, it was confirmed that the proliferation of cancer cells such as pancreatic cancer, breast cancer, and lung cancer was significantly reduced.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising a peptide having cancer cell proliferation inhibitory activity and an anticancer agent as active ingredients.

Preferably, the peptide may consist of the amino acid sequence represented by SEQ ID NO: 1.

Corresponding features may be substituted for the above-mentioned parts.

In the pharmaceutical composition for preventing or treating cancer according to the present invention, the anticancer agent may be a cytotoxic anticancer agent showing an anticancer effect by attacking cancer cells, preferably gemcitabine, capecitabine, theta Cytarabine, decitabine, enocitabine, doxorubicin, epirubicin, daunorubicin, idarubicin and mitoxantrone It may be one or two or more selected from the group consisting of, but is not limited thereto.

The composition may include the peptide and the anticancer agent in a weight ratio of (20 to 60): 1.

Preferably, in the case of pancreatic cancer, the peptide and the anticancer agent (40 to 60): 1, more preferably, may be included in a weight ratio of 50: 1, in the case of lung cancer or breast cancer, the peptide and the anticancer agent (20 to 30): 1, more preferably, may be included in a weight ratio of 25: 1, but is not limited thereto.

The peptide may enhance the anticancer activity of the anticancer agent.

The cancer may be one or two or more selected from the group consisting of pancreatic cancer, lung cancer and breast cancer, but is not limited thereto.

The pharmaceutical composition according to the present invention may be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition may be combined with a suitable pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. may be prepared further comprising have. The appropriate carrier and the like do not inhibit the activity and properties of the peptide or anticancer agent according to the present invention, and may be selected differently depending on the dosage form and formulation.

The pharmaceutical composition according to the present invention can be applied in any dosage form, and more specifically, it can be used by formulating oral dosage forms, external preparations, suppositories, and parenteral dosage forms of sterile injection solutions according to conventional methods.

The solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, gels, capsules, etc., and at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, It can be prepared by mixing gelatin, etc., and lubricants such as magnesium stearate and talc in addition to simple excipients may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.

In addition, the pharmaceutical composition according to the present invention may further add calcium or vitamins to enhance therapeutic efficacy.

The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

The effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to any animal capable of developing a cancer disease, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs. .

The pharmaceutical composition according to the present invention may be administered by an appropriate administration route according to the form of the formulation, and may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, muscle, skin application, respiratory inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. have.

The pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of cancer, or may be used in combination with surgery or other drug treatment.

The present invention provides a reagent composition for inhibiting cancer cell proliferation, comprising a peptide having cancer cell proliferation inhibitory activity as an active ingredient.

Preferably, the peptide may consist of the amino acid sequence represented by SEQ ID NO: 1.

Corresponding features may be substituted for the above-mentioned parts.

In addition, the present invention provides a method for inhibiting cancer cell proliferation, comprising the step of treating an animal other than a human with a peptide having cancer cell proliferation inhibitory activity.

Preferably, the peptide may consist of the amino acid sequence represented by SEQ ID NO: 1.

Corresponding features may be substituted for the above-mentioned parts.

Hereinafter, the present invention will be described in detail according to non-limiting examples. Of course, the following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. Therefore, what can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention.

<Preparation Example 1> Peptide design and synthesis

It is known that the 1267-1540 amino acid region of the fibronectin (FN) protein region acts as an important part for integrin binding and promotes cell adhesion, proliferation and signal transduction. Therefore, to generate a short mimetic peptide, 1,267-1,540 (274 amino acids: including RGD, which is an important site for cell adhesion, binding to several integrin receptors) amino acid sites were selected, and 10-20 amino acid sequences were randomly selected from these sites. A peptide was designed using methods such as position change, deletion, and addition of selected amino acids (without RGD) (FIG. 1).

Each designed peptide was analyzed for sequence uniqueness (https://research.bioinformatics. udel.edu/peptidematch/index.jsp) and material chemical properties (https://www.thermofisher.Com/kr/en/home) /life-science/protein-biology/eptides-proteins/custom-peptidesynthesis-services/peptide-analyzing-tool.html).

In addition, the interactions between the synthesized peptides and integrins (α5β1, αvβ3 and αIIbβ3) were analyzed using the PatchDock and FireDock protocols (FIGS. 2a-c). The designed peptide was synthesized by Peptron (Korea).

Table 2 below shows the peptide sequence, length, chemical properties, and the like.

As shown in Table 2 above, FNIN3-* and one other peptide were designed. In the case of FNIN3-*, it represents a peptide amidated at the C-terminus, and in the case of *-FNIN3, it represents a peptide acetylated at the N-terminus. In addition, *-FNIN3-* represents a peptide amidation at the C-terminus and acetylation at the N-terminus.

<Experimental Example 1> Confirmation of inhibition of proliferation of pancreatic cancer cells by FNIN3 peptide and gemcitabine treatment

(1) Effect of FNIN3 on proliferation in two-dimensional culture of pancreatic cancer cells (MIA PaCa-2)

MIA PaCa-2 (KCLB, Republic of Korea) was cultured in DMEM (Hyclone, USA) + 10% FBS (Hyclone) + 1% P/S (Hyclone) + 1% L-Glutamine, cells at passage 10-12 was used for the study. To verify the effect of the peptide, MIA PaCa-2 (5×10 ³ cells/well) was placed in a 96-well cell culture dish and adhered for 24 hours, treated with 0.1, 1, 5, 10, 25, 50 μM of FNIN3 and 72 incubated for hours. After 72 hours of culture, cell proliferation was confirmed using MTT assay.

Figure 3a shows the cell proliferation inhibitory activity, that is, the anticancer activity according to the concentration of FNIN3 in the two-dimensional culture of pancreatic cancer cells. Referring to this, it can be confirmed that FNIN3 has little effect on cell growth in the two-dimensional culture in which cells grow by attaching to the bottom of the culture dish.

(2) Effect of FNIN3 on proliferation in 3D culture of pancreatic cancer cells

MIA PaCa-2 (5×10 ³ cells/well) was placed in an Ultra Low Cluster 96-well cell culture dish, cultured for 7 days, treated with 0.1, 1, 5, 10, 25, 50 μM of FNIN3, and cultured for 72 hours. After 72 hours of culture, cell proliferation was confirmed using WST assay.

Figure 3b shows the anticancer activity according to the concentration of FNIN3 in a three-dimensional tumor model of pancreatic cancer cells. Referring to this, in the three-dimensional tumor model, it can be confirmed that FNIN3 inhibits cell proliferation according to the concentration of FNIN3 by breaking the cell-to-cell bond. This can also be confirmed through the photomicrograph and fluorescence micrograph of FIG. 4 . Green fluorescence in FIG. 4 indicates live cells, and red fluorescence indicates dead cells.

(3) Effect of Gemcitabine on Proliferation in 2D Culture of Pancreatic Cancer Cells

MIA PaCa-2 (5×10 ³ cells/well) was placed in a 96-well cell culture dish and adhered for 24 hours, treated with 0.01, 0.1, 0.5, 1, 5, 10, 50 μM of gemcitabine and cultured for 72 hours. did. After 72 hours of culture, cell proliferation was confirmed using MTT assay.

Figure 3c shows the anticancer activity according to the concentration of gemcitabine in the two-dimensional culture of pancreatic cancer cells. Referring to this, it can be observed that the proliferation of pancreatic cancer cells is inhibited by more than 50% at 0.1 μM because there is no physical barrier generated through cell-to-cell bonding in two-dimensional culture.

(4) Effect of gemcitabine on proliferation in 3D culture of pancreatic cancer cells

MIA PaCa-2 (5×10 ³ cells/well) was placed in an Ultra Low Cluster 96-well cell culture dish and incubated for 7 days, treated with 0.01, 0.1, 0.5, 1, 5, 10, 50 μM of gemcitabine for 72 hours. cultured. After 72 hours of culture, cell proliferation was confirmed using WST assay.

Figure 3d shows the anticancer activity according to the concentration of gemcitabine in a three-dimensional tumor model of pancreatic cancer cells. Referring to this, the 3D tumor model forms a physical barrier because the cell-to-cell bond is tight, so that, unlike 2D cultured cells, cell growth is inhibited less than 0.1μM in 2D culture even with 50μM gemcitabine. can confirm. This can also be confirmed through the photomicrograph and fluorescence micrograph of FIG. 4 .

(5) Inhibitory effect of pancreatic cancer in combination treatment of FNIN3 and gemcitabine in 3D culture

MIA PaCa-2 (5×10 ³ cells/well) was placed in a 96-well cell culture dish, cultured for 7 days, treated with 25 μM FNIN3, and cultured for 72 hours. After that, 0.5 μM of gemcitabine was treated and incubated for 72 hours again. Cell changes and proliferation were observed and measured through Live & Dead assay.

Figure 3e shows the difference in anticancer activity when treated with FNIN3 and gemcitabine, respectively, and when combined treatment in a three-dimensional tumor model of pancreatic cancer cells. Referring to this, it can be confirmed that the anticancer activity significantly increased during the combined treatment. In addition, a corresponding appearance can be confirmed through the micrograph and the fluorescence micrograph of FIG. 4 .

<Experimental Example 2> Confirmation of inhibition of proliferation of lung cancer cells by treatment with FNIN3 peptide and doxorubicin

(1) Effect of FNIN3 on proliferation in two-dimensional culture of lung cancer cells (A 549)

A 549 (KCLB, Republic of Korea) was cultured in RPMI (Hyclone, USA) + 10% FBS (Hyclone) + 1% P/S (Hyclone) + 1% L-Glutamine, and cells at passage 12-15 were studied. was used for To verify the effect of the peptide, A 549 (5×10 ³ cells/well) was placed in a 96-well cell culture dish and adhered for 24 hours, then treated with 0.1, 1, 5, 10, 25, 50 μM of FNIN3 and treated for 72 hours cultured. After 72 hours of culture, cell proliferation was confirmed using MTT assay.

Figure 5a shows the anticancer activity according to the concentration of FNIN3 in the two-dimensional culture of lung cancer cells. Referring to this, it can be seen that FNIN3 has little effect on cell growth in a two-dimensional culture in which cells grow by attaching to the bottom of the culture dish.

(2) Effect of FNIN3 on proliferation in three-dimensional culture of lung cancer cells

A 549 (5×10 ³ cells/well) was placed in an Ultra Low Cluster 96-well cell culture dish, cultured for 7 days, treated with 0.1, 1, 5, 10, 25, 50 μM of FNIN3, and cultured for 72 hours. After 72 hours of culture, cell proliferation was confirmed using WST assay.

Figure 5b shows the anticancer activity according to the concentration of FNIN3 in a three-dimensional tumor model of lung cancer cells. Referring to this, it can be seen that in the 3D tumor model, FNIN3 inhibits cell proliferation according to the concentration of FNIN3 by breaking the cell-to-cell bond. This can also be confirmed through the photomicrograph and fluorescence micrograph of FIG. 6 . Green fluorescence in FIG. 6 indicates live cells, and red fluorescence indicates dead cells.

(3) Effect of doxorubicin on proliferation in two-dimensional culture of lung cancer cells

A 549 (5×10 ³ cells/well) was placed in a 96-well cell culture dish and adhered for 24 hours, treated with 0.01, 0.1, 0.5, 1, 2, 5, 10 μM doxorubicin, and cultured for 72 hours. After 72 hours of culture, cell proliferation was confirmed using MTT assay.

Figure 5c shows the anticancer activity according to the concentration of doxorubicin in the two-dimensional culture of pancreatic cancer cells. In two-dimensional culture, it can be observed that the proliferation of lung cancer cells is inhibited by more than 50% at 0.1 μM because there is no physical barrier generated through cell-to-cell bonding.

(4) Effect of doxorubicin on proliferation in three-dimensional culture of lung cancer cells

A 549 (5×10 ³ cells/well) was placed in an Ultra Low Cluster 96-well cell culture dish, cultured for 7 days, treated with 0.01, 0.1, 0.5, 1, 2, 5, 10 μM doxorubicin, and cultured for 72 hours. After 72 hours of culture, cell proliferation was confirmed using WST assay.

Figure 5d shows the anticancer activity according to the concentration of doxorubicin in a three-dimensional tumor model of lung cancer cells. Referring to this, the three-dimensional tumor model forms a physical barrier because the cell-to-cell bond is firmly established, so that, unlike the two-dimensional cultured cells, cell growth is inhibited less than 0.1 µM in the two-dimensional culture even with 10 µM doxorubicin. can be checked This can also be confirmed through the photomicrograph and fluorescence micrograph of FIG. 6 .

(5) Lung cancer inhibitory effect when combined treatment of FNIN3 and doxorubicin in three-dimensional culture

A 549 (5×10 ³ cells/well) was placed in a 96-well cell culture dish, cultured for 7 days, treated with 25 μM FNIN3, and cultured for 72 hours. After that, 1 μM of doxorubicin was treated and incubated for 72 hours again. Cell changes and proliferation were observed and measured through Live & Dead assay.

Figure 5e shows the difference in anticancer activity when treated with FNIN3 and doxorubicin, respectively, and when combined treatment in a three-dimensional tumor model of lung cancer cells. Referring to this, it can be confirmed that the anticancer activity significantly increased during the combined treatment. A corresponding appearance can be confirmed through the micrograph and the fluorescence micrograph of FIG. 6 .

<Experimental Example 3> Confirmation of inhibition of proliferation of breast cancer cells by treatment with FNIN3 peptide and doxorubicin

(1) Effect of FNIN3 on proliferation in two-dimensional culture of breast cancer cells (MDA MB-231)

MDA MB-231 (KCLB, Republic of Korea) was cultured in RPMI (Hyclone, USA) + 10% FBS (Hyclone) + 1% P/S (Hyclone) + 1% L-Glutamine, passage 7-10 cells was used for the study. To verify the effect of the peptide, MDA MB-231 (7×10 ³ cells/well) was placed in a 96-well cell culture dish and adhered for 24 hours, then treated with 0.1, 1, 5, 10, 25, 50 μM of FNIN3 and 72 incubated for hours. After 72 hours of culture, cell proliferation was confirmed using MTT assay.

Figure 7a shows the anticancer activity according to the concentration of FNIN3 in the two-dimensional culture of breast cancer cells. Referring to this, it can be seen that FNIN3 has little effect on cell growth in a two-dimensional culture in which cells grow by attaching to the bottom of the culture dish.

(2) Effect of FNIN3 on proliferation in 3D culture of breast cancer cells

MDA MB-231 (9×10 ³ cells/well) was placed in an Ultra Low Cluster 96-well cell culture dish, cultured for 7 days, treated with 0.1, 1, 5, 10, 25, 50 μM of FNIN3, and cultured for 72 hours. After 72 hours of culture, cell proliferation was confirmed using WST assay.

Figure 7b shows the anticancer activity according to the concentration of FNIN3 in a three-dimensional tumor model of breast cancer cells. Referring to this, it can be seen that in the 3D tumor model, FNIN3 inhibits cell proliferation according to the concentration of FNIN3 by breaking the cell-to-cell bond. This can also be confirmed through the photomicrograph and fluorescence micrograph of FIG. 8 . In FIG. 8, green fluorescence indicates a live cell, and red fluorescence indicates a dead cell.

(3) Effect of doxorubicin on proliferation in two-dimensional culture of breast cancer cells

MDA MB-231 (7×10 ³ cells/well) was placed in a 96-well cell culture dish and adhered for 24 hours, treated with 0.01, 0.1, 0.5, 1, 2, 5, 10 μM doxorubicin, and cultured for 72 hours. . After 72 hours of culture, cell proliferation was confirmed using MTT assay.

Figure 7c shows the anticancer activity according to the concentration of doxorubicin in the two-dimensional culture of pancreatic cancer cells. Referring to this, it can be observed that the proliferation of lung cancer cells is inhibited by more than 50% at 1 μM because there is no physical barrier generated through cell-to-cell bonding in two-dimensional culture.

(4) Effect of doxorubicin on proliferation in three-dimensional culture of breast cancer cells

MDA MB-231 (9×10 ³ cells/well) was placed in an Ultra Low Cluster 96-well cell culture dish, cultured for 7 days, treated with 0.01, 0.1, 0.5, 1, 2, 5, 10 μM doxorubicin, and cultured for 72 hours. did. After 72 hours of culture, cell proliferation was confirmed using WST assay.

Figure 7d shows the anticancer activity according to the concentration of doxorubicin in a three-dimensional tumor model of breast cancer cells. Referring to this, it can be seen that the 3D tumor model forms a physical barrier because the cell-to-cell bond is firmly established, so that, unlike 2D cultured cells, cell growth is less inhibited than 1μM in 2D culture even with 10μM doxorubicin. can This can also be confirmed through the photomicrograph and fluorescence micrograph of FIG. 8 .

(5) Inhibitory effect of breast cancer in combination treatment of FNIN3 and doxorubicin in three-dimensional culture

MDA MB-231 (9×10 ³ cells/well) was placed in a 96-well cell culture dish, cultured for 7 days, treated with 25 μM FNIN3, and cultured for 72 hours. After that, 1 μM of doxorubicin was treated and incubated for 72 hours again. Cell changes and proliferation were observed and measured through Live & Dead assay.

Figure 7e shows the difference in anticancer activity when treated with FNIN3 and doxorubicin, respectively, and when combined with treatment in a three-dimensional tumor model of breast cancer cells. Referring to this, it can be confirmed that the anticancer activity significantly increased during the combined treatment. This can be confirmed through the micrograph and the fluorescence micrograph of FIG. 8 as well.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Research Cooperation Foundation of Yeungnam University <120> PEPTIDE FNIN3 INHIBITING CANCER CELL PROLIFERATION AND USES THEREOF <130> ADP-2020-0313 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> FNIN3 peptide <400> 1 Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 1 5 10 15 Pro Cys

Claims (13)

delete delete A pharmaceutical composition for enhancing the anticancer therapeutic effect of gemcitabine or doxorubicin for pancreatic cancer, lung cancer or breast cancer, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. 4. The method of claim 3,
The peptide is
A pharmaceutical composition, characterized in that it inhibits the proliferation of pancreatic cancer cells, lung cancer cells or breast cancer cells. delete delete A pharmaceutical composition for preventing or treating pancreatic cancer, lung cancer or breast cancer, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an anticancer agent gemcitabine or doxorubicin as active ingredients. 8. The method of claim 7,
The composition is
The peptide and the anticancer agent (20 to 60): A pharmaceutical composition, characterized in that it is included in a weight ratio of 1. delete delete delete delete A method for inhibiting cancer cell proliferation, comprising treating an animal other than a human with a peptide comprising the amino acid sequence represented by SEQ ID NO: 1.

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