# Patent

## Title
Composition for inducing migration of neural stem cells containing periostin as effective ingredient

## Abstract

A novel use of periostin, and more particularly a composition for inducing the migration of neural stem cells, which contains periostin or a periostin-secreting cell as an active ingredient, based on the discovery of a novel function of periostin that induces the migration of neural stem cells. Periostin induces the migration of neural stem cells, and thus the composition for inducing the migration of neural stem cells, which contains periostin or periostin-secreting cells as an active ingredient, can be used in various applications based on neural stem cells. A pharmaceutical composition containing suicide gene-expressing neural stem cells and periostin allows the neural stem cells to effectively migrate to tumor tissues, and thus can be used as a cell therapeutic agent for treating cancer. Also, the pharmaceutical composition containing neural stem cells and periostin has improved effects on the stimulation of regeneration of nerve cells and the treatment of neural diseases.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority under 35 U.S.C. 119 to Korean Patent Application No. 10-2010-0130889, filed on Dec. 20, 2010, the disclosure of which is expressly incorporated by reference herein in its entirety.
TECHNICAL FIELD
The present invention relates to the novel use of periostin, and more particularly to a composition for inducing the migration of neural stem cells, which contains periostin or periostin-secreting cells as an active ingredient, based on the discovery of a novel function of periostin that induces the migration of neural stem cells.
BACKGROUND ART
Brains can perform complex functions by generating a systemic neural network through a series of processes, including the division, differentiation, survival and death of neuronal stem cells and the formation of synapses. Such brains have a low ability to regenerate, compared to other organs, and thus damage to the brain can result in serious sequelae. Particularly, the occurrence of a tumor in the brain can cause significant damage to the brain, resulting in a significant reduction in the survival rate of the patient. The most effective conventional method for treating brain tumors is the resection of brain tumors by a surgical operation. However, depending on the type and location of brain tumor, it is impossible to perform a surgical operation, and complete resection of brain tumors by surgery presents a high risk of complications. In addition, in chemotherapy with anticancer drugs, administration of a high concentration of anticancer drugs is required due to the presence of the blood-brain barrier, thus causing serious side effects.
In attempts to overcome such problems, gene therapy methods have been proposed that use viruses to introduce genes, which inhibit the proliferation of cancer cells, directly into the cancer cells. However, it was actually impossible to introduce viruses even into a minute tumor or cancer tissue. Also, conventional methods, including viral surface modification and magnetic transfer techniques, had limitations in targeting minute cancer tissue. Additionally, the problems according to the immune toxicity of viruses are still raised.
In recent years, as the affinity of stem cells (including neural stem cells and mesenchymal stem cells) for brain tumors has been known, a possibility to stem cells as a gene delivery medium has been proposed (Aboody et al., Proc. Natl. Acad. Sci. USA, 97:12846, 2000; Brown et al., Human Gene Therapy, 14:1777, 2003; Tang et al., Human Gene Therapy,14:1247, 2003; Zhang et al., Neurolmage, 23:281, 2004; Nakamura et al., Gene Therapy, 11:1155, 2004; Zhang et al., Neurolmage, 23:281, 2004). Particularly, with respect to methods for treating brain tumors using such stem cells, it was reported that, when neural stem cells expressing cytosine deaminase (CD) that is the E. coli suicide gene were used to target glioma, excellent anticancer effects were shown (Aboody et al., Proc. Natl. Acad. Sci. USA, 97:12846, 2000; Brown et al., Human Gene Therapy, 14:1777, 2003). Also, Korean Patent Laid-Open Publication No. 10-2007-0036289 discloses a composition for treating cancer comprising mesenchymal stem cells expressing a suicide gene.
However, the above-described methods have a problem in that they are not effective for the treatment of cancer, because cancer cells are targeted at a low level. In addition, these methods are inefficient, because craniotomy should always be performed in order to introduce neural stem cells expressing a suicide gene. Meanwhile, the above-described literature showed only the affinity of stem cells for brain tumors, but did not present a method for delivering neural stem cells to brain tumor sites, based on the discovery of a substance that causes the affinity.
Meanwhile, periostin that is also called “OSF-2” is a protein originally isolated from osteoblasts and is known to be involved in the replacement, adhesion and spreading of osteoblasts (WO/2005/062055). It causes mature cardiac cells to divide, thus inducing the production of new cardiac cells. Also, it is known that periostin is tissue-specifically expressed in the periosteum and periodontal ligament and that the expression is regulated by TGF-beta (Johnson & Lancero, 1999). Also, the possibility of periostin as a cancer metastasis marker has been reported (Wei Wan & Rong Shao, J. Biol. Chem., 281(28):19700, 2006). Periostin is known as a protein that is secreted from various cancer cells. Specifically, periostin has been reported to be secreted from small intestinal and colon cancer cells (Bao et al., 2004), breast cancer cells (Shao et al., 2004), lung cancer cells (Sasaki et al., 2001), pancreas cancer cells (Baril et al., 2007) and ovarian cancer cells (Gillan et al., 2002). Also, it was reported that the expression of periostin increased to facilitate the differentiation of endothelial cells in damaged cardiac tissue and the migration of endothelial cells to the damaged site (Lindner et al., 2005) and also that periostin is involved in the metastasis, invasion, propagation and survival of cancer cells (Kanno et al., 2008). However, it has not yet been reported that periostin has a function of inducing the migration of neural stem cells.
Accordingly, the present inventors have made extensive efforts to provide a substance for inducing the migration of neural stem cells so that a cell therapeutic agent for inducing cancer cell death can be prepared, which has enhanced efficiency, and can be used in studies on neural stem cells in various fields. As a result, the present inventors have found that periostain isolated from tumor tissue has the ability to induce the migration of neural stem cells, thereby completing the present invention.
DISCLOSURE OF INVENTION
Technical Problem
It is an object of the present invention to provide a composition for inducing the migration of neural stem cells, which contains periostin or periostion-secreting cells as an active ingredient.
Another object of the preset invention is to provide a pharmaceutical composition for treating cancer or neural disease, which contains periostin or periostin-secreting cells as an active ingredient.
Technical Solution
To achieve the above objects, the present invention provides a composition for inducing the migration of neural stem cells, which contains periostin or periostion-secreting cells as an active ingredient.
The present invention also provides a pharmaceutical composition for treating cancer diseases, the composition containing: (a) neural stem cells expressing a suicide gene; and (b) periostin or periostin-secreting cells.
The present invention also provides a composition for stimulating the regeneration of nerve cells and a pharmaceutical composition for treating neural disease, each composition containing: (a) neural stem cells; and (b) periostin or periostin-secreting cells.
Advantageous Effects
The present invention is based on the discovery of a novel function of periostin that induces the migration of neural stem cells, and it provides a composition for inducing the migration of neural stem cells, which contains periostin or periostin-secreting cells as an active ingredient.
The inventive composition for inducing the migration of neural stem cells can be used to treat cancer diseases using neural stem cells and to stimulate the regeneration of nerve cells and treat neural diseases. In addition, the composition of the present invention can be widely used in various applications based on neural stem cells.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the result of quantitative analysis of 14 candidate mRNAs for inducing migration of neural stem cells, which are overexpressed in tumor cells.
FIG. 2 is a set of optical microphotographs of normal brain tissue and brain tumor tissue, taken after immunostaining in order to determine whether periostin was expressed in the tissues.
FIG. 3 is a set of graphs showing the results of measuring the number of neural stem cells, which migrated by periostin or VEGF, at various concentrations.
FIG. 4 is a set of graphs showing the results of measuring the number of neural stem cells, which migrated by periostin, at various time points.
FIG. 5 shows the results of analyzing and quantifying the expression of integrin subunits in neural stem cells.
FIG. 6 shows the results of Western blotting for each step carried out in order to determine whether the induction of migration of neural stem cells by periostin is mediated by the PI3K signaling pathway.
FIG. 7 shows the results of measuring the number of cells that migrated when treated with an MEK/ERK kinase inhibitor and a CDK5 inhibitor (FIG. 7A), and the locations and expression levels of DCX and ERK in the cells (FIG. 7B).
FIG. 8 shows a method for constructing a periostin-overexpressing cell line, and the results of analyzing vectors using restriction enzymes (FIGS. 8A and 8B), the results of ELISA assay (FIG. 8C), and the results of Western blotting (FIG. 8D).
FIG. 9 is a set of confocal laser scanning microphotographs taken in order to observe the migration of fluorescent dye-labeled neural stem cells, implanted ipsilaterally, toward periostin-expressing cells with the passage of time, and schematically shows the portion into which the neural stem cells were introduced.
FIG. 10 is a set of confocal laser scanning microphotographs taken in order to observe the migration of fluorescent dye-labeled neural stem cells, implanted contralaterally, toward periostin-expressing cells with the passage of time, and schematically shows the portion into which the neural stem cells were introduced.
FIG. 11 shows the results of analyzing the expression levels of the CD gene in F3 cells and F3-CD cells (FIG. 11A), and the activity of the enzyme by HPLC analysis (FIGS. 11B to 11D).
FIG. 12 is a set of photographs and graphs showing cell death around neural stem cells expressing a suicide gene (CD).
FIG. 13 is a set of photographs and graphs showing that suicide gene-expressing cells (F3-CD) implanted ipsilaterally into a rat brain were induced toward periostin-expressing cells and showed the in vivo bystander effect of killing the cells.
FIG. 14 is a set of photographs and graphs showing that suicide gene-expressing neural stem cells (F3-CD) implanted into a rat brain in a C6 rat glioma model were induced toward periostin-expressing cells and showed the therapeutic effect of killing glioma cells (C6).
FIG. 15 is a set of close-up photographs showing that the invasion of cancer cells was inhibited when a C6 rat glioma model introduced with periostin-expressing cells was treated with 5-FC. Specifically, FIG. 15 is a set of photographs showing a slice in 2 weeks of treatment with 5-FC after different migration period, 7 days (A) or 10 days (B), following implantation of neural stem cells (F3-CD).



BEST MODE FOR CARRYING OUT THE INVENTION
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein and the experiment methods which will be described later are well known and commonly used in the art.
The definition of main terms used in the detailed description of the invention is as follows.
As used herein, the term “stem cells” refers to cells having not only self-replication ability but also the ability to differentiate into at least two cells. The stem cells can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
As used herein, the term “neural stem cells” refers to stem cells having self-renewal ability, but also the ability to differentiate into neurons, oligodendrocytes and astrocytes, which are associated with nerve cells. In in vitro tests, such neural stem cells show characteristic cell clusters, called neurospheres, which are formed by reaction with nestin that is the marker of neural stem cells (Gage, F. H., Science, 287:1433, 2000).
As used herein, the term “suicide gene” refers to a gene having a function of converting a prodrug harmless to the human body into an anticancer substance toxic to malignant tumor cells. It allows stem cells having a suicide gene to target cancer cells, thus eliminating cancer. Herein, because stem cells having a suicide gene convert a prodrug into an anticancer substance, the suicide gene is harmless to normal cells and destroys only cancer cells, so that it can be efficiently used for cancer therapy.
In one aspect, the present invention is directed to a composition for inducing the migration of neural stem cells, which contains periostin or periostion-secreting cells as an active ingredient. In addition, the present invention includes a method of inducing the migration of neural stem cells using the composition for inducing the migration of neural stem cells.
In the present invention, periostin may comprise an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 4, and may be a protein secreted from tumor cells. Periostin that is also called OSF-2 is a protein originally isolated from osteoblasts and is known to be involved in the replacement, adhesion and spreading of osteoblasts (WO/2005/062055). Also, periostin is known as a protein which is overexpressed in tumor tissues, including lung cancer, ovarian cancer and breast cancer tissues (Wei Yan & Rong Shao, J. Biol. Chem., 281(28), 19700-19708, 2006). In the present invention, it was found that periostin is overexpressed in brain tumor tissue.
It was found that periostin is expressed only in brain tumor tissue, but not in normal brain tissue. Particularly, it was found that periostin is overexpressed only in malignant tumors which have increased penetration into normal brain tissue and increase the recurrence rate of brain cancer after brain cancer surgery.
In the present invention, periostin-secreting cells may be cells that secrete periostin in natural conditions, that is, osteoblasts, etc. Preferably, the periostin-secreting cells may be cells obtained by introducing the periostin gene into general cells. In the present invention, a cell line introduced with the periostin gene was constructed by introducing a pCL-Ampho vector and a periostin-encoding pCXbsr-POSTN vector (Shiga University, Dr. Inoue) into 293T cells.
Also, an in vitro Boyden chamber assay revealed that periostin is a substance inducing the migration of neural stem cells. It was confirmed that the ability of periostin to induce the migration of neural stem cells is higher than that of VEGF known as a substance inducing the migration of stem cells, suggesting that periostin is an important factor in the induction of migration of stem cells. Also, it was found that the ability of periostin to induce the migration of neural stem cells was the highest at a concentration of 10 μg/ml. Meanwhile, the ability of periostin to induce the migration of neural stem cells was maintained over time.
In an in vivo test using animals, it was found that stem cells migrated toward cells overexpressing periostin and also that the number of migrated stem cells increased with the passage of time. This suggests that periostin can strongly induce the migration of stem cells in both in vivo and in vitro conditions.
The present inventors have found that neural stem cells have an αvβ5 integrin receptor and also that the migration of neural stem cells to tumor tissue is mediated by integrin αvβ5. This suggests that integrin αvβ5 is a primary receptor for periostin in neural stem cells and that periostin is an important ligand in inducing the migration of neural stem cells to tumor tissue.
The present inventors investigated the activation of the PI3K pathway (PI3K-AKT-mTOR-CDK5-Nudel1, PAK1) among signaling pathways which influence cells migration in order to examine what signaling pathway in neural stem cells are involved in inducing the migration of neural stem cells. In one Example of the present invention, it was found that the downward stages of the PI3K signaling pathway are activated depending on the time of treatment with periostin.
Meanwhile, in order to examine whether periostin is associated with the CDK5 signaling and MEK/ERK signaling pathway which mediate the migration of neural stem cells, cells were treated with periostin together with either PD98059 acting as an MEK/ERK kinase inhibitor or roscovitine acting as a cyclin-dependent kinase inhibitor. The effects of the inhibitors on cell migration were measured and, as a result, it was shown that the increase in cell migration by periostin was influenced by roscovitine, suggesting that the cell migration by periostin is mediated by the CDK5 pathway.
In addition, it is known that DCX which is the downstream signaling pathway of CDK5 is a factor regulating nucleokinesis during the migration of neural stem cells which is required to form the cerebral cortex in the developmental process of the brain. In order to examine whether the same signaling pathway is also involved in inducing the migration of neural stem cells by periostin, neural stem cells were treated with periostin. As a result, it was found that the location of DCX was shifted from cytoplasm to the area around the nucleus, suggesting that the DCX signaling pathway is also involved in inducing the migration of neural stem cells by periostin.
In another aspect, the present invention is directed to a pharmaceutical composition for treating cancer diseases, the composition containing: (a) neural stem cells expressing a suicide gene; and (b) periostin or periostin-secreting cells, and can be used for treatment of various kinds of cancer diseases, including brain tumor, lung cancer, breast cancer, ovarian cancer, and the like. In addition, the present invention is directed to a method for treating cancer diseases, the method including administering (a) neural stem cells expressing a suicide gene; and (b) periostin or periostin-secreting cells.
Also, the suicide gene is used for the purpose of killing cancer cells, it will be obvious to a person skilled in the art that the suicide gene can be replaced by any gene and factor known to inhibit the growth of cancer cells, for example, PEX, Trail, IFN-b, etc.
Examples of the suicide gene that is used in the present invention include Herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD) and the like.
In recent years, as methods of treating cancer by destroying tumor cells using suicide genes, systems consisting of various combinations of suicide genes and precursors have been reported. Among these methods, the most frequently used method uses a HSV-TK suicide gene and GCV (ganciclovir; 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine). With respect to the HSV-TK/GCV system, it was reported that, when tumor cells are introduced with the HSV-TK gene and treated with GCV, the GCV inhibits the function of the DNA polymerase of the cells by the expressed HSV-TK or inserted into the cell DNA to interfere with DNA replication, thus causing necrosis of the cells (Frank et al., J. Biol. Chem., 259:1566, 1984).
Also, cytosine deaminase has a function of converting 5-FC (5-fluorocytosine) into 5-FU (5-fluorouracil) that is a highly cytotoxic anticancer agent. For this reason, when 5-FU is systemically administered, it will cause serious side effects due its high cytotoxicity, but when 5-FC is used together with a suicide gene, the concentration of 5-FU will increase only around the suicide gene, so that the anticancer effect of 5-FU will appear locally around cancer cells (Bourbeau et al., J. Gene Med., 6:1320, 2004).
The suicide gene can be introduced into neural stem cells using a viral vector containing it, preferably a retroviral vector, according to any conventional method of introducing genes into cells. Specifically, the suicide gene can be introduced into neural stem cells by inserting the suicide gene into a retroviral vector to construct an expression vector, introducing the vector into packaging cells to prepare transformants, culturing the transformants, filtering the cultured transformants to obtain a retroviral solution, and infecting neural stem cells with the retroviral solution. Then, neural stem cells that continuously express the suicide gene can be collected using the selective marker contained in the retroviral vector.
In one Example of the present invention, it was found that, when periostin-secreting cells were introduced into a brain cancer site in a rat brain cancer model and when neural stem cells introduced with a suicide gene were introduced into the brain, the size of the rat brain cancer site decreased. Also, in the rat brain cancer model, it was found that, as the amount of periostin increased, the size of the brain cancer in the rat decreased. It is generally known that the patient's prognosis is a year or less even after a surgical operation due to the recurrence of malignant tumors caused by minute malignant tumor cells which cannot be found by existing methods. However, according to Examples of the present invention, it can be seen that, as the amount of periostin increases, the number of invasive malignant tumor cells which spread from the central portion of brain cancer to normal tissue significantly decreases. Thus, the use of the inventive pharmaceutical composition for treating cancer, which contains (a) neural stem cells expressing a suicide gene and (b) periostin or periostin-secreting cells, will make it possible to inhibit the growth of a malignant tumor and inhibit the metastasis of a malignant tumor to normal surrounding tissue, thereby preventing the recurrence of a malignant tumor.
It will be obvious to a person skilled in the art that, when the inventive pharmaceutical composition for treating cancer, which contains (a) neural stem cells expressing a suicide gene and (b) periostin or periostin-secreting cells, is injected into the affected part using, for example, an osmotic pump, so that it is continuously secreted, the periostin will induce the migration of the neural stem cell so that the neural stem cell will migrate to a brain tumor cell in a more efficient manner than a conventional method and expresses the suicide gene therein, indicating that the composition of the present invention can be effectively used for the treatment of various cancer diseases, including brain tumor, lung cancer, breast cancer, ovarian cancer, and the like. Still another aspect, the present invention is directed to a composition for stimulating the regeneration of nerve cells and a pharmaceutical composition for treating neural disease, each composition containing: (a) neural stem cells; and (b) periostin or periostin-secreting cells. The periostin-secreting cell in the present invention may be any cell type present in the body, and may preferably be a nerve cell. In addition, the present invention is directed to a method for treating neural disease, the method including administering periostin or periostin-secreting cells. Preferably, neural stem cells may be further administered.
In the present invention, the term “neural disease” is meant to include nerve injury, and examples of the neural disease include brain disease, peripheral nerve injury, amyotropic laterial sclerosis and peripheral nerve disease. Herein, the “term “brain disease” is meant to include brain injury, and examples thereof include dementia, Parkinson's disease, Alzheimer's disease, Huntington's disease, epilepsy, paralysis, stroke, ischemic brain disease and degenerative brain disease.
A process in which stem cells develop and differentiate in an adult brain can be defined as a regeneration process (Johansson, C. B., et al., Cell, 96(1):25, 1999), and it is known that neural stem cells are divided into granulocytes which can differentiate into functional cells (van Praag, H, et al., Nature, 415:1031, 2002). In this sense, numerous studies focused on stimulating nerve regeneration by promoting the development and differentiation of neural stem cells has been conducted. In addition, methods for stimulating the regeneration of nerve cells are well known to a person skilled in the art, and examples thereof include a method of stimulating the differentiation of neural stem cells by overexpressing nerve growth factor, a method of administering a composition containing a substance for stimulating the differentiation of neural stem cells, for example, wogonin (for example, KR 10-2004-0013997), and a method of using neural stem cells introduced with a gene stimulating the regeneration and differentiation of neural stem cells (for example, KR 10-2006-0093269).
Thus, when nerve stem cells, preferably nerve stem cells introduced with, for example, a gene for stimulating the development and differentiation of neural stem cells as described above, are administered together with periostin or the periostin-secreting cells as disclosed in the present invention, the targeting property thereof will be improved to stimulate the regeneration of nerve cells in a tissue-specific manner. Specifically, the migration of neural stem cells will be increased by periostin, and when periostin or periostin-secreting cells are injected into a location requiring the regeneration of nerve cells, followed by introducing neural stem cells into the body, the neural stem cells will migrate toward the periostin so that the regeneration of nerve cells in the area injected with the periostin or the periostin-secreting cells will be stimulated. More preferably, when periostin is injected into the affected part using an osmotic pump or the like so that it is continuously secreted, the neural stem cells will migrate to the periostin-secreting area so that the regeneration of nerve cells in that area will be stimulated.
Meanwhile, because it is known that neural diseases are very difficult to treat and reverse, neurotransplantation has been used for the repair of injured nerve tissue and the recovery of the function thereof (Bjorklund, Nature, 362:414, 1993; Olson, Nature Med., 3:1329, 1997; Spenceret al., N. Engl. J. Med., 327:1541, 1992: Freed et al., N. Engl. J. Med., 327:1549, 1992; Kordower et al., N. Engl. J. Med., 332:1118, 1995; Defer et al., Brain, 119:41, 1996; Lopez-Lozano et al., Transp. Proc., 29:977, 1997; Rosenstein, Exp. Neurol., 33:106, 1995; Turner et al., Neurosurg., 33:1031, 1993; Kang et al., J. Neurosci., 13:5203, 1993; Andersson et al., Int. J. Dev. Neurosci., 11:555, 1993; Sanberg et al., Nature Med., 3:1129, 1997). For example, a series of human patients with Parkinson's disease have been treated by neurotransplantation of mesencephalic cells obtained from 6 to 9 week old abortuses of human fetuses (Spencer et al., N. Engl. J. Med., 327:1541, 1992: Freed et al., N. Engl. J. Med., 327:1549,1992; Kordower et al., N. Engl. J. Med., 332:1118, 1995; Defer et al., Brain 119:41, 1996; Lopez-Lozano et al., Transp. Proc., 29:977, 1997). Thus, as described above, when periostin or periostin-secreting cells and neural stem cells are injected into the affected part, the neural stem cells will migrate to the periostin-secreting area so that the regeneration of nerve cells in that area can be stimulated, indicating that this ability to stimulate the regeneration of nerve cells can be used for the treatment of neural diseases.
In the present invention, the ability of periostin to induce the migration of neural stem cells was confirmed, and as described above, the method of regenerating nerve cells using neural stem cells and the effect thereof on the treatment of neural diseases were confirmed. Thus, it will be obvious to a person skilled in the art that, when neural stem cells are administered together with periostin or periostin-secreting cells, the neural stem cells will migrate to the area requiring nerve regeneration so that the regeneration of nerve cells can be stimulated in a tissue-specific manner, indicating that the administration of neural stem cells together with periostin or periostin-secreting cells is effective for the treatment of neural diseases.
The pharmaceutical composition of the present invention may be in the form of its pharmaceutically acceptable salts, and may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
The pharmaceutical composition according to the present invention may be formulated in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrup or aerosol, skin external agents, suppositories and sterile injectable solutions. Examples of carriers, excipients and diluents, which can be contained in the composition, include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium state and mineral oil.
The composition of the present invention may be formulated with commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid formulations comprise, in addition to the composition, at least one excipient, for example, starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, and syrup, and may contain various excipients, for example, wetting agents, flavoring agents, aromatics and preservatives, in addition to water and liquid paraffin, which are frequently used simple diluents. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents or suspending agents, propylene glycol, polyethylene glycol, plant oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppositories, witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin and the like can be used.
The preferred dose of the composition of the present invention may vary depending on the condition and weight of the patient, severity of the disease, the form of drug, the route and period of administration, but can be appropriately selected by a person having ordinary skill in the art. However, for preferable effects, it is desirable to administer the composition of the present invention in an amount of 0.0001 to 500 mg/kg, and preferably 0.001 to 250 mg/kg, per day. Administration may be carried out once a day, or may be carried out several times a day. The amount of administration is not intended to limit the scope of the present invention in any way.
The most preferable method for administration of the composition of the present invention will be a method of administering periostin or periostin-secreting cells into the surgical area after tumor removal surgery to induce the migration of neural stem cells so as to stimulate the efficient removal of the remaining cancer cells.
The composition of the present invention can be administered by various routes to mammals, including rats, mice, domestic animals and humans. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrauterine, epidural or intracerebroventricular injection.
EXAMPLES
Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.
Particularly, only HB1.F3 and HB1.F5 were illustrated as neural stem cell lines in the following examples, but it will be obvious to a person skilled in the art that the use of other neural stem cells can provide the same results as the use of the illustrated cell lines.
Example 1
Isolation of Periostin and Examination of Tissue-specific Expression of Periostin
1-1: Isolation of Periostin
Brain tumor tissues from three patients (obtained from the Ajou University Hospital, Korea; provided by M.D. K. Cho) as shown in Table 1 below were treated with Trizol reagent (Invitrogen) to extract total RNAs from the tumor tissues, thus preparing RNA samples. After the RNA sample has been extracted from each tissue by treatment with TRIzol, secreted genes which were highly expressed were examined using human U 133 Plus 2.0 GeneChip Oligo Microarrays (Affymetrix, Santa Clara, Calif., USA).












TABLE 1









Up-regulated 
Down-






gene
regulated


Tumor
Gender
Age
Histology
number
gene number







1
M
32
Glioma
4491
3758


2
F
50
Schwanoma
4775
5087


3
M
63
Ependyroma
6119
6824









The examination results showed that a total of 14 candidates (CXCL10, CYR61, FN1, GLIPR1, GPX3, HLA-DQB1, POSTN, PROS1, SFRP4, TIMP1, TIMP2, TNC, TNFRSF1A and TNFSF13B) are genes which were more than 4-fold overexpressed in the tumor tissue compared to normal tissue and associated with to the process of tumor development (see Table 2)










TABLE 2










Fold change













Gene

Tumor
Tumor
Tumor


Probe ID
symbol
Gene Function
1
2
3





Hs.413924
CXCL10
lymphocyte 
3.76
3.16
4.27




trafficking





Hs.8867
CYR61
cell proliferation, 
2.08
3.23
6.43




angiogenic factor





Hs.553516
GLIPR1
cell proliferation, 
1.53
3.25
5.16




invasion,







antiapoptic effect





Hs.386793
GPX3
antioxidative
3.16
2.97
5.20




function





Hs.409934
HLA-DQB1
immune response
2.08
6.51
6.96


Hs.438102
IGFBP2
regulation of tumor 
5.04
1.66
6.07




growth and invasion





Hs.136348
POSTN
induce cell 
4.19
4.13
7.79




migration







and angiogenesis





Hs.64016
PROS1
Anti-coagulation
3.84
2.19
8.00


Hs.105700
SFRP4
inhibit cell 
2.74
3.37
4.83




proliferation





Hs.522632
TIMP1
Anti-apoptosis, 
2.25
2.26
4.38




induce cell growth





Hs.104839
TIMP2
Anti-angiogenesis
2.32
2.07
3.38


Hs.143250
TNC
cell proliferation, 
2.15
7.21
3.87




migration





Hs.279594
TNFRSF1A
Apoptosis
2.80
2.32
2.68


Hs.525157
TNFSF13B
related with 
2.69
5.13
3.09




tumor genesis gene









The resulting candidates were subjected to quantitative Real-Time PCR. For this purpose, total RNA (50 ng) was used to generate cDNA using Taqman Reverse Reagents (Applied Biosystems, Foster City, Calif., US).
Taqman Gene Expression assays which contained Taqman Universal PCR Master Mix and the primers (Applied Biosystems, US; see Table 3 below for Probe ID) of the 14 candidates and the positive control GAPDH were performed to analyze the PCR products. The PCR reactions were performed under the following conditions: 2 min at 50° C. for incubation; 10 min at 95° C. for AmpliTaq amplification and 40 cycles for the melting (95° C. for 15 sec) and annealing (60° C. for 1 min) steps. PCR reactions for each template were performed in one 96-well dish for one gene primer pair. The quantification of the expression of each gene was performed using the comparative CT method (Livak and Schmittgen, 2001), and the patient's brain tumor tissues (obtained from the Ajou University Hospital, Korea; provided by M.D. K. Cho) shown in Table 4 below were used.










TABLE 3







Gene symbol
Probe ID









CXCL10
Hs 00171042. m1



CYR61
Hs 00155479. m1



FN1
Hs 00415006. m1



GLIPR1
Hs 00199268. m1



GPX3
Hs 00173566. m1



HLA-DQB1
Hs 00109790. m1



POSTN
Hs 00170815. m1



PROS1
Hs 00165590. m1



SFRP4
Hs 00180066. m1



TIMP1
Hs 00171558. m1



TIMP2
Hs 00234278. m1



TNC
Hs 00233648. m1



TNFRSF1A
Hs 00533568. g1



TNFSF13B
Hs 00198106. m1






















TABLE 4







Tumor
Gender
Age
Histology









Tumor1
F
59
Adenoma



Tumor2
F
56
Adenoma



Tumor3
F
36
Choroidplexuspapilloma



Tumor4
M
36
Meningotheliomatousmeningioma



Tumor5
F
48
Meningioma



Tumor6
M
45
Fibroblasticmeningioma



Tumor7
F
46
Fibroblasticmeningioma



Tumor8
F
56
Meningioma



Tumor9
M
63
Schwannoma



Tumor10
M
45
Anaplasticoligodendroglioma



Tumor11
M
33
Glioblastoma










As a result, as shown in FIG. 1, periostin (POSTN) was identified as a protein which was highly expressed (about 4-fold overexpressed) specifically in the malignant tumor tissue in comparison with the positive control GAPDH.
Meanwhile, the amino acid sequence of the overexpressed protein periostin was analyzed using the genetic information determined (http://www.ncbi.nlm.nih.gov/gene/10631) by search of NCBI Pubmed (http://www.ncbi.nlm.nih.gov/pubmed). As a result, the amino acid sequence of periostin having a total of the following four isoforms was determined.








periostin isoform 1



SEQ ID NO: 1










  1
mipflpmfsl llllivnpin annhydkila hsrirgrdqg pnvcalqqil gtkkkyfstc



 


 61
knwykksicg qkttvlyecc pgymrmegmk gcpavlpidh vygtlgivga tttqrysdas


 


121
klreeiegkg sftyfapsne awdnldsdir rglesnvnve llnalhshmi nkrmltkdlk


 


181
ngmiipsmyn nlglfinhyp ngvvtvncar iihgnqiatn gvvhvidrvl tqigtsiqdf


 


241
ieaeddlssf raaaitsdil ealgrdghft lfaptneafe klprgvleri mgdkvaseal


 


301
mkyhilntlq csesimggav fetlegntie igcdgdsitv ngikmvnkkd ivtnngvihl


 


361
idqvlipdsa kqvielagkq qttftdlvaq lglasalrpd geytllapvn nafsddtlsm


 


421
dqrllklilq nhilkvkvgl nelyngqile tiggkqlrvf vyrtavcien scmekgskqg


 


481
rngaihifre iikpaekslh eklkqdkrfs tflslleaad lkelltqpgd wtlfvptnda


 


541
fkgmtseeke ilirdknalq niilyhltpg vfigkgfepg vtnilkttqg skiflkevnd


 


601
tllvnelksk esdimttngv ihvvdkllyp adtpvgndql leilnkliky iqikfvrgst


 


661
fkeipvtvyt tkiitkvvep kikviegslq piiktegptl tkvkiegepe frlikegeti


 


721
tevihgepii kkytkiidgv pveiteketr eeriitgpei kytristggg eteetlkkll


 


781
qeevtkvtkf ieggdghlfe deeikrllqg dtpvrklqan kkvqgsrrrl regrsq


 








periostin isoform 2



SEQ ID NO: 2










  1
mipflpmfsl llllivnpin annhydkila hsrirgrdqg pnvcalqqil gtkkkyfstc



 


 61
knwykksicg qkttvlyecc pgymrmegmk gcpavlpidh vygtlgivga tttqrysdas


 


121
klreeiegkg sftyfapsne awdnldsdir rglesnvnve llnalhshmi nkrmltkdlk


 


181
ngmiipsmyn nlglfinhyp ngvvtvncar iihgnqiatn gvvhvidrvl tqigtsiqdf


 


241
ieaeddlssf raaaitsdil ealgrdghft lfaptneafe klprgvleri mgdkvaseal


 


301
mkyhilntlq csesimggav fetlegntie igcdgdsitv ngikmvnkkd ivtnngvihl


 


361
idqvlipdsa kqvielagkq qttftdlvaq lglasalrpd geytllapvn nafsddtlsm


 


421
dqrllklilq nhilkvkvgl nelyngqile tiggkqlrvf vyrtavcien scmekgskqg


 


481
rngaihifre iikpaekslh eklkqdkrfs tflslleaad lkelltqpgd wtlfvptnda


 


541
fkgmtseeke ilirdknalq niilyhltpg vfigkgfepg vtnilkttqg skiflkevnd


 


601
tllvnelksk esdimttngv ihvvdkllyp adtpvgndql leilnkliky iqikfvrgst


 


661
fkeipvtvyk piikkytkii dgvpveitek etreeriitg peikytrist gggeteetlk


 


721
kllqeevtkv tkfieggdgh lfedeeikrl lqgdtpvrkl qankkvqgsr rrlregrsq


 








periostin isoform 3



SEQ ID NO: 3










  1
mipflpmfsl llllivnpin annhydkila hsrirgrdqg pnvcalqqil gtkkkyfstc



 


 61
knwykksicg qkttvlyecc pgymrmegmk gcpavlpidh vygtlgivga tttqrysdas


 


121
klreeiegkg sftyfapsne awdnldsdir rglesnvnve llnalhshmi nkrmltkdlk


 


181
ngmiipsmyn nlglfinhyp ngvvtvncar iihgnqiatn gvvhvidrvl tqigtsiqdf


 


241
ieaeddlssf raaaitsdil ealgrdghft lfaptneafe klprgvleri mgdkvaseal


 


301
mkyhilntlq csesimggav fetlegntie igcdgdsitv ngikmvnkkd ivtnngvihl


 


361
idqvlipdsa kqvielagkq qttftdlvaq lglasalrpd geytllapvn nafsddtlsm


 


421
dqrllklilq nhilkvkvgl nelyngqile tiggkqlrvf vyrtavcien scmekgskqg


 


481
rngaihifre iikpaekslh eklkqdkrfs tflslleaad lkelltqpgd wtlfvptnda


 


541
fkgmtseeke ilirdknalq niilyhltpg vfigkgfepg vtnilkttqg skiflkevnd


 


601
tllvnelksk esdimttngv ihvvdkllyp adtpvgndql leilnkliky iqikfvrgst


 


661
fkeipvtvyr ptltkvkieg epefrlikeg etitevihge piikkytkii dgvpveitek


 


721
etreeriitg peikytrist gggeteetlk kllqedtpvr klqankkvqg srrrlregrs


 


781
q


 








periostin isoform 4



SEQ ID NO: 4










  1
mipflpmfsl llllivnpin annhydkila hsrirgrdqg pnvcalqqil gtkkkyfstc



 


 61
knwykksicg qkttvlyecc pgymrmegmk gcpavlpidh vygtlgivga tttqrysdas


 


121
klreeiegkg sftyfapsne awdnldsdir rglesnvnve llnalhshmi nkrmltkdlk


 


181
ngmiipsmyn nlglfinhyp ngvvtvncar iihgnqiatn gvvhvidrvl tqigtsiqdf


 


241
ieaeddlssf raaaitsdil ealgrdghft lfaptneafe klprgvleri mgdkvaseal


 


301
mkyhilntlq csesimggav fetlegntie igcdgdsitv ngikmvnkkd ivtnngvihl


 


361
idqvlipdsa kqvielagkq qttftdlvaq lglasalrpd geytllapvn nafsddtlsm


 


421
dqrllklilq nhilkvkvgl nelyngqile tiggkqlrvf vyrtavcien scmekgskqg


 


481
rngaihifre iikpaekslh eklkqdkrfs tflslleaad lkelltqpgd wtlfvptnda


 


541
fkgmtseeke ilirdknalq niilyhltpg vfigkgfepg vtnilkttqg skiflkevnd


 


601
tllvnelksk esdimttngv ihvvdkllyp adtpvgndql leilnkliky iqikfvrgst


 


661
fkeipvtvyk piikkytkii dgvpveitek etreeriitg peikytrist gggeteetlk


 


721
kllqedtpvr klqankkvqg srrrlregrs q






1-2 : Examination of Tissue-Specific Expression of Periostin
Whether the periostin isolated as described in Example 1-1 is expressed specifically in tumor tissue compared to normal brain tissue, the following experiment was performed.
Each of the patient's brain tumor tissue and normal brain tissue (obtained from the Ajou University Hospital) was fixed with a solution of 4% paraformaldehyde in 0.1M phosphate buffer, immersed in a solution of 30% sucrose in 0.1M phosphate buffer, allowed to stand overnight 4° C., and then frozen with an OCT compound. Each of the frozen samples was sectioned to 30 μm with a cryostat (KORF Instrum 900). The sectioned samples were washed with PBS, blocked with a solution of 5% goat serum and 0.5% triton X-100 in PBS for 30 minutes, and then washed twice with a solution of 0.5% BSA (bovine serum albumin) in PBS for 15 minutes. Then, each sample was treated with a 1:100 dilution of rabbit polyclonal human Periostin antibody (BioVender), incubated overnight at 4° C., and then incubated with biotin-conjugated secondary antibody at room temperature for 2 hours. After that, periostin of each sample was stained using an ABC kit (Vector) and a diaminbenzidin peroxidase (DAB) kit.
As a result, as can be seen in FIG. 2, periostin was not expressed in the normal brain tissue, whereas it was expressed specifically in the brain tumor tissue. Particularly, it could be seen that the expression of periostin in the benign brain tumor was marked, and strongly appeared only in the malignant brain tissue, and such results were consistent with the real-time PCR results shown in FIG. 1. FIGS. 2A and 2B shows the results of observation of the normal brain tissue, FIGS. 2C to 2E show the results of observation of the malignant brain tumor tissue, and FIG. 2F shows the results of observation of the benign brain tumor tissue.
Example 2
Examination of the Ability of Periostin to Induce the Migration of Neural Stem Cells—in vitro
2-1 : Examination of Migration of Neural Stem Cells at Various Concentrations of Periostin
In order to examine the effect of periostin on the migration of neural stem cells, a Boyden chamber assay was performed using two human neural stem cell lines (HB1.F3 and HB1.F5), thereby determining whether human neural stem cells migrated in response to periostin in vitro. For controls, VEGF (vascular endothelial growth factor) was used as a positive control, and FBS-DMEM medium was used as a negative control.
Specifically, cultured neural stem cells were detached with 0.05% trypsin, washed with DMEM medium, and then re-suspended in DMEM medium, after which 5×104 cells seeded into an upper chambers (Costar Transwell) in 200 μl of 10% FBS-containing DMEM medium. After one day, a lower chamber was treated with various concentrations of periostin or VEGF. The periostin was used at concentrations of 5, 10 and 20 μg/ml, and the VEGF was used at concentrations of 5, 10 and 20 ng/ml. 10% FBS-DMEM medium was used as a negative control. After 12 hours, in order to determine the number of cells that migrated from the upper chamber through a 8-μm porous polycarbonate membrane to the lower portion of the membrane in response to the stimulation of the lower chamber, the migrated neural stem cells were stained with hematoxylin and then counted.
As a result, as can be seen in FIG. 3, it was observed that both the two neural stem cell lines migrated below the membrane in response to the periostin. As shown in FIG. 3A, in the case of the HB1.F3 cells treated with periostin, the ability to induce the migration of neural stem cells was increased by about 100% compared to when the HB1.F3 cells were not treated with periostin, and this increase was about 4 times higher than the increase in induction of migration by VEGF (25%), suggesting that the ability of periostin to induce the migration of neural stem cells is excellent compared to that of the positive control VEGF. Also, the ability of VEGF to induce the migration of neural stem cells was the highest at a concentration of 5 ng/ml, and the ability of periostin was the highest at a concentration of 10 μg/ml. In the case of the HB1.F5 cells, the ability to induce the migration of neural stem cells was similar between VEGF and periostin, but the migration of neural stem cells was increased upon treatment with periostin (FIG. 3B).
2-2: Examination of Migration of Neural Stem Cells at Various Time Points After Treatment with Periostin
In order to demonstrate the migration-inducing ability of periostin by measuring the number of migrated cells, each of the two neural stem cell lines was detached with 0.05% trypsin, washed with DMEM medium, and then re-suspended in DMEM medium, after which 5×104 cells were inoculated into an upper chamber (Costar Transwell) in 200 μl of 10% FBS-containing DMEM medium. After one day, a lower chamber was treated with 10 μg/ml of periostin, and after 4 hours, 8 hours, 12 hours and 24 hours, the migrated cells were stained with H&E (hematoxylin & eosin), counted and photographed (Olympus BX51, Japan).
As a result, as can be seen in FIG. 4, the two neural stem cell lines showed the same migration pattern with time, and the migration thereof was increased with time. As can be seen in the photographs of FIGS. 4A and 4C showing the staining results, the increase in the number of the migrated cells with the passage of time was clearly observed. In addition, the number of the migrated cells also increased (FIGS. 4B and 4D).
Example 3
Examination of Pathways Involved in the Migration of Neural Stem Cells by Periostin
3-1: Examination of Periostin Recognition Receptor
Periostin is known to bind to a complex of integrin-alpha and integrin-beta, and a complex of integrin alpha V beta 3, alpha V beta 5 and alpha 6 beta 4 is known to act as a receptor in cell migration. In order to examine what complex acts as a receptor in the migration of HB 1.F3 neural stem cells, reverse transcription-PCR (RT-PCR) and a microarray assay were performed.
For RT-PCR, total RNA was isolated from F3 neural stem cells using TRIZol reagent (Invitrogen), and the first strand cDNA was synthesized by incubating the total RNA in a reaction mixture containing Superscriptll (Invitrogen, Calif.) and 12-18mer oligo dT at 42° C. for 50 minutes, and then at 72° C. for 15 minutes. Then, PCR was performed using 50 μl of a PCR mix containing 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxyribonucleoside-triphosphate, 50 pmol primer, 1U Tag DAN polymerase (Invirogen) and 150 ng of the cDNA as a template. The PCR reaction was performed under the following conditions: 25-30 cycles of 45 sec at 94° C., 45 sec at 55° C. and 45 sec at 72° C., and final extension at 72° C. for 10 min. Primer sequences for each integrin subunit are shown in Table 5 below.










TABLE 5





Gene
Primer sequence
F/B
SEQ ID NO







Human
5′-ACT GGG AGC ACA AGG AGA ACC-3′
Forward
SEQ ID NO: 5


integrin
5′-CCG CTT AGT GAT GAG ATG GTC-3′
Backward
SEQ ID NO: 6


alpha V





 


Human
5′-CTG CAA GAA CGG GGT GAA TG-3′
Forward
SEQ ID NO: 7


integrin
5′-CAC AAT GTC TAC CAA GCC C-3′
Backward
SEQ ID NO: 8


beta 1





 


Human
5′-CAA GCT GGC TGA AAA CAA CA-3′
Forward
SEQ ID NO: 9


integrin
5′-ACT GCT CCT GGA TGC ACT CT-3′
Backward
SEQ ID NO: 10


beta 2





 


Human
5′-AGA TGC GAA AGC TCA CCA GT-3′
Forward
SEQ ID NO: 11


integrin
5′-CCG TCA TTA GGC TGG ACA AT-3′
Backward
SEQ ID NO: 12


beta 3





 


Human
5′-GCC TTC ACT TTG AGC ACT CC-3′
Forward
SEQ ID NO: 13


integrin
5′-CTG CTG TAC TCG CTT TGC AG-3′
Backward
SEQ ID NO: 14


beta 4





 


Human
5′-AGC AGC TTC CAT GTC CTG AG-3′
Forward
SEQ ID NO: 15


integrin
5′-GAA GTT GCT GGT GAG CTT CC-3′
Backward
SEQ ID NO: 16


beta 5





 


Human
5′-GAC TCC GGA AAC ATT CTC CA-3′
Forward
SEQ ID NO: 17


integrin
5′-CTG ACA GTC GCA GTT GCA TT-3′
Backward
SEQ ID NO: 18


beta 6





 


Human
5′-AGC AAT GGC CTC TAC AGT CGC AGC-3′
Forward
SEQ ID NO: 19


integrin
5′-GCT TGG AGA GAA ACC CAG AAA GTC-3′
Backward
SEQ ID NO: 20


beta 7





 


Human
5′-TTC ATC ATT TTC ATA GTT ACA TTC-3′
Forward
SEQ ID NO: 21


integrin
5′-CAT TAA GTG TTT AAA AAT CTT TTT-3′
Backward
SEQ ID NO: 22


beta 8









The amplified PCR products were loaded onto 1% agarose gel and then electrophoreses, and the resulting bands were observed. A microarray assay was performed using a probe for each subunit according to the Trizol method. For this purpose, neuronal stem cells were treated with Trizol reagent (Invitrogen) to extract total RNA, thus preparing an RNA sample. After the RNA sample has been extracted, the expressions of integrin subunits were examined using human U 133 Plus 2.0 GeneChip Oligo Microarrays (Affymetrix, Santa Clara, Calif., USA).
As a result, as shown in FIG. 5, among the subunits, integrin alpha V, beta 1 and beta 5 were highly expressed, and such results were consistent with the RT-PCR results. Thus, it was concluded that the receptor involved in the migration of neural stem cells is alpha V beta 5. Receptors for periostin include three types: integrin alpha V beta 3, alpha V beta 5, and alpha 6 beta 4. Among them, beta 3 and beta 4 do not exist, and thus alpha V beta 5 can be considered as a potential receptor for periostin.
3-2: Examination of Whether PI3 Kinase Signaling Pathway is Involved
The FAK signaling pathway is linked to kinases as its downstream pathways and transduces signals. With respect to cell migration, the PI3K/AKT signaling pathway (PI3K-AKT-mTOR-CDK5-Nudel1, PAK1) is frequently used as the downstream pathway of FAK, and thus the following experiment was performed in order to determine the kinase activation pathway.
First, neural stem cells treated with periostin were washed with PBS and lysed with PMSF and a protease inhibitor in RIPA buffer (0.5% sodiumdeoxycholate, 0.1% sodiumdodecyl sulfate, 1% NP-40, PBS). Protein concentration was determined by a Bio-Rad-DC protein assay (Bio-Rad, Hercules, Calif., US). The samples were adjusted to the same protein concentration, after 30 μg of each protein was separated on SDS-PAGE gel. Then, protein samples were transferred to an immobilon-P membrane (Millipore Corp., MA, US). For protein detection, the membrane was incubated in TBS-T 5% skim milk at room temperature for 30 minutes and washed three times with TBS-T for 15 minutes each time. Then, the membrane was incubated with primary antibodies of FAK, pFAK, PAK and pPAK (1:1000 dilution; Epitomics) in 5% BSA-TBS-T overnight at 4° C. Then, the membrane was washed three times with TBS-T for 15 minutes each time. Then, the membrane was incubated with secondary anti-rabbit antibody (1:2000 dilution; Zymed, Calif., USA) at room temperature for 1 hour. The membrane was washed three times with TBS-T, after which the antibody binding protein was added to an ECL Western blotting detection solution (Amersham Pharmacia Biotechnology, buckinghamahire, UK) for 1 minute and exposed to Kodak X-ray firm for detection. The Western blotting analysis was performed at various time points between 0 and 12 hours after treatment with periostin.
As can be seen in FIG. 6, the results of Western blotting indicated that FAK was highly activated from 30 minutes after treatment with periostin. Meanwhile, 1 hour after treatment with periostin, AKT and mTOR were activated, and then CDK5 was activated and the activation thereof was maintained up to 12 hours after treatment with periostin. Meanwhile, PAK1 started to be activated 1 hour after periostin treatment, and the activation thereof was maintained up to 6 hours. Nudel1 was activated at PAK1-activation decreased time point, and the activation thereof was maintained for 12 hours. Such results indicate that cell migration signaling induced by periostin is made through the FAK signaling pathway and occurs through the AKT signaling pathway and the downward pathway of CDK.
3-3: Examination of Nucleokinesis Induced by Periostin
DCX (doublecortin) together with Nudle1 and PAK1 is well known as a factor that regulates cell migration using actin and tubulin.
It has been reported that, in the development of the central nervous system, neural stem cells migrate from the subventricular zone to cortex through radial glial cells using the ERK (extracellular signal regulated kinase) and CDK5 signaling pathways, and in the neuronal migration, DCX participated in nucleus translocation, neucleokinesis (Niethammer et al., 2000). Thus, in order to examine the role of ERK, CDK5, and particularly DCX, in the migration of neural stem cells induced by periostin, neural stem cells were treated with the MEK/ERK inhibitor PD98057 or the CDK5 inhibitor roscovitine and subjected to an in vitro migration assay and an immunohistochemical assay as follows.
3-3-1: In vitro Migration Assay Using ERK Pathway and CDK5 Pathway Inhibitors
Cultured neural stem cells were detached with 0.05% trypsin, washed with DMEM medium and re-suspended in DMEM medium, after which 5×104 cells were inoculated into an upper chamber (Costar Transwell) in 200 μl of 10% FBS-containing DMEM medium. After one day, a lower chamber was treated with each of combined solution that composite with POSTN (R&D), VEGF, 20 nM PD98059 and 20 nM Roscovitine (Calbiochem).
After 12 hours, in order to determine the number of cells that migrated from the upper chamber through a 8-μm porous polycarbonate membrane to the lower portion of the membrane in response to the stimulation of the lower chamber, the migrated neural stem cells were stained with hematoxylin & eosin and counted.
As a result, as can be seen in FIG. 7A, the migration of neural stem cells induced by periostin was significantly inhibited by roscovitine (94% inhibited) or PD98059 (40% inhibited). Also, in the control group not treated with periostin, the migration of neural stem cells was inhibited by roscovitine or PD98059, suggesting that the cell migration signaling induced by periostin passes through the ERK pathway and the CDK5 pathway. Particularly, it could be seen that the inhibitory effect of the ERK inhibitor PD98059 on the migration of neural stem cells was lower than that of the DCX inhibitor roscovitine. This indicates that the DCX signaling pathway plays an important role in the process of inducing the migration of neural stem cells by periostin.
3-3-2: Immunohistochemical Analysis of ERK and DCX
To analyze the ERK and DCX affection of migrated cells, transwell membrane immunostaining was performed using ERK and DCX antibodies (Cell signaling tech., Danver, Mass.).
Cultured neural stem cells were detached with 0.05% trypsin, washed with DMEM medium and re-suspended in DMEM medium, after which 5×104 neural stem cells were inoculated into an upper chamber (Costar Transwell) in 200 μl of 10% FBS-containing DMEM medium. After one day, a lower chamber was treated with each of VEGF (10 ng/ml), periostin (10 μg/ml) and periostin+inhibitor. After 12 hours, the changes in locations of DCX and ERK in the cells that migrated from the upper chamber through the 8-μm porous polycarbonate membrane to the lower portion of the membrane in response to the stimulation of the lower chamber were examined.
Next, the membrane was washed with PBS, blocked twice with a solution of 5% goat serum and 0.5% triton X-100 in PBS for 30 minutes, and then washed with a solution of 0.5% BSA (bovine serum albumin) in PBS for 15 minutes. After this, the membrane was incubated with a 1:500 dilution of goat polyclonal human DCX antibody and a 1:1000 dilution of rabbit polyclonal human ERK antibody at 4° C. overnight, and then washed twice with PBS for 15 minutes each time. Next, the membrane was incubated with biotin-conjugated anti-rabbit secondary antibody at room temperature for 1 hour and washed trice with PBS, after which periostin was detected by staining the membrane using an avidin-biotin kit (Vector, Calif.) and a diaminbenzidin peroxidase (DAB) kit (Vector, Calif.).
As a result, as can be seen in FIG. 7B, periostin and VEGF induced cell migration, and DCX was concentrated around the nuclei in the periotin-treated environment (see the small boxes of FIG. 7B). When the CDK5 inhibitor was added, the concentration of DCX around the nuclei was inhibited.
Example 4
Examination of the Ability of Periostin to Induce the Migration of Neural Stem Cells—In vivo
4-1: Construction of NIH3T3 Cell Line Overexpressing Periostin
In order to construct a cell line overexpressing periostin, 293T cells were transfected with a pCL-Ampho vector expressing the amphotropic envelope gene and with a pCXbsr vector (Shiga University, Dr. Inoue) encoding the blasticidin S-resistant gene and the periostin gene, in which each of the vectors was digested with restriction enzymes in order to confirm whether the periostin gene and the amphotropic envelope gene were present in the vectors.
Specifically, the two vectors were transfected with lipofectamin and Plus reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. After 2 days, the culture medium was collected and treated with 5 μg/ml of blasticidin S (Invitrogen, Japan) for 1 week. Next, it was replaced with a fresh medium containing no blasticidin S and incubated for 3 days, after which the medium was incubated with NIH3T3 cells for 2 days. Then, a blasticidin S-containing medium was added thereto and incubated for 1-2 weeks, and cells introduced with the genes were selected. Cells forming colonies were culture in each well of a 96-well plate (FIGS. 8A and 8B).
The amounts of the secreted periostin in the recombinant periostin-expressing cell lines were analyzed by ELISA (FIG. 8C) and Western blotting (FIG. 8D), and the 1-8 cell line showing the highest expression level of periostin was selected and named “P-NIH3T3 cell line”.
4-2: Ipsilateral Implantation of Neural Stem Cells and NIH3T3 Cells
Neural stem cells labeled with DiI were implanted into the cerebral hemispheric cortex ipsilaterally to the normal NIH3T3 cells or periostin-overexpressing NIH3T3 cells (P-NIH3T3) labeled with Dyecycle Green.
Specifically, 1×106 P-NIH3T3 cells labeled with Dyecycle green were implanted into the right striatum, and NIH3T3 cells were used as a control group. The location of implantation was AP +0.4, ML −2.3, DV −4.5 mm from the bregma. After 5 days, 1×106 neural stem cells labeled with DiI were implanted into the ipsilateral hemispheric cortex at a location of AP +0.4, ML −2.3, DV −2.0 from the bregma.
3 μl of HBSS (Hanks' balanced salt solution) or 1×106 P-NIH3T3 (periostin-overexpressing NIH3T3) cells labeled with Dyecycle Green were implanted into the right striatum. The location of implantation was AP +0.4, ML −2.3, DV −4.5 mm from the bregma. After 5 days, 1×106 neural stem cells labeled with DiI were implanted into the ipsilateral hemispheric cortex at a location of AP +0.4, ML −2.3, DV −2.0 from the bregma.
The cells and the HBSS were implanted at a rate of 0.2 μl/min using a 26-gauge Hamilton syringe (Hamilton, Nev., US) equipped with an automated microinjector (KD scientific INC, MA, US).
After implantation, the needle was maintained at that location for 15 minutes, and then pulled out slowly.
After 2-4 weeks, the rat's brain was fixed with 4% paraformaldehyde in 0.1M cold phosphate buffer. After 4 hours, the brain was immersed in 30% sucrose in 0.1M phosphate buffer, and then allowed to stand overnight at 4° C. Next, the brain was frozen using an OCT compound and sliced to 30 μm with a cryostat. The slice was mounted using Vectashield® Hard SetTM mounting medium (Vector, Calif., US) and measured using an Olympus IX71 confocal laser scanning microscope (Olympus, Tokyo, Japan).
As a result, as shown in FIG. 9, the DiI-labeled neural stem cells showed no tropism for the normal NIH3T3 cells before 2 weeks after implantation. However, for the P-NIH3T3 cells continuously expressing periostin, it was observed that the neural stem cells passed through the corpus callosum to express the Vybrant Dyecycle Green label (FIG. 9B). 2 weeks after implantation of the neural stem cells, it was observed that the neural stem cells were dispersed throughout the P-NIH3T3 cells implanted around the striatum tissue and were mixed with the P-NIH3T3 cells (FIG. 9D). It was observed that the P-NIH3T3 cells continued to express periostin even 4 weeks after implantation of the neural stem cells and had the ability to induce migration of the neural stem cells (FIGS. 9C and 9E).
4-3: Contralateral Implantation of Neural Stem Cells and NIH3T3 Cells
DiI-labeled neural stem cells (HB1.F3) were implanted into the hemispheric cortex contralaterally to the hemispheric cortex implanted with periostin-overexpressing NIH3T3 cells (P-NIH3T3), in the same manner as Example 4-2, except that the location of implantation was AP +0.4, ML +2.3, DV −2.0 mm from the bregma.
As a result, it could be seen that the neural stem cells surrounded the p-NIH3T3 cells (FIGS. 10A and 10B) along the corpus callosum (FIG. 10C), even though the two types of cells were significantly spaced apart from each other. Also, it was observed that the neural stem cells migrated to the p-NIH3T3 cells along the corpus callosum pathway even 2 weeks after implantation of the cells (FIGS. 10F to 10H).
The in vivo expression of periostin was observed by immunohistochemical analysis using a DAB kit (diaminbenxidine peroxidase kit; Vecter, Calif.). Specifically, the slice was washed three times with PBS and blocked with a solution of 0.5% Triton X-100 and 5% BSA (bovine serum albumin) in PBS for 30 minutes. Then, the slice was washed with 0.5% BSA for 15 minutes and incubated with rabbit polyclonal human periostin antibody (1:500 duilution; Abcam, Cambridge, UK) overnight at 4° C. The slice was washed three times with PBS for 15 minutes each time and incubated with secondary anti-rabbit antibody (1:1000 dilution; Vector, Calif., US) at room temperature for 1 hours. Then, periostin was observed either by detection with a DAB kit (FIGS. 10D and 10I) or by fluorescence staining (FIGS. 10E and 10J).
As can be seen in FIGS. 10D and 10I or from the fluorescence staining results (FIGS. 10E and 10J), the p-NIH3T3 cells continuously secreted periostin during 1-2 weeks after implantation. In the figures, the red color indicates neural stem cells, and the green color indicates p-NIH3T3 cells.
Example 5
Examination of Therapeutic Effects of Periostin-expressing Cells (p-NIH3T3) and Suicide Gene-expressing Cells (F3-CD) in Rat Glioma Model
5-1: Analysis of Cytotoxic Effect of F3-CD Cells
5-1-1: Determination of Whether F3-CD Cells Produce CD, and Analysis of Activity Thereof
To determine the bystander effect of suicide gene-expressing neural stem cells on tumor cells, an F3-CD cell line expressing cytosine deaminase was made and whether the cell line expresses the CD gene and protein was determined.
For CD, the following primers were used.









SEQ ID NO: 23:










5′- GAGTCACCGCCAGCCACACCACGGC-3′:
Forward



 









SEQ ID NO: 24:










5′- GTTTGTAATCGATGGCTTCTGGCTGC-3′:
Backward






Total RNA was isolated from neural stem cells using TRIZol reagent (Invitrogen). The first strand cDNA was prepared by incubating a reaction mixture containing SuperscriptII (Invitrogen, Calif.), 12-18mer oligo dT and the isolated total RNA. Herein, the reaction mixture was incubated at 42° C. for 50 minutes, and then at 72° C. for 15 minutes. Next, PCR was performed using 50 μl of a PCR mix containing 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxyribonucleotide-triphosphate, 50 pmol primer, 1U Tag DAN polymerase (Invirogen) and 150 ng of the cDNA as a template. The PCR reaction was performed under the following conditions: 25-30 cycles of 5 sec at 94° C., 60 sec at 60° C. and 90 sec at 72° C.; and final extension at 72° C. for 10 min.
The RT-PCR results showed clear bands. Thus, it could be seen that the CD gene was not expressed in the control F3 cell, but was expressed in the F3-CD cells (FIG. 11A).
5-1-2: HPLC Analysis of CD Protein
Through enzyme activity analysis and HPLC analysis, the activity of the CD protein was measured. The CD protein has the activity of converting 5-FC (5-fluorocytosine) into 5-FU (5-flurouracil) that is a highly toxic anticancer agent. For HPLC analysis, 2×105 F3-CD cells were dispensed into each well of a 12-well plate and cultured with 1 mM 5-FC in 1 ml of medium for 48 hours. 50 μl of the culture medium was extracted using 500 μl of a mixture of ethyl acetate: isopropanol: acetic acid (84:15:1 [v:v:v]), and the organic fraction was re-suspended in 500 μl of a mixture of water: methanol (4:1 [v:v]). HPLC of the resulting solution was performed using a Kromasil 100-5C-19 column (Kromasil, Bohus, Sweden) at 270 nm. 5-FC and 5-FU were separated at a flow rate of 1 ml/min by dissolution in 40 mM KH2PO4-containing isocratic mobile phase, adjusted to a pH of 7.0 by addition of 10% KOH. The residence time was 3.4 min for 5-FC, 3.9 min for 5-FU, and 8.9 min for 5-bromouracil (Sigma), and the quantitative analysis of 5-FC and 5-FU was performed using 5-bromouracil as an internal standard.
Meanwhile, 50 μg of the protein from the cell lysate was incubated at 37° C. for 8 hours in the presence of 1 mM 5-FC and subjected to HPLC analysis in the same manner as described above.
As a result, it was found that 2.9 μg of 5-FU was produced in the lysate by the CD and that the production of 5-FU was not observed in the control F3 cells (FIG. 11B). Also, the supernatant of the F3-CD cells cultured with 5-FC showed two peaks corresponding to 5-FC and 5-FU at an UV absorbance of 270 nm. Also, the quantification of HPLC showed 93 μM, indicating that the F3-CD cells convert 5-FC into 5-FU (FIGS. 11C and 11D). Accordingly, it was confirmed that the F3-CD cells encoding the CD gene can produce the CD protein having normal enzymatic activity and that the converted 5-FU is continuously accumulated in culture medium. It is considered the secreted 5-FU plays an important role in exhibiting a bystander effect for the surrounding cells.
5-1-3: Analysis of Cytotoxic Effect of F3-CD Cells on C6 Cells—in vitro
To determine the in vitro cytotoxic effect of F3-CD cells on rat glioma C6 cells (provided from Myung-Ae Lee, Ajou University) by co-culture of rat glioma C6 cells with F3-CD cells in the presence of 5-FC, the following experiment was performed.
Specifically, C6 cells and F3-CD cells were dispensed into each well of a 96-well plate at various ratios (C6:F3-CD=100:5-200) and cultured for one day, after which the cells were cultured in 5-FC—containing fresh medium (100 μg/ml, Sigma). After 3 days, each well was washed with PBS, and MTT solution (0.5 mg/ml, Sigma) was added thereto. After 4 hours, the MTT was replaced with 200 μl of DMSO, and each well was further incubated for 1 hour. Then, the supernatant of each well was collected and transferred to a fresh 96-well plate, after which the number of the C6 cells was counted using a microplate spectrophotometer (Bio Tek instrument Inc.) at 550 nm and 630 nm as a reference filter.
As a result, as can be seen in FIG. 12, as the number of F3-CD cells co-cultured with C6 cells was increased, the cytotoxic effect increased. Also, when the ratio of F3-CD cells to the total cells was 10%, C6 cells started to die, and when the ratio of F3-CD cells reached 50%, the death of 80% of C6 cells was induced.
5-2: Cytotoxic Effect of F3-CD Cells on p-NIH3T3 Cells—in vivo
In order to determine the bystander effect of F3-CD cells on p-NIH3T3 cells in in vivo conditions, the following bystander effect assay was performed.
0.5-2×106 p-NIH3T3 cells labeled with Dyecycle green together with rat glioma C6 cells were implanted into the right striatum of rats. After 5 days, for each group, 1×106 neural stem cells labeled with DiI were implanted into the right hemispheric cortex. After implantation, the needle was allowed to stand for 15 minutes, and then was removed slowly. After 7-10 days, 5-FC was administered by intraperitoneal injection to the rats at different concentrations (250, 500 and 1000 μg/kg) everyday for 2 weeks.
Then, brains were collected from the rats and fixed with 4% paraformaldehyde in 0.1M cold phosphate buffer. After 4 hours, the brains were immersed in 30% sucrose in 0.1M phosphate buffer and allowed to stand overnight at 4° C. Then, the brains were frozen with an OCT compound and sliced to 30 μm with a cryostat. The slices were mounted using a gel mount (Biomeda corp, Foster City, Calif.). The cancer cells and the size thereof were measured using a BMF stereo investigator program (MicroBrightField Inc., Williston, USA).
The survival rate of periostin-overexpressing NIH3T3 cells was stereologically measured. As a result, it was found that, when F3-CD cells were implanted into the animals having a high concentration of 5-FC, the survival rate of p-NIH3T3 cells was significantly decreased (44%; FIG. 13) compared to when the control F3 cells were implanted. The graph of FIG. 13B shows the results of quantifying the number of p-NIH3T3 cells in each rat (administered with 5-FC) using the stereo investigator program. In the in vivo assay, the bystander effect of the F3-CD cells seriously appeared in the group administered with 5-FC, particularly at a concentration of 500 μg/kg. Such results indicate that, even when F3-CD cell line and the periostin-overexpressing cell line are administered to different areas, the F3-CD cells migrates from the original area of implantation by periostin, so that the F3-CD cells exhibit sufficient cytoxicity on the periostin-overexpressing cells in vivo.
5-3: Examination of Therapeutic Effects of Periostin-expressing Cells (p-NIH3T3) and Suicide Gene-expressing Cells (F3-CD) in Rat Glioma Model
In order to examine whether the use of periostin-expressing cells (p-NIH3T3) and suicide gene-expressing cells (F3-CD) can induce the death of cancer cells around the periostin-expressing cells to reduce the size of tumor tissue, thus exhibiting therapeutic effects, the following experiment was performed.
F3-CD cells, constructed in Example 4-1 and labeled with DiI, were implanted into the cerebral cortex of rats contralaterally to C6 glioma cells alone or a combination of C6 cells and p-NIH3T3 cells, and then treated with 5-FC for 2 weeks. Implantation of each type of cells was performed in the same manner as Example 4-3, and the rat brain was collected at the end of treatment with 5-FC.
As a result, as can be seen in FIG. 14, the direction of migration of F3-CD cells was regulated by the introduction of p-NIH3T3 cells, and the ability to induce the migration of F3-CD cells increased in a manner dependent on the number of p-NIH3T3 cells, so that the F3-CD cells migrated toward the C6 glioma cells. Also, the tumor size of the animal implanted with F3-CD cells decreased to 40% of that of the animal implanted with C6 cells alone, indicating that the growth of brain tumor was significantly inhibited. In addition, the tumor size decreased in proportion to the number of p-NIH3T3 cells inducing F3-CD cells to the tumor mass.
Meanwhile, in the case of the animal implanted with the same number of p-NIH3T3 cells, the tumor size decreased more significantly with time (60%; FIG. 14B). It is believed that the decrease in the tumor size resulted from the inhibition of invasion of C6 glioma cells. In the case of the group implanted with C6 cells alone, it was observed that the invasion of C6 cells occurred so that the C6 cells propagated far from the original area of implantation. However, in the case implanted with the p-NIH3T3 cells, the C6 cells did not propagate far away from the original site of implantation.
Also, as shown in FIG. 15, the results of H&E staining support that the invasion of rat C6 glioma cells in the animal implanted with the p-NIH3T3 cells was inhibited. FIG. 15 shows the slice of rat brain treated with 5-FC treatment for 2 weeks at 7 (FIG. 15A) or 10 (FIG. 15B) days after F3.CD cells were transplanted.
As described above, the inhibition of invasion of glioma cells is enhanced by the increase in the number of p-NIH3T3 cells and the period of migration of F3-CD cells. Such results indicate that, as the amount of periostin secreted from p-NIH3T3 cells increases, cancer cell targeting of suicide gene-expressing F3 cells can be enhanced, thereby significantly inhibiting the growth of cancer cells. Also, such results suggest that, as the amount of periotin increases, the invasion of cancer cells into normal tissue, which is an important factor in the recurrence of brain cancer, can be effectively inhibited, thereby maximizing the effect of treating tumors.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Sequence Listing
The electronic file was attached.

## Claims

1. A method of treating a glioma the method comprising:
(a) administering to the area around the glioma a periostin-overexpressing and over-secreting cell transfected by the periostin gene, wherein the transfected cell is a fibroblast or HEK cell;
(b) administering to the area around the glioma a human neural stem cells transfected with a cytosine deaminase gene, wherein the neural stem cells express cytosine deaminase;
(c) administering to the area around the glioma 5-fluorocytosine, wherein the cytosine deaminase converts the 5-fluorcytosine to 5-fluoruracil; wherein the oversecreted periostin enhances targeting of the human neural stem cells, and wherein the 5-fluorouracil is in an amount that will kill glioma cells.

2. The method of claim 1, wherein the periostin comprises an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 4.