KR102312414B1 - Composition for preventing or treating cancer comprising TRAIL-secreting mesenchymal stem cells and compound C - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, kit, or anticancer adjuvant for preventing or treating cancer comprising mesenchymal stem cells and compound C secreting TRAIL.
When the mesenchymal stem cells secreting TRAIL of the present invention and compound C are combined, the expression of proteins related to apoptosis of cancer cells is reduced compared to the case of treating TRAIL-secreting mesenchymal stem cells or compound C alone, respectively. It is possible to significantly increase apoptosis of cancer cells by increasing and reducing the expression of anti-apoptotic proteins, thereby obtaining a high anticancer effect.

Description

A pharmaceutical composition for preventing or treating cancer comprising TRAIL-expressing mesenchymal stem cells and compound C TECHNICAL FIELD

The present invention relates to a pharmaceutical composition, kit, or anticancer adjuvant for preventing or treating cancer comprising mesenchymal stem cells and compound C secreting TRAIL.

Glioblastoma multiforme (GBM) is one of the most aggressive and devastating types of cancer among gliomas. GBM has penetrating properties into functional brain tissue and resistance to chemotherapy and radiation therapy. Conventional treatments such as surgery, radiation therapy and chemotherapy have a low average survival rate, and it is almost impossible to completely remove GBM surgically, especially because the boundary between brain cells and tumor cells is not clear. Therefore, new strategies to increase therapeutic efficacy are needed.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively activates apoptosis in cancer cells, not normal cells. However, when TRAIL is used alone to treat cancer including glioma, its toxicity and short protein half-life are problematic. In particular, most malignant glioma cells are resistant to TRAIL-induced cytotoxicity despite the expression of surface apoptosis-inducing TRAIL receptors.

AMPK (AMP-activated protein kinase) is a key energy sensor that regulates cellular energy homeostasis and is associated with cell cycle and apoptosis. Compound C, also called dorsomorphin, is commonly used as an AMPK inhibitor to effectively block the action of AMPK.

Republic of Korea Patent Publication No. 10-2015-0012136 "Improved anticancer use according to the combination of TRAIL-secreting stem cells and MK886" aims to provide a more enhanced anti-cancer effect by combining TRAIL-secreting stem cells and MK886.

However, there is a need for additional research on a method that more significantly increases the sensitivity of TRAIL-induced apoptosis in cancer, particularly brain cancer such as glioblastoma multiforme, and guarantees clinical effects.

As a result of the present inventors' earnest efforts to provide a more effective anticancer composition by increasing the sensitivity of cancer cells to apoptosis induced by TRAIL, when both the stem cells secreting TRAIL and compound C (compound C) are treated in cancer cells By significantly increasing the apoptosis ability of TRAIL, it was confirmed that it can provide a high cancer cell inhibitory effect, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition, kit or anticancer adjuvant for preventing or treating cancer comprising mesenchymal stem cells and compound C secreting TRAIL.

The present invention provides a pharmaceutical composition, kit or anticancer adjuvant for preventing or treating cancer comprising mesenchymal stem cells and compound C secreting TRAIL.

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be derived from any one selected from the group consisting of humans, cattle, pigs and horses.

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be derived from any one selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, hair follicles and skin.

According to a preferred embodiment of the present invention, the cancer may be any one or more selected from the group consisting of melanoma, brain cancer, lung cancer, breast cancer, testicular cancer and kidney cancer.

According to a preferred embodiment of the present invention, the mesenchymal stem cells, compound C or a pharmaceutical composition comprising the mesenchymal stem cells and compound C may be administered parenterally.

When the mesenchymal stem cells secreting TRAIL of the present invention and compound C (compound C) are combined, compared to the case of treating TRAIL-secreting mesenchymal stem cells or compound C alone, it is related to apoptosis of cancer cells. By increasing the expression of proteins and reducing the expression of proteins related to anti-apoptosis, apoptosis of cancer cells can be significantly increased, thereby obtaining a high anticancer effect.

1 shows the main experimental method of the present invention.
Figure 2 shows the results of confirming the viability of glioma (glioma) cells (U87, U138) after treatment with TRAIL and / or compound C (compound C). A represents the cell viability according to the treatment concentration of TRAIL or Compound C, and B represents the cell viability according to the treatment with TRAIL (10ng/ml) and/or Compound C (10 μM). TRAIL alone does not show sensitivity to U87 cells, and when TRAIL and Compound C were combined, it was confirmed that cell viability decreased compared to the case of treatment alone.
Figure 3 shows the results of confirming the expression levels of cFLIP, XIAP (A) and PARP (B) after treating TRAIL and / or compound C (compound C) in glioma cells (U87, U138). When TRAIL and Compound C were combined, it was confirmed that the expression of cFLIP and XIAP, which are anti-apoptosis-related proteins, decreased, and the cleavage of PARP, an apoptosis-related protein, increased compared to the case of treatment alone, respectively.
4 shows glioma cells (U87, U138) after treatment with MSC-TRAIL and/or compound C (A) to the expression level of BAX, BCL2, cFLIP or XIAP (B) shows the result of checking . When MSC-TRAIL and Compound C were treated in combination, the survival rate of glioma cells was reduced compared to the case of treatment alone, pro-apoptotic protein (BAX) increased, and anti-apoptotic proteins (BCL2, cFLIP and XIAP) ) was found to decrease.
Figure 5 shows the results of administering MSC-TRAIL and / or compound C (compound C) to a mouse model of glioma. When MSC-TRAIL and Compound C were combined, it was confirmed that bioluminescence expression was reduced (A) and the growth of gliomas was significantly inhibited (B) compared to the case of each treatment alone (magnification: 1x. dashed line: tumor edge).
Figure 6 shows the results of histological analysis after administration of MSC-TRAIL and / or compound C (compound C) to a glioma mouse model. When MSC-TRAIL and Compound C were combined, it was confirmed that the number of killed glioma cells was significantly increased compared to the case of treatment alone (TUNEL: red at the top, cleaved caspase 3: red at the bottom) , DAPI: blue).

Hereinafter, the present invention will be described in more detail.

As described above, since TRAIL alone cannot obtain a sufficient effect on apoptosis of cancer cells, additional research on a method for activating cancer cell apoptosis that can be expected to have clinical effects while increasing the sensitivity of cancer cells to apoptosis induced by TRAIL is required.

When the mesenchymal stem cells secreting TRAIL of the present invention and compound C (compound C) are treated in combination, the cleavage rate of PARP compared to the case of treating TRAIL, mesenchymal stem cells secreting TRAIL or compound C alone, respectively. , increases the expression level of BAX, a pro-apoptotic protein, and decreases the expression level of BCL2, cFLIP, or XIAP, which is an anti-apoptotic protein. Therefore, the combined treatment of TRAIL-secreting mesenchymal stem cells and Compound C can induce cancer cell growth inhibition or apoptosis, thereby providing an effective anticancer effect.

The present invention may provide a pharmaceutical composition for preventing or treating cancer comprising mesenchymal stem cells and compound C secreting TRAIL.

According to a preferred embodiment of the present invention, the mesenchymal stem cells (MSC) may be derived from any one selected from the group consisting of humans, cattle, pigs, and horses, preferably human-derived it is preferable

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be derived from any one selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, hair follicles and skin, preferably bone marrow. It is preferably derived.

The mesenchymal stem cells secreting TRAIL of the present invention can be produced by transforming the stem cells with a nucleic acid (polynucleotide) encoding TRAIL or a vector (recombinant vector) containing the same. Specifically, the TRAIL-secreting stem cells (a) inserting a nucleic acid encoding TRAIL into an expression vector; and (b) introducing the expression vector into stem cells.

The 'nucleic acid' or 'polynucleotide' refers to polymers of nucleotides of all lengths, such as deoxyribonucleotides as well as ribonucleotides. This term refers only to the primary structure of a molecule, and thus to double or single-stranded DNA or RNA. It also includes known types of modification, e.g., one or more naturally occurring nucleotide substitutions of a label, methylation, caps, analogues known in the art, debonding (e.g., methyl phosphonate, phosphotriester, phosphoamidate, carbarnate, etc.). ) and binding (e.g. phosphorothioate, phosphorodithioate, etc.), proteins (e.g. nucleases, toxins, antibodies, signal peptides, poly-Llysine, etc.); inserts (e.g. acridine, Psolalen, etc.), chelates (eg, metal elements, radioactive metal elements, boron, metal oxides, etc.) , etc.), and also means internucleotide modifications, including unmodified polynucleotides.

The 'vector' refers to a nucleic acid molecule capable of transporting another nucleic acid to its associated place. An 'expression vector' includes a plasmid, cosmid or phage capable of synthesizing a target protein encoded by each recombinant gene carried by the vector.

The nucleic acid encoding the TRAIL may be used without limitation as long as it has a nucleotide sequence encoding the TRAIL known in the art. In addition, the nucleic acid may have a nucleotide sequence encoding a functional equivalent of TRAIL.

The 'functional equivalent' means at least 70%, preferably 80%, more preferably 90% or more sequence homology to TRAIL as a result of addition, substitution or deletion of amino acids, and substantially to TRAIL of the present invention. Refers to a polypeptide that exhibits the same physiological activity. Here, 'substantially homogenous physiological activity' means an activity inducing the death of tumor cells. Such functional equivalents include, for example, amino acid sequence variants in which some of the amino acids of TRAIL are substituted, deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions for naturally occurring amino acids are; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) ) and sulfur-containing amino acids (Cys, Met). The scope of functional equivalents of TRAIL includes polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of TRAIL and its physiological activity. For example, structural alterations to alter the stability, shelf life, volatility or solubility of the polypeptide of the present invention are included therein. In addition, the nucleic acid encoding the TRAIL can be prepared by a genetic recombination method known in the art. Also, in the present invention, the term 'expression vector' refers to a plasmid, viral vector or other medium known in the art into which a nucleic acid encoding TRAIL can be inserted and the nucleic acid can be expressed in a host cell. Preferably, it may be a viral vector.

The viral vector may be any one selected from the group consisting of a retroviral vector, an adenoviral vector, a herpes virus vector, an avipox virus vector, a lentiviral vector, and a herpes virus vector.

The expression vector containing the nucleic acid according to the present invention may be prepared by a method known in the art, for example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation. Methods, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation ), a gene gun and other known methods for introducing a nucleic acid into a cell may be introduced into a stem cell.

According to a preferred embodiment of the present invention, the cancer may be any one or more selected from the group consisting of melanoma, brain cancer, lung cancer, breast cancer, testicular cancer and kidney cancer, preferably brain cancer. The brain cancer may be glioma, glioblastoma, glioblastoma, glioblastoma multiforme, meningioma, pituitary adenoma or schwannoma.

According to a preferred embodiment of the present invention, the pharmaceutical composition may be a formulation for parenteral administration.

The formulation for parenteral administration includes a sterile aqueous solution, a non-aqueous solution, a suspension solution, an emulsion, a freeze-dried formulation, or a suppository. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

The pharmaceutical composition of the present invention may be administered parenterally, and when administered parenterally, a method known in the art in the form of an injection for injection into tumor, intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular It can be formulated according to

In the case of the injection, it must be sterile and protected from contamination by microorganisms such as bacteria and fungi. For injection, examples of suitable carriers include, but are not limited to, water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof, and/or a solvent or dispersion medium containing vegetable oil. can More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanolamine or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may further include various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In addition, in most cases, the injection may further contain isotonic agents such as sugars or sodium chloride.

The pharmaceutical composition for preventing or treating cancer of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the level of an effective amount may vary depending on the patient's disease type, severity, drug activity, drug sensitivity, and administration. Time, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field may be determined. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. That is, the total effective amount of the composition of the present invention may be administered to a patient as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. . In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the pharmaceutical composition of the present invention may be increased or decreased according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention may provide a kit for preventing or treating cancer comprising mesenchymal stem cells secreting TRAIL and compound C (compound C).

Since the mesenchymal stem cells and Compound C secreting the TRAIL are the same as the concepts used in the pharmaceutical composition, the description is replaced with the description thereof.

The kit of the present invention may provide a TRAIL expression vector, mesenchymal stem cells, and Compound C, respectively, or may provide mesenchymal stem cells and Compound C transfected with a TRAIL expression vector. The expression vector can be prepared by introducing TRAIL into a viral vector, preferably an adenoviral vector.

According to a preferred embodiment of the present invention, the mesenchymal stem cells and Compound C secreting the TRAIL may be administered parenterally simultaneously or sequentially.

Since the parenteral administration is the same as the concept used in the pharmaceutical composition, the description is replaced with the description thereof. Preferably, the mesenchymal stem cells secreting the TRAIL may be administered directly to cancer cells, and the compound C may be administered intraperitoneally.

In addition, the present invention can provide an anticancer adjuvant comprising mesenchymal stem cells and compound C (compound C) secreting TRAIL.

Since the mesenchymal stem cells and Compound C secreting the TRAIL are the same as the concepts used in the pharmaceutical composition, the description is replaced with the description thereof.

The anticancer adjuvant of the present invention refers to any form for enhancing the anticancer effect of an anticancer agent or suppressing or improving the side effects of an anticancer agent. The anti-cancer adjuvant of the present invention can be administered in combination with various types of anti-cancer agents or anti-cancer adjuvants, and when administered in combination, even when the anti-cancer agent is administered at a level lower than the dosage of a conventional anti-cancer agent, the same level of anti-cancer therapeutic effect can be exhibited, so it is safer anti-cancer treatment can be performed.

The administration route of the anticancer adjuvant may be administered through any general route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, orally, intrapulmonary administration, or rectal administration, as desired. In addition, the anticancer adjuvant may be administered by any device capable of transporting an active substance to a target cell.

The anticancer adjuvant of the present invention may be preferably formulated as an anticancer adjuvant by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Carriers, excipients or diluents that may be included in the anticancer adjuvant of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

According to a preferred embodiment of the present invention, the anticancer adjuvant may be a preparation for parenteral administration, and since the preparation for parenteral administration is the same as the concept used in the pharmaceutical composition, the description is replaced with the description thereof.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Experiment preparation

<1-1> Reagents and cell culture

Human bone marrow-derived mesenchymal stem cells (MSCs) were obtained from the Catholic Cell Therapy Research Institute (CIC, Seoul, Korea). MSCs were cultured in low-glucose DMEM (Dulbecco's modified Eagle's medium; Thermo Fisher Scientific, Waltham, MA, USA) in 20% fetal bovine serum (Wisent Bioproducts, St-Bruno, QC, Canada) and antibiotic-antibiotic solution (Gibco, Carlsbad, CA, USA) were co-cultured. MSCs were used for experiments after passages 5-8. An adenovirus having a secretable trimeric form of the TRAIL gene (Ad-TRAIL) was prepared, and a recombinant replication-deficient adenovirus vector encoding an enhanced green fluorescent protein (Ad-EGFP) was used in the AdEasy vector system. (Quantum Biotechnologies) was used to design. Cell-permeable peptide-mediated adenoviral transduction was performed to prepare TRAIL-secreting MSCs (MSC-TRAIL). Human glioma cells (U87, U138; American Type Culture Collection) were cultured in high-glucose DMEM (Thermo Fisher Scientific). Firefly luciferase (Luc)-expressing U87 cells (U87-Luc) were stably transduced using lentivirus-expressing Luc. All cells were cultured in a humidified atmosphere at 37° C. containing 5% CO _{2 .}

<1-2> Manufacture of orthotopic xenograft mouse model

The use of nude mice (6-8 weeks old, male; Charles River Laboratories, Wilmington, MA, USA) was approved by the Catholic University Institutional Animal Care and Use Committee (approval number: 2017-0211-04). An intracranial xenograft mouse model of human glioma was anesthetized by intraperitoneal injection of ketamine / xylazine into the mouse, and then a Hamilton syringe using a micro-infusion pump (Harvard Apparatus) (Hamilton Company) ) through stereotactic inoculation of 1×10 ⁵ U87-Luc cells (3 μl PBS) into the right frontal lobe (2.5 mm deep from the skull base, 2 mm lateral to bregma and 1 mm anterior). did.

Glioma cell killing effect of TRAIL and/or compound C

To determine the effect of TRAIL and / or compound C on the survival of glioma cells.

Specifically, cell viability is dependent on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (3-(4,5-dimethyl-2 It was determined using the Sigma-Aldrich Co., St. Louis, MO, USA); -thiazolyl) -2,5-diphenyl-2 H -tetrazolium bromide, MTT. Detection with water-soluble tetrazolium salts reduced by dehydrogenase in living cells gives a colored product (formazan). Glioma cells U87 or U138 cells were seeded in 96-well or 24-well plates to measure TRAIL-induced cytotoxicity and Compound C-mediated apoptosis. Cells were treated with TRAIL (0, 5, 10, 50 or 300 ng/ml; R&D Systems, Minneapolis, MN, USA) and/or Compound C (0, 5, 10 or 20 μM; Selleckchem, Houston, TX, USA). Treatment and analysis 24 hours later. Each well was measured for optical density (OD) at 570 nm using a Spectramax Plus 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

As a result, as shown in A of [Fig. 2], TRAIL alone increased U87 apoptosis, but did not increase U138 apoptosis, indicating no sensitivity to TRAIL. On the other hand, compound C increased apoptosis in U87 and U138 cells. In addition, as shown in B of [FIG. 2], when both TRAIL and Compound C were treated, it was confirmed that U87 and U138 cells were significantly killed compared to the case of treatment alone, respectively. This indicated that Compound C enhanced TRAIL-induced apoptosis.

Effect of combined TRAIL and Compound C treatment on glioma cells

The purpose of this study was to determine whether the combined treatment of TRAIL and Compound C affects glioma cells with respect to the expression of apoptosis-related proteins.

In glioma cells U87 and U138 cells treated with TRAIL (10 ng/ml) and/or Compound C (10 μM) in the same manner as in <Example 2>, the expression levels of cFLIP and XIAP, which are anti-apoptotic proteins, The expression level of PARP, an apoptosis-related protein, was confirmed by Western blotting. Western blotting is Trans-BlotProteins were transferred to a nitrocellulose membrane (Invitrogen) using an SD Semi-Dry Transfer Cell (BIO-RAD) followed by cellular FLICE inhibitory protein (cFLIP; Alexis), an X-linked inhibitor of apoptosis (XIAP) and poly (ADP). -ribose) was incubated with a primary antibody against polymerase (PARP). β-actin (Sigma) was used as a loading control.

As a result, as shown in A of [FIG. 3], the expression of anti-apoptotic proteins such as cFLIP and XIAP was significantly reduced when TRAIL and Compound C were combined treatment compared to the case of treatment alone. Increased apoptosis of glioma cells was confirmed by cleavage of PARP, a major apoptosis protein, in glioma cells, which was further increased when TRAIL and Compound C were combined. This indicates that Compound C enhanced TRAIL-induced apoptosis in glioma cells.

Glioma cell killing effect of MSC-TRAIL and/or compound C (in vitro)

To determine the effect of MSC-TRAIL and / or combined treatment with Compound C on the survival of glioma cells.

Specifically, glioma cells (5×10 ⁴ ) were seeded into the lower wells of a 24-well plate, and MSC-TRAIL (1×10 ⁴ ) was seeded on trans well inserts (0.4 μm pores). Then, after treatment with Compound C (10 μM), the co-culture was performed, and the viability was confirmed by MTT analysis after 24 hours. In addition, expression levels of B-cell lymphoma 2 (BCL2)-related X protein (BAX), BCL2 (Cell Signaling Technology, Beverly, MA, USA), cFLIP and XIAP proteins in glioma cells in the same manner as in <Example 3> was confirmed.

As a result, as shown in A of [Fig. 4], the combined treatment of MSC-TRAIL and Compound C increased the death of glioma cells significantly increased compared to the case of treatment alone, respectively, and it can be confirmed that it has a therapeutic effect. there was. In addition, as shown in B of [FIG. 4], the combined treatment of Compound C and MSC-TRAIL on glioma cells increases pro-apoptotic protein (BAX) compared to the case of treatment alone, respectively, and anti- It was confirmed that apoptosis proteins (BCL2, cFLIP and XIAP) were reduced. It was determined that the combined treatment of MSC-TRAIL and Compound C increases the therapeutic efficacy for gliomas by increasing apoptosis through enhancement of apoptotic protein and reduction of anti-apoptotic protein.

Effect of MSC-TRAIL and Compound C Combination Treatment on Glioma Treatment Efficiency

The purpose of this study was to confirm the therapeutic effect of the combined treatment of MSC-TRAIL and Compound C in an intracranial xenograft mouse model.

^{MSC-TRAIL (2×10 5} cells/5 μl of PBS) was intracranially implanted 10 days after cancer inoculation into the orthotopic xenograft mouse model (n=5/group) prepared in Example <1-2>. . Then, 10 mg/kg of Compound C was intraperitoneally injected with a saline mixture for 5 days. The luciferase substrate, D-luciferin (150 mg of luciferin/kg body weight; Xenogen, CA, USA) was analyzed 10 min prior to visualization using an optical in vivo imaging system-IVIS Lumina XRMS (PerkinElmer Inc., Waltham, MA, USA). was injected intraperitoneally. In vivo bioluminescence imaging analysis was used to monitor cancer growth every 7 days post tumor inoculation.

In addition, to evaluate cancer growth inhibition by histological analysis, mice were sacrificed 20 days after cancer inoculation during imaging experiments. Mouse brains were perfused with 4% paraformaldehyde under deep anesthesia. The fixed tissue was frozen section (14-μm thick) and stained with hematoxylin and eosin (hematoxylin, eosin, H&E). Images were captured using Pannoramic MIDI.

As a result, as shown in A of [Fig. 5], bioluminescence expression was significantly reduced when MSC-TRAIL and Compound C were combined treatment compared to the case of treatment alone. In addition, it was confirmed that H & E staining significantly inhibited cancer growth in mice treated with MSC-TRAIL and Compound C as compared to the PBS or single treatment group. These results indicate that combined treatment of MSC-TRAIL and Compound C reduced cancer growth in glioma-bearing mice.

Effect of MSC-TRAIL and Compound C combined treatment on apoptosis of glioma cells

The purpose of this study was to determine whether the apoptotic effect of the combined treatment of MSC-TRAIL and Compound C is involved in anticancer activity.

The mouse brain section of <Example 5> was subjected to terminal deoxyribonucleotidyl transferase-.mediated dUTP nick end labeling (TUNEL) analysis kit (Roche, Mannheim, Germany). It was detected through staining using Cy3-conjugated streptavidin (Cy3-conjugated streptavidin, Invitrogen). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma).

As a result, as shown in A of [Fig. 6], the staining of TUNEL and cleaved caspase 3 was significantly increased in the number of killed cells when MSC-TRAIL and Compound C were combined, compared to when treated alone, respectively. showed that These results implied that the anticancer activity of the combined treatment of MSC-TRAIL and Compound C was involved in apoptosis and enhanced apoptosis in intracranial gliomas.

[statistical analysis]

All data are expressed as mean ± SD (standard deviation). Statistical differences between test conditions were determined using one-way ANOVA with Bonferroni multiple correction. Probability values less than p <0.05 were considered significant.

Claims (11)

A pharmaceutical composition for preventing or treating brain cancer comprising mesenchymal stem cells and compound C secreting TRAIL.
The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are derived from any one selected from the group consisting of humans, cattle, pigs and horses.
The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are derived from any one selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, hair follicles and skin.
delete The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a preparation for parenteral administration.
A kit for preventing or treating brain cancer comprising mesenchymal stem cells and compound C that secrete TRAIL.
The kit according to claim 6, wherein the mesenchymal stem cells are derived from any one selected from the group consisting of humans, cattle, pigs and horses.
The kit according to claim 6, wherein the mesenchymal stem cells are derived from any one selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, hair follicles and skin.
The kit according to claim 6, wherein the mesenchymal stem cells and Compound C secreting TRAIL are administered parenterally.
delete An anticancer adjuvant for brain cancer comprising mesenchymal stem cells secreting TRAIL and compound C.

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