KR102231066B1 - Use of active androgen receptor antagonist for treating cancer - Google Patents

Abstract

The present invention relates to the use of an active androgen receptor antagonist for the treatment of cancer. More specifically, oligonucleotide KilGreene according to the present invention comprises a Kil sequence and a distal glucocorticoid response element (GRE), in which an active androgen receptor binds to the GRE, and a signaling protein interacting with the active androgen receptor binds to the Kil sequence to remove a complex of the active androgen receptor and the signaling protein interacting therewith. Therefore, the KilGreene exhibits the effect of inhibiting the growth of cancer cells by blocking the transcriptional activity of the active androgen receptor through removing the active androgen receptor that has migrated into the nucleus through androgen-independent signaling by EGFR in EGFR-positive non-small cell lung cancer cells. Accordingly, the KilGreene can be usefully used as an active ingredient in androgen receptor signaling-related cancer treatment as a gene therapy.

Description

Use of active androgen receptor antagonist for treating cancer

The present invention relates to the use of an active androgen receptor antagonist for the treatment of cancer, specifically as an active androgen receptor antagonist for the treatment of cancer, and the androgen receptor and signal protein complex formed by the interaction between the androgen receptor and a signal factor. It relates to the use of the oligonucleotide KilGreene to form a operative double strand.

Androgen receptor (AR) belongs to the group of steroid receptors, and has a function as a transcription factor. Upon binding of the ligand binding domain (LBD) of the androgen receptor to steroids such as androgens, the androgen receptor is released from Hsp90 and migrates to the nucleus. Androgen receptors that have migrated into the nucleus activate the transcription of genes by binding to the androgen responsive element (ARE) in the promoter.

Prostate cancer, the most common tumor disease in men and the second leading cause of cancer-related deaths in men, is mainly caused by the differentiation of prostate lumen epithelial cells, and androgen receptors are known to be involved in the differentiation of these prostate lumen epithelial cells. . Androgen receptors control cell survival through mechanisms that are not yet clearly defined. In addition, in addition to prostate cancer, it is associated with the etiology of other cancers including breast cancer (Cato, A.C., et al. 1998. Trends Endocrinol Metab 9:150-154).

Cancerous prostate cells continue to express androgen receptors. In addition, the survival of these cells depends on the presence of androgens, which makes androgen deficiency a method of choice for patients with advanced prostate cancer. Anti-androgen treatments, for example using the inhibitors flutamide and casodex, are generally effective initially, but rarely cure completely. Prostate cancer recurs in most patients treated with this treatment method, which leads to androgen-independence chemotherapy-resistant tumors with poor prognosis.

Androgen-independent prostate cancer is typically characterized by an increase in androgen receptor activity due to activation of receptor tyrosine kinase or expression of androgen receptor mutations responsive to non-androgen ligands (Chen, Y., et al. 2008. Curr Opin Pharmacol 8:440-448).

This means that androgen receptors are both androgen-dependence and androgen-independent cancers, and other androgen receptor positive cancer types, such as "triple negative" (ER-, PR-, Her2-) breast cancer. , Bladder cancer, and salivary gland cancer. For example, androgen receptor antagonists enzalutamide and bicalutamide have been used to treat prostate cancer (Minyong Kang and Ja Hyeon Ku. 2017, Translational Cancer Research 6 (Suppl 4): S702-S707; P Li., et al. 2017. Cancers (Basel) 9(2): E20; Khoo., et al. 2017. Oncotarget 5(44): 75893-75903; CM Chiu., et al. 2007. PNAS 104(8):2571-2578; L. Gerratana., et al. 2018.Cancer treatment reviews 68: 102-110).

These androgen receptor antagonists stimulate cancer cells by disrupting or reducing the function of androgen receptors, including androgen receptor cell transport, such as androgen-androgen receptor binding, androgen receptor transcriptional activity, or cytoplasm-to-nuclear translocation. It is expected to be reduced. However, the current treatment modalities are quite ineffective against androgen receptor-positive cancers, and thus, the development of new androgen receptor antagonists for the treatment of androgen receptor-positive cancer cells is constantly required.

On the other hand, non small cell lung cancer (NSCLC) is a disease that accounts for an important cause of malignant tumor-related death worldwide, and accounts for 75 to 88% of lung cancer. In many non-small cell lung cancer cells, EGFR (Epidermal Growth Factor Receptor), which is involved in the proliferation and differentiation of cancer cells, is expressed as a receptor tyrosine kinase. Recently, treatment of non-small cell lung cancer using an antibody targeting EGFR has been attempted, but most are used in combination with chemotherapeutic agents. Therefore, it is necessary to find therapeutic targets other than EGFR in order to obtain more satisfactory results. In this attempt, androgen receptors have been suggested (Sean Brennan., et al. 2017. Cancer Res 77 (13 Suppl); Abstract 4121).

Accordingly, the present inventors have endeavored to develop cancer therapeutics that can be used in androgen receptor-positive cancers, including androgen-dependent cancers and androgen-independent cancers. The effect of inhibiting the growth of cancer cells was confirmed by removing the complex of signal factors. Particularly, in EGFR-positive non-small cell lung cancer cells, the transcriptional activity of the active androgen receptor by removing the complex of the active androgen receptor and the signaling protein that interacts with the active androgen receptor moved into the nucleus through androgen-independent signaling by EGFR, which instructs cancer cell division. It was confirmed that it exhibits an effect of inhibiting the growth of cancer cells by blocking.

Accordingly, the oligonucleotide KilGreene according to the present invention may be usefully used in the treatment of cancer related to androgen receptor signaling as a gene therapy.

Numerous attempts have been made to inhibit cancer cell growth by blocking Tyrosine kinase of the Epidermal Growth Factor Receptor (EGFR) receptor in non-small cell lung cancer. However, countless attempts to block EGFR as antibodies or small molecules at the cell membrane level have not been successful. The tyrosine kinase action of EGFR, which initiates cancer cell growth in non-small cell lung cancer cells, goes through a signal pathway to reach the androgen receptor in the nucleus.

The interaction between the active androgen receptor and the signaling protein is recognized as a novel anticancer agent, and as a small molecule that binds to the N-terminal region of the androgen receptor, it is being attempted to treat refractory prostate cancer.

An object of the present invention is to eliminate the interaction between the signaling protein and the androgen receptor through the oligonucleotide KilGreene according to the present invention, thereby blocking androgen receptor signaling, which is the stage of EGFR tyrosine kinase signaling in non-small cell carcinoma, as a gene therapy for androgen receptor signaling related cancer. It is to provide a pharmaceutical composition for prophylaxis or treatment.

In order to achieve the object of the present invention, the present invention contains as an active ingredient an oligonucleotide in which the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 are complementarily bonded to each other to form a double strand, It provides a pharmaceutical composition for preventing or treating cancer.

In addition, the present invention

a) transfection preparations containing vegetable oils; And

b) Provides a pharmaceutical composition for preventing or treating cancer, containing oligonucleotides that form double strands by complementary bonding of the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 as an active ingredient. .

The oligonucleotide according to the present invention has an effect of inhibiting the growth of cancer cells by binding with a complex of an androgen receptor and a signaling protein interacting therewith in non-small cell lung cancer cells. More specifically, the oligonucleotide according to the present invention binds to the androgen receptor and signal protein complex formed by interacting with the androgen receptor moved into the nucleus through androgen-independent signaling by EGFR in EGFR Tyrosine Kinase-positive non-small cell lung cancer cells, and the androgen receptor. Since it has the effect of inhibiting the growth of cancer cells by blocking the transcriptional activity of, it can be usefully used in the treatment of cancer related to androgen receptor signaling as a gene therapy.

1 is a diagram showing the degree of cell death of each of six non-small cell lung cancer cell lines transfected with an oligonucleotide (KilGreene) prepared in an embodiment of the present invention:
1: NCI-H596 cells;
2: Calu-3 cells;
3: SK-MES-1 cells;
4: NCI-H520 cells;
5: SK-LU-1 cells; And
6: NCI-H1793 cells.
Figure 2 is a diagram confirming the action of the Kil sequence contained in KilGreene through EMSA and a signal factor in the nuclear protein extracted from six types of non-small cell lung cancer cell lines:
1: NCI-H596 cells;
2: Calu-3 cells;
3: SK-MES-1 cells;
4: NCI-H520 cells;
5: SK-LU-1 cells; And
6: NCI-H1793 cells.
Figure 3 is a diagram illustrating the signal protein binding to the kil sequence in KilGreene and the androgen receptor binding to GRE by performing EMSA using the NCI-H1793 cell line nuclear extract and the reaction product of KilGreene:
1: Reactant of nuclear protein and KilGreene; And
2: Reactant of KilGreene and nuclear protein from which androgen receptor was removed by immunoprecipitation
4 is a diagram illustrating the removal of the androgen receptor in the nuclear protein extracted from the NCI-H1793 cell line by immunoprecipitation, and confirming the removed androgen receptor:
1: Protein A-agarose bead washing solution obtained after immunoprecipitation; And
2: Immunoprecipitation product obtained after washing protein A-agarose beads obtained after immunoprecipitation.
FIG. 5A is a diagram illustrating changes in tumor volume in a mouse model transfected with NCI-H1793 (Control) or KilGreene transfected with a non-small cell lung cancer cell line.
5B is a diagram illustrating changes in tumor volume in a non-small cell lung cancer cell line NCI-H520 (Control) or an NCI-H520 (KilGreene) transplanted mouse model transfected with KilGreene.
6 is a diagram confirming the stability in flaxseed oil of KilGreene.
7 is a diagram illustrating intracellular beta-galactosidase expression after intracellular transfection of the pCMB-LaZ beta galactosidase vector using flaxseed oil.
Figure 8 is a diagram showing the expression of luciferase in the crude after intravenous administration of a mixture of flaxseed oil and luciferase plasmid to rats.
9 is a diagram showing whether or not KilGreene was transduced and anticancer effect in lung tissue after intravenous administration of flaxseed oil and KilGreene mixture to an animal model inducing lung cancer.
10 is a diagram schematically illustrating the mechanism of action of KilGreene.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating cancer, containing as an active ingredient an oligonucleotide in which the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 are complementarily bound to each other to form a double strand do.

The oligonucleotide used in the present invention is functionally equivalent to the nucleic acid molecule constituting it, for example, even if some nucleotide sequences of the oligonucleotide are modified by deletion, substitution, or insertion, the oligonucleotide can have a functional equivalent function. It is a concept that includes variants. For example, the oligonucleotide of the present invention may exhibit 80% or more homology with the base sequence of each corresponding sequence number, and specifically, may include those exhibiting 90% or more, more specifically 95% or more homology. Such homology can be readily determined by comparing the sequence of a nucleotide to the corresponding portion of the target gene using computer algorithms well known in the art, for example Align or BLAST algorithms.

In addition, the oligonucleotides used in the present invention can be isolated or prepared using standard molecular biology techniques, for example, chemical synthesis methods or recombinant methods.

In the present invention, the oligonucleotide may be provided in a form trapped in a carrier for introduction into a cell. The carrier is, for example, G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymer, cationic micelle, cationic emulsion or Liposomes may be mentioned, but the present invention is not limited thereto, and any one that functions as a delivery system may be used without limitation. In addition, biocompatible polymers such as polyethylene glycol may be conjugated to increase intracellular absorption.

In one embodiment of the present invention, a vegetable oil containing a large amount of unsaturated fatty acids such as linolenic acid, linoleic acid, and oleic acid is used as a carrier for introducing the oligonucleotide according to the present invention into cells. It was confirmed that it can be. The unsaturated fatty acid increases the permeability and fluidity by changing the structure of the cell membrane. These changes change the activity of receptors in the cell membrane of cancer cells, thereby facilitating the introduction of anticancer agents into cells.

In the present invention, the oligonucleotide is an upstream (-204) to (-188) site based on the transcription start site (+1) of a mouse mammary tumor virus (MMTV) promoter gene. Includes.

In addition, the oligonucleotide includes upstream (-186) to (-170) sites based on the transcription start site (+1) of the mouse mammary tumor virus promoter gene.

The upstream (-204) to (-188) sites based on the transcription start site (+1) of the mouse mammary tumor virus promoter gene are known as the Kil sequence or the Kil site, and the upstream ( Sites -186) to (-170) are known as distal glucocorticoid response elements (GREs). In particular, the Kil sequence is known as an enhancer in which the regulatory proteins of steroid hormone receptors, such as GR (glucocorticoid receptor), PR (progesterone receptor), and ER (estrogen receptor), act on the distal GRE (EMBO Genebank x16475). . This was confirmed by foot printing reaction of nuclear extract and MMTV LTR gene. It was not confirmed in the reaction with whole cell extract. Through this, it was confirmed that proteins that do not exist in the cytoplasm but only exist in the nucleus act. Examples of the protein include transcription factors such as NF kappa B, Oct-1, Sp-1, SW1, and STAT3.

In addition, it is known that these sites act as an enhancer by binding to cell factors that respond by a steroid hormone response (KI Lee., et al. 1995. JBC 270(41): 24502-24508).

In the present invention, the oligonucleotide binds to a steroid hormone receptor, specifically an androgen receptor, and a signal factor that interacts therewith in the nucleus, including the Kil sequence and the distal GRE. More specifically, the oligonucleotide binds to the androgen receptor and signal protein complex formed by the interaction between the activated androgen receptor and the signal protein, wherein the activated androgen receptor binds to the distal GRE, and the signal protein binds to the Kil sequence. .

It is known that the androgen receptor is activated by several signaling pathways. For example, in some cancers, androgen receptors are known to be activated through two signaling pathways, specifically an androgen-dependence signaling pathway and an androgen-independence signaling pathway to promote the growth of cancer cells. . The androgen-dependent signaling pathway is a signaling pathway in which androgen receptors are activated by binding of androgen receptors to dihydrotestosterone produced by conversion of androgens by 5α-reductase in cells. The androgen-independent signaling pathway is a signaling pathway in which PI3K is activated by phosphorylation of receptor tyrosine kinase such as EGFR, thereby activating Akt to activate androgen receptor. Activated androgen receptors promote gene expression by binding to signaling proteins such as androgen receptor elements in the nucleus (L. Gerratana., et al. 2018. Cancer treatment reviews 68: 102-110).

Accordingly, the oligonucleotide binds to a complex of an androgen receptor activated through an androgen-dependent or androgen-independent signaling pathway and a signaling protein that interacts with it, thereby inhibiting the transcriptional activity of the androgen receptor. More specifically, the oligonucleotide inhibits the growth of cancer cells by inhibiting the transcriptional activity of the androgen receptor.

In the present invention, the cancer is preferably androgen receptor positive cancer. Examples of the cancer include, but are not limited to, non-small cell lung cancer, bladder cancer, breast cancer, liver cancer, prostate cancer, colon cancer, thyroid cancer, salivary gland cancer, glioblastoma, or astrocytoma.

More specifically, the cancer may include androgen receptor-positive non-small cell lung cancer, bladder cancer, breast cancer, liver cancer, prostate cancer, colon cancer, thyroid cancer, salivary gland cancer, glioblastoma, or astrocytoma, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors prepared an oligonucleotide according to the present invention using the MMTV promoter region, and named it "KilGreene".

In addition, the present inventors confirmed that apoptosis is induced in most EGFR-positive Non Small Lung Cancer (NSLC) cell lines transduced with KilGreene.

In addition, the present inventors confirmed that the KilGreene binds to a complex of androgen receptors in the nucleus of EGFR-positive non-small cell lung cancer cell lines in which apoptosis is induced and a signal protein interacting therewith, and specifically, EGFR-positive at the distal GRE site of KilGreene. It was confirmed that the activated androgen receptor in the nucleus of the non-small cell lung cancer cell line binds, and a signaling protein that interacts with the activated androgen receptor binds to the Kil sequence site to inhibit the transcriptional activity of the androgen receptor, thereby inducing apoptosis.

Through this, as shown in the schematic diagram of FIG. 10, cancer cell growth occurs by transcriptional action through the signaling pathway from EGFR in the cell membrane of EGFR-positive non-small cell lung cancer cells through the action of signaling proteins and androgen receptors in the nucleus, and the KilGreene is the androgen receptor and the intracellular membrane. It was confirmed that cross-talking of the tyrosine kinase EGFR, which instructs the existing cell growth, was blocked.

In addition, as a result of transduction of KilGreene into non-small cell lung cancer cells exhibiting the apoptosis effect and transplantation into mice, it was confirmed that tumor growth was inhibited.

Therefore, the present inventors confirmed the effect of inhibiting the growth of cancer cells by binding the oligonucleotide according to the present invention to the androgen receptor activated in the nucleus through the androgen-independent signaling pathway by EGFR in cancer cells and a signaling protein that interacts with it. Therefore, the oligonucleotide according to the present invention can be usefully used as an active ingredient in a pharmaceutical composition for the treatment of cancer related to androgen receptor signaling as a gene therapy.

The composition of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with a carrier.

The pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound. For example, acceptable pharmaceutical carriers in a composition formulated as a liquid solution, as sterilization and biocompatible, include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, Glycerol, ethanol, and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. It can also be formulated in the form of a solution or suspension (eg, microparticles, liposomes, or integrated with cells).

The composition of the present invention may be applied in any formulation containing it as an active ingredient, and may be prepared and administered in an oral or parenteral formulation. Administration refers to introducing the composition of the present invention into a patient by any suitable method, and includes implantation of cells expressing a nucleic acid molecule. The route of administration of the composition of the present invention may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. For example, oral administration, transdermal administration, topical administration, inhalation administration, intrapulmonary administration, intrathecal administration, intranasal administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, or subcutaneous administration may be made, but It is not limited.

The compositions and treatment methods of the present invention are applicable to any animal that may develop cancer, and the animals include humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs and cats.

It is obvious to those skilled in the art that the range of the effective amount of the composition of the present invention or a suitable total daily amount can be determined by the treating physician within the range of correct medical judgment. A specific therapeutically effective amount for a particular patient is the type and extent of the reaction to be achieved, the specific composition, including whether or not other agents are used in some cases, the patient's age, weight, general health status, sex and diet, administration time, It is preferable to apply differently depending on various factors including the route of administration and the secretion rate of the composition, the treatment period, and the amount of radiation to be irradiated, and similar factors well known in the medical field. For example, it may be used as 0.001 μg/kg-100 mg/kg (body weight) per day, but is not limited thereto. It is preferable to determine the effective amount of a pharmaceutical composition suitable for the purposes of the present invention in consideration of the above.

In addition, the present invention provides a transfection formulation comprising a vegetable oil.

In the present invention, the vegetable oil is flaxseed oil, corn oil, borage oil, safflower oil, soybean oil, perilla oil, evening primrose oil, sunflower oil, blackcurrant oil (blackcurrant pip oil), malt oil (wheatgerm oil), hemp oil (hemp oil) or western pumpkin seed oil (marrow seed oil) may be, specifically, may be flaxseed oil, but is not limited thereto.

In addition, the vegetable oil may contain 70 to 99% by weight (w/w) _{of a C 18} fatty acid based on the total weight of the oil _{, and examples of the C 18} fatty acid include linolenic acid and linoleic acid. ) And oleic acid, but are not limited thereto.

In the present invention, the transfection agent can be used for in vitro or in vivo transfection through the following steps:

1) mixing and stirring the transfection agent and the oligonucleotide;

2) allowing the stirred mixture to stand at room temperature;

3) Transfecting the left mixture in vitro or in vivo.

In the present invention, the transfection agent: oligonucleotide may be mixed in a weight ratio of 1 to 5: 1, specifically 1 to 3: 1 may be mixed in a weight ratio, more specifically 1 to 2: 1 weight ratio It may be mixed with, but is not limited thereto.

In addition, the stirring in step 1) may be performed for 30 seconds to 120 seconds, more specifically 50 seconds to 90 seconds using a vortex device, but is not limited thereto.

In addition, in step 2), the mixture may be performed for 30 seconds to 120 seconds, more specifically 50 seconds to 90 seconds, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors confirmed that the oligonucleotide including the oligonucleotide according to the present invention was transfected into cells and tissues using flaxseed oil as a vegetable oil harmless to the human body, thereby showing excellent transfection efficiency.

Accordingly, the present inventors have confirmed that vegetable oil harmless to the human body can be used as a delivery vehicle for introducing the oligonucleotide containing the oligonucleotide according to the present invention in vitro or in vivo, so that the vegetable oil containing the oligonucleotide according to the present invention It can be usefully used as an active ingredient of a transfection agent for transfecting oligonucleotides in vitro or in vivo.

In addition, the present invention

a) a transfection agent comprising the vegetable oil; And

b) An oligonucleotide in which the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 are complementarily bonded to each other to form a double strand

It provides a pharmaceutical composition for preventing or treating cancer, containing as an active ingredient.

In the present invention, the types of oligonucleotides and cancers are the same as those described in the pharmaceutical composition for preventing or treating cancer, which contains the oligonucleotide as an active ingredient, so the specific description refers to the above, and hereinafter, vegetable oils and oligos Only the unique composition of a pharmaceutical composition for preventing or treating cancer containing nucleotides as an active ingredient will be described.

In the present invention, the pharmaceutical composition may be administered orally or parenterally. Specifically, oral administration, transdermal administration, topical administration, inhalation administration, intrapulmonary administration, intrathecal administration, intranasal administration, intravenous administration, intramuscular administration, intradermal administration, or intraperitoneal administration may be performed. In particular, when administered by inhalation, the delivery efficiency of oligonucleotides to lung tissue can be improved.

In a specific embodiment of the present invention, the present inventors can introduce the oligonucleotide according to the present invention into cells and tissues using flaxseed oil as a vegetable oil harmless to the human body, and the present invention introduced into lung tissue using flaxseed oil. It was confirmed that the oligonucleotide according to was exhibiting excellent anticancer effect.

Therefore, the present inventors have confirmed that a vegetable oil harmless to the human body can be used as a carrier for introducing the oligonucleotide according to the present invention into a cell, so that the oligonucleotide and vegetable oil according to the present invention are used in pharmaceuticals for the treatment of cancer related to androgen receptor signaling. It can be usefully used as an active ingredient of the composition.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Experimental Examples.

<Example 1> Preparation of KilGreene

Oligonucleotides consisting of the base sequence represented by SEQ ID NO: 1 (5'-AGCTTCCCAGGGTTTAAATAAGTTTAATGGTTACAAACTGTTCTTAAAACGCT-3') and the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 2 (5'-AGGGTCCCAAATTTATTCAAATTACCAATGTTTGACAAGAATTTTGCGAGATC-3') Bioneer, Korea). Then, the synthesized oligonucleotides are mixed in equal amounts, heated at 100°C, and then slowly left to room temperature, so that the oligonucleotides of SEQ ID NO: 1 and the oligonucleotides of SEQ ID NO: 2 are complementary to each other to form a double strand. I did. Thereafter, the double-stranded oligonucleotide was separated on a 7% SDS PAGE gel. In addition, the double-stranded oligonucleotide prepared above was named "KilGreene".

At this time, KilGreene is from -204 to upstream based on the transcription start site (+1) of the mouse mammary tumor virus (MMTV) promoter gene known as the Kil sequence (Kil sequence or Kil site). -188 site (SEQ ID NO: 3: 5'-GGGTTTAAATAAGTTTA-3') and -186 to -170 site known as distal glucocorticoid response element (GRE) (SEQ ID NO: 4: 5'-GGTTACAAACTGTTCT-3').

<Experimental Example 1> Confirmation of anticancer effect of KilGreene in Non Small Lung Cancer cells

In order to investigate the effect of KilGreene on EGFR (epidermal growth factor receptor) positive Non Small Lung Cancer (NSCLC), various types of non-small cell lung cancer cells that are positive or negative for EGFR were treated with KilGreene and treated with MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis was performed to confirm the degree of cell death.

Specifically, NCl-H596, Calu-3, SK-LU-1, SK-MES-1 and NCl-H1793, known as EGFR-positive non-small cell lung cancer cell lines, and NCI-H520, known as EGFR-negative non-small cell lung cancer cell lines, were used by the US Cell Line Bank. _{Obtained from (American tissue and cell collection) and cultured at 5% CO 2} , 37° C. using 10% FBS-MEM medium or 10% FBS-RPMI1640 medium. Then, each of the cells ^{was inoculated into a 60 mm culture dish with a number of 1×10 5} cells, and 10 μl of 100 μg of KilGreene prepared in Example 1 was added to 10 μl of lipofectamine (Thermo Fisher Scientific). Then, it was incubated for 3 weeks after transfection according to the manufacturer's procedure.

After incubation, the culture solution was removed, 100 μl of a new culture solution was added, and MTT dissolved in 75% isopropanol containing 0.2 N NaCl was added, followed by incubation at 37° C. for 2 hours. Then, 100 μl of a reaction stop solution, SDS-HCl solution (1 g SDS/10 ml 0.01 M HCl) was added and incubated at 37° C. for 4 hours, and OD was measured at 570 nM using a microplate leader. % Cell death was calculated by comparing the OD of control cells not transfected with KilGreene. As a control, an NIH 3T3 cell line was used, and MTT analysis was performed in the same manner as described above.

As a result, as shown in FIG. 1, it was confirmed that remarkably high apoptosis appeared in the case of EGFR-positive non-small cell lung cancer cell lines treated with KilGreene except for SK-MES-1. In addition, it was confirmed that apoptosis was insufficient in the case of an EGFR-negative non-small cell lung cancer cell line treated with KilGreene.

<Experimental Example 2> Confirmation of signal protein and non-small cell lung cancer apoptosis action acting on the Kil sequence in KilGreen

It is known that the Kil sequence in KilGreene acts by the regulatory protein of the steroid hormone receptor. To find out the relationship between the apoptosis of EGFR-positive cancer cells by KilGreene and the factor that interacts with the steroid hormone receptor, that is, a signal factor. For this, electrophoretic mobility shift assay (EMSA) analysis was performed using nuclear proteins extracted from various types of non-small cell lung cancer cells that are positive or negative for epidermal growth factor receptor (EGFR) and the Kil sequence of KilGreene.

Specifically, nuclear proteins were extracted from NCl-H596, Calu-3, SK-LU-1, NCl-H1793, SK-MES-1 and NCI-H520 of 1 ^{×10 9 cells.} The following solutions were used as the nuclear protein solution: Buffer A (10 mM Tris-HCl (pH 7.5), 2 mM MgCl ₂ , 3 mM NaCl, 0.2 mM Nonidet p-40 (Nonidet p-40), 0.5 mM PMSF) , 2 ug/ml leupeptin, 2 ug/ml aprotinin, buffer B (10 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.5 mM PMSF), buffer C (10 mM Tris- HCl (pH 7.5), 2 mM MgCl ₂ , 0.5 mM DTT, 0.1 mM PMSF), buffer D (10 mM Tris-HCl (pH 7.5), 80 mM NaCl, 3 mM MgCl ₂ , 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol) In addition, KI Lee., et al. (JBC 1995 270(41): 24502-24508) [α- ³² P]dCTP labeled Kil sequence was obtained in the same manner as disclosed in I did.

Then, 2 ug of the extracted nuclear protein and the [α- ³² P]dCTP-labeled Kil sequence were mixed with a 30 ul binding buffer containing 1 ug poly(dI-dC) (10 mM Tris-HCl (pH 7.5), 80 mM NaCl.3 mM MgCl ₂ , 0.1 mM EDTA, 1 mM PMSF, 5% glycerol) and reacted at room temperature for 1 hour, and then the DNA-protein reactant was subjected to electrophoresis on a 6% polyacrylamide gel. Separated through.

As a result, as shown in Fig. 2, in NCI-H596, Calu-3, SK-LU-1, and NCI-H1793 cells, signal proteins that bind to the Kil sequence were identified. On the other hand, in SK-MES-1 and NCI-H520 cells, a signal protein binding to the Kil sequence was not identified. In addition, it was confirmed that the degree of apoptosis and the amount of signal protein binding to the Kil sequence were proportional.

From the above results, it can be seen that the anticancer action of KilGreene depends on the level of the signal protein acting on the Kil sequence.

<Experimental Example 3> Confirmation of signal protein and androgen receptor acting on KilGreene

It is known that the expression of the androgen receptor adversely affects the survival rate in non-small cell lung cancer (L. Grant., et al. 2018. Horm Cancer 9(4): 288-294). Therefore, in order to find out the relationship between the signaling protein acting on KilGreene and the androgen receptor, using the Kil sequence of KilGreene and the nuclear protein extracted from EGFR-positive non-small cell lung cancer cells with the best apoptosis effect by KilGreene treatment, EMSA (electrophoretic mobility shift assay) analysis was performed.

Specifically, nuclear proteins were extracted from NCl-H1793 ^{of 1×10 9} cells, an EGFR-positive non-small cell lung cancer cell line showing the best effect by KilGreene as shown in FIG. 1 by the same method as described in <Experimental Example 2>. . In addition, in KilGreene prepared in <Example 1>, KI Lee., et al. ^{[Α- 32} P]dCTP was labeled in the same manner as disclosed in (JBC 1995 270(41): 24502-24508).

Then, in order to remove the androgen receptor in the extracted nuclear protein, immunoprecipitation was performed as follows. 200 ug of rabbit anti-androgen receptor antibody (Sigma A4719) was added to 875 ul of the extracted nuclear protein, and reacted with stirring at 4° C. for 30 minutes. After the reaction, 100 ul of protein A-Agarose beads were added and re-reacted in the same manner as described above. After completion of the reaction, the bead and the supernatant were separated by centrifugation for 2 minutes. The supernatant and the 2 ug [α- ³² P] dCTP-labeled by KilGreene 1 ug poly 30 ul binding buffer (10 mM Tris-HCl (pH 7.5 containing (dI-dC)), 80 mM NaCl. 3 mM MgCl ₂ , 0.1 mM EDTA, 1 mM PMSF, 5% glycerol) and reacted at room temperature for 1 hour, and then the DNA-protein reaction product was separated by electrophoresis on a 6% polyacrylamide gel. EMSA analysis was performed in the same manner as described above using the extracted nuclear protein as a control (FIG. 3).

In addition, in order to confirm that the androgen receptor in the supernatant was removed, the beads were recovered and immunoprecipitation buffer (1% Tritonx-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4)), 1 mM EDTA, 1 mM EGTA (pH 8.0), protease inhibitor), and then centrifuged at 4° C. for 2 minutes to separate into beads and washing liquid, and each of them was recovered. 50 ul of 0.1 M glycine (pH 2.5) was added to the recovered beads, mixed, and then reacted with stirring at 4° C. for 10 minutes. next. Centrifugation was performed at 4° C. for 2 minutes to obtain a supernatant. The obtained supernatant (immunoprecipitation product) and the recovered washing liquid were subjected to electrophoresis on a 7% SDS PAGE gel (FIG. 4).

Through the above method, the nuclear extract and the nuclear extract from which the androgen receptor were removed were obtained, and EMSA analysis and electrophoresis were performed to identify signal proteins and androgen receptors acting on KilGreene.

As a result, as shown in Figs. 3 and 4, the gastric band reaction strongly shown in Fig. 3 1 was dramatically reduced in the reaction with the nuclear extract confirmed to remove the androgen receptor through immunoprecipitation in Fig. 4 2. This confirmed that the androgen receptor was present in the upper band. On the other hand, it was confirmed that the function of the signal protein acting on the Kil sequence was increased when the androgen receptor was removed by the antibody in 2 of FIG. 4.

Through this, since the reaction of the gastric band reactant strongly reacting in 1 of FIG. 3 decreases sharply in the reaction with the androgen receptor-removing nuclear extract of 2 of FIG. 3, the gastric band reactant is an androgen receptor that binds to the distal GRE, but rather an increased response. It was confirmed that the reaction band shown below is a signal protein that binds to the kil sequence.

Through the above results, as shown in FIG. 10, in androgen receptor and EGFR-positive non-small cell lung cancer cells, KilGreene binds to androgen receptor activated in the nucleus through androgen-independent signaling by EGFR and a signal protein that interacts with the androgen receptor. It can be seen that it inhibits the transcriptional activity of. In addition, it can be seen that the transcriptional activity of the androgen receptor is inhibited, thereby inhibiting the growth of cancer cells.

<Experimental Example 4> Confirmation of the anticancer effect of KilGreene in a non-small cell lung cancer cell transplant mouse model

The anticancer effect of KilGreene was confirmed in a non-small cell lung cancer cell transplant mouse model.

Specifically, as shown in FIG. 1, NCI-H1973, an EGFR-positive non-small cell lung cancer cell line, showing the best effect by KilGreene, and NCI-H520, an EGFR-negative non-small cell lung cancer cell line, showing the most excellent effect by KilGreene, were obtained ^{at 2×10 6 cells.} Then, 10 µl of 100 ug of KilGreene prepared in Example 1 was transfected using 10 µl of lipofectamine according to the manufacturer's procedure. Then, the cells transfected with KilGreene were injected subcutaneously into the flanks of a 4-week-old BALB/C female nude mouse obtained from Charles river Laboratories and given once a week for 6 weeks to measure the volume of the tumor using a caliper at time. I did. The volume of the tumor was calculated as follows by measuring the length (a), area (b) and height (c): tumor volume = tumor length (a) × tumor width (b) × tumor height (c)/2.

As a control, cells not transfected with KilGreene were injected subcutaneously into nude mice in the same manner as described above, and then the volume of the tumor was measured.

As a result, as shown in FIGS. 5A and 5B, it was confirmed that only the mouse group transplanted with KilGreene-transfected NCI-H1793 decreased the tumor volume compared to the control group.

<Experimental Example 5> Confirmation of KilGreene transfection efficiency using vegetable oil

<5-1> Confirmation of KilGreene stability in vegetable oil

Flaxseed, which is rich in linolenic acid, linoleic acid, and oleic acid, as vegetable oils, to find out whether KilGreene can be transfected into cells or tissues using vegetable oils that are harmless to the human body. oil), the stability of KilGreene was confirmed.

Specifically, 100 ul of PBS and 100 ul of flaxseed oil were mixed and well mixed with a vortex device, and 50 ul (50 ug) of KilGreene prepared in Example 1 was added and mixed well with a vortex device. Then, after allowing to stand at room temperature for 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days, electrophoresis was performed in the same manner as described in <Experimental Example 3>.

As a result, as shown in FIG. 6, it was confirmed that KilGreene was stably maintained without being decomposed in flaxseed oil for about 40 days.

<5-2> Confirmation of DNA transfection efficiency using vegetable oil in cells and tissues

In order to find out whether KilGreene can be transfected into cells or tissues using vegetable oil, the transfection efficiency of DNA in cells and tissues was confirmed using flaxseed oil.

Specifically, to confirm transfection of intracellular DNA, 50 ul of PBS and 50 ul of flaxseed oil were mixed, mixed well with a vortex device, 5 ul of pCMV-LacZ vector was added, and mixed well with a vortex device. Then, after standing at room temperature for 1 minute, NIH 3T3 cells were treated and transfected. At this time, NIH 3T3 cells were cultured in a culture medium from which BSA was removed one day before transfection. Then, beta-galactosidase analysis was performed according to the manufacturer's procedure using a beta-galactosidase staining kit (abcam) (FIG. 7).

In addition, 500 ul of PBS and 500 ul of flaxseed oil were mixed with 500 ul of flaxseed oil in order to confirm the transfection of the DNA in the tissue, and 500 ul of luciferase plasmid was added after mixing well with a vortex device, followed by mixing well with a vortex device. Then, after allowing to stand at room temperature for 1 minute, it was injected into the tail vein of F344/N rats, and after 24 hours, luciferase expression in the tissue was measured using a luciferase assay kit (Promega) according to the manufacturer's procedure (FIG. 8) ).

As a result, as shown in Figs. 7 and 8, it was confirmed that DNA in cells and tissues can be transfected using flaxseed oil. In addition, it was confirmed that DNA transfection using flaxseed oil was most excellent in the lungs of the tissues.

<5-3> Confirmation of KilGreene transfection efficiency using vegetable oil in tissues

Intra-tissue transfection of KilGreene was confirmed using vegetable oil.

Specifically, F344/N rats were divided into a control group, a KilGreene-free group, and a KilGreene-treated group, and the RR1022 cells, which are rat sarcoma cells, were 7×10 ⁶ cells in PBS. After addition to 500 ul, it was injected into the tail vein to induce lung cancer for 7 days.

In addition, 200 ul of PBS and 200 ul of flaxseed oil were mixed, mixed well with a vortex device, 100 ul (2 mg) of KilGreene prepared in Example 1 was added, and mixed well with a vortex device to prepare a KilGreene transfection formulation. I did. Then, the KilGreene-treated group was injected with the KilGreene transfection agent once every two weeks into the tail vein 3 times for 6 weeks from 7 days later. After 6 weeks, the lung tissue was removed, stained with Indian ink, and visually observed. In the KilGreene untreated group, 500 ul of PBS was injected into the tail vein.

As a result, as shown in FIG. 9, in the case of the lung tissue of the KilGreene-treated group, it was confirmed that the normal lung tissue of the control group showed a similar staining pattern, unlike the non-KilGreene-treated group.

From the above results, it can be seen that KilGreene can be administered using a vegetable oil that is harmless to the human body in clinical practice.

<110> LEE, Kyong il <120> Use of androgen receptor antagonist for treating cancer <130> PB2019-222 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligonucleotide <400> 1 agcttcccag ggtttaaata agtttaatgg ttacaaactg ttcttaaaac gct 53 <210> 2 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligonucleotide <400> 2 agggtcccaa atttattcaa attaccaatg tttgacaaga attttgcgag atc 53 <210> 3 <211> 17 <212> DNA <213> Mouse mammary tumor virus <400> 3 gggtttaaat aagttta 17 <210> 4 <211> 16 <212> DNA <213> Mouse mammary tumor virus <400> 4 ggttacaaac tgttct 16

Claims (14)

A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an oligonucleotide in which the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 are complementary to each other to form a double strand.
The method of claim 1, wherein the oligonucleotide is a mouse mammary tumor virus (MMTV) promoter gene transcription start site (+1) upstream from the reference (-204) to (-188). ) Site (Kil sequence: Kil sequence) and upstream (-186) to (-170) site (far GRE: distal glucocorticoid response element), characterized in that it comprises a, cancer prevention or treatment pharmaceutical composition.
The method according to claim 1 or 2, wherein the oligonucleotide binds to a complex of an androgen receptor and a signal protein formed by an interaction between an activated androgen receptor and a signal factor. Pharmaceutical composition.
The pharmaceutical composition for preventing or treating cancer according to claim 3, wherein the activated androgen receptor binds to the distal GRE, and the signal protein binds to the Kil sequence.
The method of claim 1, wherein the oligonucleotide binds to a complex of an androgen receptor and a signal protein activated through an androgen-dependence or androgen-indepdence signaling pathway to activate the transcriptional activity of the androgen receptor. A pharmaceutical composition for preventing or treating cancer, characterized in that it inhibits.
The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the oligonucleotide inhibits the transcriptional activity of the androgen receptor to inhibit cancer cell growth.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is an androgen receptor positive cancer.
The method of claim 1, wherein the cancer is selected from the group consisting of non-small cell lung cancer, bladder cancer, breast cancer, liver cancer, prostate cancer, colon cancer, thyroid cancer, salivary gland cancer, glioblastoma and astrocytoma, or Pharmaceutical composition for treatment.
a) transfection preparations containing vegetable oils; And
b) An oligonucleotide in which the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 are complementarily bonded to each other to form a double strand
Containing as an active ingredient, a pharmaceutical composition for preventing or treating cancer.
The method of claim 9, wherein the vegetable oil is flaxseed oil, corn oil, borage oil, safflower oil, soybean oil, perilla oil, evening primrose oil, sunflower oil, blackcurrant. Blackcurrant pip oil (blackcurrant pip oil), malt oil (wheatgerm oil), hemp oil (hemp oil), and amber seed oil (marrow seed oil), characterized in that selected from the group consisting of, cancer prevention or treatment pharmaceutical composition.
The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the vegetable oil comprises 70 to 99% by weight (w/w) _{of C 18 fatty acids based on the total weight of the oil.}
The pharmaceutical composition for preventing or treating cancer according to claim 11, wherein the C ₁₈ fatty acid is selected from the group consisting of linolenic acid, linoleic acid, and oleic acid.
delete The group consisting of oral administration, transdermal administration, topical administration, inhalation administration, intrapulmonary administration, intrathecal administration, intranasal administration, intravenous administration, intramuscular administration, intradermal administration, and intraperitoneal administration. A pharmaceutical composition for preventing or treating cancer, characterized in that it is administered by any one or more administration methods selected from.

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