KR101338473B1 - Compositions for Preventing or Treating Cancers Comprising Exosomal ADAM15 as Active Ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising microvesicular ADAM15 as an active ingredient. According to the present invention, it is possible to provide a microvesicle ADAM15 composition from human immune cells, and to provide new cellular regulatory mechanisms mediated by ADAM proteins through the functional study of microvesicle ADAM15. In addition, the present invention can provide a novel anticancer agent using microvesicles ADAM15 to inhibit the adhesion, proliferation and migration of tumor cells.

Description

Composition for Preventing or Treating Cancers Comprising Exosomal ADAM15 as Active Ingredient

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising microvesicular ADAM15 as an active ingredient.

A Disintegrin And Metalloprotease (ADAM) protein is a membrane composed of propeptide, metalprotease, disintegrin-like, cysteine-rich, epidermal growth factor, transmembrane, and cytoplasmic domains. -Membrane-anchored glycoprotein (Mochizuki and Okada, 2007; Wolfsberg et al., 1995). Human ADAM15 is the only member of the ADAM family that contains the Arg-Gly-Asp (RGD) motif in the disintegrin-like domain, which is expressed in various tissues in vivo and is reported to be involved in tumor development and inhibition ( Herren et al., 1997; Kratzschmar et al., 1996). ADAM15 is highly expressed in several tumors and has been associated with tumor development and metastasis through ectodomain shedding of cadherin, Tumor Growth Factor (TGF) -α and amphiregulin (Najy et. al., 2008a; Najy et al., 2008b; Schafer et al., 2004a; Schafer et al., 2004b). In contrast, some studies have shown that overexpression of ADAM15 inhibits tumor development (Chen et al., 2008; Toquet et al., 2010; Ungerer et al., 2010). It is also described that the recombinant disintegrin-like domain of ADAM15 binds to integrins ανβ3 and α5β1 to inhibit tumor growth and metastasis (Nath et al., 1999; Zhang et al., 1998; Lu et al., 2007; Trochon-Joseph et al., 2004; Wu et al., 2009a; Wu et al., 2008; Zibert et al., 2011). However, the functional mechanism of ADAM15 is not clear.

 Plasma membrane proteins can be secreted out of cells by ectodomain shedding and release of microvesicles (van Kilsdonk et al., 2010). Ectodomain shedding is involved in proteolytic cleavage of the cell surface. Numerous growth factors, receptors and adhesion molecules are released from the plasma membrane through the ectodomain shedding process and the cut form has various biological functions (Arribas and Borroto, 2002). The vesicles are also released from the cell membrane and consist of shedding vesicles and exosomes (Cocucci et al., 2009; Thery et al., 2009). Unlike shedding-vesicles budding from the plasma membrane, microvesicles produce multivesicular bodies through the endocytic pathway and are released by fusion of the plasma membrane with the polyves. Microvesicles have a conservative size (40-60 nm) and density (1.11-1.18 g / ml), while shedding-vesicles have a heterogeneous size (up to 1 μm) (Cocucci et al., 2009; Thery et al. , 2009). The importance of vesicle function is increasing in the transmission, regulation and alteration of cellular genetic information (Cocucci et al., 2009; Hawari et al., 2004; Thery et al., 2009; Valadi et al., 2007 ).

In the present invention, ADAM15 is released from the cells as a component of microvesicles, and the release of microvesicles ADAM15 is enhanced by various phorbol 12-myristate 13-acetate (PMA) PKC (Protein Kinase C) activator in various tumor cells (Choi et al., 2006). Moreover, human mononuclear leukocyte-derived macrophages released significant levels of microvesicles ADAM15 without stimulation of PMA. The functional study of microvesicles ADAM15 has identified a novel cellular regulator mediated by ADAM proteins.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors sought to identify their biological significance in the tumor suppression mechanism related to the immune function of ADAM15, a component of microvesicles, and to develop an anticancer composition using the same. As a result, the present invention was completed by effectively inhibiting tumor growth in the case of a composition comprising microvesicle ADAM15 as an active ingredient.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising human cell-derived microvesicles ADAM15 as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising human cell-derived microvesicles ADAM15 as an active ingredient.

The present inventors sought to identify their biological significance in the tumor suppression mechanism related to the immune function of ADAM15, a component of microvesicles, and to develop an anticancer composition using the same. As a result, in the case of a composition containing microvesicles ADAM15 as an active ingredient, it was confirmed that the growth of the tumor effectively inhibited.

As used herein, the term “microcystic ADAM15” refers to ADAM15 located on the surface of microvesicles.

Preferably, the human cell is an immune cell.

In the present specification, the term 'immune cell' refers to a cell derived from hematopoietic cells and is involved in an immune response. Immune cells include lymphocytes such as B cells and T cells, natural killer cells, myeloid cells (eg, mononuclear leukocytes, macrophages, coral leukocytes, mast cells and basophils) and granulocytes.

Preferably, the human cells are macrophages, more preferably, the macrophages are cells differentiated from human mononuclear leukocytes, and even more preferably, the differentiation is PPMA (phorbol-12-myristate-13). Acetate), 1-oleoyl-2-acetylglycerol, 1-stearoyl-2-arachidonoyl-sn-glycerol, 1,2-didecanoylglycerol, 1,2-dioctanoyl-sn-glycerol , (2S, 5S) -8-decylbenzolactam V, 6- (N-decylamino) -4-hydroxymethylindole, 7-octylindolactam V, 1,2-di-O-octanoyl-3- O-β-D-galactopyranosilane-glycerol, 12-dioxyphorball 13-phenylacetate, 1-oleoyl-2-acetyl-sn-glycerol (OAG), 1-oleoyl-2-O- Acetyl-3-β-D-galactopyranosyl sn glycerol, 1-stearoyl-2-linoleoyl-sn-glycerol, 8-hydroxy- [S- (E, Z, Z, Z)]- 5,9,11,14-eicosatetranoic acid, arachidonic acid, cholesterol 3-sulfate, oleic acid, Leubol 12,13-diacetate (PDA), phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diedecanoate (PDD), 4α-phorball-12,13-diedecanoate , L-α-phosphatidyl inositol-3,4-bisphosphate, L-α-phosphatidyl inositol-4,5-bisphosphate, L-α-phosphatidyl inositol-3,4,5-triphosphate, 1-stearoyl 2-arachidonoyl-sn-glycerol, 5-chloro-N-heptylnaphthalin-1-sulfonamide, resiniferatoxin (RTX), resiniferronol 9,13,14- ortho-phenylacetate ( ROPA), 12-dioxyphorball 13-phenylacetate 20-acetate, indolactam V, 20-dibenzoate, phorbol 12, 13-dihexanoate, phorbol-12,13-dietecanoate , 1-hexyl indolactam-V10, 6,11,12,14-tetrahydroxy-avieta-5,8,11,13-tetraene-7-one (coleon U), 8-octyl-benzolactam -V9, acetyl-L-carnitine, chloroform, retinoic acid or phorbol ester 12-O-tetradecano - not using the phorbol-13-acetate, and is not limited thereto.

In the present specification, the term 'macrophages' are leukocytes produced by differentiating mononuclear leukocytes. Macrophages are involved in specific immune mechanisms (acquired immunity) as well as non-specific defenses (innate immunity). Macrophages phagocytosis against cell debris and pathogens, and stimulate lymphocytes and other immune cells with defense against pathogens.

Preferably the macrophages are cells treated with an immunoactivator, more preferably the immunoactivator is a liposome, lipopolysaccharide (LPS) or immunostimulatory oligonucleotide. Immunostimulatory oligonucleotides (eg, CpG oligonucleotides) used in the present invention include any immunostimulatory oligonucleotide known in the art. The immunostimulatory oligonucleotides can be, for example, certain palindroms, CpG motifs, CpT motifs, multiple G domains or other known immunostimulatory sequences (ISSs) that form hairpin secondary structures. For example, immunostimulatory oligonucleotides used in the present invention include immunostimulatory oligonucleotides disclosed in US Patent Application Publication Nos. 20080045473, WO 2006/063152 and WO 1998/18810. As used herein, the term “CpG oligonucleotide” refers to a base sequence containing at least two or more unmethylated or methylated cytosine-guanines to activate an immune response. Synthetic oligonucleotides containing CpG motifs (CpG-ODNs) activate various types of immune cells, including macrophages, dendritic cells, NK cells, and B lymphocytes, thereby boosting immunity similar to the direct immunostimulatory effect of native bacterial DNA. Effect.

According to the present invention, PMA increases the release of microvesicles ADAM15 and binds to integrin ανβ 3 and integrin ανβ3 Inhibits the interaction of Vitronectin and Fibronectin. Integrin ανβ3 and Inhibition of the interaction of vitronectin and fibronectin inhibits the growth of tumors by inhibiting cell adhesion, proliferation and migration. In addition, microvesicles ADAM15 do not cause apoptosis.

Preferably, the composition inhibits the attachment, migration and proliferation of cells.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and preparations are described in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or transdermal administration.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention can provide a pharmaceutical composition for preventing or treating microvesicles of ADAM15 from mammalian cells.

(b) The present invention can provide a novel cellular regulatory mechanism mediated by the ADAM protein through the functional study of microvesicles ADAM15.

(c) The present invention can provide a novel anticancer agent using microvesicles ADAM15 that inhibits the adhesion, proliferation and migration of tumor cells.

1 shows the results of confirming the release of ADAM15 out of cells. Figure 1a is confirmed by Western blotting of the antibody to the extracellular domain of ADAM15 in the conditioned medium. MCF-7 cells were cultured in serum-free RPMI1640 medium supplemented with 5 ng / ml PMA, where P represents pro-ADAM15 and M represents Mature ADAM15. The lower panel is the mean and standard deviation of 4 experiments, and the Student's t test shows a confidence of * p <0.01. 1B shows the cell lysates analyzed by western blotting using the specified antibodies. Cells were treated as indicated in the figure, with each arrow pointing to pro-ADAM15 or Mature ADAM15. Figure 1c is the result of analyzing the ADAM15 mRNA of MCF-7 cells. Semi-quantitative RT-PCR analysis of MCF-7 cells treated with 5 ng / ml PMA for the indicated time.
2 shows that release ADAM15 is associated with microvesicles and results in the secretion of microvesicle-mediated ADAM15 by PKC activation by PMA stimulation. FIG. 2A shows the following sequential centrifugation of treated or untreated 5 ng / ml PMA for 24 hours in conditioned media of MCF-7 cells:
300 × g (P1), 1,200 × g (P2), 10,000 × g (P3), 100,000 × g (P4) and 100,000 × g, and after centrifugation, 0.22 μm pore size filter (P4 ′) Shows. Precipitates of each step were analyzed by Western blotting using anti-ADAM15 intracellular domain antibodies. Each arrow points to either pro-ADAM15 or Mature ADAM15. Figure 2b shows the results of Western blotting using each antibody of the fraction after the sucrose density difference fractionation method. The vesicles obtained from the conditioned medium of MCF-7 cells treated with 5 ng / ml PMA were subjected to sucrose density difference centrifugation, and each fraction was concentrated, followed by SDS-PAGE. Figure 2c is a result confirming the secretion of microvesicles ADAM15 by PMA stimulation in various cancer cells. After 100,000 × g ultrafast centrifugation, the precipitate is shown by immunoblotting analysis with an antibody against the ADAM15 intracellular domain. Figure 2d is a result confirming that PKC inhibitors affect the secretion of microvesicles ADAM14. MDAH-2774 cells treated with PMA were treated for 24 hours at the concentration specified by calphostin C, and then microvesicles were separated and immunoblotted with ADAM15 antibody to show the results. Figure 2e shows the results of analysis of flow cytometry of ADAM15 expression of cells and microvesicles. MCF-7 cells were treated with or without 5 ng / ml PMA for 24 hours. The change in fluorescence intensity due to PMA treatment shows the presence of ADAM15.
Figure 3 shows that ADAM15-rich microvesicles have a high binding force to integrin ανβ3 and inhibit the interaction between integrin ανβ3 and Vitronectin. Figure 3a shows the results of analysis of microvesicles obtained from HEK-293F cells expressing a control vector (Exo-CON), ADAM15 (Exo-WT), ADAM15 D66E mutant (Exo-D66E) and ADAM15 E350A mutant (Exo-E350A) to be. 3B and 3C show microvesicles isolated from cells treated or untreated with 5 ng / ml PMA for 24 hours, followed by immunoblotting analysis with the indicated antibodies. The microvesicles were reacted with purified integrin ανβ3 (FIG. 3B) or α5β1 (FIG. 3C), followed by ultracentrifugation to show immunoblotting analysis. The graph is a graph showing the relative immunoblotting results. 3D shows microvesicles obtained from HEK-293F cells expressing control vectors (Exo-CON), ADAM15 (Exo-WT), ADAM15 D66E mutant (Exo-D66E), and ADAM15 E350A mutant (Exo-E350A) and integrin ανβ3 This is the result of analyzing the effect on the binding reaction of Vitronectin. Microvesicles were detected with anti-integrin αvβ3 antibodies by reaction with integrin αvβ3 in purified Vitronectin-coated plates. The graph shows the mean and standard deviation for the results of the six experiments. Figure 3e is a bit of microvesicles obtained from HEK-293F cells transfected with control vectors (Exo-CON), ADAM15 (Exo-WT), ADAM15 D66E mutant (Exo-D66E) and ADAM15 E350A mutant (Exo-E350A). Analysis of the effect on cell adhesion to Nectic. After treatment with microvesicles in MDAH-2774 cells in a Vitronectin-coated plate and incubated for 2 hours, the number of attached cells was confirmed by absorbance at 540 nm by MTT analysis. The results are graphs calculated from the mean values and standard deviations of six experiments. The right panel shows the value of plasma membrane-associated integrin αvβ3 of MDAH-2774. Through the Student's t test, * means a significant difference at p <0.05 compared to the control, and ** at p <0.01 compared to the control.
4 shows that microvesicles ADAM15 derived from transfected cells reduce vitronectin and fibronectin-induced cell proliferation and migration. Figure 4a is a result confirming the effect of microvesicles ADAM15 on MCF-7 cell proliferation. RPMI1640 medium containing 1% FBS and 0.5 μg microvesicles was used to prepare 0.5 × 10 ⁴ cells in 96-well coated with 1% BSA (control), fibronectin (10 μg / ml) or Vitronectin (8 μg / ml). The microvesicles were cultured in a plate and treated with microvesicles obtained from 293F cells transfected with a vector (Exo-CON or Exo-A15) and cultured for 48 hours, and the number of cells was confirmed at an absorbance of 540 nm by MTT analysis. . Figure 4b is the result confirming the effect of microvesicles ADAM15 on the proliferation of MDAH-2774 (2774) and NCI-460 (460) cells. The result is a graph calculated from the average value and the standard deviation of three experiments. Figure 4c is a result of confirming the functional effect of the RGD motif on the anti-proliferative effect of microvesicle ADAM15, control microvesicles (Exo-CON), ADAM15 (Exo-WT), ADAM15 R66E (Exo-R66E) or ADAM15 E350A ( MCF-7 cells treated with microvesicles containing Exo-E350A) were cultured in Vitronectin or fibronectin-coated 96-well plates to confirm cell proliferation by MTT assay. The result is a graph calculated from the average value and the standard deviation of three experiments. 4D shows the results of analyzing the effect of microvesicles ADAM15 on cell migration. MDAH-2774 (2774) and NCI-460 (460) cells were incubated with microvesicles in a transwell chamber coated with Vitronectin or Fibronectin, and the number of migrated cells was calculated by optical microscopy and the average value of four experiments and The graph shows the standard deviation. Figure 4e is a result confirming the induction of apoptosis of microvesicles ADAM15. MCF-7 cells were incubated with microvesicles in Vitronectin-coated plates, and after 48 hours, apoptotic cells were identified by fluorescence microscopy in Tunnel (TUNEL) assay. Cells were treated with DNase as a positive control of apoptosis. Through the Student's t test, * means a significant difference at p <0.05 compared to the control, and ** at p <0.01 compared to the control.
5 shows the results of confirming the function of microvesicles ADAM15 derived from tumor cells by in vitro and in vivo experiments. FIG. 5A shows microvesicles isolated from MDAH-2774 cells (Exo) or PMA-stimulated MDAH-2774 cells (Exo-PMA), and confirmed by microscopic vesicles by electron microscopy and immunoblotting analysis. Scale bar indicates 100 nm. Figure 5b is a result confirming the effect of tumor-derived microvesicle ADAM15 on cell proliferation. MTT analysis of cell proliferation with and without anti-ADAM15 extracellular domain antibody by incubating 0.5 μg microvesicles in 0.5 × 10 ⁴ cells with 0.5 μg microvesicles and incubating in 96-well plates coated with Vitronectin (8 μg / ml) This is the result confirmed by absorbance at 540 nm. The results are graphs calculated from the mean and standard deviation of five experiments. Figure 5d is the result of confirming the in vivo xenograft tumor formation. Injecting 20 μg microvesicles isolated from MDAH-2774 cells (Exo) or PMA-stimulated MDAH-2774 cells (Exo-PMA) into mice with MDAH-2774 cells and confirming inhibition of microvesicle functional ADAM15 For this purpose, microvesicles were injected with ADAM15 antibody pre-reaction (Exo-PMA-Ab) prior to injection of mice (n = 5 / group) and tumor images and volume comparison graphs after 20 days. Through the Student's t test, * means a significant difference at p <0.05 compared to the control, and ** at p <0.01 compared to the control.
6 shows the results of confirming the release of microvesicles ADAM15 from human macrophages. 6A shows Western blotting analysis of THP-1 microvesicles and cell lysates. After PMA-differentiated THP-1 cells were washed, the cells were cultured for another 2-4 days, and THP-1 mononuclear leukocytes and differentiated macrophages were cultured in serum-free medium for 24 hours, and microvesicles were separated, followed by Western blotting. . Each arrow points to either pro-ADAM15 or Mature ADAM15. Figure 6b shows FACS results analyzed by dual staining of THP-1 mononuclear leukocytes and differentiated macrophages with CD11b and ADAM15 antibodies. Figure 6c shows the results of Western blotting of microvesicles isolated from primary human mononuclear leukocytes and differentiated macrophages incubated for 16 hours in serum-free medium. The lower panel is a graph of the average and standard deviation of three experiments. Through the Student's t test, * means a significant difference at p <0.01 compared to the control. 6D shows FACS analysis of primary human mononuclear leukocytes and differentiated macrophages.
Figure 7 shows the results confirming the in vitro and in vivo tumor suppression ability of macrophage-derived microvesicle ADAM15. 7A and 7B confirm the effects of the effects of differentiated macrophages-derived microvesicle ADAM15 on tumor proliferation and migration. Microvesicles are isolated and treated in THP-1 mononuclear leukocytes (Exo-Mo) and differentiated macrophages (Exo-MΦ) and show the effect on MDAH-2774 cell proliferation and migration with or without ADAM15 ectodomain antibody. The results are graphs calculated from the mean values and standard deviations of six experiments. Figure 7c is a result confirming the effect of macrophages-derived microvesicles on macrophage proliferation and migration. The results are graphs calculated from the mean values and standard deviations of six experiments. Figure 7d shows the results of measuring the tumor volume by intradermal injection of macrophage microvesicles and MDAH-2774 cells into mice in an experiment confirming the in vivo xenograft tumor formation ability. To confirm the functional inhibition of microvesicle ADAM15, macrophages microvesicles were pre-reacted with ADAM15 antibody and then injected into mice (Exo-MΦ-Ab). Through the Student's t test, * means a significant difference at p <0.05 compared to the control, and ** at p <0.01 compared to the control.
8 shows the results confirming that microvesicles ADAM15 induce apoptosis. MCF-7 cells were cultured in control microvesicles (Exo-CON) or ADAM-rich microvesicles (Exo-A15) in fibronectin- or vitronectin-coated 96-well plates for 48 hours, and the apoptotic cells were tunnel analyzed. Show results. Cells were treated with DNase as a positive control of apoptosis.
9 is a result confirming that microvesicles ADAM15 is released by LPS stimulation in differentiated THP-1 macrophages. 9A shows Western blotting results of microvesicles and cell lysates after 24 hours of THP-1 cells treatment with PMA in serum-free medium. Each arrow points to either pro-ADAM15 or Mature ADAM15. Total protein bands of microvesicles were identified by coomassieblue staining (middle panel). The right panel is a graph showing the mean and standard deviation calculated from three experiments. 9B shows that PMA-differentiated THP-1 cells were washed with PBS and incubated for 2-3 days, and THP-1 macrophages were cultured in serum-free medium added with LPS for 18 hours, and then cell lysates were isolated to prevent anti- Western blotting results in response to ADAM15 are shown. Each arrow points to either pro-ADAM15 or Mature ADAM15. Total protein bands of microvesicles were identified by coomassieblue staining (middle panel). The right panel graphically shows the mean and standard deviation calculated from three experiments. 9C shows Western blotting results of microvesicles after 19 hours of incubation in serum-free medium with or without THP-1 cells treated or untreated with PMS and / or LPS. The arrow points to Mature ADAM15, and the right panel shows the graph of the mean and standard deviation from three experiments. Through the Student's t test, * means a significant difference at p <0.05 compared to the control, and ** at p <0.01 compared to the control.
10 shows the results of experiments showing that plasma membrane-related ADAM15 influences the interaction of cell-extracellular matrix (ECM). 10A shows the effect of ADAM15 expression on cell adhesion to fibronectin, vitronectin, collagen I and collagen IV. Prior to cell adhesion assays, MCF-7 cells were transfected with control (CON), ADAM15 (A15), control siRNA (si-CON) or ADAM15 siRNA (si-A15) and immunoblotting and expression levels of ADAM15 were expressed. This is confirmed by FACS analysis. MCTT-7 (5 × 10 ⁴ ) was incubated for 2 hours in 96-well plates coated with fibronectin (FN), vitronectin (VN), collagen I (COL-I) and collagen IV (COL-IV) for MTT The assay counted the number of cells attached. The results show the mean and standard deviation calculated from three experiments. FIG. 10B shows the results of flow cytometry confirming cell surface expression of integrin ανβ3 and integrin α5β1 in transfected MCF-1 cells. Samples that did not react with the primary antibody were used as controls.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Experimental material

The experimental materials used in this experiment are as follows:

Mouse Monoclonal Anti-ADAM15 Extracellular Domain Antibody (MAB935, R & D System, USA), Mouse Monoclonal Anti-Integrin ανβ3 Antibody (MAB1976Z, R & D System, USA), Mouse Monoclonal Anti-Integrin αν Antibody (MAB1930, R & D System, USA), mouse monoclonal anti-integrinα5β1 antibody (MAB1969, R & D system, USA), mouse monoclonal anti-integrinβ1 antibody (MAB1965, R & D system, USA), mouse monoclonal anti-integrinβ3 antibody (MAB1932, R & D system , USA), GAPDH (glyceraldehyde 3-phosphate dehydrogenase, MAB374, R & D system, USA). Rabbit polyclonal anti-ADAM15 cytoplasmic domain antibody (AB19036, Chemicon, USA), mouse monoclonal anti-CD11b (FCMAB178P, Millipore, USA), mouse monoclonal anti-CD9 antibody (sc-13118, santa cruz, USA), mouse Monoclonal anti-TSG101 antibody (sc-7964, santa cruz, USA), mouse monoclonal anti-ADAM10 antibody (sc-28358, santa cruz, USA), mouse monoclonal anti-ADAM15 antibody (sc-73686, santa cruz, USA), mouse monoclonal anti-E-cadherin (BD610181, BD Biosciences, USA) and PMA (Phorbol 12-Myristate 13-Acetate, Sigma-Aldrich, USA)

Expression plasmids and site-directed mutagenesis

The complete sequence of ADAM15 was obtained from the National Center for Biotechnology Information (NCBI) (accession number: NM_003815.3). PCR was performed to amplify ADAM15 cDNA using primers 5'-ATGCGGCTGGCGCTGCTCTGG-3 'and 5'-TCAGAGGTAGAGCGAGGACAC-3' from cDNAs of human fetal liver (marathon cDNA, Clontech, USA). PCR products were cloned into TOPO cloning vector (invitrogen, USA). B was re-cloned into the vector (Invitrogen, USA) - the ORF (Open Reading Frame) Xho I / Bam H1 cut with Xho I / Bam cut into H1 pcDNA3.1 / myc-His expression plasmid to produce a complete ADAM15 () . All mutants used in this study were constructed by site-specific mutagenesis induction (Intron Biotechnology, South Korea).

Cell Culture, Transfection, and MTT Analysis

MCF-7 (human breast cancer, HTB-22, ATCC, USA), NCI-H460 (human lung cancer, HTB-177, ATCC, USA) and THP-1 (human mononuclear leukocytes, TIB-202, ATCC, USA) cells 10% FBS (Fetal bovine serum) was added to RPMI 1640 culture medium. MDAH 2774 (human ovarian cancer, CRL-10303, ATCC, USA), A549 (human lung cancer, CCL-185, ATCC, USA), SK-MEL-28 (human melanoma, HTB-72, ATCC, USA) and HEK293F Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium with 10% Fetal bovine serum (FBS). Transfection of the plasmids and siRNAs (small interfering RNAs) was performed using lipofectamine 2000 (invitrogen, USA) according to the manufacturer's instructions. 500 μg / ml Hygromycin B (invitrogen, USA) was used to prepare cell lines. The cells were reacted with 50 μg / ml MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) for 2 hours to determine the number of viable cells. Absorbance was measured at 595 nm with 100 μl / well of DMSO (Dimethyl sulfoxide).

Western blotting

Cells were lysed and centrifuged at 4 ° C. in buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100 and Protease Inhibitor Cocktail (Roche Applied Science, USA) for 1 hour. The water soluble portion of the cell lysate was subjected to western blotting, and the reduced or non-reduced sample was separated by SDS-PAGE, and electroblotted to NCM to confirm antibody binding and then confirmed by ECL (enhanced chemiluminescence, amersham biosiences, USA).

Separation of Microvesicles by Continuous Centrifugation and Density Difference

Cells were incubated for 24 hours in serum-free medium with or without PMA or with medium containing 5% FBS. Bovine microvesicles were removed by ultrafast centrifugation prior to use of FBS. Conditioning medium was centrifuged at 300 × g 10 minutes, 1200 × g 20 minutes, and 10000 × g 30 minutes to remove cell debris. The supernatant was centrifuged at 10000 × g for 1 hour using a Beckman 70 Ti rotor. To completely remove the treated PMA, the pellet was washed with PBS. Microvesicle pellets were suspended in 100-150 μl PBS and stored at -80 ° C. The supernatant was filtered with a 0.22-μm size filter (Millipore, USA) by the method of removing shedding-vesicles from the microvesicles mentioned previously, prior to ultrafast centrifugation (Valadi et al., 2007). Protein concentration was determined by the Bradford (Bio-Rad Laboratories, USA) assay. Sucrose density difference fractionation was performed to obtain exosomes by the previously mentioned method (Gutwein et al., 2005). Microvesicles were suspended in 0.25 M sucrose and loaded on top of 2, 1.3, 1.16, 0.8, 0.5 and 0.25 M density difference sucrose layers. The sample was subjected to ultra high-speed centrifugation for 10 hours × g for 2.5 hours with Beckman SW 55 Ti to obtain a fraction by density difference, and analyzed microvesicles containing ADAM15.

Electron microscope

Immunoelectron microscopy was observed (Hawari et al., 2004). Microvesicles were placed on a formvar carbon-coated nickel grid and fixed with 4% paraformaldehyde. Samples were reacted with 5 mm gold-conjugated goth anti-mouse antibody (abcam, USA) after reaction with anti-ADAM15 extracellular domain monoantibody. Images were observed with transmission electron microscope (JEM-2010).

Flow cell  Analyzer

Flow cytometry analysis of microvesicles was performed by modifying the previously mentioned method (Thery et al., 2001). Prepare microvesicle-coated beads by reacting 5 μg microvesicles and aldehyde / sulfate latex beads (4% (w / v), 4 μm) in 100 μl PBS for 20 minutes and then shaking in 1 ml PBS for an additional 2 hours. It was. The reaction was stopped by treating the mixture with 100 mM glycine for 30 minutes and washing three times with PBS. For analysis of cell surface ADAM15, cells were fixed for 30 minutes with 4% formaldehyde and separated from 6-well plates using PBS with 5 mM EDTA. Cells and microvesicle-coated beads were reacted with anti-ADAM15 extracellular domain monoantibody at 4 ° C. for 2 hours. Cells and microvesicle-coated beads were washed and reacted for 2 hours by treatment with fluorescein isothiocyanate (FITC) -conjugated goth anti-mouse antibody (Chemicon, USA). Flow cytometry was performed with a FACS caliber (Calibur, BD Biosciences, USA) and data was analyzed using WinMDI software version 2.8 (The Scripps, USA).

Microvesicles - Integrin  Bond analysis

Microvesicles (5 μg) were reacted with 100-200 ng purified Integrin ανβ3 or α5β1 (R & D systems, USA) in 1 mL PBS with 200 μM Mn ²⁺ for 4 hours at 16 ° C. The reaction was diluted with 5 ml of PBS and subjected to ultra high speed centrifugation for 100000 × g, 1 hour. The precipitate was analyzed by western blotting in non-reducing conditions.

Integrin - Vitronectin  Bond analysis

Purified integrin αvβ3 was reacted for 1.5 hours in a Vitronectin (8 μg / ml) coated 96 well plate containing 100 μl serum free RPMI 1640 medium with or without microvesicles (5 μg / ml). After four washes with serum-free RPMI 1640 medium, bound integrin αvβ3 was reacted with anti-integrin αv monoantibody and then with HRP-conjugated goth anti-mouse antibody. The binding reaction was visualized by TMB (tetramethylbenzidine), and the absorbance was measured at 450 nm.

Cell migration assay

Cell invasion assays were performed using a transwell mount (Costar, USA) with well bottoms of 6.5-mm diameter polycarbonate 8-μm microporous membranes. The outer membrane of the transwell mount was coated with Vitronectin or Fibronectin (1 μg / well). Cells (3.0 × 10 ⁴ ) were precultured in 100 μl serum free medium containing 2 μg microvesicles and then placed in the upper chamber. Next, 600 μl DMEM containing 10% FBS was placed in the lower chamber. After 16 hours, the cells remaining on the inner membrane were removed with a cotton swab. Transwells were fixed with 4% formaldehyde and stained with 10% Geimsa. Cell number was determined by light microscopy.

Xenograft Oncogenesis Analysis

BALB / c (5 week old, magnetic) nu / nu mice were purchased from Orient (Korea) and bred under germ-free conditions. MDA-2774 cells (0.5 × 10 ⁶ ) with or without 20 μg microvesicles were injected into the flanks of mice. Tumor volume was measured by caliper and volume was calculated by the following formula:

Volume = 0.52 * length X height ²

The animal testing process was approved under the Yonsei University Animal Ethics Committee, and was conducted according to animal testing principles.

invite( primary ) Human Monocyte and Macrophage Preparation

PBMCs were centrifuged in Ficoll-Paque (GE, USA) to separate the buffy coat fraction of blood. Human primary mononuclear leukocytes were isolated with 98% purity using a mononuclear leukocyte separation kit (MilteniBiotec, UK). Macrophages were obtained from mononuclear leukocytes cultured for 5 days in RPMI 1640 medium with 20% FBS and 50 ng / ml MCSF (R & D systems, USA).

Statistical analysis

All experiments were repeated three more times. Data are presented as mean ± standard deviation values of independent experiments. Statistical analysis was performed by Student's t test. p value is 0.05 or less, and statistical significance is recognized.

result

ADAM15 of Extracellular  Release confirmation

An excellent study reports that various membrane proteins are secreted extracellularly through ectodomain shedding or microvesicle release (van Kilsdonk et al., 2010). The inventors tested whether the ADAM15 protein was released from human breast cancer MCF-7 cells into the conditioned medium. The detection of mature ADAM15 in the conditioned medium was confirmed by immunoblotting analysis using an antibody against the ADAM15 extracellular domain (FIG. 1A). ADAM15 release is increased due to stimulation with PMA, a variety of membrane protein-releasing activators (Arribas et al., 1996; Hahn et al., 2003; Vecchi et al., 1996). Immature and mature ADAM15 was detected in MCF-7. Immature and mature ADAM15 decreased over time after PMA stimulation and detected up to 24 hours (FIG. 1B). However, other membrane proteins of MCF-7 such as ADAM10 and E-cadherin showed no change in PMA stimulation, and ADAM15 was specifically reduced (FIG. 1B). After PMA stimulation, mRNA levels of ADAM15 were measured. 1C shows that ADAM15 mRNA levels did not change due to PMA, which shows that PMA stimulates extracellular release of plasma membrane-related ADAM15 protein, but not ADAM15 mRNA synthesis. The inventors studied whether released ADAM15 was produced by ectodomain shedding. Mature ADAM15 was detected by immunoblotting assay with ADAM15 cytoplasmic domain antibody in conditioned medium, indicating that ADAM15 release was not associated with proteolytic cleavage of the extracellular domain.

PMA On by Microvesicles ADAM15 Creation of

Since released ADAM15 is not in shed form, it was assumed that ADAM15 would be present in the microvesicles. Conditioned medium of MCF-7 cells was subjected to sequential centrifugation and immunoblotted with anti-ADAM15 antibody. Mature ADAM15 was detected in 10000 × g precipitates containing microvesicles (FIG. 2A). The vesicles consisting of shedding-vesicles and microvesicles were examined to include ADAM15. Before ultrafast centrifugation, the conditioned medium was shedding-vesicles with a 0.22-μm pore size filter (Valadi et al., 2007). As can be seen in Figure 2a, ADAM15 was mainly observed in the filtered precipitate, indicating that ADAM15 is associated with microvesicles. Microvesicle release of ADAM15 was confirmed by continuous sucrose density difference ultrafast centrifugation. As shown in Figure 2b, it was confirmed that the release of ADAM15 detected by the microvesicle markers CD9 and TSG101 in the range of 1.11 to 1.16 g / ㎖ is a component of the microvesicles. The amount of microvesicles ADAM15 increased with PMA stimulation (FIG. 2A, lower panel). Similar results were seen in other cancer cells (FIG. 2C). We also tested whether ADAM15 release was associated with PKC activation by PMA. As shown in FIG. 2D, PMA-stimulated ADAM15 release is inhibited by calphostin C, known as a PKC specific inhibitor, indicating that activation of PKC is involved in the release of ADAM15 (Linden and Connor, 1991). . Flow cytometry measured the amount of ADAM15 on the surface of microvesicles, and showed that PMA stimulation reduced plasma membrane-related ADAM15 but increased microvesicle ADAM15 produced by PMA-mediated PKC activation (FIG. 2E).

Microvesicles ADAM15 of Integrin ανβ3 binding

Microvesicle surface molecules interact with receptors in target cells and are important for many biological and pathological processes (Anand, 2010; Thery et al., 2009). ADAM15 disintegrin-like domains interact with integrin αvβ3 and α5β1 (Nath et al., 1999; Zhang et al., 1998). The binding affinity of microvesicular ADAM15 to this integrin was measured. In the binding assay, microvesicles obtained by continuous centrifugation of conditioned medium of HEK293F cells transfected with the empty vector, ADAM15 or ADAM15 mutants were observed. The ADAM15 mutant substituted the amino acids of the RGD motif active sites of the ADAM15 disintegrin-like domain (D66E) and metalloproteinase domain, respectively. As can be seen in FIG. 3A, the released microvesicles ADAM15 increased after transfection of ADAM15 and enriched for microvesicle proteins by PMA stimulation. The reaction of ADAM15-rich microvesicles with purified integrin ανβ3 and α5β1 was precipitated with anti-integrin antibody by ultra-fast centrifugation to perform Western blotting analysis. It was confirmed that the binding affinity of integrin α5β1 was not changed (FIGS. 3B and 3C). Moreover, microvesicle binding to integrinανβ3 was inhibited by mutant RGD (D66E) in the disintegrin-like domain of ADAM15, showing that the RGD motif is essential for this binding (FIG. 3B). Taken together, these results show that the binding affinity of microvesicles ADAM15 to integrin αvβ3 is RGD-dependent.

Microvesicles ADAM15 of Integrin ανβ3 and Of vitronectin  Interaction Suppression

Integrin ανβ3 interacts with vitronectin and fibronectin and is involved in cell attachment, migration and proliferation, which interaction is inhibited by RGD-containing disintegrin (Brooks et al., 1994; Brooks et al., 1995; Landen et al., 2008). The interaction between integrin ανβ3 and vitronectin was investigated by microvesicle ADAM15. Recombinant integrin αvβ3 was reacted with various microvesicles in Vitronectin-coated plates. As shown in FIG. 3D, microvesicular ADAM15 inhibited the binding of recombinant integrin αvβ3 and vitronectin. In this example, microvesicle ADAM15 inhibited the vitronectin adhesion of MDAH-2774 cells expressing a lot of integrin ανβ3 (FIG. 3E). This inhibitory effect was lost after mutation of the ADAM15 RGD motif (D66E) (FIGS. 3D and 3E). These experimental results make it clear that the RGD motif of microvesicular ADAM15 is essential for inhibiting the interaction between integrinανβ3 and vitronectin.

Microvesicles ADAM14 of Vitronectin - and Fibronectin Inhibition of induced cell proliferation and induction

To investigate the functional role of microvesicles ADAM15, microvesicles ADAM15 were isolated from PMA stimulated or unstimulated ADAM15-transfected 293F cells. MCF-7 cells were cultured in Vitronectin and Fibronectin-coated plates resulting in increased proliferation of cells cultured in Vitronectin and fibronectin-coated plates compared to BSA controls. Vitronectin- and fibronectin-induced cell proliferation was slightly reduced in microvesicles ADAM15 isolated from unstimulated cells and this inhibitory effect was slightly increased by ADAM15-rich microvesicles isolated from PMA-stimulated cells (FIG. 4A). Similar results were observed in other cancer cells, such as human lung cancer cells NCI-H460 and human ovarian cancer cells MDAH-2774 cells (FIG. 4B). To confirm the functional role of the metalloproteinase and RGD motif of ADAM15 in cell proliferation, ADAM15 D66E and catalytically inactive ADAM15 E350A mutations were prepared. When cells were cultured in the presence of microvesicles comprising wild type ADAM15 and inactive ADAM15 (E350A), cell proliferation was significantly reduced (FIG. 4C). In contrast, the ADAM15 D66E mutation did not affect cell proliferation, indicating that RGD motifs that make up the disintegrin-like domain, but not metalloproteinase activity, are involved in cell proliferation inhibition. Microvesicle ADAM15 also strongly inhibits Vitronectin- and fibronectin-induced cell migration, so microvesicles ADAM15 may control cancer progression (FIG. 4D). Whether ADAM15-rich microvesicles could induce apoptosis was confirmed using TdT-mediated dUTP Nick-End Labeling (TUNEL) analysis. Tunnel results show no apoptosis effect on cells treated with ADAM15-rich microvesicles (FIGS. 4E and 6). Overall, the experimental results show anti-cancer effects by inhibiting the interaction of microvesicle ADAM15 with the extracellular matrix (ECM).

Tumor-Derived Microvesicles ADAM15 In vivo tumor growth inhibition of

To confirm the function of tumor-derived microvesicle ADAM15, tumor microvesicles were isolated from PMA stimulated or unstimulated MDAH-2774 cells. In the isolated microvesicles, ADAM15 was confirmed by electron microscopy and immunoblotting analysis (FIG. 5A). MDAH-2774 cell proliferation and migration assays were performed on Vitronectin. ADAM15-rich microvesicles effectively reduced cell proliferation and migration and inhibited inhibitory effects by ADAM15 extracellular domain-specific antibodies (FIGS. 5B and 5C). Of interest is the increased cell proliferation and migration by microvesicles derived from non-irritating tumor cells (FIGS. 5B and 5C). However, the stimulatory effect was not affected by ADAM15 antibody.

In immunodeficient mouse models, ADAM15-rich microvesicles effectively inhibited in vivo tumor growth compared to control microvesicles (FIG. 5D). To confirm the functional role of in vivo microvesicles ADAM15, ADAM15-rich microvesicles were pre-reacted with ADAM15 antibodies and separated by ultra-fast centrifugation followed by flow cytometry to measure antibody blocking (FIG. 5D). As shown in FIG. 5D, antibody-blocking microvesicles in the immunodeficient mouse model did not inhibit in vivo tumor growth. These results suggest that microvesicles ADAM15 play a critical role in tumor suppression mechanisms.

Of human mononuclear leukocyte-derived macrophages Microvesicles ADAM15  Release

Based on the tumor growth inhibitory function of microvesicle ADAM15, it was investigated whether tumor suppressor microvesicles ADAM15 were released from human macrophages. First, THP-1 monocytes were differentiated into macrophages with PMA, a stimulant to differentiate into macrophages (Daigneault et al., 2010). PMA stimulation of THP-1 cells differentiated monocytes into macrophages, effectively increasing the release of microvesicles ADAM15 (FIG. 9A). To fully differentiate THP-1 cells and rule out the direct effects of PMA, PMA-differentiated THP-1 cells were washed with PBS and further cultured for 2-3 days (Daigneault et al., 2010; Whatling et al., 2004 ). It was confirmed by flow cytometry that the level of CD-11b was increased that fully differentiated THP-1 cells were differentiated into macrophages (FIG. 6B). As in FIGS. 6A and 6B, maturation and surface expression of ADAM15 were significantly increased and microvesicles ADAM15 continued to be released into differentiated macrophages. In addition, the release of microvesicles ADAM15 in macrophages was increased by lipopolysaccharide (LPS), known as an activator of anti-tumor immune responses (Hibbs et al., 1982) (FIGS. 9B and 9C). Release of microvesicles ADAM15 is associated with monocyte / macrophage differentiation and activity.

The release of microvesicles ADAM15 in cultured human primary macrophages was investigated. Primary cultured human monocytes were isolated from buffy coat of normal blood and differentiated into macrophages (FIG. 6D). As with THP-1 macrophages, ADAM15 surface expression was increased in primary cultured human macrophages and microvesicle ADAM15 was released. This shows that the release of microvesicles occurs physiologically in the natural immune system (FIGS. 6C and 6D).

Macrophage-derived Microvesicles ADAM15 Tumor suppression function

To confirm the tumor suppressor effect of macrophage-derived microvesicle ADAM15, cancer proliferation and migration assays were analyzed by treatment of THP-1 monocytes or differentiated macrophage derived microvesicles cultured in Vitronectin-coated plates. As can be seen in Figure 7a, microvesicles released from differentiated macrophages inhibited the proliferation and migration of ovarian cancer MDAH-2774 cells. This inhibitory effect is inhibited by the treatment of ADAM15 extracellular domain antibodies, demonstrating the tumor suppressor function of macrophage-derived microvesicle ADAM15. In contrast, monocyte-derived microvesicles slightly stimulated cancer proliferation and migration, and this effect was not affected by the treatment of ADAM15 antibodies, which is consistent with previous results (FIGS. 5B and 5C). To confirm that microvesicles ADAM15 released from differentiated macrophages without stimulation of PMA have persistent tumor suppressor function, macrophage-derived microvesicles were purified by the specified method. Inhibitory effect of macrophage-derived microvesicle ADAM15 on tumor cell proliferation and migration was observed (FIG. 7B). Interestingly, the proliferation and migration of macrophages was stimulated by microvesicles themselves, indicating the anti-tumor response of human macrophages (FIG. 7C).

In addition, in the immunodeficient mouse model, microvesicles of macrophages significantly inhibited tumor growth, and the inhibitory effect was reduced by microvesicle ADAM15 pre-reacted with ADAM15 antibody prior to injection (FIG. 7D). These results make it clear that macrophage-derived microvesicle ADAM15 inhibits the growth of in vitro and in vivo tumors.

debate

The experimental data of the present invention show some scientific findings. First, ADAM15 is released extracellularly as a component of microvesicles, and release of microvesicles ADAM15 in various tumor cells is induced by decreasing plasma membrane-related ADAM15 by PMA-mediated PKC activation. Second, microvesicular ADAM15 binds to integrin α v β 3 and inhibits the interaction of integrin α v β 3 and vitronectin. Third, microvesicular ADAM15 inhibits tumor proliferation and migration and in vivo tumor growth with Vitronectin- and Fibronectin-induced in vitro . Fourth, the RGD motif-containing disintegrin-like domain, but not the metalloproteinase activity of ADAM15, is a major component of the tumor suppressor function of microvesicular ADAM15. Finally, human macrophages are functionally activated to release microvesicles ADAM15, and macrophages-derived microvesicles ADAM15 effectively function as tumor suppressors.

The present invention confirms that the tumor is highly released microvesicle ADAM15 while plasma membrane-related ADAM15 is reduced by PMA stimulation. Recently, it has been reported that elements of microvesicles are separately classified into microvesicles by PMA-mediated PKC activation (Abache et al., 2007). Therefore, ADAM15 will be one of the microvesicle elements classified as microvesicles by PKC activation. PMA-mediated PKC activation regulates tumor growth and survival (Choi et al., 2006; Feuerstein et al., 1984; Oh et al., 2005; Tahara et al., 2009). Some studies have shown that vesicles are involved in cell regulatory mechanisms. For example, microvesicle-like vesicles comprising TNF receptors are secreted from human vascular endothelial cells, and TNF signals are negatively regulated by the released TNF receptors (Hawari et al., 2004). It was also confirmed that the β-catenin-mediated Wnt signal was inhibited by the release of microvesicles β-catenin (Chairoungdua et al., 2010). In the present invention, one can assume a novel regulatory mechanism of ADAM15-mediated tumorigenesis regulated by the release of microvesicle ADAM15.

Some reports have demonstrated that the disintegrin-like domain of ADAM15 binds more specifically to integrinανβ3 than to integrinα5β1, depending on cell type, but binds to integrinανβ3 and α5β1 (Nath et al., 1999; Zhang et al. , 1998). Examples of the present invention show that microvesicles ADAM15 specifically interact with RGD-dependent integrin αvβ3.

Disintegrins with RGD motifs have been shown to inhibit tumor growth through inhibition of integrin-mediated cell-ECM interactions (Chung et al., 2003; Lu et al., 2007; McLane et al., 2004; Trochon- Joseph et al., 2004; Wu et al., 2008). However, it was found that ADAM15 located in the plasma membrane does not regulate cell-ECM interaction or expression of ECM receptors on the cell surface (FIGS. 9A and 9B). Released microvesicular ADAM15 regulates cell-ECM interactions associated with tumor suppression mechanisms. These results indicate that release of microvesicles ADAM15 is a critical process of ADAM15-mediated tumor suppression.

The example suggests that non-irritating cancer cell-derived microvesicles induce tumor proliferation and migration without affecting ADAM15 antibodies, suggesting the presence of other microvesicle elements other than ADAM15 (FIGS. 5B and 5C). For example, microvesicles have a variety of EGFR ligands that are involved in tumor growth (Higginbotham et al., 2010).

Tumor-suppressive microvesicles ADAM15 released from differentiated human macrophages without PMA stimulation were found. Differentiation of monocytes into macrophages leads to the activation and translocation of sustainable PKCs (Aihara et al., 1991; Monick et al., 1998). Therefore, release of ADAM15 will be associated with sustained PKC activation of differentiated macrophages. Macrophages play a key role in the immune response to infection or tumors (Adams and Snyderman, 1979; Bock et al., 1991; Braun et al., 1993; Hibbs et al., 1982; Vicetti Miguel et al., 2010; Wu et al., 2009b; Young et al., 1990). Mononuclear leukocyte-derived macrophages are known to rapidly congregate at tumor site and inhibit tumor growth (Bock et al., 1991; Braun et al., 1993; Vicetti Miguel et al., 2010; Wu et al., 2009b; Young et al., 1990). The physiological and pathological significance of the function of the vesicles in the immune response is increasing. Many studies have shown that microvesicles derived from dendritic cells or macrophages activate innate and adaptive immunity via antigen or Major Histocompatibility Complex (MHC) -peptide complexes (Lynch et al. , 2009; Segura et al., 2007; Taieb et al., 2006; Zeelenberg et al., 2008). The inventors have proposed a novel anticancer immune function that directly inhibits tumor growth without triggering other immune cells of macrophage-derived microvesicle ADAM15. It was found that macrophages-derived microvesicles proliferate and migrate macrophages themselves, in contrast to the results of tumor cells (FIG. 7B). Macrophages can destroy tumorigenic cells through tumor cytotoxic factors that do not affect non-tumor cells (Sone and Fidler, 1981; Sone et al., 1982).

The present invention confirms the release of ADAM15, a component of microvesicles, and explains its biological significance in tumor suppression mechanisms associated with immune function.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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Claims (8)

A pharmaceutical composition for preventing or treating cancer comprising microvesicles comprising ADAM15 derived from human cells as an active ingredient.
The composition of claim 1, wherein the human cell is an immune cell.
The composition of claim 1, wherein the human cell is a macrophage.
4. The composition of claim 3 wherein said macrophages are differentiated from human mononuclear leukocytes.
The method according to claim 4, wherein the differentiation is PMA (phorball-12-myristate-13-acetate), 1-oleoyl-2-acetylglycerol, 1-stearoyl-2-arachidonoyl-sn-glycerol, 1,2-didecanoylglycerol, 1,2-dioctanoyl-sn-glycerol, (2S, 5S) -8-decylbenzolactam V, 6- (N-decylamino) -4-hydroxymethylindole, 7-octylindolactam V, 1,2-di-O-octanoyl-3-O-β-D-galactopyranosilane-glycerol, 12-dioxyphorball 13-phenylacetate, 1-oreoyl- 2-acetyl-sn-glycerol (OAG), 1-oreoyl-2-O-acetyl-3-β-D-galactopyranosyl sn glycerol, 1-stearoyl-2-linoreoyl-sn-glycerol , 8-hydroxy- [S- (E, Z, Z, Z)]-5,9,11,14-eicosatetranoic acid, arachidonic acid, cholesterol 3-sulfate, oleic acid, phorbol 12,13- Diacetate (PDA), phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diedecanoate (PDD), 4α-formol-12,13-diedecanoate, L-α-phosphatidylinositol-3,4-bisphosphate, L-α-phosphatidylinositol-4,5-bisphosphate, L-α-phosphatidylinositol-3,4,5-triphosphate, 1-stearoyl- 2-arachidonoyl-sn-glycerol, 5-chloro-N-heptylnaphthalin-1-sulfonamide, resiniferatoxin (RTX), resiniferronol 9,13,14- ortho-phenylacetate (ROPA ), 12-dioxyphorball 13-phenylacetate 20-acetate, indolactam V, 20-dibenzoate, phorbol 12, 13-dihexanoate, phorbol-12,13-dietecanoate, 1-hexylindolactam-V10, 6,11,12,14-tetrahydroxy-avieta-5,8,11,13-tetraene-7-one (coleon U), 8-octyl-benzolactam- A composition characterized by the addition of V9, acetyl-L-carnitine, chloroform, retinoic acid or phorbol ester 12-O-tetradecanoyl- phorbol-13-acetate.
The composition of claim 3, wherein the macrophages are cells treated with an immunoactivator.
7. The composition of claim 6, wherein said immunoactivator is a liposome, lipopolysaccharide (LPS) or immunostimulatory oligonucleotide.
The composition of claim 1, wherein the composition inhibits cell adhesion, migration, and proliferation.

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