Composition comprising extracellular matrix derived from skin keratinocytes as active ingredient for preventing or treating cancer by controlling tumor microenvironment

Abstract

The present invention relates to a composition comprising a skin keratinocyte-derived extracellular matrix as an active ingredient as an active ingredient for the prevention, alleviation, or treatment of cancer. The composition for preventing, alleviating, or treating cancer of the present invention contains an extracellular matrix derived from skin keratinocytes as an active ingredient, thereby significantly reducing only the proliferation of cancer cells without exhibiting side effects such as cytotoxicity in normal cells such as fibroblasts constituting the dermis of normal skin. In addition, the composition for preventing, alleviating, or treating cancer of the present invention was observed to: have the effect of suppressing the proliferation of MDA-MB-231, which cannot be expected to be greatly responsive to targeted therapies and currently lacks effective treatments, in addition to MCF7 among breast cancer cells; be able to remarkably inhibit the proliferation of Caki-1 and Caki-2, which are clear cell carcinomas classified as intractable cancer among kidney cancer cells and known to have many distant metastasis recurrences; and significantly reduce the proliferation of PANC-1, which is a pancreatic cancer cell line classified as refractory cancer that is difficult to detect early among cancer cells and is found in the late stage when surgery and treatment are limited, as well as having low treatment responsiveness to the latest immunotherapy drugs and low treatment responsiveness to targeted therapy and traditional chemotherapy. Furthermore, the composition for preventing, alleviating, or treating cancer of the present invention can be applied not only to breast cancer, kidney cancer, and pancreatic cancer, but also to the treatment of various carcinomas and solid cancers occurring on the skin. Moreover, the composition for preventing, alleviating, or treating cancer of the present invention can be used as a therapeutic agent in combination with other anticancer agents. Additionally, the composition for preventing, alleviating, or treating cancer of the present invention can be used in a treatment method for reducing the size of cancer before surgery without side effects, unlike traditional chemotherapy, through intratumoral injection.

Description

A composition for preventing or treating cancer by controlling the tumor microenvironment, containing an extracellular matrix derived from skin keratinocytes as an effective ingredient.

[0001] The present invention relates to a composition for preventing or treating cancer by controlling the tumor microenvironment, which comprises an extracellular matrix derived from skin keratinocytes as an effective ingredient.

[0002] As modernization accelerates, Korea's diet has become more Westernized. With rising awareness, modern people are increasingly working harder and experiencing stress from various sources in an effort to lead better lives. Consequently, many adverse effects are emerging, including changes in disease patterns and causes of death. According to Statistics Korea's Cause of Death Survey, cancer ranked first in Korea in 2010, with 1,444 deaths per 100,000 people, and the rate continues to rise. Lung cancer was the most common cancer, followed by stomach cancer, liver cancer, colon cancer, and pancreatic cancer.

[0003] Cancer refers to a group of abnormal cells that develop through continuous division and proliferation due to the disruption of the balance between cell division and death due to various causes. It is also called a tumor or neoplasm. It typically occurs in over 100 different parts of the body, including organs, white blood cells, bones, and lymph nodes, and progresses to serious symptoms through invasion of surrounding tissues and metastasis to other organs (WHO, 2006). The causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, as well as internal factors such as congenital genetic mutations (Klaunig & Kamendulis, Annu Rev Pharmacol Toxicol 2004, 44:239-267).

[0004] Despite remarkable progress in controlling the cell cycle and apoptosis, and identifying new targets, including oncogenes and tumor suppressor genes, to combat cancer, the incidence of cancer continues to rise. Current cancer treatment options rely on surgery, radiation therapy, and chemotherapy using highly cytotoxic anticancer drugs. Approximately 60 different anticancer drugs are currently in use, and with increasing knowledge of cancer development and cell characteristics, research into the development of new anticancer drugs is actively underway. Many patients suffer from the side effects of anticancer drugs, and their toxicity limits their use. Anticancer drugs currently in clinical use affect both cancer cells and normal cells, and repeated administrations often lead to treatment failures, side effects, and drug resistance. Research is underway to develop new anticancer therapeutics that can overcome the side effects of existing anticancer drugs, stemming from the diversity of cancers and their diversified pathogenesis.

[0005] Meanwhile, the cell-derived extracellular matrix (ECM) serves as the fundamental environment for cells to survive. It consists of protein structures and polymers and inorganic substances supplied by body fluids. These macromolecules form a complex network surrounding the cell exterior, occupying the space between cells and determining tissue morphology, providing an environment for cells to function normally.

[0006] In Korean Patent No. 10-0816395, an extracellular matrix derived from chondrocytes was collected, dried, and then an extracellular matrix membrane was manufactured. This not only replaces the periosteum or artificially manufactured collagen membranes used in cartilage regeneration, but also enables reimplantation of bone dura mater and repair of skin defects. In addition, Korean Patent Publication No. 10-2003-0093009 proposes a biodegradable polymer matrix for manufacturing artificial organs and a method for manufacturing the same, which can reduce immune rejection by culturing cells on a biodegradable polymer matrix and then removing the cells, leaving only the extracellular matrix secreted by the cells. However, these are materials that can be used for artificial organs using extracellular matrix, and there have been no cases of their use for the purpose of anticancer treatment.

[0007] Accordingly, the inventors of the present invention have made extensive efforts to solve the above-mentioned problems, cultured skin keratinocytes, and confirmed the effect of inhibiting cancer cell proliferation in the extracellular matrix obtained therefrom, thereby completing the present invention.

[0008] One aspect is to provide a pharmaceutical composition for preventing or treating cancer, which comprises an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0009] Another aspect is to provide a pharmaceutical composition for inhibiting angiogenesis comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0010] Another aspect is to provide a pharmaceutical composition for inhibiting cancer metastasis, which comprises an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0011] Another aspect is to provide a health functional food composition for preventing or improving cancer, which contains an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0012] Another aspect is to provide a feed composition for preventing or improving cancer, which contains an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0013] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for preventing or treating cancer.

[0014] Another aspect provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a composition comprising an effective amount of an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0015] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of cancer.

[0016] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for inhibiting angiogenesis.

[0017] Another aspect provides a method for inhibiting angiogenesis, comprising administering to a subject in need thereof a composition comprising an effective amount of an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0018] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicament for inhibiting angiogenesis.

[0019] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for inhibiting cancer metastasis.

[0020] Another aspect provides a method for inhibiting cancer metastasis, comprising administering to a subject in need thereof a composition comprising an effective amount of an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0021] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicament for inhibiting cancer metastasis.

[0022] One aspect provides a pharmaceutical composition for preventing or treating cancer, comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0023] The term "extracellular matrix (ECM)" as used herein refers to a complex aggregate of biopolymers that fills the space within or outside of tissues, and is composed of various molecules that are synthesized by cells, secreted outside the cells, and accumulated outside the cells. For example, the constituent molecules of the extracellular matrix include, but are not limited to, fibrous proteins such as collagen and elastin; complex proteins called proteoglycans and glycosaminoglycans; and cell adhesion glycoproteins such as fibronectin, laminin, and vitronectin. The constituent molecules of the extracellular matrix have multivalent binding sites, so that the same molecules aggregate with each other or compete with other molecules to build a large matrix. The extracellular matrix contains various types of proteins necessary for cell adhesion and cell signaling, and growth factors and cytokines essential for regulating stem cell differentiation may also be present.

[0024] Meanwhile, the extracellular matrix performs physical roles such as supporting and binding tissues, forming physical boundaries, absorbing force, and maintaining elasticity in structures such as the skeleton, teeth, tendons, and skin. Furthermore, the extracellular matrix also plays a physiochemical role in regulating cell proliferation, migration, differentiation, intracellular metabolism, and cell morphology from the outside of the cell. The extracellular matrix is ​​also involved in development, aging, cancer metastasis, tissue remodeling, wound healing, and biological defense, and transmits cellular information within the cell through integrins.

[0025] Regulation of cell function by the extracellular matrix is ​​achieved through a mechanism in which cells respond to the extracellular matrix by ultimately regulating gene expression as extracellular matrix signals are transmitted into the cell through the cell membrane.

[0026] Interactions between cells and the extracellular matrix play a crucial role in regulating various biological phenomena. For example, normal cell growth and proliferation require cell attachment and spreading to the extracellular matrix. Conversely, in the absence of cell-extracellular matrix interactions, extracellular matrix receptor expression acts as a negative regulator of cell growth.

[0027] The above extracellular matrix can be formed in a generally used cell flat culture method, and preferably can be extracted before cornification proceeds through the differentiation stage.

[0028] In one specific example, the extracellular matrix can be obtained by culturing skin keratinocytes. Specifically, the extracellular matrix can be obtained by culturing skin keratinocytes in agarose gel, collagen gel, laminin, fibronectin, or matrigel, but is not limited thereto.

[0029] In one specific example, the extracellular matrix may be obtained by culturing skin keratinocytes and then decellularizing the same. For example, the extracellular matrix derived from skin keratinocytes may be obtained by culturing skin keratinocytes in vitro and then decellularizing them. An extracellular matrix obtained through a decellularization process may be more effective in terms of anticancer efficacy.

[0030] The term "decellularization" as used herein means removing cellular components other than the extracellular matrix, such as the nucleus, cell membrane, nucleic acids, etc., from a cell or tissue.

[0031] The term "decellularized extracellular matrix" as used herein refers to the extracellular matrix remaining after cellular components such as the nucleus, cell membrane, and nucleic acids are removed from a tissue or cell. For example, the decellularized extracellular matrix may be obtained by removing only the nucleus and cell membrane of cells from a cell population, and when only the nucleus and cell membrane of cells are removed from a cell population, the entire extracellular matrix component can be utilized. The extracellular matrix obtained by removing only the nucleus and cell membrane of cells from a cell population can utilize the entire extracellular matrix component, thereby providing a more natural biomimetic microenvironment for cell growth and differentiation. In addition, the decellularized extracellular matrix has a structure in which nano- or micro-sized fiber bundles are formed in a consistent pattern.

[0032] In one specific example, the decellularized extracellular matrix may be obtained from cells cultured in vitro. For example, it may be obtained by culturing skin keratinocytes in vitro and decellularizing the cultured skin keratinocyte population. When the extracellular matrix obtained by decellularizing the in vitro cultured skin keratinocyte population is used in the composition for preventing, improving, or treating cancer according to the present invention, it may be more effective in terms of anticancer efficacy.

[0033] In addition, the extracellular matrix obtained by decellularizing the above skin keratinocyte population not only has superior anticancer efficacy compared to the extracellular matrix derived from skin tissue formed within an individual, but also, when the decellularized extracellular matrix is ​​obtained from cells cultured in vitro, the supply problem, which is a disadvantage of tissue-derived extracellular matrix, can be solved, and autologous cells can be used, so there is an advantage in that the possibility of an immune rejection reaction is low.

[0034] In one specific example, the decellularized extracellular matrix may be obtained by a method comprising: a step of culturing skin keratinocytes into a cell population; a step of primary decellularization by adding a solution containing a nonionic surfactant to the cell population; and a step of secondary decellularization by adding DNase and RNase A to the solution in which the primary decellularization is completed. The decellularized extracellular matrix of the present invention obtained by the above method has cancer cell proliferation inhibitory activity.

[0035] The decellularization method for obtaining the above-described decellularized extracellular matrix can be performed by a known method or an appropriate modification thereof. The decellularization method is intended to remove the remaining cellular components except for the extracellular matrix, and may use a physical method, a chemical method, or a mixed method thereof, and specifically, a chemical method may be used. The chemical method may be a process of immersing cultured cells in a decellularization solution for a certain period of time. The decellularization solution may be an acidic solution, an alkaline solution, a non-ionic detergent, or an ionic detergent, but is preferably a non-ionic detergent.

[0036] Specifically, the decellularization method may be a treatment with one or more selected from an ionic detergent, a nonionic detergent, a denaturant, a hypotonic solution, DNase, RNase, and ultrasound, and may be performed at a temperature range of 0 to 50° C. For example, keratinocytes may be cultured in a flask to form an extracellular matrix derived from keratinocytes, and then treated with Triton X-100, DNase, or RNase to obtain an extracellular matrix derived from keratinocytes.

[0037] In one specific example, the cancer is oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, bone cancer, non-small cell bone cancer, pancreatic cancer, skin cancer (melanoma, etc.), basal cell carcinoma, head cancer, cervical cancer, skin cancer, cervical cancer, ovarian cancer, ovarian germ cell tumor, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, salivary gland cancer, tongue cancer, esophageal cancer, duodenal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma, pseudomyxoma, urethral cancer, ureteral cancer, penile cancer, testicular cancer, prostate cancer, chronic leukemia, acute leukemia, multiple It may be at least one selected from myeloma, lymphocytic lymphoma, pediatric lymphoma, kidney cancer, renal pelvic cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, vulvar cancer, thymic cancer, central nervous system tumor, primary central nervous system lymphoma, neuroblastoma, astrocytoma, meningioma, choroidal melanoma, ampulla of Vater cancer, peritoneal cancer, small cell carcinoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, and preferably at least one solid cancer selected from breast cancer, kidney cancer, pancreatic cancer, lung cancer, and prostate cancer, and more preferably breast cancer.

[0038] In one specific example, a pharmaceutical composition for preventing or treating cancer, which includes the extracellular matrix derived from skin keratinocytes as an active ingredient, may be for animal use.

[0039] The above animal means any animal capable of harboring cancer, and may be, for example, a mammal including a human, and more specifically, may be, but is not limited to, a rat, mouse, dog, cat, livestock, etc.

[0040] In one specific example, a pharmaceutical composition for preventing or treating cancer, which includes the extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient, may inhibit the proliferation of cancer cells.

[0041] In one specific example, the composition may control the tumor microenvironment (TME).

[0042] The term "tumor microenvironment" as used herein may refer to the environment comprised of tissues and cells surrounding a tumor. For example, it may include, but is not limited to, tumor cells themselves, as well as surrounding blood vessels, extracellular matrix (surrounding tissue), immune cells, and inflammatory mediators.

[0043] In one specific example, controlling the tumor microenvironment may mean suppressing at least one selected from the group consisting of angiogenesis, inflammatory response, cell migration, and immunosuppression within the tumor microenvironment. For example, controlling the tumor microenvironment may involve regulating the expression of genes related to angiogenesis, inflammatory response, cell migration, or immunosuppression within the tumor microenvironment.

[0044] In one specific example, a pharmaceutical composition for preventing or treating cancer, which includes the extracellular matrix (ECM) derived from the skin keratinocytes as an active ingredient, may include 0.001 to 10.0 (w / v)% of the decellularized extracellular matrix.

[0045] For example, the pharmaceutical composition may contain the decellularized extracellular matrix in an amount of 0.001 to 10.0 (w / v)%, preferably 0.01 to 5.0 (w / v)%, more preferably 0.05 to 2.0 (w / v)%, and even more preferably 0.05 to 1.0 (w / v)%. When the concentration of the decellularized extracellular matrix contained in the composition of the present invention is within the above range, there is an advantage in that the anticancer effect by the decellularized extracellular matrix is ​​sufficiently exhibited, while not exhibiting toxicity or side effects to a living organism or its cells. When the content of the decellularized extracellular matrix of the present invention contained in the composition is less than the lower limit, the anticancer effect is not exhibited, and when it exceeds the upper limit, safety problems may arise, and it is not economical.

[0046] In one specific example, the pharmaceutical composition may be administered intratumorally.

[0047] As used herein, the term "administration" means introducing a given substance into a subject by an appropriate method. In the present invention, the pharmaceutical composition may be administered orally or parenterally during clinical administration. In the case of parenteral administration, it may be administered by intratumorally injection, intravenously injection, intradermally injection, subcutaneously injection, intramuscular injection, intrapulmonary injection, intranasal injection, intrarectally injection, intraperitoneally injection, intraspinal injection, intracardiac injection, intrathoracic injection, intraarterial injection, intraosseous injection, intraarticular injection, transdermally administration, etc., and is preferably administered intratumorally, but is not limited thereto. For injection, it may be formulated with a pharmacologically suitable buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. At this time, the ECM derived from skin keratinocytes may be processed into a patch form, gel form, dispersion form, etc. and included in a pharmaceutical composition for preventing or treating cancer.

[0048] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0049] The dosage of the pharmaceutical composition, medicament, veterinary pharmaceutical composition, or veterinary medicament may vary depending on the age, sex, and weight of the patient or animal to be treated, and will depend, among other things, on the condition of the subject to be treated, the specific category or type of disease to be treated, the route of administration, and the properties of the therapeutic agent used. The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of treatment for cancer.

[0050] The above pharmaceutical composition, medicine, veterinary pharmaceutical composition or veterinary medicine is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age and weight, sex and condition of the patient or animal to be treated, the severity of the disease to be treated, etc., but the daily dosage may be 0.0001 to 200 mg / kg, preferably 0.0001 to 100 mg / kg, more preferably 0.001 to 100 mg / kg, more preferably 0.01 to 100 mg / kg, more preferably 0.1 to 50 mg / kg. However, the actual dosage of the active ingredient may be determined in consideration of various related factors such as the amount of cells of the target tissue to be differentiated and proliferated, the route of administration, the body weight of the patient, the body weight of the animal, age, sex, health and ecology, diet, administration time, administration method, excretion rate and the severity of the disease, and may be administered once or several times a day in divided doses. Therefore, the above dosage does not limit the scope of the present invention in any form.

[0051] The above pharmaceutical composition, medicine, veterinary pharmaceutical composition or veterinary medicine may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0052] In one specific example, the pharmaceutical composition may be administered in combination with another anticancer agent.

[0053] The term "combination administration" as used herein refers to any form of simultaneous or concurrent treatment using substances useful for the treatment of at least two separate diseases. The components of the combination may be administered simultaneously, sequentially, in reverse order, or in any order. The components may be administered in different dosages, at different frequencies, or via different routes, as appropriate.

[0054] The term "administered simultaneously" as used herein is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

[0055] The term "administered sequentially" as used herein is not particularly limited and means that the components of the combination therapy are administered not simultaneously, but one after another or in clusters with a specific time interval between administrations. The time intervals may be the same or different between the administrations of each of the components of the combination therapy, and may be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

[0056] A combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy, as measured by, for example, the degree of response, the rate of response, the time to disease progression, or the duration of survival, is therapeutically superior to the efficacy obtained by administering one or more of the components of the combination therapy at their usual doses. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the components alone. In particular, a synergistic effect is considered to exist if the usual dose of the pharmaceutical composition or anticancer agent can be reduced without compromising one or more of the degree of response, the rate of response, the time to disease progression, and the survival data, particularly without compromising the duration of response, and with reduced and / or fewer problematic side effects than when each component is used at its usual dose.

[0057] The above other anticancer agents may include standard therapy, immune checkpoint inhibitors, immunotherapeutics, etc., and may also be therapeutic compositions and adjuvant therapeutic compositions for enhancing the therapeutic effect of standard therapy, but are not limited thereto. As described above, when the pharmaceutical composition of the present invention is administered in combination with other anticancer agents, the anticancer efficacy may be further enhanced compared to when the ECM derived from skin keratinocytes and the other anticancer agents are administered alone.

[0058] Another aspect is to provide a pharmaceutical composition for inhibiting angiogenesis comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0059] The term "angiogenesis" as used herein refers to the process of creating new blood vessels. Although it rarely occurs under normal biological conditions, it is a process that inevitably accompanies embryogenesis, corpus luteum formation, or wound healing. Generally, it is formed by the reorganization of blood vessels through the degradation of the vascular basement membrane by proteases, the migration and proliferation of vascular endothelial cells, and the formation of lumen by vascular endothelial cell differentiation by stimulation of angiogenic factors, thereby creating new capillaries. This angiogenic process plays an important role in the growth and metastasis of cancer, and by inhibiting angiogenesis, the amount of blood supplied to cancer cells is reduced, thereby inhibiting cancer growth.

[0060] The above-mentioned inhibition of angiogenesis can be achieved by regulating the expression of genes involved in the inhibition of angiogenesis. For example, the inhibition of angiogenesis within the tumor microenvironment can be achieved by increasing the expression of any one or more genes selected from the group consisting of PTGS2, CCL2, RHOB, CYP1B1, ANGPTL4, NRP2, CXCL8, and ZC3H12A compared to before treatment with a composition comprising an extracellular matrix derived from skin keratinocytes.

[0061] In one specific example, the inhibition of angiogenesis may mean inhibition of angiogenesis within a tumor microenvironment.

[0062] Another aspect is to provide a pharmaceutical composition for inhibiting cancer metastasis, which comprises an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0063] As used herein, the term "cancer metastasis" refers to the phenomenon in which cancer cells break away from the primary tumor, invade surrounding blood vessels or lymphatic vessels, and travel long distances to other parts of the body, forming new tumors. Inhibiting cancer metastasis can contribute to suppressing cancer progression by blocking the cancer cell migration pathways or preventing the formation of new tumors.

[0064] The above-mentioned inhibition of cancer metastasis can be achieved by regulating the expression of genes closely related to tumor growth, invasion, and metastasis within cancer cells. For example, the above-mentioned inhibition of cancer metastasis can be achieved by increasing the expression of any one or more genes selected from the group consisting of CCL2, CSF1, RHOB, IL1B, DNER, ITGA1, CYP1B1, TGFBR3, NRP2, IL6, PLAT, NTN4, CXCL8, ITGA2, JUP, CXCL1, and SAA1 compared to before treatment with a composition comprising an extracellular matrix derived from skin keratinocytes.

[0065] The above “pharmaceutical composition”, “medicine”, “veterinary pharmaceutical composition” or “veterinary medicine” may further include, in addition to the extracellular matrix derived from skin keratinocytes as an active ingredient, an appropriate carrier, excipient and diluent commonly used in the manufacture of pharmaceutical compositions, etc.

[0066] The term "pharmaceutical composition" as used herein means a mixture comprising one or more active ingredients and various ingredients that deliver or assist them for a specific therapeutic, preventive or diagnostic purpose.

[0067] The above "carrier" is a compound that facilitates the addition of a compound into cells or tissues. The above "excipient" is a compound added to formulate an active ingredient by imparting it with an appropriate form or to increase its quantity for convenient use. The above "diluent" is a compound that not only stabilizes the biologically active form of the target compound but also dilutes it in water, thereby dissolving the compound.

[0068] The carrier, excipient and diluent need not be particularly limited, but examples thereof include lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0069] As used herein, the term "treatment" may refer to the cure of cancer or other conditions in a shorter period of time compared to natural healing. Such treatment may include the improvement and / or palliation of cancer. Furthermore, such treatment may refer to the cure and / or recovery of symptoms caused by cancer.

[0070] As used herein, the term "prevention" refers to a method of partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its associated symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. For example, the term "prevention" refers to any action that inhibits or delays the development of cancer by administering a composition according to the present invention.

[0071]

[0072] Another aspect provides a health functional food composition for preventing or improving cancer, which contains an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0073] Another aspect provides a health functional food composition for inhibiting angiogenesis, which contains an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0074] A health functional food composition for inhibiting cancer metastasis is provided, which contains an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0075] Since the “decellularized extracellular matrix derived from skin keratinocytes,” “cancer,” “angiogenesis,” and “cancer metastasis” of the present invention have already been described above, their descriptions are omitted to avoid excessive duplication.

[0076] The term "health functional food" used herein refers to a food manufactured (including processed) in accordance with legal standards using raw materials or ingredients with functionality useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The term "health functional food" may vary in terminology and scope depending on the country, but may correspond to "dietary supplement" in the United States, "food supplement" in Europe, "health functional food" or "food for special health use (FoSHU)" in Japan, and "health food" in China. The health functional food may refer to a food manufactured or processed using a specific ingredient as a raw material or a specific ingredient contained in a food raw material by extracting, concentrating, refining, mixing, etc. for the purpose of health supplementation, and refers to a food designed and processed so that the above ingredients can sufficiently exert bioregulatory functions such as biological defense, biological rhythm regulation, disease prevention and recovery, etc. on the body. The health functional food may perform functions related to the prevention and improvement of cancer, etc.

[0077] The "health functional food composition" of the present invention may include, in addition to the decellularized extracellular matrix as an active ingredient, food raw materials that can be used as food as described in the standards and specifications for food commonly used in food manufacturing ("Food Code"), and food additives described in the Food Additive Code.

[0078] There are no special restrictions on the types of the above foods. Foods to which the composition according to one aspect can be added include, for example, various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, health functional foods, etc. More specifically, the composition includes formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and includes all health foods in the conventional sense.

[0079] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0080] As used herein, the term "food-acceptable" means that the compound exhibits the property of being non-toxic to cells, humans or animals exposed to the compound.

[0081] The above health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of a carrier, diluent, excipient, and additive.

[0082] In addition to containing the above-mentioned effective ingredient, the above-mentioned health functional food may contain other ingredients as essential ingredients without special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0083] In addition to the above, health functional foods according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0084] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0085] When preparing a food composition using the above decellularized extracellular matrix as an effective ingredient, the effective ingredient does not need to be particularly limited as long as it has an anticancer effect, but may be included in an amount of, for example, 0.001 to 10.0 (w / v)%, preferably 0.01 to 5.0 (w / v)%, more preferably 0.05 to 2.0 (w / v)%, and even more preferably 0.05 to 1.0 (w / v)%.

[0086] In the above health functional food composition, the decellularized extracellular matrix derived from skin keratinocytes, which is an active ingredient, may vary depending on the condition, weight, presence or severity of the disease, and duration of the ingestion, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 0.0001 to 200 mg / kg, preferably 0.0001 to 100 mg / kg, more preferably 0.001 to 100 mg / kg, more preferably 0.01 to 100 mg / kg, and even more preferably 0.1 to 50 mg / kg. The number of administrations need not be particularly limited, but may be adjusted by a person skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, the dosage may be below the above range.

[0087] The above health functional food may be provided in combination with a health functional food for preventing or improving cancer known in the past or another existing health functional food, and the other health functional food for preventing or improving cancer may be a health functional food for preventing or improving cancer known in the past, an existing health functional food, or a newly developed health functional food.

[0088] When the above health functional food contains other health functional foods that have a cancer prevention or improvement effect, it is important to mix them in an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

[0089] In addition, the decellularized extracellular matrix can be added to general foods to provide anticancer functionality. The foods to which it can be added are not particularly limited, but for example, it can be added to confectionery, bread or rice cakes, cocoa products or chocolates, meat or egg products, fish products, tofu or jelly products, noodles, tea, coffee, beverages, special-purpose foods, soy sauce, seasoned foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, dried foods, and other foods listed in the standards and ingredient specifications for livestock products listed in Article 4 of the Livestock Products Sanitation Management Act. In addition, it can be added to dairy products, processed meat products, packaged meat products, and egg products listed in the processing standards and ingredient specifications for livestock products listed in Article 4 of the Livestock Products Sanitation Management Act.

[0090]

[0091] Another aspect provides a feed composition for preventing or improving cancer, which contains an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0092] Another aspect provides a feed composition for inhibiting angiogenesis comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0093] Another aspect provides a feed composition for inhibiting cancer metastasis, which comprises an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0094] Since the "decellularized extracellular matrix derived from skin keratinocytes", "cancer", "inhibition of angiogenesis", and "cancer metastasis" of the present invention have already been described above, their descriptions are omitted to avoid excessive duplication.

[0095] The above "feed composition" may use, in addition to the decellularized extracellular matrix derived from skin keratinocytes as an active ingredient, food raw materials usable as food as described in the Food Standards and Specifications ("Food Code") and food additives described in the Food Additives Code, and even if they are not food raw materials or food additives usable as food, raw materials falling within the scope of single feed in Appendix 1 of the "Standards and Specifications for Feed, etc." and raw materials falling within the scope of supplementary feed in Appendix 2 may be used.

[0096] The above “feed composition” may be an extractant among supplementary feeds according to the “Standards and Specifications for Feed, etc.”, and may be a compound feed containing the supplementary feed.

[0097] When preparing the above feed composition, the decellularized extracellular matrix does not need to be particularly limited as long as it has an anticancer effect, but may be included in an amount of, for example, 0.001 to 10.0 (w / v)%, preferably 0.01 to 5.0 (w / v)%, more preferably 0.05 to 2.0 (w / v)%, and even more preferably 0.05 to 1.0 (w / v)%.

[0098] In the above feed composition, the decellularized extracellular matrix derived from skin keratinocytes, which is an effective ingredient, may vary depending on the condition, weight, presence or severity of disease, and duration of the ingesting animal, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 0.0001 to 200 mg / kg, preferably 0.0001 to 100 mg / kg, more preferably 0.001 to 100 mg / kg, more preferably 0.01 to 100 mg / kg, and even more preferably 0.1 to 50 mg / kg. The number of administrations need not be particularly limited, but may be adjusted by a person skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, the dosage may be below the above range.

[0099]

[0100] Another aspect provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a composition comprising an effective amount of an extracellular matrix (ECM) derived from keratinocytes as an active ingredient.

[0101] The method for treating the cancer may be to administer the composition intratumorally to a human or a non-human animal, particularly a mammal, for example, to administer the composition intratumorally to a subject having cancer.

[0102] The dosage, administration method, and number of administrations for the above treatment may refer to the dosage, administration method, and number of administrations of the pharmaceutical composition, medicine, veterinary pharmaceutical composition, or veterinary medicine.

[0103] Another aspect provides a method for inhibiting angiogenesis, comprising administering to a subject in need thereof a composition comprising an effective amount of an extracellular matrix (ECM) derived from keratinocytes as an active ingredient.

[0104] Another aspect provides a method for inhibiting cancer metastasis, comprising administering to a subject in need thereof a composition comprising an effective amount of an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

[0105] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of cancer.

[0106] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicament for inhibiting angiogenesis.

[0107] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicament for inhibiting cancer metastasis.

[0108] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for preventing or treating cancer.

[0109] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from keratinocytes as an active ingredient for inhibiting angiogenesis.

[0110] Another aspect provides the use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for inhibiting cancer metastasis.

[0111] The terms and methods described for the above inventions apply equally to each invention.

[0112] The composition for preventing, improving or treating cancer of the present invention contains an extracellular matrix derived from skin keratinocytes as an active ingredient, and thus can significantly reduce only the proliferation of cancer cells without causing side effects such as cytotoxicity to normal cells such as fibroblasts that constitute the dermis of normal skin.

[0113] In addition, the composition for preventing, improving or treating cancer of the present invention is expected to be very effective not only in MCF7 breast cancer cells but also in targeted therapy, etc., and has the effect of inhibiting the proliferation of MDA-MB-231, which currently has no clear treatment, and can significantly inhibit the proliferation of Caki-1 and Caki-2, which are clear cell carcinomas known to be intractable cancers among renal cancer cells and have a high rate of distant metastasis recurrence, and it was confirmed to significantly reduce the proliferation of PANC-1, a pancreatic cancer cell line, which is not only difficult to detect early and is often found at a late stage when surgery and treatment are limited, but also has a low therapeutic response to the latest immunotherapy drugs and a low therapeutic response to targeted therapy and traditional chemotherapy. In addition, the composition for preventing, improving or treating cancer of the present invention can be applied to the treatment of various carcinomas and solid tumors occurring in the skin as well as the breast cancer, renal cancer and pancreatic cancer.

[0114] In addition, the composition of the present invention for preventing, improving or treating cancer can be used as a combination treatment with other anticancer agents.

[0115] In addition, the composition for preventing, improving or treating cancer of the present invention can be used in a treatment method for reducing the size of cancer before surgery without side effects, unlike traditional chemotherapy, through intratrumoral injection.

[0116] Figure 1 is a graph showing the cytotoxicity of extracellular matrix derived from skin keratinocytes (HaCaT_ECM) according to one aspect of the present invention against human dermal fibroblasts (HDF) constituting the dermis of normal skin and various cancer cells (MDA-MB-231, MCF7, Caki-1, Caki-2, PANC-1, MiaPaCa-2, LNCaP, PC3, A549). *, **, and *** indicate significant differences (Bonferroni post hoc test) at p<0.05, p<0.01, and p<0.001, respectively, compared to each concentration treatment group.

[0117] Figure 2 is a graph showing the cytotoxicity of exosomes derived from UC-MSCs (Umbilical Cord-derived Mesenchymal Stem Cells), decellularized extracellular matrix (dECM) derived from UC-MSCs, and decellularized extracellular matrix (dECM) derived from porcine skin against HDFn (human dermal fibroblast), Caki-2, MDA-MB-231, A549, MiaPaCa-2, and PC3.

[0118] Figure 3 shows the results of RNA base sequence analysis after treating MDA-MB-231 cells with HaCaT_ECM according to one aspect. Figure 3a shows genes that changed by more than twofold by gene type compared to the control group that was not treated with HaCaT_ECM, categorized by gene type, Figure 3b shows the proportion of genes that showed increase and decrease in each gene category, and Figure 3c shows the genes that showed such changes by more than twofold in a scatter plot. In Figure 3c, blue dots indicate decrease, red dots indicate increase, and gray dots indicate genes that did not show a difference by more than twofold.

[0119] Figure 4 shows the results of RNA base sequence analysis of gene expression changes after treating MDA-MB-231 cells with HaCaT_ECM according to one aspect. Figure 4a shows changes in the expression of genes related to angiogenesis, Figure 4b shows changes in the expression of genes related to inflammation, Figure 4c shows changes in the expression of genes related to extracellular matrix, Figure 4d shows changes in the expression of genes related to cell migration, Figure 4e shows changes in the expression of genes related to apoptosis, and Figure 4f shows changes in the expression of genes related to immune checkpoint.

[0120] Figures 5a and 5b show the results of DAVID Functional Annotation analysis on 162 genes selected at the level of Fold change 2, Normalized Data 4 (Log 2), and p-value 0.05 through RNA base sequence analysis on MDA-MB-231 cells treated with one aspect of HaCaT_ECM.

[0121] Figure 6 shows the results of RNA base sequence analysis after treating MCF7 cell lines with HaCaT_ECM according to one aspect. Figure 6a shows genes that changed by more than twofold by gene type compared to the control group that was not treated with HaCaT_ECM, categorized by gene type, Figure 6b shows the proportion of genes that showed increase and decrease in each gene category, and Figure 6c shows the genes that showed such changes by more than twofold in a scatter plot. In Figure 6c, blue dots indicate decreases, red dots indicate increases, and gray dots indicate genes that did not show a difference by more than twofold.

[0122] Figure 7 shows the results of RNA base sequence analysis of gene expression changes after treating MCF7 cells with HaCaT_ECM according to various aspects. Figure 7a shows changes in the expression of DNA repair-related genes, Figure 7b shows changes in the expression of cell cycle-related genes, Figure 7c shows changes in the expression of apoptosis-related genes, Figure 7d shows changes in the expression of cell senescence-related genes, and Figure 6e shows changes in the expression of RNA splicing-related genes.

[0123] Figures 8a and 8b show the results of DAVID Functional Annotation analysis on 1403 genes selected at the level of Fold change 2, Normalized Data 4 (Log 2), and p-value 0.05 through RNA base sequence analysis on MCF7 cells treated with HaCaT_ECM according to one aspect.

[0124] Figure 9 is a photograph showing a mouse tumor 3 weeks after administration of HaCaT_ECM according to the daily pattern.

[0125] Figure 10 is a graph measuring changes in tumor size according to HaCaT_ECM administration according to daily aspect.

[0126] Figure 11 is a graph measuring the change in body weight of mice according to HaCaT_ECM administration according to daily aspects.

[0127] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0128]

[0129] Example 1. Obtaining decellularized extracellular matrix (HaCaT_ECM) derived from skin keratinocytes

[0130] After culturing skin keratinocytes (HaCaT), extracellular matrix (HaCaT_ECM) was obtained through a decellularization process.

[0131] Specifically, 2 Х 10 skin keratinocytes (HaCaT) were cultured in a T-175 flask. 4 cells / cm 2 The cells were seeded at a density of 10 μg / mL and cultured under the conditions of Dulbecco's Modified Eagle medium, 10% fetal bovine serum, and 1% antibiotics. When the cell confluency reached 80%, the cells were washed with phosphate-buffered saline (PBS) and then lysed with a mixture of 0.25% Triton X-100 and 10 mM NH4OH at 37°C for 90 seconds (Cell lyse). Afterwards, DNase (Deoxyribonuclease °) and RNase A at a concentration of 50 U / mL were added and reacted for 90 minutes to remove nucleic acids. The obtained extracellular matrix was washed with PBS and the final product was freeze-dried at -80°C to obtain decellularized extracellular matrix derived from skin keratinocytes (HaCaT_ECM).

[0132]

[0133] Experimental Example 1: Confirmation of the cancer cell proliferation inhibition ability of decellularized extracellular matrix (HaCaT_ECM) derived from skin keratinocytes.

[0134] It was confirmed through CCK-8 assay whether the decellularized extracellular matrix (HaCaT_ECM) derived from skin keratinocytes obtained by the method of Example 1 exhibits cytotoxicity against skin fibroblasts (HDFn; human dermal fibroblasts) and various cancer cells.

[0135] Figure 1 is a graph showing the cytotoxicity of extracellular matrix derived from keratinocytes (HaCaT_ECM) according to the daily aspect against human dermal fibroblasts (HDF) that constitute the dermis of normal skin and various cancer cells.

[0136] As shown in Fig. 1, it was specifically confirmed that HaCaT_ECM obtained by the method of Example 1 significantly inhibited the proliferation of breast cancer cell lines (MCF7), pancreatic cancer cell lines (PANC-1, MiaPaCa-2), renal cancer cell lines (Caki-1, Caki-2), prostate cancer cell lines (LNCaP, PC3), and lung cancer cell lines (A549), including hormone receptor triple-negative breast cancer cell lines (MDA-MB-231), which are representative refractory solid cancers, without any effect on normal human dermal fibroblast (HDFn) cells at all concentrations.

[0137] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient significantly reduces the proliferation of various cancer cells without affecting normal cells, thereby selectively acting on cancer cells and exhibiting an anticancer effect.

[0138]

[0139] Experimental Example 2. Evaluation of the cancer cell proliferation inhibition ability of UC-MSC-derived exosomes, UC-MSC-derived decellularized extracellular matrix, and porcine skin-derived decellularized extracellular matrix.

[0140] The decellularized extracellular matrix (HaCaT_ECM) derived from skin keratinocytes obtained by the method of Example 1, the exosomes derived from UC-MSCs (Umbilical Cord-derived Mesenchymal Stem Cells), the decellularized extracellular matrix (dECM) derived from UC-MSCs, and the decellularized extracellular matrix (dECM) derived from porcine skin were treated to renal cell carcinoma cell lines (Caki-2), hormone receptor triple-negative breast cancer cell lines (MDA-MB-231), lung cancer cell lines (A549), pancreatic cancer cell lines (MiaPaCa-2), and prostate cancer cell lines (PC3), respectively, and the cytotoxicity against cancer cells was confirmed through the CCK-8 assay.

[0141] Figure 2 is a graph showing the cytotoxicity of exosomes derived from UC-MSCs (Umbilical Cord-derived Mesenchymal Stem Cells), decellularized extracellular matrix (dECM) derived from UC-MSCs, and decellularized extracellular matrix (dECM) derived from porcine skin against HDFn (human dermal fibroblast), Caki-2, MDA-MB-231, A549, MiaPaCa-2, and PC3.

[0142] As shown in Fig. 2, it was confirmed that renal cell carcinoma cell line (Caki-2) and hormone receptor triple-negative breast cancer cell line (MDA-MB-231) treated with UC-MSC-derived exosomes, UC-MSC-derived decellularized extracellular matrix (dECM), and porcine skin-derived decellularized extracellular matrix (dECM) showed cell proliferation at a level similar to that of normal cells, HDFn (Human derml fibroblast), at all concentrations.

[0143] Meanwhile, it was confirmed that lung cancer cell line (A549) treated with UC-MSC-derived decellularized extracellular matrix (dECM) at a concentration of 10 to 3000 μg / mL showed more active cell proliferation than normal cells, HDFn (Human derml fibroblast).

[0144] In addition, it was confirmed that pancreatic cancer cell lines (MiaPaCa-2) treated with 10 to 100 μg / mL of UC-MSC-derived exosomes, 10 to 100 μg / mL of UC-MSC-derived decellularized extracellular matrix (dECM), and 100 to 3000 μg / mL of porcine skin-derived decellularized extracellular matrix (dECM) showed more active cell proliferation than normal cells (HDFn) (Human delaml fibroblast).

[0145] In addition, it was confirmed that prostate cancer cell line (PC3) treated with 100 to 3000 μg / mL of UC-MSC-derived exosomes, 3000 μg / mL of UC-MSC-derived decellularized extracellular matrix (dECM), and 10 to 3000 μg / mL of porcine skin-derived decellularized extracellular matrix (dECM) showed more active cell proliferation than normal cells HDFn (Human delaml fibroblast).

[0146] These experimental results imply that UC-MSC-derived exosomes, UC-MSC-derived decellularized extracellular matrix (dECM), and porcine skin-derived decellularized extracellular matrix (dECM) not only do not have an inhibitory effect on cancer cell proliferation, but may even promote cancer cell proliferation.

[0147]

[0148] Experimental Example 3. Analysis of gene expression changes in cancer cells before and after HaCaT_ECM treatment.

[0149] 3.1 Confirmation of gene expression changes in MDA-MB-231 cells

[0150] 3.1.1 Identifying gene groups with significant expression changes

[0151] First, MDA-MB-231 cells, a hormone receptor triple-negative breast cancer cell line, were treated with HaCaT_ECM obtained by the method of Example 1 at a concentration of 3000 μg / mL and cultured for 24 hours. Total RNA was extracted from the culture using the RNeasy Mini kit (Qiagen), and the concentration was measured using NanoVue (GE HealthCare, USA), after which mRNA sequence analysis was requested (ebiogen, Republic of Korea).

[0152] Figure 3 shows the results of RNA base sequence analysis after treating MDA-MB-231 cells with HaCaT_ECM according to one aspect: Figure 3a shows genes that changed by more than twofold by gene type compared to the control group that was not treated with HaCaT_ECM, categorized by gene type, Figure 3b shows the proportion of genes that showed increase and decrease in each gene category, and Figure 3c shows the genes that showed such two-fold or more changes in a scatter plot. In Figure 3c, blue dots indicate decreases, red dots indicate increases, and gray dots indicate genes that did not show a difference of more than twofold.

[0153] As shown in Fig. 3, it was confirmed that in MDA-MB-231 treated with HaCaT_ECM according to an embodiment of the present invention, there was a notable change in the expression of genes related to angiogenesis, inflammatory response, extracellular matrix, cell migration, apoptotic process (cell death), and immune response (an expression level difference of more than two times compared to before HaCaT_ECM treatment).

[0154]

[0155] 3.1.2 Confirming changes in the expression of specific genes

[0156] The changes in gene expression after treating MDA-MB-231 cells, a hormone receptor triple-negative breast cancer cell line, with HaCaT_ECM obtained by the method of Example 1 were specifically observed through RNA base sequence analysis.

[0157]

[0158] Angiogenesis-related genes

[0159] Figure 4a shows the results of analyzing changes in the expression of genes related to angiogenesis in MDA-MB-231 cells treated with HaCaT_ECM according to Example 1.

[0160] As shown in Fig. 4a, when HaCaT_ECM was treated in MDA-MB-231 cells, the expression of PTGS2, CCL2, RHOB, CYP1B1, ANGPTL4, NRP2, CXCL8, and ZC3H12A genes, which are genes related to angiogenesis, increased by more than 2-fold compared to before HaCaT_ECM treatment.

[0161] The genes whose expression increased more than twice compared to before HaCaT_ECM treatment are related to angiogenesis in the tumor microenvironment (TME). In particular, PTGS2 is an enzyme involved in prostaglandin (PG) synthesis, which promotes angiogenesis and affects tumor growth and metastasis. CCL2 is a chemokine that induces an inflammatory response related to angiogenesis in the TME and also affects extracellular matrix (ECM) remodeling. RHOB is a gene of the Rho GTPase family and is involved in regulating angiogenesis through the interaction between tumor cells and the extracellular matrix (ECM), and CYP1B1 is involved in promoting inflammation and angiogenesis by generating ROS (reactive oxygen species) in response to various environmental stimuli.

[0162] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient exhibits an anticancer effect through tumor microenvironment (TME) control by not only inhibiting tumor cell proliferation but also regulating the expression of genes related to angiogenesis.

[0163]

[0164] Inflammatory response-related genes

[0165] Figure 4b shows the results of analyzing changes in the expression of genes related to inflammatory response in MDA-MB-231 cells treated with HaCaT_ECM according to Example 1.

[0166] As shown in Fig. 4b, when HaCaT_ECM was treated in MDA-MB-231 cells, the expression of PTGS2, CCL2, CSF1, C3, IL1B, IL1A, ELF3, IL6, SAA2, CXCL8, ZC3H12A, VNN1, CXCL1, and SAA1 genes, which are genes related to inflammatory response, increased by more than 2-fold compared to before HaCaT_ECM treatment.

[0167] The genes whose expression increased more than twice compared to before HaCaT_ECM treatment are related to the inflammatory response in the tumor microenvironment (TME). In particular, PTGS2 (COX-2) is involved in the inflammatory response, pain, fever, and promotion of angiogenesis, and CCL2 is a chemokine that is involved in the inflammatory response of tumors and the influx of immune cells. CSF1 is involved in the recruitment and activation of macrophages, playing an important role in the immune response and progression of tumors. When activated, C3 is decomposed into C3a and C3b. C3a promotes inflammation, and C3b binds to the surface of tumor cells and is involved in the removal (opsonization) of tumor cells by immune cells.

[0168] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient exhibits an anticancer effect through tumor microenvironment (TME) control by not only inhibiting tumor cell proliferation but also regulating the expression of genes related to inflammatory responses.

[0169]

[0170] extracellular matrix-related genes

[0171] Figure 4c shows the results of analyzing changes in the expression of extracellular matrix-related genes in MDA-MB-231 cells treated with HaCaT_ECM according to Example 1.

[0172] As shown in Fig. 4c, when HaCaT_ECM was treated in MDA-MB-231 cells, the expression of extracellular matrix-related genes, GDF15, MMP1, ANGPTL4, TFP12, TGFBR3, VASN, PLAT, TIMP3, NTN4, SERPINE2, ANGPTL2, and S100A4, increased by more than 2-fold compared to before HaCaT_ECM treatment.

[0173] The genes whose expression increased more than twice compared to before HaCaT_ECM treatment are related to extracellular matrix remodeling. In particular, GDF15 is a gene belonging to the TGF-beta superfamily, and its expression increases under inflammation and stress, and is involved in tumor growth and metastasis. MMP1 is an enzyme that decomposes collagen, a major component of the extracellular matrix (ECM), and is involved in ECM remodeling and tumor metastasis. ANGPTL4 is involved in angiogenesis and ECM remodeling, tumor growth and metastasis, and TFP12 regulates the interaction between tumor cells and surrounding cells (fibroblasts, immune cells), and is involved in ECM remodeling, inflammation, and immune cell infiltration.

[0174] These experimental results indicate that the composition of the present invention, which contains the extracellular matrix derived from skin keratinocytes as an active ingredient, exhibits an anticancer effect through tumor microenvironment (TME) control by not only inhibiting tumor cell proliferation but also regulating the expression of genes related to extracellular matrix remodeling.

[0175]

[0176] Cell migration-related genes

[0177] Figure 4d shows the results of analyzing changes in the expression of genes related to cell migration in MDA-MB-231 cells treated with HaCaT_ECM according to Example 1.

[0178] As shown in Fig. 4d, when HaCaT_ECM was treated on MDA-MB-231 cells, it was confirmed that the expression of CCL2, CSF1, RHOB, IL1B, DNER, ITGA1, CYP1B1, TGFBR3, NRP2, IL6, PLAT, NTN4, CXCL8, ITGA2, JUP, CXCL1, and SAA1 genes, which are genes related to cell migration, increased by more than 2-fold compared to before HaCaT_ECM treatment.

[0179] The genes whose expression increased more than twice compared to before HaCaT_ECM treatment are related to cell migration, which is closely related to tumor growth, invasion, and metastasis. In particular, CCL2 is involved in tumor growth and metastasis by promoting macrophage and T cell infiltration in the TME, and CSF1 is involved in tumor inflammatory response and immune evasion by regulating the function of macrophages in the TME. RHOB is involved in regulating the interaction between tumor cells and the ECM, thereby regulating tumor cell migration and invasion. IL1B is involved in promoting inflammatory cell migration in the TME and promoting tumor cell migration and invasion. DNER is a protein related to the Notch signaling pathway and is involved in tumor cell migration and invasion.

[0180] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient exhibits an anticancer effect through tumor microenvironment (TME) control by not only inhibiting tumor cell proliferation but also regulating the expression of genes related to cell migration.

[0181]

[0182] apoptosis-related genes

[0183] Figure 4e shows the results of analyzing changes in the expression of genes related to apoptosis (apoptotic process, cell death) in MDA-MB-231 cells treated with HaCaT_ECM according to Example 1.

[0184] As shown in Fig. 4e, when HaCaT_ECM was treated in MDA-MB-231 cells, the expression of TNFSF15, RHOB, IL1B, IL24, LCN2, CYP1B1, ANGPTL4, IL1A, IL6, NFKBIA, SOD2, MAP3K5, IER3, SQSTM1, ZC3H12A, BCL3, and CARD6 genes, which are genes related to apoptosis, increased by more than 2-fold compared to before HaCaT_ECM treatment.

[0185] The genes whose expression increased more than twice compared to before HaCaT_ECM treatment are related to apoptosis. In particular, TNFSF15 is involved in the activation of immune cells and regulation of inflammatory responses, and RHOB is involved in the death and migration of tumor cells. IL1B induces apoptosis and cell death through regulation of inflammation and immune responses, IL24 promotes cell death by inducing inflammation, apoptosis, and autophagy, and LCN2 is involved in the regulation of inflammation and immune responses and in apoptosis and autophagy. ANGPTL4 is involved in lipid metabolism, affecting the metabolism and growth of tumor cells, and SOD2 is involved in suppressing oxidative stress and cell protection.

[0186] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient exhibits an anticancer effect through tumor microenvironment (TME) control by not only inhibiting tumor cell proliferation but also regulating the expression of genes related to apoptosis.

[0187]

[0188] immune checkpoint-related genes

[0189] Figure 4f shows the results of analyzing changes in the expression of immune checkpoint-related genes in MDA-MB-231 cells treated with HaCaT_ECM according to Example 1.

[0190] As shown in Fig. 4f, when HaCaT_ECM was treated in MDA-MB-231 cells, the expression of the C10ORF54 (VISTA) and DIDO1 genes, which are genes related to the immune checkpoint, increased compared to before HaCaT_ECM treatment, and the expression of the CD276 (B7-H3) and CD274 (PD-L1) genes was the same or decreased compared to before HaCaT_ECM treatment.

[0191] C10ORF54 (VISTA), a gene whose expression increased compared to before HaCaT_ECM treatment, is involved in suppressing T cell activation through immunosuppressive function, and DIDO1 creates an immunosuppressive environment by regulating the tryptophan metabolic pathway.

[0192] CD276 (B7-H3), a gene whose expression changed minimally compared to before HaCaT_ECM treatment, suppresses T cell activation and promotes immune evasion of tumors.

[0193] CD274 (PD-L1), a gene whose expression was reduced compared to before HaCaT_ECM treatment, is expressed in tumor cells and immune cells, and PD-1 (PDCD1) is expressed in T cells, and PD-L1 / PD-1 is involved in immune evasion.

[0194] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient exhibits an anticancer effect through tumor microenvironment (TME) control by not only inhibiting tumor cell proliferation but also regulating the expression of genes related to the immune checkpoint.

[0195]

[0196] 3.1.3 DAVID - Functional Annotation Analysis

[0197] DAVID Functional Annotation analysis was performed on 162 genes selected at the level of Fold change 2, Normalized Data 4 (Log 2), and p-value 0.05 through RNA base sequence analysis of MDA-MB-231 cells treated with HaCaT_ECM according to Example 1, and the results are shown in Figs. 5a and 5b.

[0198] Figures 5a and 5b show the results of DAVID Functional Annotation analysis on 162 genes selected at the Fold change 2, Normalized Data 4 (Log 2), and p-value 0.05 levels through RNA sequencing of MDA-MB-231 cells treated with one aspect of HaCaT_ECM. Specifically, genes related to the IL-17 signaling pathway, TNF signaling pathway, hematopoietic cell lineage, and cytokine-cytokine receptor interaction are well known to be closely related to the tumor microenvironment. Increased gene expression in the rheumatoid arthritis, legionellosis, and leishmaniasis pathways indicates activation of the immune response. In addition, increased gene expression in the lipid and atherosclerosis and fluid shear stress and atherosclerosis pathways indicates increased gene expression related to lipid metabolism, which implies that it can induce growth inhibition or apoptosis of tumor cells. The fluid shear stress pathway is associated with cellular stress and can induce TME changes and cell death, suggesting that increased gene expression in the alcoholic liver disease pathway can induce cell death through cytotoxic rebound and oxidative stress.

[0199] As shown in FIGS. 5a and 5b, when MDA-MB-231 cells were treated with HaCaT_ECM obtained by the method of Example 1, not only was there an inhibitory effect on tumor cell proliferation in the tumor microenvironment (TME), but also changes in genes related to the IL-17 signaling pathway involved in angiogenesis and immunosuppression; activation of signal transduction related to tumor necrosis and regulation of inflammation and immune response due to an increase in NFKB1A, IL6, CSF1, IL1B, CCL2, CXCL1, PTGS2 (COX-2) and MAP3K5 (ASK1) of the TNF signaling pathway; changes in expression between pro-inflammatory and anti-inflammatory factors such as IL1A, IL6, CXCL8 and CSF3 related to cytokine-cytokine receptor interaction; It was found that it exhibits an antitumor effect by regulating the expression of C10ORF54 (VISTA), DIDO1, CD276 (B7-H3), and CD274 (PD-L1) genes related to the immune checkpoint.

[0200]

[0201] 3.2 Confirmation of gene expression changes in MCF7 cells

[0202] 3.2.1 Identifying gene groups with significant expression changes

[0203] First, MCF7 cells, a breast cancer cell line, were treated with HaCaT_ECM according to the example at a concentration of 3000 μg / mL and cultured for 24 hours. Total RNA was extracted from the culture using the RNeasy Mini kit (Qiagen), the concentration was measured using NanoVue (GE HealthCare, USA), and then mRNA base sequence analysis was requested (ebiogen, Republic of Korea).

[0204] Figure 6 shows the results of RNA base sequence analysis after treating MCF7 cells with HaCaT_ECM according to Example 1: Figure 6a shows genes that changed by more than twofold by gene type compared to the control group that was not treated with HaCaT_ECM, categorized by gene type, Figure 6b shows the proportion of genes that showed an increase and a decrease in each gene category, and Figure 6c shows the genes that showed such a two-fold or more change in a scatter plot. In Figure 6c, blue dots indicate a decrease, red dots indicate an increase, and gray dots indicate genes that did not show a two-fold or more difference.

[0205] As shown in Figures 6a to 6c, it was confirmed that in MCF7 cells treated with HaCaT_ECM, there was a notable change in the expression of genes related to DNA repair, cell cycle, apoptosis, aging, and RNA splicing (an expression level difference of more than two times compared to before HaCaT_ECM treatment).

[0206]

[0207] 3.2.2 Changes in the expression of specific genes

[0208] The changes in gene expression after treating MCF7 cells, a breast cancer cell line, with HaCaT_ECM obtained by the method of Example 1 were specifically observed through RNA base sequence analysis.

[0209]

[0210] DNA repair related genes

[0211] Figure 7a shows the results of analyzing changes in the expression of genes related to DNA repair in MCF7 cells treated with HaCaT_ECM according to Example 1.

[0212] As shown in Fig. 7a, when HaCaT_ECM was treated in MCF7 cells, the expression of MCM3, FEN1, HMGB1, UNG, MCM4, PTTG1, BRCA2, HMGB2, MCM2, CLSPN, BRIP1, and CDK1 genes, which are genes related to DNA repair, decreased by more than 4 times compared to before HaCaT_ECM treatment. Among the DNA repair-related genes whose expression decreased by more than 4 times compared to before HaCaT_ECM treatment, CDK1 (cyclin-dependent kinase 1) in particular regulates the entry from G2 phase to M phase and cell division in relation to cell cycle control. Inhibition of CDK1 expression causes cells to arrest in G2 phase, which buys time to repair damaged DNA, or promotes apoptosis if repair is impossible. BRIP1 (BRCA 1 interacting protein C-terminal helicase 1) is a protein involved in DNA repair by interacting with BRCA1. Inhibition of BRIP1 expression reduces DNA repair capacity, promoting cell death. CLSPN (claspin) is a protein that plays a crucial role in the DNA repair checkpoint. When CLSPN expression is suppressed, the G2 / M checkpoint function is weakened, the cell cycle is arrested, and genetic instability increases, leading to cell death and suppressing cancer cell proliferation.

[0213] Decreased expression of genes involved in DNA repair can lead to disruption of the DNA repair process, leading to accumulated DNA damage that can promote cellular stress responses and apoptosis and trigger immune responses in the tumor microenvironment (TME). Furthermore, the stress response to DNA damage can influence the expression of inflammatory cytokines and growth factors, thereby influencing changes in the extracellular matrix (ECM) of the tumor microenvironment (TME).

[0214] These experimental results indicate that the composition of the present invention, which contains the extracellular matrix derived from skin keratinocytes as an active ingredient, not only inhibits tumor cell proliferation but also promotes cancer cell death by regulating the expression of genes related to DNA repair, and exhibits an anticancer effect by controlling the tumor microenvironment (TME).

[0215]

[0216] cell cycle-related genes

[0217] Figure 7b shows the results of analyzing changes in the expression of cell cycle-related genes in MCF7 cells treated with HaCaT_ECM according to Example 1.

[0218] As shown in Fig. 7b, when HaCaT_ECM was treated in MCF7 cells, the expression of SMC2, TUBA1B, PRC1, KIP20B, MCM3, FEN1, TPX2, TOP2A, STMN1, CENPF, MKI67, BRIP1, MYBL2, and ASPM genes, which are cell cycle-related genes, decreased by more than 4 times compared to before HaCaT_ECM treatment, and the expression of DDIT3 and CDKN1A genes increased by more than 7 times compared to before HaCaT_ECM treatment.

[0219] Among the cell cycle-related genes that were decreased by more than 4 times compared to before the HaCaT_ECM treatment, in particular, MKI67 (marker of proliferation Ki-67) is a gene that is mainly expressed when cells divide and is a major indicator of cell proliferation. In other words, the decrease in the expression of MKI67 indicates that cell proliferation is decreasing. BRIP1 (BRCA1 Interacting Protein C-terminal Helicase 1) is a protein that plays an important role in the DNA damage repair process. When the expression of BRIP1 is decreased, DNA repair efficiency decreases, cell survival becomes difficult, and apoptosis is induced. MYBL2 (MYB proto-oncogene like 2) is a transcription factor that is important for cell cycle regulation and cell proliferation. When the expression of MYBL2 is decreased, cell cycle progression is inhibited and cell proliferation is suppressed. ASPM (abnormal spindle microtubule assembly) is a gene involved in cell cycle progression and cell division. When the expression of ASPM is decreased, cell division is inhibited and the cell proliferation rate is lowered.

[0220] When HaCaT_ECM treatment reduces the expression of these cell cycle-related genes, cancer cell proliferation is suppressed, apoptosis is induced, immune cell infiltration into the tumor microenvironment (TME) is promoted, and an inflammatory response is induced. Furthermore, by activating the tumor microenvironment (TME) in an immune-activated state, anti-tumor immune responses can be enhanced.

[0221] Meanwhile, among the cell cycle-related genes that increased more than 7-fold compared to before the HaCaT_ECM treatment, CDKN1A (p21, cyclin-dependent kinase inhibitor 1A) in particular is a cell cycle inhibitor induced by the p53 tumor suppressor protein. Increased CDKN1A expression inhibits the activity of the cyclin-CDK complex, blocks cell cycle progression at the G1 / S or G2 / M checkpoint, and induces cell death. DDIT3 (DNA damage inducible transcript 3) is a transcription factor induced under endoplasmic reticulum (ER) stress or various cellular stress situations, and increased DDIT3 expression induces cell death.

[0222] When the expression of the above cell cycle-related genes is increased by HaCaT_ECM treatment, an anti-tumor effect can be exhibited that suppresses cancer cells through inhibition of each cell cycle and induction of cell death. In addition, antigens released from dead cancer cells can promote an anti-cancer immune response that activates immune cells in the tumor microenvironment (TME). In addition, the increased expression of stress response genes such as DDIT3 can affect other cells such as fibroblasts and endothelial cells within the tumor microenvironment (TME), thereby suppressing the survival and proliferation of cancer cells within the tumor microenvironment (TME).

[0223] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient not only inhibits tumor cell proliferation but also promotes cancer cell death by regulating the expression of genes related to the cell cycle and exhibits an anticancer effect through control of the tumor microenvironment (TME).

[0224]

[0225] apoptosis-related genes

[0226] Figure 7c shows the results of analyzing changes in the expression of genes related to apoptosis (apoptotic process) in MCF7 cells treated with HaCaT_ECM according to Example 1.

[0227] As shown in Fig. 7c, when HaCaT_ECM was treated in MCF7 cells, the expression of HMGB2, TPX2, and TOP2A genes, which are genes related to apoptosis, decreased by more than 4 times compared to before HaCaT_ECM treatment, and the expression of DDIT4, NUPR1, CDKN1A, BBC3, HSPA5, CYP1B1, DDIT3, SQSTM1, JUN, and CEBPB genes increased by more than 5 times compared to before HaCaT_ECM treatment.

[0228] Among the apoptosis-related genes whose expression was reduced by more than 4 times compared to before the HaCaT_ECM treatment, HMGB2 (high mobility group box 2) in particular plays an important role in cell growth, differentiation, and DNA damage response by regulating the structure of chromatin and regulating gene expression. When HMGB2 expression is reduced, DNA repair ability is reduced, which reduces cell survival ability. TOP2A (topisomerase ±) plays a role in unwinding the helical structure of DNA during DNA replication and transcription. When TOP2A expression is reduced, DNA replication and segregation are not performed properly, leading to cell cycle arrest or cell death. TPX2 (targeting protein for Xklp2) is a microtubule-associated protein of the cell and is essential for spindle formation. When TPX2 expression is reduced, errors in the cell division process are caused, leading to cell death.

[0229] When HaCaT_ECM treatment reduces the expression of these apoptosis-related genes, cell cycle progression and DNA repair capacity of cancer cells are impaired, leading to apoptosis. Increased apoptosis in cancer cells can activate immune cells within the tumor microenvironment (TME), enhancing the anti-tumor immune response.

[0230] Among the apoptosis-related genes whose expression increased by more than 5 times compared to before the HaCaT_ECM treatment, DDIT4 (DNA damage inducible transcript 4) in particular increases in expression under cellular stress conditions such as hypoxia or nutrient deficiency, and inhibits cell growth and induces apoptosis by inhibiting the mTORC1 (mammalian Target of Rapamycin Complex 1) pathway. NUPR1 (nuclear protein 1) expression increases under cellular stress conditions, and if the increase in NUPR1 expression continues, apoptosis is induced. CDKN1A (cyclin-dependent kinase inhibitor 1A) is induced by the p53 tumor suppressor protein, and when the expression of CDKN1A increases, the cell cycle and cell proliferation are inhibited, and cell senescence and apoptosis are promoted.

[0231] When the expression of the above apoptosis-related genes increases through HaCaT_ECM treatment, anticancer immunity responses can be promoted by stimulating dendritic cells and macrophages, which are immune cells within the tumor microenvironment (TME).

[0232] These experimental results indicate that the composition of the present invention, which contains the extracellular matrix derived from skin keratinocytes as an active ingredient, not only inhibits tumor cell proliferation but also promotes cancer cell death by regulating the expression of genes related to cell death, and exhibits an anticancer effect by controlling the tumor microenvironment (TME).

[0233]

[0234] aging-related genes

[0235] Figure 7d shows the results of analyzing changes in the expression of aging-related genes in MCF7 cells treated with HaCaT_ECM according to Example 1.

[0236] As shown in Fig. 7d, when HaCaT_ECM was treated in MCF7 cells, the expression of IGFBP5, TYMS, GFRA1, and AURKB genes, which are aging-related genes, decreased by more than 4 times compared to before HaCaT_ECM treatment, and the expression of ASS1, SOD2, LONP1, JUND, ATP2B1, FOXO3, GCLM, and NFE2L2 genes increased by more than 2 times compared to before HaCaT_ECM treatment.

[0237] Among the aging-related genes whose expression decreased by more than fourfold compared to before HaCaT_ECM treatment, TYMS (thymidylate synthase) is particularly involved in thymidine synthesis during DNA synthesis. Since DNA synthesis is essential for cell proliferation, decreased TYMS expression inhibits cell division and proliferation. GFRA1 (GDNF family receptor alpha 1) interacts with glial cell-derived neurotrophic factor (GDNF) to transmit cell survival signals. GFRA1 is primarily involved in cell survival and growth in the nervous system, but activation of this pathway in some cancer cells is known to promote cancer cell survival and proliferation. Decreased GFRA1 expression prevents cancer cells from receiving sufficient survival signals, lowering their survival rate and promoting cell death under stressful situations. AURKB (aurora kinase B) is involved in chromosome alignment and segregation during mitosis. Decreased AURKB expression inhibits cell division and proliferation.

[0238] When the expression of these aging-related genes is reduced by HaCaT_ECM treatment, cancer cell proliferation is suppressed, which not only promotes immune activation within the tumor microenvironment (TME), but also increases the availability of resources that can be utilized by immune cells and fibroblasts. In other words, the reduction in the expression of these aging-related genes by HaCaT_ECM treatment changes the overall characteristics of the tumor microenvironment (TME), creating an environment more unfavorable to cancer cell survival.

[0239] Meanwhile, among the aging-related genes that increased more than twice compared to before HaCaT_ECM treatment, ASS1 (aginosuccinate synthase 1) is a key enzyme in the amino acid arginine biosynthesis pathway. Arginine is essential for protein synthesis, nitrogen metabolism, cell growth, and survival, and many cancer cells are dependent on arginine. While increased ASS1 expression may increase arginine synthesis in cancer cells, this may lead to an imbalance in amino acid metabolism, which may ultimately lead to cellular stress and aging. SOD2 (superoxide dismutase 2) is an antioxidant enzyme that detoxifies reactive oxygen species (ROS) in mitochondria. While it protects cells from oxidative damage, it can also promote cellular aging. Aged cells have decreased proliferative capacity and are also associated with the activation of tumor suppressor mechanisms. LONP1 (lon peptidase 1) is a protease that degrades damaged proteins within mitochondria. Impairment of mitochondrial function can induce cellular aging, which is related to the inhibition of cancer cell proliferation.

[0240] The increased expression of these aging-related genes by HaCaT_ECM treatment allows cells to better withstand environmental stresses such as oxidative stress, but also promotes cellular aging. Aged cancer cells exhibit anti-tumor effects, either inhibiting proliferation or leading to cell death. Furthermore, the accumulation of aging cells within the tumor microenvironment (TME) modulates the TME's inflammatory response, thereby activating the anti-cancer immune response.

[0241] These experimental results indicate that the composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient not only inhibits tumor cell proliferation but also promotes cancer cell death by regulating the expression of genes related to aging, and exhibits an anticancer effect through controlling the tumor microenvironment (TME).

[0242]

[0243] RNA splicing-related genes

[0244] Figure 7e shows the results of analyzing changes in the expression of RNA splicing-related genes in MCF7 cells treated with HaCaT_ECM according to Example 1.

[0245] As shown in Fig. 7e, when HaCaT_ECM was treated in MCF7 cells, the expression of SNRPF, SNRPB, SNRPD3, PSIP1, BCLAF1, HNRNPR, FUS, HSPA8, SNRPE, SNRPD1, HNRNPA2B1, HNRNPM, SRSF1, SRSF3, SFPQ, and SRSF2 genes, which are genes related to RNA splicing, decreased by more than 2-fold compared to before HaCaT_ECM treatment, and the expression of ESRP1 gene increased by more than 2-fold compared to before HaCaT_ECM treatment. Among the RNA splicing-related genes whose expression was reduced by more than twofold compared to before the HaCaT_ECM treatment, SRSF1, 2, and 3 (serine and arginine rich splicing factors) were particularly involved in the process of removing introns from transcribed RNA and joining exons to form the final mRNA, and thus have a significant impact on protein expression, function, and various physiological processes of cells. The decreased expression of SRSF1, 2, and 3 results in abnormal splicing patterns in cancer cells, inducing abnormal protein expression and dysfunction in tumor cells, resulting in an antitumor effect.

[0246] HNRNPM (heterogeneous nuclear ribonucleoprotein M) is an RNA-binding protein involved in splicing, RNA stability, and translation regulation. Decreased HNRNPM expression affects cancer cell growth, differentiation, and apoptosis, thereby inhibiting tumor growth.

[0247] When the expression of the above RNA splicing-related genes is reduced by HaCaT_ECM treatment, the immune cells of the TME are activated, resulting in an anti-tumor effect.

[0248] Meanwhile, among the RNA splicing-related genes that increased by more than twice compared to before the HaCaT_ECM treatment, ESRP1 (epithelial splicing regulatory protein 1) in particular maintains the epithelial cell-specific splicing pattern in epithelial cells, which plays a role in regulating the epithelial and epithelial-mesenchymal transition (EMT) processes of the cells. When the expression of ESRP1 is increased to induce the maintenance of epithelial cell characteristics, the epithelial-mesenchymal transition (EMT) is suppressed, thereby reducing the metastatic ability of cancer cells.

[0249] ESRP1 influences cell-cell interactions and interactions with the extracellular matrix, playing a crucial role in maintaining epithelial cell characteristics. Increased ESRP1 expression enhances cell-cell adhesion and interactions, potentially reducing tumor metastasis and invasion within the tumor microenvironment (TME). Furthermore, ESRP1 is associated with epithelial maintenance, creating an environment in the tumor microenvironment (TME) where immune cells can better recognize and attack tumor cells.

[0250] These experimental results indicate that the composition of the present invention, which contains the extracellular matrix derived from skin keratinocytes as an active ingredient, not only inhibits tumor cell proliferation but also promotes cancer cell death by regulating the expression of genes related to RNA splicing and exhibits an anticancer effect through controlling the tumor microenvironment (TME).

[0251]

[0252] 3.2.3 DAVID - Functional Annotation Analysis

[0253] DAVID Functional Annotation analysis was performed on 1403 genes selected at the level of Fold change 2, Normalized Data 4 (Log 2), and p-value 0.05 through RNA base sequence analysis of MCF7 cells treated with HaCaT_ECM according to Example 1, and the results are shown in Figs. 8a and 8b.

[0254] Figures 8a and 8b show the results of DAVID Functional Annotation analysis on 1,403 genes selected at the level of Fold change 2, Normalized Data 4 (Log 2), and p-value 0.05 through RNA base sequence analysis on MCF7 cells treated with HaCaT_ECM according to Example 1. Specifically, inhibition of the expression of genes involved in the cell cycle, DNA replication, and p53 signaling pathway inhibits cell division and growth and DNA replication, and increases apoptosis, thereby activating immune cells within the tumor microenvironment (TME). Therefore, inhibition of gene expression of the cell cycle, DNA replication, and p53 signaling pathway can induce an anti-tumor immune response and reduce the mobility and invasiveness of tumor cells. When the expression of genes involved in DNA repair pathways such as base excision repair, homologous recombination, and mismatch repair is reduced, cells proliferate with damaged DNA, which can induce cancer cell death and increase sensitivity to anticancer treatment. Genes in the Fanconi anemia pathway play a crucial role in the repair of DNA cross-link damage. Reduced expression of genes related to the Fanconi anemia pathway induces tumor cell death through DNA damage accumulation and induces an anti-tumor immune response within the tumor-like stromal membrane (TME). Reduced expression of genes associated with oocyte meiosis affects cell division and growth. Reduced expression of genes associated with cellular senescence is associated with tumor cell aging and apoptosis.

[0255] As shown in FIGS. 8a and 8b, when HaCaT_ECM according to Example 1 was treated on MCF7 cells, the expression of CCDN1, CHEK1, and FBXO5 genes in the cell cycle pathway was reduced, thereby inhibiting the cell cycle and suppressing tumor cell proliferation ability. In addition, the expression of genes in the DNA replication pathway including RFC5, FEN1, RFC3, PCNA, LIG1, MCM7, RFC2, PRIM1, and RPA2 was reduced, thereby affecting the reduction in tumor cell proliferation. In addition, the expression of FOXM1, CCNB2, CCDN1, RNBBP4, CHEK1, and E2F1 genes involved in cellular senescence was reduced, thereby regulating the expression of genes in cell senescence, tumor suppression, and the p53 signal pathway, thereby exhibiting an anti-tumor effect.

[0256]

[0257] Experimental Example 4. Confirmation of the anticancer effect of HaCaT_ECM administration in vivo.

[0258] To confirm the anticancer effect of HaCaT_ECM treatment in vivo, tumors were created by injecting MDA-MB231 cells into mice, and the decellularized extracellular matrix (HaCaT_ECM) derived from skin keratinocytes obtained by the method of Example 1 was administered to observe the results.

[0259] Specifically, MDA-MB231 cells 2Х10 7 Cells / 200 μL were injected subcutaneously into the axillary region of 6-week-old female Balb / c nude mice. Tumor sizes averaged 100 mm 2 When this was done, HaCaT_ECM was dissolved in PBS, prepared at 1% and 3%, and administered intratumorally once a week for 3 weeks.

[0260] Figure 9 is a photograph showing a mouse tumor 3 weeks after administration of HaCaT_ECM according to the daily pattern.

[0261] Figure 10 is a graph measuring changes in tumor size according to HaCaT_ECM administration according to daily aspect.

[0262] Figure 11 is a graph measuring the change in body weight of mice according to HaCaT_ECM administration according to daily aspects.

[0263] As shown in Figures 9 to 11, it was confirmed that tumor growth was inhibited when HaCaT_ECM was administered.

[0264] These experimental results indicate that a composition containing the extracellular matrix derived from skin keratinocytes according to the present invention as an active ingredient exhibits an antitumor effect in vivo.

[0265]

[0266] Although the present invention has been described with reference to the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. Furthermore, the appended claims encompass such modifications and variations as fall within the spirit of the present invention.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

2. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the extracellular matrix is ​​obtained by culturing skin keratinocytes and then decellularizing the skin keratinocytes.

3. In paragraph 1, The above cancers are oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, bone cancer, non-small cell bone cancer, pancreatic cancer, skin cancer (melanoma, etc.), basal cell carcinoma, head cancer, cervical cancer, skin cancer, cervical cancer, ovarian cancer, ovarian germ cell tumor, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, salivary gland cancer, tongue cancer, esophageal cancer, duodenal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma, pseudomyxoma, urethral cancer, ureteral cancer, penile cancer, testicular cancer, prostate cancer, chronic leukemia, acute leukemia, multiple myeloma, lymphocytic A pharmaceutical composition for preventing or treating cancer, wherein the cancer is at least one selected from among lymphoma, pediatric lymphoma, kidney cancer, renal pelvic cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, vulvar cancer, thymic cancer, central nervous system tumor, primary central nervous system lymphoma, neuroblastoma, astrocytoma, meningioma, choroidal melanoma, ampulla of Vater cancer, peritoneal cancer, small cell carcinoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

4. In paragraph 1, The above composition is a pharmaceutical composition for preventing or treating cancer, which inhibits the proliferation of cancer cells.

5. In paragraph 1, The above composition is a pharmaceutical composition for preventing or treating cancer, which controls the tumor microenvironment (TMM).

6. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the control of the tumor microenvironment (TMM) is to suppress at least one selected from the group consisting of angiogenesis, inflammatory response, cell migration, and immunosuppressive action within the tumor microenvironment.

7. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the composition comprises 0.001 to 10.0 (w / v)% of the decellularized extracellular matrix.

8. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the composition is administered intratumorally.

9. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition is administered in combination with another anticancer agent.

10. In paragraph 7, A pharmaceutical composition for preventing or treating cancer, wherein the other anticancer agent is a standard therapy, an immune checkpoint inhibitor, or an immunotherapy agent.

11. A pharmaceutical composition for inhibiting angiogenesis comprising extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

12. A pharmaceutical composition for inhibiting cancer metastasis, comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

13. A health functional food composition for preventing or improving cancer, comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

14. A feed composition for preventing or improving cancer, comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient.

15. Use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for preventing or treating cancer.

16. A method for preventing or treating cancer, comprising administering to a subject in need thereof a composition containing an effective amount of extracellular matrix (ECM) derived from skin keratinocytes as an effective ingredient.

17. Use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a medicine for the prevention or treatment of cancer.

18. Use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for inhibiting angiogenesis.

19. A method for inhibiting angiogenesis, comprising a step of administering to a subject in need thereof a composition containing an effective amount of extracellular matrix (ECM) derived from skin keratinocytes as an effective ingredient.

20. Use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a pharmaceutical agent for inhibiting angiogenesis.

21. Use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for inhibiting cancer metastasis.

22. A method for inhibiting cancer metastasis, comprising administering to a subject in need thereof a composition containing an effective amount of extracellular matrix (ECM) derived from skin keratinocytes as an effective ingredient.

23. Use of a composition comprising an extracellular matrix (ECM) derived from skin keratinocytes as an active ingredient for use in the manufacture of a drug for inhibiting cancer metastasis.

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