KR101994957B1 - Pharmaceutical composition containing a RUNX3 protein or RUNX3 gene for enhancing the anti-cancer effect of TRAIL - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for promoting the anticancer effect of TRAIL, which comprises RUNX3 protein or a RUNX3 gene encoding the RUNX3 protein as an active ingredient, and reduces resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) .
The pharmaceutical composition for enhancing the anticancer effect of TRAIL according to the present invention can be effectively used for the prevention or treatment of cancer showing tolerance to TRAIL by using the RUNX3 protein capable of overcoming TRAIL resistance or the RUNX3 gene encoding the same.

Description

[0001] The present invention relates to a pharmaceutical composition for enhancing the anticancer effect of TRAIL comprising a RUNX3 protein or a RUNX3 gene encoding the RUNX3 protein,

The present invention relates to a pharmaceutical composition for promoting the anticancer effect of TRAIL, which comprises RUNX3 protein or a RUNX3 gene encoding the RUNX3 protein as an active ingredient, and reduces resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) .

Cancer is one of the greatest threats to human health, a disease that occurs when a cell line undergoes a series of mutagenic processes and is immortalized in an unlimited, unregulated way. The causes of cancer are environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital gene mutations (Klaunig & Kamendulis, Annu Rev Pharmacol Toxicol. , 44: 239-267 , 2004).

Surgery, radiation therapy, chemotherapy, and other treatments, however, are becoming a major problem in the early detection of cancer. In the case of terminal cancer or metastatic cancer, life is terminated without any special treatment. In addition, various biochemical mechanisms related to cancer have been identified and therapies have been developed, but no fundamental treatment methods for cancer have yet been proposed.

TRAIL (Tumor necrosis factor-related apoptosis inducing ligand) is a kind of tumor necrosis factor (TNF), which is known as a ligand for inducing autologous cell death of tumor cells. TRAIL has four receptors. Death receptors (DR4, DR5) in the receptors are overexpressed in cancer cell lines and decoy receptors (Decoy receptor, DcR1, DcR2) are overexpressed in normal cell lines. The TRAIL may bind to death receptors such as DR4 and DR5 on the surface of tumor cells to induce the death of tumor cells. In the case of the decoy receptor, there is no death domain at the terminal of the c-terminal, and cell death signaling is not transmitted to the cell. Therefore, TRAIL-based therapy is a highly effective next-generation chemotherapeutic agent because it is toxic to normal cells and induces toxicity only for various cancer cell lines.

However, when TRAIL was continuously treated with cancer cells, it became clear that cancer cells that were susceptible to TRAIL gradually became resistant to TRAIL. Specifically, many cancer cells exhibit reduced death receptors and cellular FLICE-like inhibitory proteins (c-FLIP (L)), B-cell lymphoma-2, 2), anti-apoptotic proteins such as B-cell lymphoma-extra large (Bcl-xL) or myeloid cell leukemia-1, Mcl-1 , Which are resistant to TRAIL-induced apoptosis through a variety of mechanisms.

Therefore, there is a need to study a method for preventing cancer cell proliferation and promoting apoptosis by overcoming tolerance of TRAIL which can selectively induce apoptosis in cancer cells.

KR 10-0475649 B

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer which can enhance the anti-cancer effect by overcoming resistance of TRAIL which selectively induces apoptosis of cancer cells without affecting normal cells and inducing apoptosis of cancer cells effectively .

In order to achieve the above-mentioned object, the present invention provides an anti-cancer effect of TRAIL, which comprises RUNX3 protein or RUNX3 gene encoding the RUNX3 protein as an active ingredient, and reduces tolerance of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) Thereby providing a pharmaceutical composition for enhancing blood circulation.

The present invention also relates to a method for the treatment and / or prophylaxis of TRAIL (TRAIL), which comprises the RUNX3 gene-containing vector or the RUNX3-overexpressing cell transformed with said vector as an active ingredient, and which reduces the tolerance of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) A pharmaceutical composition for promoting the anti-cancer effect.

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In addition, the present invention provides a cancer cell death promoting method, which comprises administering RUNX3 protein or a RUNX3 gene encoding the RUNX3 protein and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to provide.

The pharmaceutical composition for enhancing the anticancer effect of TRAIL and the method for promoting cancer cell death according to the present invention include RUNX3 protein capable of overcoming TRAIL resistance or RUNX3 gene coding therefor and thus being useful for prevention or treatment of cancer showing resistance to TRAIL Can be used.

FIG. 1 is a schematic diagram showing a cell death pathway by TRAIL.
2 is a graph showing cell viability by TRAIL in colon cancer cells (HCT116, DLD-1, Colo205, SNU283, HT29).
FIG. 3 is a graph of cell viability showing the degree of overexpression of RUNX3 affecting cell growth of colon cancer. FIG.
FIG. 4 is a graph showing the effect of RUNX3 overexpression on the sensitivity of TRAIL in colon cancer cell lines (HT29, Colo205, DLD-1).
FIG. 5 is a graph showing the effect of RUNX3 overexpression on TRAIL sensitivity in gastric cancer cell line (AGS, MKN45) or breast cancer cell line (MCF-7, MDA-MB 231).
FIG. 6 shows the results of Western blot analysis at the protein level that RUNX3 overexpression and TRAIL-induced apoptosis factor expression were increased in HT29 cell line.
FIG. 7 is a graph showing that TRAIL-induced apoptosis is significantly increased in the RUNX3 overexpressing colorectal cancer cell line.
Figure 8 shows that RUNX3 overexpression in the HT29 cell line and the sensitivity of TRAIL, which was increased by RUNX3, were eliminated when the caspase inhibitor was treated with TRAIL.
FIG. 9 shows Western blotting results showing that DR5 (Death receptor 5), which is known to be important for TRAIL sensitivity, is increased in RUNX3 overexpression.
FIG. 10 shows the results of fluorescence immunostaining that DR5 is increased by overexpression of RUNX3.
Figure 11 shows western blot that RUNX3 overexpression in HT29 cell line and DR5 siRNA treatment with TRAIL abolished TRAIL sensitivity, which was increased by RUNX3.
Figure 12 shows through FACS that RUNX3 overexpression in HT29 cell line and the TRAIL sensitivity that was increased by RUNX3 when DR5 siRNA was treated with TRAIL was lost.
FIG. 13 is a Western blot test result for confirming whether ER stress-related factors are increased by RUNX3 overexpression.
Figure 14 shows the results of measuring the ROS generated to determine whether RUNX3 increases ER stress through ROS.
FIG. 15 shows that ROS was increased by RUNX3 overexpression by immunofluorescence staining.
Figure 16 shows the results of treatment with NAC, an ROS inhibitor, as a result of which TRAIL sensitivity, which was increased by RUNX3, was blocked by NAC.
FIG. 17 shows the inhibition of the expression of SOD3, which inhibits apoptosis of cancer cells upon RUNX3 overexpression.
FIG. 18A is a photograph showing the result of comparing the size of tumors according to presence or absence of RUNX3 through xenograft, FIG. 18B is a photograph of a 5-week-old nude mouse injected subcutaneously with a colon cancer cell line HT29 and RUNX3- In the case of the second embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

The inventors of the present invention have been studying a method of overcoming resistance to TRAIL capable of selectively inducing apoptosis in cancer cells. When the RUNX3 protein or the RUNX3 gene encoding the same and TRAIL are co-administered to cancer cells, The overexpression of RUNX3 increases the expression of DR5, a cytotoxic receptor, resulting in an increase in the cell death of TRAIL-induced cancer cells, thereby completing the present invention.

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising RUNX3 protein or RUNX3 gene encoding the RUNX3 protein and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient.

In the present invention, the TRAIL includes TRAIL in which some base sequences are mutated or engineered.

According to one embodiment of the present invention, when the cancer cell is treated with the RUNX3 protein or the RUNX3 gene encoding the same and TRAIL, apoptosis is increased. Specifically, it was confirmed that overexpression of RUNX3 increased the expression of DR5, a type of cell death receptor for TRAIL, and that DR5, which is increased in expression, promoted TRAIL-induced cell death sensitivity. Therefore, the RUNX3 protein or the RUNX3 gene encoding the RUNX3 protein can be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating cancer together with TRAIL.

The pharmaceutical composition may comprise 1 to 50% by weight of the RUNX3 protein or the RUNX3 gene thereof and 50 to 99% by weight of TRAIL.

 The RUNX3 protein preferably has the following amino acid sequence of 1) to 3), but is not limited thereto.

1) an amino acid sequence represented by SEQ ID NO: 1;

2) an amino acid sequence represented by a part of SEQ ID NO: 1; And

3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.

The RUNX3 gene is preferably RUNX3 cDNAM or RUNX3 RNA.

Preferably, the RUNX3 protein is a TRAIL sensor sensitizer.

In the present invention, the RUNX3 gene comprises a nucleotide sequence encoding the RUNX3 protein, and the nucleotide sequence includes both DNA, cDNA, and RNA sequences.

In the present invention, the RUNX3 protein can be prepared using DNA recombinant technology, and methods known in the art can be used for this purpose. Specifically, the RUNX3 protein can be produced by a genetic engineering method in which the RUNX3 gene is injected recombinantly into bacteria, yeast, plant cell lines and animal cell lines.

The RUNX3 protein or the RUNX3 gene encoding the RUNX3 protein is preferably used together when treating cancer using TRAIL.

It is preferable that the cancer is one selected from the group consisting of colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, bladder cancer and blood cancer.

The composition may contain at least one active ingredient that exhibits the same or similar function in addition to the RUNX3 protein or the RUNX3 gene encoding the RUNX3 protein.

The pharmaceutical compositions according to the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions.

Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The composition can be administered orally or parenterally at the time of clinical administration and can be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, And may be used in the form of a general pharmaceutical preparation.

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

The daily dose of the composition is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and it is preferable to administer the composition one time or several times a day. However, , The time of administration, the administration method, the excretion rate, and the severity of the disease.

The effective dose of RUNX3 cDNA is 0.002 to 1 mg / kg, preferably 0.02 to 0.2 mg / kg, and the effective dose of RUNX3 RNA is 0.001 to 0.5 mg / kg, preferably 0.01 to 0.1 mg / kg , The effective dose of the RUNX3 protein is 0.04 to 4 mg / kg, preferably 0.4 to 1 mg / kg.

The composition of the present invention can be administered in various forms of parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, do. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered for human or animal use.

The present invention also relates to a method for the prevention and treatment of cancer comprising as an active ingredient a vector comprising the RUNX3 gene, or a RUNX3-overexpressing cell transformed with the vector or a culture thereof, and a TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) To provide a pharmaceutical composition.

The RUNX3 gene is preferably RUNX3 cDNA or RUNX3 RNA, and may be introduced into any expression vector selected from the group consisting of a plasmid DNA and a recombinant viral vector by a known method.

The vector contained in the pharmaceutical composition of the present invention refers to a vector having the RUNX3 gene inserted therein, and the vector is preferably any one selected from the group consisting of plasmid DNA and recombinant viral vectors known in the art.

The recombinant virus may be any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus.

The vectors according to the present invention can be introduced into target cells using methods known in the art. For example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acid into cells. Can be introduced.

The cells are preferably selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells.

The RUNX3 protein produced by the vector according to the present invention can increase the expression of DR5, a cytotoxic receptor of TRAIL, and thereby increase the susceptibility to TRAIL-induced apoptosis. Therefore, a vector containing the RUNX3 gene, a RUNX3-overexpressing cell transformed with the vector, or a culture thereof may be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating cancer together with TRAIL.

The pharmaceutical composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the intended method, and the dose may be determined depending on the patient's body weight, age, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, more preferably administered once to several times a day.

In addition, in the case of RUNX3-overexpressing cells transformed with the vector, the composition of the present invention preferably contains 10 ³ to 10 ^8, more preferably 10 ⁴ to 10 ⁷ , but is not limited thereto.

For the case of vector effective dose of the composition weight 1 ㎏ each containing a vector or cell comprising the RUNX3 gene as an active ingredient is 0.05 to 12.5 ㎎ / ㎏, recombinant virus, the 10 ⁷ to 10 ¹¹ viral particles (10 ⁵ to 10 ⁹ IU) / ㎏, if the cells 10 ³ to 10 ⁶ cells / ㎏, preferably when the vector is 0.1 to 10 ㎎ / ㎏, when the recombinant virus is 10 ⁸ to 10 ¹⁰ particles (10 ⁶ for ⁸ to 10 IU) / ㎏, cells, and 10 ² to 10 ⁵ cells / ㎏, it may be administered twice or three times a day. Such composition is not necessarily limited to this, but may vary depending on the condition of the patient and the degree of neurological disease.

The present invention also provides a pharmaceutical composition for promoting the anticancer effect of TRAIL, which comprises the RUNX3 protein or the RUNX3 gene encoding the RUNX3 protein as an active ingredient, and reduces tolerance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) to provide.

The RUNX3 protein preferably has the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The RUNX3 protein is preferably a TRAIL sensor sensitizer, but is not limited thereto.

The cancer is preferably any one selected from the group consisting of colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, bladder cancer and blood cancer, , Breast cancer or colorectal cancer, but is not limited thereto. It is preferable that all of the cancer resistant to TRAIL are included.

The present invention also provides a cancer cell death promoting method, which comprises administering a RUNX3 protein or a RUNX3 gene encoding the RUNX3 protein and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to a cancer cell of a mammal other than a human, to provide.

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Example

Experimental Example  1. In colorectal cancer cells TRAIL and RUNX3  Effect on cell growth

In colorectal cancer Of Runx3  Confirmation of increase in expression level - western blotting

We transfected pFlag-c1 or pFlag-c1 RUNX3 into colon cancer cells (HCT116, DLD-1, Colo205, SNU283, HT29) and constructed a stably overexpressed stable cell line. Cell proliferation was measured using a stable cell line overexpressing RUNX3. After 24 hours, the MTS solution was added, and after 4 hours of reaction, the absorbance at 550 nm was measured to confirm cell viability, which is shown in FIG. 2 and FIG.

FIG. 2 is a graph showing cell viability by TRAIL in colorectal cancer cells (HCT116, DLD-1, Colo205, SNU283, HT29), and FIG. 3 is a graph showing the cell viability of TRAIL- Cell viability < / RTI >

3, HCT116 showed the highest sensitivity to TRAIL among colon cancer cells (HCT116, DLD-1, Colo205, SNU283, and HT29). In addition, as shown in FIG. 3, it was confirmed that the change of cell growth due to overexpression of RUNX3 had no significant effect.

Experimental Example  2. RUNX3's  Overexpression TRAIL  Combined effect

RUNX3  In an over-expressing cell line On TRAIL  - Increased sensitivity to the increase in the number of patients - Flow  cytometry

To investigate whether RUNX3 overexpression affects TRAIL sensitivity in colon cancer cell lines (HT29, Colo205, DLD-1).

For this purpose, colon cancer cell lines (HT29, Colo205, DLD-1) and RUNX3-overexpressing cell lines were seeded. After 24 hours, TRAIL (20 ng / ml) was treated and after 6 hours, cells were harvested. The cells were stained with Annexin V-FITC, PI (propidium iodide), and the number of viable cells was measured using a flow cytometry measuring machine.

FIG. 4 shows that TRUN-induced sensitivity to RUNX3 overexpressed cells is higher.

We also investigated whether RUNX3 overexpression affects TRAIL sensitivity in gastric cancer cell line (AGS, MKN45) and breast cancer cell line (MCF-7, MDA-MB 231).

For this purpose, the gastric cancer cell line (AGS, MKN45) and the breast cancer cell line (MCF-7, MDA-MB 231) and the RUNX3 overexpressing cell line were seeded, respectively. After 24 hours, TRAIL (20 ng / ml) was treated and after 6 hours, cells were harvested. The cells were stained with Annexin V-FITC, PI (propidium iodide), and the number of viable cells was measured using a flow cytometry measuring machine.

FIG. 5 shows that TRUN-induced sensitivity to RUNX3 over-expressing gastric cancer cells and breast cancer cells is higher.

RUNX3  In an over-expressing cell line On TRAIL  The sensitivity of the protein to the increase in protein level - Western blot

Colorectal cancer cell line (HT29) and RUNX3 overexpressing cell line were seeded and treated with TRAIL (20 ng / ml) after 24 hours. After 6 hours, the cells were harvested and lysed to collect proteins and subjected to western blotting. And, the change of expression was confirmed using a marker related to apoptosis.

FIG. 6 shows the results of Western blotting at the protein level that the expression of Apoptosis factors is increased when RUNX3 is overexpressed and TRAIL is treated in HT29 cell line. 6, the expression of PARP, Caspase-3, Caspase-9 and Caspase-8, which are apoptosis factors, was increased by overexpressing RUNX3 and treating TRAIL with HT29 cell line.

FIG. 7 is a graph showing that TRAIL-induced apoptosis is significantly increased in the RUNX3 overexpressing colorectal cancer cell line. 7, the expression of TRAIL-induced apoptosis was increased by 2% in the HT29 control cell line, and that of TRAIL-induced apoptosis was increased by 29% in the RUNX3-overexpressing cell line.

To investigate whether apoptosis is caspase-dependent, caspase inhibitors were treated under the same conditions.

Figure 8 shows that RUNX3 overexpression in the HT29 cell line and the sensitivity of TRAIL, which was increased by RUNX3, were eliminated when the caspase inhibitor was treated with TRAIL.

These results suggest that the increase of TRAIL sensitivity by RUNX3 occurs through caspase.

Experimental Example  3. In colorectal cancer cells RUNX3  Increased expression of DR5 in overexpression

RUNX3  Overexpression Death receptor  Confirmation of protein expression

RUNX3 overexpression, we examined the expression of pro-apoptotic and anti-apoptotic proteins.

For this purpose, colon cancer cell line (HT29) and RUNX3 overexpressing cell line were seeded. After 24 hours, the cells were harvested and lysed to collect proteins and subjected to western blotting, as shown in FIG.

FIG. 9 shows that DR5 (Death receptor 5), which is known to be important for TRAIL sensitivity upon RUNX3 overexpression, is significantly increased.

RUNX3  Overexpression of DR5 expression changes - Immunofluorescence

Colorectal cancer cell line (HT29) and RUNX3 overexpressing cell line were seeded. After 24 hours, the plate was immersed in 3.7% formaldehyde, and the permeability of the antibody was increased with triton x 100, followed by the addition of a primary antibody and a secondary antibody. After staining the nuclei through DAPI staining, the DR5 expression was confirmed by confocal microscopy.

FIG. 10 shows the results of fluorescence immunostaining that DR5 is increased by overexpression of RUNX3.

RUNX3's  Increased DR5 by overexpression TRAIL  Impact on Sensitivity

And to confirm whether the increase of DR5 by RUNX3 overexpression affects the TRAIL sensitivity.

DR5 expression was inhibited by using DR5 siRNA.

Figure 11 shows western blot that RUNX3 overexpression in HT29 cell line and DR5 siRNA treatment with TRAIL abolished TRAIL sensitivity, which was increased by RUNX3. In Fig. 11, it can be confirmed that cell death is induced by overexpression of PARP.

Figure 12 also shows through FACS that RUNX3 overexpression in HT29 cell line and the TRAIL sensitivity that was increased by RUNX3 in the treatment of DR5 siRNA with TRAIL disappear.

These results demonstrate that increased DR5 by RUNX3 overexpression enhances TRAIL-induced apoptosis.

Experimental Example  4. In colorectal cancer cells RUNX3  Overexpression ROS  increase

DR5 is known to be increased by ROS-ER stress. To confirm whether the increase of DR5 by RUNX3 is due to ROS-ER stress.

For this purpose, ROS was induced by RUNX3 overexpression in colorectal cancer cells by flow cytometry using 2,7-dicholorofluorescein diacetate (DCFH-DA) staining.

First, colorectal cancer cell line (HT29) and RUNX3-overexpressing cell line were seeded. After staining with fluorescent probe DCFH-DA, cells were stained with 10,000 cells through flow cytometry.

FIG. 13 is a Western blot test result for confirming whether ER stress-related factors are increased by RUNX3 overexpression.

FIG. 13 shows that RUNX3 induces ER stress through IRE1α by increasing ER stress-related factors p-IRE1α, p-JNK, and XBP1S by RUNX3 overexpression. It was also found that DR5 was increased by the ER stress.

Figure 14 shows the results of measuring the ROS generated to determine whether RUNX3 increases ER stress through ROS. 14, it was confirmed that ROS increased more than twice by RUNX3 overexpression.

FIG. 15 shows that ROS was increased by RUNX3 overexpression by immunofluorescence staining.

Figure 16 shows the results of treatment with NAC, an ROS inhibitor, as a result of which TRAIL sensitivity, which was increased by RUNX3, was blocked by NAC. That is, it can be confirmed that cell death is induced by overexpression of PARP. FIG. 16 shows that TRAIL sensitivity by RUNX3 is ROS-dependent.

These results indicate that RUNX3 not only increases DR5 through ROS induction but also increases TRAIL sensitivity.

Experimental Example  5. In colorectal cancer cells RUNX3  Increased cell death upon overexpression

RUNX3  Overexpression Of SOD3  Identification of expression changes - Western blot , Immunofluorescence

Experiments were carried out to examine changes in the expression of factors related to apoptosis at the protein level during overexpression of RUNX3.

For this purpose, colon cancer cell line (HT29) and RUNX3 overexpressing cell line were seeded first. Twenty-four hours later, TRAIL (5 ng / ml) was treated, harvested 6 hours later, and lysed to collect western blotting. In addition, the expression of ROD3 was confirmed by immunofluorescence at the time of overexpression of RUNX3.

FIG. 17 shows the inhibition of the expression of SOD3, which inhibits apoptosis of cancer cells upon RUNX3 overexpression.

Thus, when SOD3 expression is inhibited, ROS is increased and TRAIL sensitivity by RUNX3 is increased.

RUNX3  Overexpression On TRAIL  Induced cytotoxicity - Increased sensitivity to tumor size, xenograft

Based on the previous results, we tried to confirm that overexpression of RUNX3 promotes TRAIL-induced apoptosis sensitivity through animal experiments.

First, colorectal cancer cells (HT29) and RUNX3-overexpressing cell lines were injected subcutaneously into 5-week-old Balb / c-nude mice and grown until the tumors grew. Tumor size was measured every 2-3 days and TRAIL was injected into peritoneal cavity at a constant size (100 mm ³ ). TRAIL was injected into the peritoneal cavity at intervals of 3 days, and xenograft was used to confirm the difference in tumor size. FIG. 18A is a photograph showing the result of comparing the size of tumors according to presence or absence of RUNX3 through xenograft, FIG. 18B is a photograph of a 5-week-old nude mouse injected subcutaneously with a colon cancer cell line HT29 and RUNX3- In the case of the second embodiment of the present invention.

5-week-old nude mice were injected subcutaneously with HCT116 and RUNX3-overexpressing colon cancer cell lines, and tumor size was measured. When RUNX3 was overexpressed, tumors were less formed and TRUN-treated RUNX3- (Fig. 18B).

As a result of comparing the sizes of intact tumors through xenograft, the size of tumors treated with TRAIL was the smallest in the RUNX3 overexpressed condition (Fig. 18A).

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

<110> Korea University Research and Business Foundation <120> Pharmacological composition containing a RUNX3 protein or RUNX3          gene for the prevention and treatment of cancer <130> HPC-7677 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Human RUNX3 <400> 1 Met Arg Ile Pro Val Asp Pro Ser Thr Ser Arg Arg Phe Thr Pro Pro   1 5 10 15 Ser Pro Ala Phe Pro Cys Gly Gly Gly Gly Gly Gly Lys Met Gly Glu Asn              20 25 30 Ser Gly Ala Leu Ser Ala Gln Ala Ala Val Gly Pro Gly Gly Arg Ala          35 40 45 Arg Pro Glu Val Arg Ser Ser Val Asp Val Leu Ala Asp His Ala Gly      50 55 60 Glu Leu Val Arg Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu Pro  65 70 75 80 Ser His Trp Arg Cys Asn Lys Thr Leu Pro Val Ala Phe Lys Val Val                  85 90 95 Ala Leu Gly Asp Val Asp Gly As Val Val Thr Val Met Ala Gly             100 105 110 Asn Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn Ala Ser Ala Val Met         115 120 125 Lys Asn Gln Val Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg Ser     130 135 140 Gly Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn Pro 145 150 155 160 Thr Gln Val Ala Thr Tyr His Ale Ile Lys Val Thr Val Asp Gly                 165 170 175 Pro Arg Glu Pro Arg Arg His Arg Gln Lys Leu Glu Asp Gln Thr Lys             180 185 190 Pro Phe Pro Asp Arg Phe Gly Asp Leu Glu Arg Leu Arg Met Arg Val         195 200 205 Thr Pro Ser Thr Pro Ser Pro Arg Gly Ser Leu Ser Thr Thr Ser His     210 215 220 Phe Ser Ser Gln Pro Gln Thr Pro Ile Gln Gly Thr Ser Glu Leu Asn 225 230 235 240 Pro Phe Ser Asp Pro Arg Gln Phe Asp Arg Ser Phe Pro Thr Leu Pro                 245 250 255 Thr Leu Thr Glu Ser Arg Phe Pro Asp Pro Arg Met His Tyr Pro Gly             260 265 270 Ala Met Ser Ala Ala Phe Pro Tyr Ser Ala Thr Pro Ser Gly Thr Ser         275 280 285 Ile Ser Ser Leu Ser Val Ala Gly Met Pro Ala Thr Ser Arg Phe His     290 295 300 His Thr Tyr Leu Pro Pro Pro Tyr Pro Gly Ala Pro Gln Asn Gln Ser 305 310 315 320 Gly Pro Phe Gln Ala Asn Pro Ser Pro Tyr His Leu Tyr Tyr Gly Thr                 325 330 335 Ser Ser Gly Ser Ser Gly Phe Ser Met Val Ala Gly Ser Ser Ser Gly             340 345 350 Gly Asp Arg Ser Ser Pro Thr Arg Met Leu Ala Ser Cys Thr Ser Ser Ala         355 360 365 Ala Ser Val Ala Gly Asn Leu Met Asn Pro Ser Leu Gly Gly Gln     370 375 380 Ser Asp Gly Val Glu Ala Asp Gly Ser His Ser Asn Ser Pro Thr Ala 385 390 395 400 Leu Ser Thr Pro Gly Arg Met Asp Glu Ala Val Trp Arg Pro Tyr                 405 410 415

Claims (8)

A pharmaceutical composition for promoting the anticancer effect of TRAIL, which comprises RUNX3 protein or a RUNX3 gene encoding the RUNX3 protein as an active ingredient, and reduces tolerance of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand). The method according to claim 1,
Wherein the RUNX3 protein comprises the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the cancer is any one selected from the group consisting of colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, bladder cancer and blood cancer. A method for promoting the anticancer effect of TRAIL, which comprises the RUNX3 gene-containing vector or the RUNX3-overexpressing cell transformed with the vector as an active ingredient, and which reduces resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) A pharmaceutical composition. 5. The method of claim 4,
Wherein said vector is any one selected from the group consisting of plasmid DNA or recombinant viral vectors. 5. The method of claim 4,
Wherein the cell is any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells. delete delete

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