KR20200118931A - A composition for inhibiting metastasis cancer under hypoxic condition - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition which can inhibit cancer metastasis caused in a hypoxic environment by containing an AROS inhibitor; and a method for preventing or treating cancer metastasis comprising a step of treating the composition to an individual concerned with metastasis of cancer in the hypoxic environment. The composition containing the AROS inhibitor of the present invention can effectively inhibit cancer metastasis caused in the hypoxic environment, thereby being able to be widely used for preventing cancer recurrence by preventing cancer metastasis.

Description

Composition for inhibiting metastasis cancer under hypoxic condition

The present invention relates to a composition for inhibiting metastasis of cancer induced in a hypoxic environment, and more specifically, the present invention comprises an AROS inhibitor, a pharmaceutical composition capable of inhibiting metastasis of cancer induced in a hypoxic environment, and the composition It relates to a method for preventing or treating cancer metastasis comprising the step of treating an individual concerned with metastasis of cancer in a hypoxic environment.

According to data published by the American Cancer Society, 7.6 million people, or 17% of the world's deaths in 2008, died of cancer in 2008, and it is expected to increase to 9 million in 2015 and 11.4 million in 2030. Cancer remains the number one cause of death in Korea, and the number of deaths in 2016 was 28,827, the highest ever since statistics were compiled. Cancer was the first among the top 10 deaths, and the number of deaths from cancer per 100,000 population is 153, increasing every year. Accordingly, the social cost is enormous, and the need for developing anticancer drugs with low toxicity and high effectiveness is increasing. In addition, in recent years, due to the enormous social cost of cancer, the development of drugs that can be treated at low cost is required.

One of the biggest causes of life-threatening cancer is the metastasis of cancer cells. Currently, surgical surgery is a common cancer treatment method, but because cancer cells metastasize to various places other than the primary cancer site, a cure through surgery can be expected only in the early stages. In most cases, cancer develops in one organ (lung, liver, kidney, stomach, colon, rectum, etc.) and then spreads from the first organ, which is the primary site, to other tissues, and spreading from the primary site to other tissues is metastasis ( metastasis). Metastasis is a phenomenon accompanying the progression of a malignant tumor. As malignant tumor cells proliferate and the cancer progresses, new genetic traits necessary for metastasis are acquired, infiltrating into blood vessels and lymph glands, circulating along blood and lymph, and then settling in other tissues. After doing it, it will multiply.

Like cancer incidence, such cancer metastasis is known to be made by a combination of various genes and factors involved in the movement and invasion of cancer cells, and the movement of cancer cells plays an important role in the cancer metastasis. For example, when cancer cells move from the initial primary cancer site to the blood vessel through the extracellular matrix (ECM) or from the second metastasis tissue to the outside of the blood vessel, vascular endothelial cells move from the new blood vessel. When to get involved. In migrating cells, polarity is induced by signal receptors activated by cell migration inducers. In addition, the cell membrane in front of the cell is extended forward by polymerization of actin, and the cell adheres to the interstitial via integrin. At this time, a strong contraction force is formed between the actin polymers by myosin bound to the actin polymer, and a strong contraction force is imparted to the entire cell. Therefore, the direction of cell movement is determined by the difference in adhesion between the front part and the back part of the cell, and the cell moves.

Many factors are involved in the migration of cancer cells. Among them, a protrusion called filopodia is formed that extends from the cytoskeleton or the fibrous tissue called lamellipodia around the cell. These protrusions help normal healthy cells move their position in the tissues they belong to. However, in malignant cancer, normal healthy cell function is often transformed into a destructive excess, resulting in excessive production of lamelipodia and phylopodia. It is known that suppressing this phenomenon can fundamentally prevent the movement of cancer cells, and studies to develop substances that inhibit metastasis of cancer cells are being actively conducted using this. For example, Korean Patent Registration No. 10-1323728 discloses a method of effectively inhibiting cancer metastasis by blocking cancer cell migration and inhibiting neovascularization by using a drug called benproperine, which is commercially used as an antitussive expectorant. . However, in order for these drugs that inhibit the migration of cancer cells to show an effect, a high level of treatment is required, and a problem that side effects occur due to such treatment amount has been newly raised, but a solution to this has not been reported. Actually.

Under this background, the present inventors have made intensive research efforts to develop a safer and more effective method of inhibiting metastasis of cancer. As a result, when inhibiting the action of AROS generally expressed in cancer cells, the level of intracellular HIF-1α is reduced. , As a result, it was confirmed that metastasis of cancer was suppressed, and the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for inhibiting metastasis of cancer comprising an AROS inhibitor.

Another object of the present invention is to provide a method for inhibiting cancer metastasis comprising treating the composition to an individual other than humans who are concerned about metastasis of cancer.

One aspect of the present invention for achieving the above object is a pharmaceutical composition for inhibiting metastasis of cancer comprising an AROS (active regulator of SIRT1) inhibitor.

The term "AROS (active regulator of SIRT1)" of the present invention means a direct regulator of SIRT1 (sirtuin-1), by promoting SIRT1-mediated deacetylation to p53/TP53, p53/TP53-mediated transcriptional activity It is known to play a role in suppressing The amino acid sequence thereof or the polynucleotide sequence encoding the same can be obtained from a known database such as NCBI, for example, GenBank Accession No. It can be a protein represented by NP_919307.1.

The term "AROS inhibitor" of the present invention is used collectively for agents that reduce the expression or activity of AROS, and specifically, in a manner such as acting directly on AROS or indirectly acting on its ligand, Any agent that reduces the expression level of AROS or its activity by reducing the expression of AROS at the transcriptional level or interfering with its activity may be included.

The AROS inhibitor may be used without limitation in its form, such as a compound, nucleic acid, peptide, virus, or vector containing the nucleic acid that can target AROS and inhibit the expression or activity of AROS. The AROS inhibitor is not limited thereto, but specifically, may be an oligonucleotide that inhibits the expression of AROS mRNA, or an antibody that inhibits the activity of an AROS protein or a ligand protein for AROS, an antigen-binding fragment or peptide thereof, and more specifically The oligonucleotide that inhibits the expression of AROS mRNA may be an antisense oligonucleotide, aptamer, miRNA, shRNA or siRNA specific to AROS mRNA, and more specifically, the siRNA is the oligonucleotide of SEQ ID NO: 1 And it may be a double-stranded oligonucleotide having a complementary sequence thereof.

The term "antisense oligonucleotide" of the present invention is a DNA, RNA or derivative thereof containing a nucleic acid sequence that is complementary to a specific mRNA sequence, which binds to a complementary sequence in the mRNA and inhibits translation of the mRNA into a protein. It works.

In the present invention, the antisense oligonucleotide sequence may be a DNA or RNA sequence that is complementary to the AROS mRNA and capable of binding to the mRNA. This can inhibit the translation, translocation into the cytoplasm, maturation or all other essential activity of the overall biological function of the AROS mRNA. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. The antisense oligonucleotide may be synthesized in vitro by a conventional method and administered in vivo, or the antisense oligonucleotide may be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the multicloning site (MCS). It is preferable that the antisense RNA has a translation stop codon in the sequence so that it is not translated into a peptide sequence. The design of the antisense oligonucleotide that can be used in the present invention can be easily prepared according to a method known in the art by referring to the base sequence of AROS.

The term "aptamer" of the present invention refers to a nucleic acid molecule having a size of about 20 to 60 nucleotides as a single-stranded oligonucleotide and having binding activity to a predetermined target molecule. It has various three-dimensional structures depending on the sequence, and can have high affinity with a specific substance, such as an antigen-antibody reaction. The aptamer can inhibit the activity of a predetermined target molecule by binding to a predetermined target molecule.

In the present invention, the aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof that binds to AROS and inhibits the activity of AROS, and may also be in a linear or cyclic form. Such an aptamer can be prepared from the sequence of AROS by a method known to those skilled in the art.

The term "miRNA" of the present invention refers to a non-cording RNA of up to 22 base sequences that can regulate gene expression by degrading target mRNA or inhibiting the transcription of target mRNA. Means.

The terms "siRNA" and "shRNA" of the present invention are nucleic acid molecules capable of mediating RNA interference or gene silencing, and are efficient gene knockdown methods or genes because they can inhibit the expression of target genes. Used as a treatment method. shRNA is a single stranded oligonucleotide in which a hairpin structure is formed by binding between complementary sequences, and in vivo, the shRNA is a small RNA fragment of 21 to 25 nucleotides in size while being cleaved by a dicer. It becomes siRNA, a double-stranded oligonucleotide, and can inhibit expression by specifically binding to mRNA having a complementary sequence. Therefore, which means of shRNA and siRNA to be used can be determined by the choice of those skilled in the art, and similar expression reduction effects can be expected if the mRNA sequences they target are the same.

For the purposes of the present invention, the "miRNA", "siRNA" or "shRNA" specifically acts on AROS to cleave AROS mRNA molecules to induce RNA interference (RNAi), thereby inhibiting the AROS. It can be interpreted as a formulation. These may be synthesized chemically or enzymatically, and the method for preparing them is not particularly limited, and methods known in the art may be used. For example, a method of directly chemically synthesizing "miRNA", "siRNA" or "shRNA", a method of synthesizing "miRNA", "siRNA" or "shRNA" using in vitro transcription, in vitro (in In vitro) method of cleaving long double-stranded RNA synthesized by transcription using an enzyme, expression method through intracellular delivery of "miRNA", "siRNA" or "shRNA" expression plasmid or viral vector, and PCR (polymerase chain) reaction) induction The expression method through intracellular delivery of “miRNA”, “siRNA” or “shRNA” expression cassettes, but is not limited thereto.

In particular, the siRNA against AROS of the present invention may be composed of a double strand of an oligonucleotide of SEQ ID NO: 1 and an oligonucleotide having a complementary sequence thereof, but is not limited thereto.

The term "antibody" of the present invention refers to a substance that reacts when an external substance, which is an antigen, invades while circulating in blood or lymph in the immune system of a living body, and is a globulin-based protein formed in lymphatic tissues and is also called immunoglobulin. Antibodies are proteins that B cells produce and flow into body fluids. They specifically bind to an antigen. One antibody molecule has two heavy chains and two light chains, each of which is a heavy chain. And the light chain have a variable region at the end of its N-terminal. Each variable region is composed of three complementarity determining regions (CDRs) and four framework regions (FRs), and the complementarity determining regions determine the antigen binding specificity of the antibody and the structure of the variable region. It exists as a relatively short peptide sequence that is maintained at the forming sites.

For the purposes of the present invention, the antibody may be an antibody capable of inhibiting the activity of AROS by binding to AROS or a ligand protein of AROS.

The term "ligand" of the present invention refers to a substance that forms a complex with a biomolecule to bring about a biological reaction.

For the purposes of the present invention, the ligand may be a protein that activates or increases the activity of AROS by binding to AROS.

The term "cancer" of the present invention refers to a disease related to the regulation of cell death, and refers to a disease caused by excessive proliferation of cells when the normal apoptosis balance is broken. These abnormally proliferated cells, in some cases, invade surrounding tissues and organs, form masses, and destroy or deform normal structures in the body, a condition called cancer. In general, a tumor means a mass that has grown abnormally due to the autonomous overgrowth of body tissues, and can be classified into a benign tumor and a malignant tumor. Malignant tumors grow faster than benign tumors, and metastasis occurs as they infiltrate the surrounding tissues, which is life-threatening. These malignant tumors are commonly referred to as cancer.

In the present invention, the cancer is not particularly limited as long as intracellular HIF-1α is stabilized by AROS and metastasis can be induced thereby, but as an example, cerebrospinal cancer, head and neck cancer, lung cancer, breast cancer, thymoma , Mesopores, esophageal cancer, brittle cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, colon cancer, lymphoma, acute leukemia , Chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.

The term "metastasis" of the present invention refers to a condition in which a malignant tumor has spread to other tissues away from the diseased organ. As a malignant tumor that started in one organ progresses, it spreads from the first primary site, the organ, to other tissues, and this spreading from the primary site to other tissues can be called before. Metastasis can be said to be a phenomenon accompanying the progression of a malignant tumor. As the malignant tumor cells proliferate and the cancer progresses, metastasis can occur while acquiring new genetic traits. Metastasis can occur when tumor cells that have acquired new genetic traits invade blood vessels and lymph lines, circulate along blood and lymph, and settle and proliferate in other tissues.

Depending on the tissue where metastasis occurs, various cancer diseases such as liver cancer, kidney cancer, lung cancer, gastric cancer, colon cancer, rectal cancer, and pancreatic cancer can be caused. The composition of the present invention can prevent and treat cancer spreading by inhibiting metastasis.

The term "inhibition" of the present invention refers to all actions of inhibiting the cancer metastasis by administration of the composition according to the present invention.

According to an embodiment of the present invention, AROS can regulate the expression of HIF-1α (Fig. 1), AROS is involved in the processing after HIF-1α is expressed (Fig. 2), and inhibition of AROS expression is ubiquitin. Induces the degradation of HIF-1α by (Fig. 3), and AROS is involved in the regulation of the intracellular level of HIF-1α by a mechanism of action related to proline hydroxylation (Fig. 4), and the deficiency of HIF-1α by AROS It was confirmed that the level of cancer cells decreased metastatic activity was decreased (FIG. 5).

Therefore, it was found that suppressing the expression of the AROS gene in cancer cells can inhibit metastatic activity of cancer cells.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may be an unnatural carrier. Compositions containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, or lactose in one or more compounds. (lactose), gelatin, etc. can be mixed to prepare. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

In addition, the pharmaceutical composition of the present invention is not limited thereto, but tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations And it may have any one formulation selected from the group consisting of suppositories.

Another aspect of the present invention is a method of inhibiting metastasis of cancer comprising administering to an individual a pharmaceutical composition comprising an AROS inhibitor as an active ingredient.

The term "individual" of the present invention means all animals including humans who have or have developed a cancer disease of the present invention, and may be an individual other than humans.

By administering the pharmaceutical composition of the present invention to an individual, metastasis of cancer including cervical cancer can be suppressed.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

The term "administration" of the present invention refers to the act of introducing the pharmaceutical composition of the present invention to a subject by any suitable method, and the route of administration may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. have.

The pharmaceutical composition may be appropriately administered to a subject according to a conventional method, route of administration, and dosage used in the art, depending on the purpose or need. Examples of the route of administration may be oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, an appropriate dosage and number of administrations may be selected according to methods known in the art, and the amount and number of administrations of the pharmaceutical composition of the present invention to be actually administered are the type of symptom to be treated, route of administration, sex, health condition, It can be appropriately determined by various factors such as diet, age and weight of the individual, and the severity of the disease.

The term "pharmaceutically effective amount" of the present invention means an amount sufficient to inhibit or alleviate the increase in vascular permeability at a reasonable benefit/risk ratio applicable to medical use, and the effective dose level is the type and severity of the subject, age, Sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

Since the composition containing the AROS inhibitor of the present invention can effectively inhibit metastasis of cancer caused in a hypoxic environment, it will be widely used to prevent cancer recurrence by preventing metastasis of cancer.

1 is a Western blot analysis photograph showing the results of measuring the expression level of HIF-1α by inhibiting the expression of the AROS gene in cancer cells cultured in a hypoxic environment.
2 is a Western blot analysis photograph showing the results of measuring the expression level of HIF-1α by inhibition of AROS gene expression and inhibition of proteolysis in cancer cells cultured in a hypoxic environment.
3 is a photograph of Western blot analysis showing the results of clarifying the relationship between the inhibition of AROS gene expression and the ubiquitination of HIF-1α protein in cancer cells cultured in a hypoxic environment.
4 is a photograph of Western blot analysis showing the results of measuring the expression level of HIF-1α by inhibition of AROS gene expression and inhibition of hydroxylation in cancer cells cultured in a hypoxic environment.
5 is a micrograph showing the result of comparing the level of migration after culturing cancer cells in which the expression of the AROS gene is suppressed in a hypoxic environment.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Reduction of accumulation of HIF-1α by inhibition of AROS expression in hypoxic environment

HeLa cells (1.5 X 10 ⁵ ), which are cervical cancer cells, were inoculated into a 60mm dish and cultured to attach the cancer cells to the floor. It was treated with a concentration of 20 nM. Then, after 48 hours elapsed, the culture was further cultured for 9 hours in a normal state or hypoxic state. After the culture was completed, the levels of HIF-1α, AROS and β-actin expressed in the cultured cancer cells were measured through Western blot analysis (FIG. 1). At this time, β-actin was used as an internal control.

1 is a Western blot analysis photograph showing the results of measuring the expression level of HIF-1α by inhibiting the expression of the AROS gene in cancer cells cultured in a hypoxic environment.

As shown in Figure 1, when the expression of the AROS gene was not suppressed in a hypoxic environment (control-siRNA treatment group), the expression of HIF-1α was increased, but when the expression of the AROS gene was suppressed even in a hypoxic environment, the expression of HIF-1α was It was confirmed that there was no increase.

From the results of FIG. 1, it was found that AROS can regulate the expression of HIF-1α.

Example 2: Identification of the function of AROS on HIF-1α

Since it was confirmed that AROS regulates the expression of HIF-1α in Example 1, it was attempted to investigate at what stage AROS is involved in the expression of HIF-1α.

Example 1, except that, after each siRNA was treated, 20 uM of MG132 (an agent that inhibits protein degradation by inhibiting the action of 26S proteasome) was treated, and incubated for an additional 9 hours only in hypoxia. By performing the same method as, the levels of HIF-1α, AROS, and β-actin expressed in cancer cells were measured through Western blot analysis (FIG. 2 ).

2 is a Western blot analysis photograph showing the results of measuring the expression level of HIF-1α by inhibition of AROS gene expression and inhibition of proteolysis in cancer cells cultured in a hypoxic environment.

As shown in FIG. 2, when the expression of the AROS gene is suppressed in a hypoxic environment, the expression of HIF-1α is not increased, but when the expression of the AROS gene and proteolytic activity are suppressed in a hypoxic environment, the expression of HIF-1α is increased. It was confirmed that it was maintained.

From the results of FIG. 2, when the proteolytic activity was suppressed, even if the expression of the AROS gene was suppressed, the expression level of HIF-1α increased by the hypoxic environment was maintained as it is, so that the AROS gene is HIF-1α expressed. It was analyzed to be involved in the post-translation modification step. For example, the AROS gene was predicted to perform a function of increasing the stability of translated HIF-1α.

Example 3: Identification of the function of AROS on the expressed HIF-1α

Since it was confirmed from the results of Example 2 that AROS is involved in the expressed HIF-1α, it was attempted to determine which stage of the post-translation modification step of HIF-1α is involved in AROS.

HeLa cells (1.5 X 10 ⁵ ) were inoculated into a 60mm dish and cultured to attach the cancer cells to the bottom, and then AROS-siRNA and control-siRNA were treated at a concentration of 20 nM. Subsequently, after 24 hours, Flag-tagged HIF-1α (Flag-tagged HIF-1α; Flag-HIF-1α) and HA-tagged ubiquitin (HA-Ub) bound with a Flag label were added. Treated with. Then, 20 uM of MG132 was treated and further incubated for 9 hours in a hypoxic environment.

After the cultivation was completed, the cultured cancer cells were disrupted to obtain an extract containing a protein. After the obtained extract was treated with an anti-Flag antibody, it was reacted for 6 hours, and then IgG beads were added to bind the IgG beads to the anti-Flag antibody. Then, Western blot analysis was performed to measure the level of ubiquitin bound to HIF-1α protein (FIG. 3).

3 is a photograph of Western blot analysis showing the results of clarifying the relationship between the inhibition of AROS gene expression and the ubiquitination of HIF-1α protein in cancer cells cultured in a hypoxic environment.

As shown in Figure 3, when the expression of the AROS gene was not suppressed in a hypoxic environment, HIF-1α was not detected, but when the expression of the AROS gene was suppressed in a hypoxic environment, not only Flag-HIF-1α, but also HA-Ub It was confirmed that HIF-1α bound was detected.

In general, it is known that ubiquitin is bound to a protein that is not required by cells, and these conjugates pass through the 26S proteasome and are degraded through the action of various proteolytic enzymes. 3, when the expression of the AROS gene is suppressed in a hypoxic environment, it was confirmed that HIF-1α bound with HA-Ub was detected, from which the inhibition of the expression of AROS prevented the degradation of HIF-1α by ubiquitin. I could see that it was induced.

Example 4: Identification of other functions of AROS on expressed HIF-1α

After HIF-1α was expressed, we tried to confirm whether AROS is also involved in hydroxylation, which is known as one of the processing processes.

In a hypoxic environment, hydroxylation of proline residues is inhibited, and stabilization of HIF-1α protein is induced. In order to confirm whether the regulation of HIF-1α expression by AROS is related to proline hydroxylation, we tried to measure the effect of AROS on HIF-1α expression after treatment with DMOG (dimethyloxalyglycine; Proline hydroxylase inhibitor). At this time, in order to know whether it has the same mechanism as SIRT1, which is known as the main target of AROS, the SIRT1 deficiency condition was also added and tested.

Roughly, except for treating SIRT1-siRNA in addition to AROS-siRNA, treating these siRNAs, and then treating 1 uM of DMOG (dimethyloxalyglycine; Proline hydroxylase inhibitor), the same method as in Example 1 was performed. , The levels of HIF-1α, SIRT1, AROS and β-actin expressed in cancer cells were measured through Western blot analysis (FIG. 4).

4 is a photograph of Western blot analysis showing the results of measuring the expression level of HIF-1α by inhibition of AROS gene expression and inhibition of hydroxylation in cancer cells cultured in a hypoxic environment.

As shown in FIG. 4, when AROS or SIRT1 expression is suppressed in a hypoxic environment, HIF-1α is decreased, but when DMOG is treated in a hypoxic environment, HIF-1α is not decreased even when AROS expression is suppressed, SIRT1 It was confirmed that HIF-1α decreased only when the expression of was inhibited.

From the results of FIG. 4, it can be seen that AROS is involved in the expression of HIF-1α by a mechanism of action completely different from that of SIRT1, and the AROS regulates the intracellular level of HIF-1α by an action mechanism related to the hydroxylation of proline. Was analyzed to be involved in.

Example 5: Anticancer effect by inhibition of AROS expression

From the results of Examples 1 to 4, it was confirmed that inhibition of AROS expression induces a decrease in HIF-1α, wherein HIF-1α induces resistance during anticancer treatment using an anticancer agent or radiation, and glucose absorption, blood vessels of cancer cells Since it is known as a major causative factor of cancer recurrence that promotes invasion and metastasis, we tried to determine how the AROS expression inhibition has an effect on cancer cells.

HeLa cells (1.5 X 10 ⁵ ), which are cervical cancer cells, were inoculated into a 60mm dish and cultured to attach the cancer cells to the bottom, and then treated with AROS-siRNA at a concentration of 20 nM, and cultured. After the cultivation was completed, the cultured cancer cells (1.5 X 10 ⁴ ) were placed in an invasion chamber coated with Matrigel, and further cultured for 48 hours in a normal environment or a hypoxic environment. After the cultivation was completed, the cancer cells moved outside the infiltration chamber were stained using a staining buffer, and observed under a microscope (FIG. 5).

5 is a micrograph showing the result of comparing the level of migration after culturing cancer cells in which the expression of the AROS gene is suppressed in a hypoxic environment.

As shown in FIG. 5, it was confirmed that the level of migration of cancer cells increased in a hypoxic environment, but suppressing the expression of the AROS gene did not increase the level of migration even in a hypoxic environment.

From the results of FIG. 5, it was found that the metastatic activity of cancer cells in which the level of HIF-1α was reduced due to AROS deficiency was decreased.

Taking the results of Examples 1 to 5 together, it was found that suppressing the expression of the AROS gene in cancer cells can suppress the metastatic activity of cancer cells.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

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Claims (6)

A pharmaceutical composition for inhibiting metastasis of cancer comprising an AROS (active regulator of SIRT1) inhibitor as an active ingredient.
The method of claim 1,
The AROS inhibitor is an oligonucleotide that inhibits the expression of AROS mRNA; Or, the AROS protein or an antibody that inhibits the activity of a ligand protein against AROS, an antigen-binding fragment or peptide thereof.
The method of claim 2,
The oligonucleotide that inhibits the expression of AROS mRNA is an antisense oligonucleotide specific for AROS mRNA, aptamer, miRNA, shRNA or siRNA.
The method of claim 3,
The siRNA is a composition consisting of double strands of an oligonucleotide of SEQ ID NO: 1 and an oligonucleotide having a complementary sequence thereof.
The method of claim 1,
The cancers are cerebrospinal cancer, head and neck cancer, lung cancer, breast cancer, thymoma, mesopores, esophageal cancer, brittle cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, Cervical cancer, endometrial cancer, colon cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma or skin cancer.
Comprising the step of administering a pharmaceutical composition comprising an active regulator of SIRT1 (AROS) inhibitor as an active ingredient to an individual other than a human. How to inhibit cancer metastasis.

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