KR101618518B1 - Pharmaceutical composition comprising tumor suppressor ap5m1 for enhancing anti-cancer effect for cervical cancer - Google Patents

Abstract

The present invention relates to a composition for enhancing an anti-cancer treating effect of uterine cervical cancer, which comprises AP5M1 protein inducing apoptosis or genes encoding the same as an active ingredient. The present invention also relates to a pharmaceutical composition for preventing or treating uterine cervical cancer, and a method for screening agents for treating uterine cervical cancer. As an active ingredient according to the present invention, AP5M1 protein or the genes encoding the same are shown to be effective for inducing apoptosis of cancer cells and inhibiting growth of cancer cells. Thus, the active ingredient according to the present invention can be used for enhancing the effect of the existing agent for treating uterine cervical cancer and for preventing or treating uterine cervical cancer, can be used as a novel biomarker of uterine cervical cancer, and thus is expected to be useful for a method for screening agents for treating uterine cervical cancer. In addition, the active ingredient according to the present invention can be used for developing a kit for effective diagnosis of uterine cervical cancer, and can be used as a target for development of new drugs effective for healthy female genital organs.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for enhancing the anticancer therapeutic effect of cervical cancer comprising, as an active ingredient, a tumor suppressor AP5M1.

The present invention relates to a composition for promoting an anticancer therapeutic effect of cervical cancer comprising an AP5M1 (adaptor-related protein complex 5, mu 1 subunit) protein or a gene encoding the same as an active ingredient, a pharmaceutical composition for preventing or treating cervical cancer, And a method for screening a cervical cancer therapeutic agent.

The uterus is composed of the corpus and the cervix. The malignant tumor that occurs in the cervix connected to the vagina is called cervical cancer.

Cervical cancer accounts for 95% of the total cervical cancer incidence, and it is the second most common cancer among women with breast cancer worldwide. It is the fourth most common cancer in Korea. Despite the advances in modern medicine, there are about 500,000 cervical cancer patients worldwide and about 50,000 deaths each year. Especially in developing countries such as Asia, South America and Africa, and 80% of all deaths occur in developing countries. In cervical cancer patients, the likelihood of death within 5 years after cancer is 34% in developed countries and 50% in developing countries, with high mortality rates in developing countries. The reason for this is that it is not easy to diagnose cancer at the early stage, to accurately diagnose advanced cancer, or to observe the prognosis of the newly developed therapeutic agent as well as the problem of cancer treatment method.

Causes of cervical cancer include human papillomavirus (HPV), race, unsanitary environment, premature experience, frequent births, smoking, spouse's unclean sex, and the main cause of HPV infection , And more than 99.7% of cervical cancer patients are reported to have high-risk HPV infection.

Recent clinical tests for cervical cancer include Pap test, colposcopy, biopsy, conization, loop electrosurgical excision procedure (LEEP), cervical cytology, . However, the diagnosis of cancer by the above-mentioned method has a problem that it can be performed only when the number of cancer cells is 1 billion or more and the diameter of cancer is 1 cm or more. In this case, the cancer cells already have the ability to metastasis, and at least half of them have already metastasized. In addition, tumor markers for monitoring substances produced directly or indirectly from cancer are used for cancer screening. However, even when cancer is present, more than half of the cancer markers are normal, and even when cancer is absent, they are often positive. There is a limitation in the present invention.

When diagnosed as cervical cancer by the above-described method, chemotherapy, immunotherapy, and gene therapy are used as treatment methods depending on each stage, such as surgery, administration of anticancer drugs, radiation therapy, administration of chemotherapeutic drugs and radiation therapy simultaneously. Currently, about 90% of chemotherapy or radiotherapy, which is the current cervical cancer treatment method, ultimately uses a mechanism that induces or promotes the apoptosis of cancer cells. Recent research has been actively conducted Is a major research and development field.

Recently, new gene targeting methods have been actively pursued for discovering new target genes, introducing them efficiently into target cells to induce long-term expression, and improving gene transfer efficiency when used in clinical treatments. However, Due to the problem, there is a limit in the diagnosis or treatment using such a target protein because the ability to transfer in the tissue is lost and a diagnostic or therapeutically effective amount is not delivered into the cells.

In addition, the major cause of failure in chemotherapy is resistance to therapeutic agents, and it is known that the cause of resistance is defective in the apoptosis mechanism of cancer cells. The main mechanism of cancer cells that cause resistance to cancer treatment is that the cancer cells are resistant to apoptosis. Thus, there is a need for research and development of cancer drugs using cell death in order to prevent cancer recurrence.

The present invention was conceived to overcome the above-mentioned problems in the prior art. It was confirmed that AP5M1 expression in the cervical cancer patients was decreased at the protein and mRNA level, and the AP5M1 apoptosis effect, tumor suppressor And the cell killing effect was significantly increased in combination with the existing cervical cancer therapeutic agent. Accordingly, it is an object of the present invention to provide a pharmaceutical composition for enhancing the anticancer therapeutic effect of cervical cancer which is co-treated with an anticancer agent.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cervical cancer, which comprises an AP5M1 protein or a gene coding therefor as an active ingredient.

In addition, the object of the present invention is to provide a screening method for treating cervical cancer.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for enhancing the anticancer therapeutic effect on cervical cancer treated in combination with an anticancer agent, wherein the composition comprises AP5M1 (adapter-related protein complex 5, mu 1 subunit) A protein or a gene coding therefor as an active ingredient, and the anticancer agent is carboplatin.

In one embodiment of the present invention, the composition is administered simultaneously, separately, or sequentially with the anticancer agent.

The present invention also provides a pharmaceutical composition for preventing or treating cervical cancer, comprising an AP5M1 (adaption-related protein complex 5, mu 1 subunit) protein or a gene coding therefor as an active ingredient.

In one embodiment of the present invention, the AP5M1 protein may consist of the amino acid sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the gene encoding AP5M1 may comprise the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention provides a screening method for treating cervical cancer comprising the steps of:

a) treating the test subject material to cervical cancer tissue or cells;

b) measuring the mRNA or protein expression pattern of AP5M1 (adapter-related protein complex 5, mu 1 subunit); And

c) screening the test subject for which the AP5M1 mRNA or protein level has been elevated compared to the control without the subject substance.

Furthermore, the present invention provides a method for enhancing the therapeutic effect of an anticancer agent comprising the step of using, in combination, an anticancer agent and a composition comprising the AP5M1 protein or a gene encoding the same as an active ingredient.

In addition, the present invention provides a method of preventing or treating cervical cancer comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically effective amount of AP5M1 protein or a gene encoding the same.

In addition, the present invention provides a composition for preventing or treating cervical cancer, comprising an AP5M1 protein or a gene coding therefor as an active ingredient.

The composition according to the present invention not only inhibits proliferation of cancer cells through interaction with FAS, which is a cytotoxic receptor, but also induces apoptosis. Therefore, the composition of the present invention can be used in combination with existing cervical cancer therapeutic agents to improve the effect of chemotherapy, Or for therapeutic use.

In addition, the AP5M1 protein or the gene encoding the AP5M1 protein can be used as a new biomarker for cervical cancer, and can be used for efficiently screening a cervical cancer therapeutic agent using the composition. Can be used to develop kits that can be easily, effectively and accurately diagnosed. In addition, the biomarkers are expected to be used as targets for the development of new medicines for the health of female reproductive organs.

FIG. 1A shows Western blotting analysis results for the detection of AP5M1 and caspase proteins by overexpressing AP5M1. FIG. 1B shows cell viability through AP5M1 and caspase inhibitor. FIG. 1C shows AP5M1 expression in knock- Down to determine the apoptotic activity of AP5M1 on the death receptor by flow cytometry. In addition, FIG. 1D shows knockdown of AP5M1 in a cervical cancer cell line and analysis of cell viability by FAS.
FIG. 2A shows Western blotting analysis results for the detection of AP5M1 and FAS proteins in normal human and cervical cancer tissues. FIG. 2B shows the results of real-time PCR for detecting mRNA expression of AP5M1 and FAS in normal human and cervical cancer tissues. time PCR analysis.
FIG. 3A shows AP5M1 knocked down to analyze the cell apoptosis and AP5M1-related cervical cancer therapeutic agent. FIG. 3B shows the cell viability of AP5M1 and cervical cancer in combination.
FIG. 4 shows the function of AP5M1 as a tumor suppressor using a cervical cancer tumor transplantation model. FIG. 4A shows binding of antisense oligonuclelides of AP5M1 to metal nanoparticles to form gold nanoparticle-AP5M1 antisense oligonucleotide complex FIG. 4B shows the time-varying tumor volume and weight change by the injection of the gold nanoparticle-AP5M1 antisense oligonucleleotide complex in the cervical cancer tumor transplantation model, and FIG. 4C shows the change in tumor volume and weight during the cervical cancer tumor implantation Inhibition of AP5M1 protein expression was confirmed by immunohistochemical analysis through AP5M1 antisense oligonucleotide delivery of tumor tissue by gold nanoparticle-AP5M1 antisense oligonucleolide complex in the model. 4D shows the inhibition of AP5M1 protein expression by Western blot analysis through AP5M1 antisense oligonuclelide transfer of tumor tissue by gold nanoparticle-AP5M1 antisense oligonucleolide complex in a cervical cancer tumor transplantation model.

The present inventors confirmed that the AP5M1 protein or the gene encoding the same as the active ingredient of the present invention is decreased in cervical cancer cells and that the AP5M1 protein induces apoptosis, inhibits the proliferation of cancer cells, The inventors of the present invention have confirmed that the cytotoxic activity of the cervical cancer treated with the therapeutic agent for cervical cancer is increased and thus a pharmaceutical composition for promoting the effect of chemotherapy for cervical cancer treated in combination with an anticancer agent is completed.

In one embodiment of the present invention, it was confirmed that AP5M1 has cytotoxicity using caspase, which plays an essential role in apoptosis (see Example 2), and the expression level of AP5M1 protein and mRNA in cervical cancer tissues Analysis showed that AP5M1 protein and mRNA expression was decreased in cervical cancer patients (see Example 3), and AP5M1 and cervical cancer treatment were shown to be excellent in apoptosis (Example 4). In addition, the cervical cancer animal model was injected with a gold nanoparticle-RNAI-antisense AP5M1 complex and the tumor volume and weight were measured to confirm the tumor suppression effect of the AP5M1 protein or gene (see Example 5).

Accordingly, the present invention provides a pharmaceutical composition for enhancing the anticancer therapeutic effect on cervical cancer treated in combination with an anticancer agent, wherein the composition comprises an AP5M1 (adaptor-related protein complex 5, mu 1 subunit) protein or a gene coding therefor as an active ingredient , And the anticancer agent is carboplatin.

The composition may be administered simultaneously, separately, or sequentially with the anti-cancer agent.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of cervical cancer by administration of the pharmaceutical composition of the present invention.

The anticancer agent that improves the therapeutic effect of the composition of the present invention is a generic name of a chemotherapeutic agent used for the treatment of malignant tumor. The anticancer agent is carboplatin, cisplatin, paclitaxel, May be selected from the group consisting of staurosporine, preferably carboplatin, but is not limited thereto.

In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cervical cancer, comprising an AP5M1 (adaption-related protein complex 5, mu 1 subunit) protein or a gene coding therefor as an active ingredient.

The AP5M1 protein may be an amino acid sequence of SEQ ID NO: 1, and the AP5M1-encoding gene may be a nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

As used herein, the term "prophylactic " means any act that inhibits or delays the development of cervical cancer by the administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. Herein, pharmaceutically acceptable carriers are those conventionally used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, The type of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight may be administered daily or every other day, or one to three divided doses per day. However, the dose may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and therefore the dosage is not limited in any way.

In another embodiment of the present invention, the present invention provides a method for treating cervical cancer comprising the step of administering the pharmaceutical composition to a subject. The term " individual "as used herein refers to a subject in need of treatment of a disease, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse, do.

In another aspect of the present invention, there is provided a screening method for treating a cervical cancer comprising the steps of:

a) treating the test subject material to cervical cancer tissue or cells;

b) measuring the mRNA or protein expression pattern of AP5M1 (adapter-related protein complex 5, mu 1 subunit); And

c) screening the test subject for which the AP5M1 mRNA or protein level has been elevated compared to the control without the subject substance.

Furthermore, as another aspect of the present invention, there is provided a method for providing information for diagnosis of cervical cancer comprising the steps of:

a) treating an agent for measuring mRNA or protein level of AP5M1 gene in a cervical tissue derived from a subject;

b) measuring the mRNA or protein expression pattern of said AP5M1; And

c) determining that the expression level of AP5M1 is cervical cancer when the level of expression is decreased as compared with normal cervical tissue.

The agent for measuring the mRNA level of step a) may be a primer pair or a probe specific to the AP5M1 gene, and the agent for measuring the protein level of step a) may be an antibody specific to the protein of the AP5M1 gene, But is not limited to.

In addition, the degree of expression of the step b) may be measured by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, and protein chips, but the present invention is not limited thereto.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Method

1-1. Animal cell culture

Human cervical carcinoma cells HeLa and KB cells were cultured in DMEM (Dulbecco's modified Eagle's medium). All media contained 10% inactivated fetal bovine serum (Caisson, USA) and 1% penicillin-streptomycin (Welgene, Korea).

1-2. Cell viability analysis

HeLa cells were inoculated into 96-well plates and cultured overnight to allow attachment of the cells. Cells were overexpressed with HA-AP5M1 using pcDNA6.a and pcDNA6.a or knocked down with AP5M1 using siRNA oligo and then cell viability was measured.

1-3. Preparation of gold nanoparticle-RNAI-antisense AP5M1 complex (AuNP-RNAI-antisense AP5M1 complex)

The gold nanoparticle-RNAI conjugate was pre-incubated at 80 ° C for 5 minutes to prevent the formation of a secondary structure. The gold nanoparticle-RNAI conjugate (1 nM) and antisense AP5M1 (100 nM) were reacted at room temperature for 10 minutes with 1x PBS buffer containing 5 mM MgCl ₂ (pH 7.2) After centrifugation, the supernatant was removed. The gold nanoparticle-RNAI-antisense AP5M1 complex was washed using TBST (Tris-Buffered Saline and Tween 20) supplemented with 500 mM NaCl and 1 M KCl.

1-4. Animal preparation

18-20 g of 5-week-old immunodeficient BALB / c-nu / nu mice (Central Lab Animal Inc, Korea) were used. The animal breeding room maintained a temperature of 30 ± 40% humidity and 22 ± 1 ℃. Indoor illumination was made with a 12-h light / dark cycle. Animal studies were approved by the Institutional Animal Case and Use Committee of the University of Dawn, and animals were treated according to the protocol guidelines.

1-5. In vivo mouse xenograft model

HeLa cells (1 x 10 ⁷ ) were subcutaneously injected into 18-20 g 6-week-old immunodeficient BALB / c-nu / nu mice to establish a tumor transplantation model. In established tumor transplant mouse models, gold nanoparticle-RNAI-antisense control or gold nanoparticle-RNAI-antisense AP5M1 complexes were injected directly into the tumor volume (~ 0.1 cm ³ ) and the length of the long and short axis of the tumor was measured daily Respectively. Mice were sacrificed 30 days after the initial injection of the complex, and tumor samples were collected for further analysis.

Results were expressed as the mean ± SEM of tumors in 7 mice in each group, and the * sign shows a significant value when compared with the corresponding control group (P < 0.05).

1-6. Immunohistochemical analysis

Tumor sections were prepared and immunostained. A monoclonal anti-AP5M1 antibody (1: 100) was used in an antibody diluent (Dako, USA).

1-7. Western blotting

Tumor samples and patient samples were collected, proteins were extracted from tissues using PRO-prep solution (Intron, Korea), and BCA protein analysis (Bicinchoninic acid protein assay) was performed to quantify the extracted proteins. The proteins were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After transferring to a PVDF membrane (polyvinylidene fluoride membrane), the cells were incubated with an antibody specific to each protein to be identified. After incubation with a secondary antibody, the protein was identified using a ChemDoc instrument.

1-8. Real-time PCR analysis

Trizol solution was used to extract RNA from patient samples and real-time PCR was performed using cDNA synthesized from the separated RNA. The real-time PCR results were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, folded relative expression, and the result (mean ± SEM) Was obtained from the results obtained. Statistically significant values were expressed as P <0.05.

1-9. Statistical analysis

Comparisons of the measured values were made by the Student-Newman-Keuls test (SAS, USA) and significant values were analyzed using Student's t-test (SAS). Data were presented as mean ± SEM, P <0.05 was considered statistically significant and P values of significant results were expressed.

Example 2. Analysis of apoptosis of AP5M1

2-1. Expression of AP5M1 and caspase proteins

In HeLa cells, pcDNA6.a and pcDNA6.a HA-AP5M1 were overexpressed after transfection using electroporation. Next, the expression of AP5M1 and caspase proteins was firstly cultured with a Western blotting method, that is, with AP5M1 and caspase antibodies, cultured with a secondary antibody, and detected by ECL (enhanced chemoluminescence) .

Caspase is a proteolytic enzyme that plays an essential role in apoptosis, necrosis, and inflammation. Abnormal caspase activation results in cell apoptosis or cell apoptosis failure, It may also cause illness.

As a result, as shown in FIG. 1A, when caspase-8, caspase-9 and caspase-3 were cleaved upon AP5M1 protein overexpression, it was confirmed that caspase-dependent cell death was induced I could.

2-2. Cell viability of AP5M1 by caspase inhibitor

In order to investigate the caspase specificity and the cell survival of AP5M1 protein, pcDNA6.a HA-AP5M1 was overexpressed in HeLa cells, and the caspase inhibitors z-VAD-fmk and z-IETD-fmk were pretreated And then cell viability was confirmed using a cell viability assay kit.

As a result, as shown in Fig. 1B, cell viability was low in the group in which AP5M1 was overexpressed, and AP5M1 alone was treated in the group treated with the caspase inhibitor (z-VAD-fmk, z-IETD-fmk) Cell survival rate was higher than that of one group.

Therefore, the AP5M1 protein shows specificity for the caspase inhibitor through cell viability, and the caspase inhibitor has the ability to regenerate apoptosis. Thus, it is confirmed that AP5M1 protein is involved in caspase-dependent apoptosis I could.

2-3. Through the death receptor AP5M1 Flow cell  analysis( flow cytometry )

AP5M1 was knocked down using siRNA in the HeLa cell line to confirm that the AP5M1 protein is involved in the activation of the death receptor. Next, the cells were treated with FASL (Fas ligand), TRAIL (TNF-related apoptosis inducing ligand) and TNF-α (Tumor necrosis factor-α) for 12 hours and then annexin V / PI staining And the cell death was confirmed by a flow cytometer.

Annexin v staining is used to detect cells that have undergone cell death, and is a method of staining using cell membrane permeability changes in apoptotic cells. More specifically, in normal living cells, phosphatidylserine (PS) is located inside the cell membrane, whereas in cells where apoptosis has started, phosphatidylserine is exposed to the outside of the cell membrane. Annexin v binds to the exposed phosphatidylserine, And the cell death is observed.

As a result, as shown in Fig. 1C, it was found that the cytotoxicity of FASL (CH-11) treated cell death suppressors was remarkably inhibited.

Therefore, it was confirmed that the AP5M1 protein is associated with the FAS signaling system.

2-4. Cervical carcinoma  In the cell line AP5M1 and  Cells of FAS death receptor Survival  Confirm

AP5M1 protein was knocked down in HPV 18 positive human cervical cancer cell line (HeLa, KB), and FASL (CH-11) was treated for 12 hours to confirm cell viability.

As a result, as shown in Fig. 1D, cell viability was increased in HeLa and KB cells knocked down by AP5M1 protein, and cell viability by FASL (CH-11) was also inhibited and FAS induction AP5M1 was involved in the signal transduction of apoptotic cells.

Example 3. Analysis of expression of AP5M1 in cervical cancer patients

3-1. AP5M1 expression pattern in cervical cancer tissue

In order to confirm the expression pattern of AP5M1 protein in cervical cancer, AP5M1 protein was extracted from a normal cervix and a cervical cancer-embedded paraffin-embedded slide tissue, and AP5M1 expression level was measured by Western blotting Respectively.

As a result, as shown in FIG. 2A, it was confirmed that the expression amount of AP5M1 in the tissues of cervical cancer patients was remarkably reduced.

3-2. AP5M1 mRNA expression pattern in cervical cancer tissue

The expression of AP5M1 protein was significantly reduced in the cervical cancer patient tissues, which was confirmed in Example 3-1. In addition, in order to determine whether the expression was regulated from the mRNA stage, Changes were investigated.

As a result, as shown in FIG. 2B, it was confirmed that the expression of AP5M1 and FAS gene was decreased in a manner similar to that of AP5M1 protein in cervical cancer patients.

Example 4. Confirmation of concurrent treatment effect of AP5M1 with cervical cancer treatment agent

4-1. AP5M1 mediates cervical cancer treatment

In order to confirm the correlation between the therapeutic effect of carboplatin, cisplatin, paclitaxel, and staurosporine, which are anticancer drugs used for cervical cancer treatment, and the cervical cancer treatment effect of AP5M1 protein, Cell viability experiments were performed. AP5M1 was knocked down and the cell viability was confirmed by treatment of carboplatin, cisplatin, paclitaxel, and staurosporine in HeLa cells for 12 hours.

As a result, as shown in FIG. 3A, it was found that AP5M1 knockdown-treated cervical cancer cells were inhibited in cell apoptosis in the group treated with carboplatin.

4-2. AP5M1 and cervical cancer treatment

To confirm the effect of AP5M1 in combination with cervical cancer treatment, AP5M1 was overexpressed in HeLa cells, and then cell viability was confirmed by treatment with carboplatin, cisplatin, paclitaxel, and staurosporine for 12 hours.

As a result, as shown in Fig. 3B, it was confirmed that AP5M1 and carboplatin were excellent in cytotoxicity when treated with cervical cancer alone.

Example 5. Confirmation of tumor activity by AuNP-RNAI-AS AP5M1 using cervical cancer tumor suppression model

5-1. Fabrication of AP5M1 antisense using gold nanoparticles

A gold nanoparticle-RNAI-antisense DNA complex (AuNP-RNAI-AS AP5M1 complex) was prepared by binding RNAi (cargo DNA) and antisense DNA (AS DNA) to gold nanoparticles. A schematic diagram is shown in FIG. 4A same.

5-2. Check tumor volume and weight

HeLa cells were injected into a nude mouse to induce cervical cancer and 0.5 mg / kg gold nanoparticle-RNAI-antisense AP5M1 complex and FASL (CH-11) were injected into xenograft tumors xenograft tumors) and the tumor volume (cm ³ ) over time was calculated using the following equation: length × width ² × π) / 6, and the tumor volume was plotted. Controls were injected with a gold nanoparticle-RNAI-Antisense Control complex. On the 33rd day after injection of each complex, mice were sacrificed and the tumor weight was measured.

As a result, as shown in FIG. 4B, it was found that the volume and the weight of the tumor were increased in the mice injected with the gold nanoparticle-RNAI-antisense AP5M1 complex as compared with the control. Furthermore, in the group treated with FASL (CH-11), it was found that the growth-inhibited tumor was not inhibited by the antisense AP5M1.

Therefore, it was confirmed that the AP5M1 protein is a key protein that inhibits tumor growth.

5-3. Immunohistochemical examination of tumor tissue

Immunohistochemical examination was performed to confirm the expression pattern of AP5M1 protein after the injection of gold nanoparticle-RNAI-antisense control complex and gold nanoparticle-RNAI-antisense AP5M1 complex into tumor tissue. More specifically, the tumor tissue was fixed with formalin, and the paraffin-embedded storage tissue was used as a tissue specimen to make a 5 μm thick continuous section. One of the sections was used for immunohistochemical staining. Immunohistochemical staining was performed by first filling the Coplin container with the diluted antigenic determinant recovery solution, placing it in a thermostat, covering the lid, pre-heating at 95 to 99 ° C, and culturing the tissue for 40 minutes. The slide was removed and allowed to cool for 20 minutes at room temperature, followed by incubation with peroxidase-blocking reagent and buffer for 5 minutes. After washing, AP5M1 antibody was incubated for 30 minutes, and then antibody specific immunohistochemical staining was performed using DAKO staining reagent kit. The sections were then photographed with an Olympus CKX41 microscope.

As a result, as shown in Fig. 4C, a significantly reduced level of AP5M1 protein could be identified in tumors injected with the gold nanoparticle-RNAI-antisense AP5M1 complex.

5-4. Protein expression pattern in tumor tissue

After the injection of gold nanoparticle-RNAI-antisense control complex and gold nanoparticle-RNAI-antisense AP5M1 complex into the tumor tissue, Western blotting was performed to confirm the expression pattern of AP5M1 protein.

As a result, as shown in FIG. 4D, it was confirmed that the expression of AP5M1 protein was decreased in the tumor injected with the gold nanoparticle-RNAI-antisense AP5M1 complex, as in the result of Example 5-3.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> PHARMACEUTICAL COMPOSITION COMPRISING TUMOR SUPPRESSOR AP5M1 FOR          ENHANCING ANTI-CANCER EFFECT FOR CERVICAL CANCER <130> CAU20151808KR_PB15-12507 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 490 <212> PRT <213> AP5M1 of Homo sapiens <400> 1 Met Ala Gln Arg Ala Val Trp Leu Ile Ser His Glu Pro Gly Thr Pro   1 5 10 15 Leu Cys Gly Thr Val Arg Phe Ser Arg Arg Tyr Pro Thr Val Glu Lys              20 25 30 Arg Ala Arg Val Phe Asn Gly Ala Ser Tyr Val Pro Val Pro Glu Asp          35 40 45 Gly Pro Phe Leu Lys Ala Leu Leu Phe Glu Leu Arg Leu Leu Asp Asp      50 55 60 Asp Lys Asp Phe Val Glu Ser Arg Asp Ser Cys Ser Arg Ile Asn Lys  65 70 75 80 Thr Ser Ile Tyr Gly Leu Ile Gly Gly Glu Glu Leu Trp Pro Val                  85 90 95 Val Ala Phe Leu Lys Asn Asp Met Ile Tyr Ala Cys Val Pro Leu Val             100 105 110 Glu Gln Thr Leu Ser Pro Arg Pro Pro Leu Ile Ser Val Ser Gly Val         115 120 125 Ser Gln Gly Phe Glu Phe Leu Phe Gly Ile Gln Asp Phe Leu Tyr Ser     130 135 140 Gly Gln Lys Asn Asp Ser Glu Leu Asn Thr Lys Leu Ser Gln Leu Pro 145 150 155 160 Asp Leu Leu Leu Gln Ala Cys Pro Phe Gly Thr Leu Leu Asp Ala Asn                 165 170 175 Leu Gln Asn Ser Leu Asp Asn Thr Asn Phe Ala Ser Val Thr Gln Pro             180 185 190 Gln Lys Gln Pro Ala Trp Lys Thr Gly Thr Tyr Lys Gly Lys Pro Gln         195 200 205 Val Ser Ile Ser Ile Thr Glu Lys Val Lys Ser Met Gln Tyr Asp Lys     210 215 220 Gln Gly Ile Ala Asp Thr Trp Gln Val Val Gly Thr Val Thr Cys Lys 225 230 235 240 Cys Asp Leu Glu Gly Ile Met Pro Asn Val Thr Ile Ser Leu Ser Leu                 245 250 255 Pro Thr Asn Gly Ser Pro Leu Gln Asp Ile Leu Val His Pro Cys Val             260 265 270 Thr Ser Leu Asp Ser Ala Ile Leu Thr Ser Ser Ile Asp Ala Met         275 280 285 Asp Asp Ser Ala Phe Ser Gly Pro Tyr Lys Phe Pro Phe Thr Pro Pro     290 295 300 Leu Glu Ser Phe Asn Leu Cys Phe Tyr Thr Ser Gln Val Pro Val Pro 305 310 315 320 Pro Ile Leu Gly Phe Tyr Gln Met Lys Glu Glu Glu Val Gln Leu Arg                 325 330 335 Ile Thr Ile Asn Leu Lys Leu His Glu Ser Val Lys Asn Asn Phe Glu             340 345 350 Phe Cys Glu Ala His Ile Pro Phe Tyr Asn Arg Gly Pro Ile Thr His         355 360 365 Leu Glu Tyr Lys Thr Ser Phe Gly Gln Leu Glu Val Phe Arg Glu Lys     370 375 380 Ser Leu Leu Ile Trp Ile Ile Gly Gln Lys Phe Pro Lys Ser Met Glu 385 390 395 400 Ile Ser Leu Ser Gly Thr Val Thr Phe Gly Ala Lys Ser His Glu Lys                 405 410 415 Gln Pro Phe Asp Pro Ile Cys Thr Gly Glu Thr Ala Tyr Leu Lys Leu             420 425 430 His Phe Arg Ile Leu Asp Tyr Thr Leu Thr Gly Cys Tyr Ala Asp Gln         435 440 445 His Ser Val Gln Val Phe Ala Ser Gly Lys Pro Lys Ile Ser Ala His     450 455 460 Arg Lys Leu Ile Ser Ser Asp Tyr Ile Trp Asn Ser Lys Ala Pro 465 470 475 480 Ala Pro Val Thr Tyr Gly Ser Leu Leu Leu                 485 490 <210> 2 <211> 1473 <212> DNA <213> AP5M1 of Homo sapiens <400> 2 atggcgcagc gggcagtgtg gctcataagc cacgaaccgg gaactccact ttgtggcacc 60 gtgagattct ccagacggta tccaactgtt gaaaaacgag ccagagtctt caatggagca 120 agttatgtgc ctgttcctga agatggtccc tttcttaaag cactgctctt tgaacttaga 180 ttattggatg atgataaaga cttcgttgag agtcgtgata gctgttcacg catcaataaa 240 acatccattt atggactcct gataggaggt gaagaactct ggccagttgt tgcttttctg 300 aagaatgaca tgatatatgc ttgtgttcca ctagttgaac aaactctgtc ccctcgtccg 360 ccactaatta gtgtcagtgg agtttcacaa ggctttgaat ttctttttgg gatacaggat 420 tttctttatt caggtcaaaa aaatgactct gagctgaata caaaattgag ccagttgcct 480 gacttgcttc tgcaggcttg tccatttggt actttattag atgccaactt acagaattca 540 ttagataata ccaattttgc atctgtgact cagccacaga aacagccagc ttggaaaact 600 gggacgtaca aaggaaaacc acaagtttct atttctatca ctgaaaaggt aaaatccatg 660 caatatgata aacagggtat agcagataca tggcaagttg ttggaacagt gacttgcaag 720 tgtgatttgg aaggaatcat gccaaatgtt accatcagct tgagtctccc caccaatgga 780 tctccacttc aggatattct agttcaccct tgtgtaactt ctcttgactc tgcaattctg 840 acttctagta gtattgatgc aatggatgac tctgcattta gtgggcctta caaatttcca 900 ttcactccac ctttagagtc attcaactta tgcttctaca cttcccaggt ccctgtccca 960 ccaattttgg gtttttatca aatgaaggag gaagaagtac aactaagaat aaccattaat 1020 ttaaaacttc atgaaagtgt gaaaaataat tttgaattct gtgaagccca tatacctttt 1080 tacaatagag gtccaattac acatttggaa tacaaaacta gttttggcca gcttgaagta 1140 tttcgagaga aaagcttatt gatctggatt attggccaga agttcccaaa atcaatggaa 1200 attagtcttt ctggaactgt aacttttgga gccaagagcc atgagaagca gccatttgac 1260 ccaatttgta ctggagaaac agcatattta aagcttcatt ttaggatctt agattacaca 1320 cttactggat gttatgcaga tcagcattca gttcaagttt ttgcatcagg aaaaccaaaa 1380 ataagtgcac accggaaact aatttcttct gattattaca tctggaattc taaagcccct 1440 gctccagtaa catatggatc attattattg taa 1473

Claims (6)

A pharmaceutical composition for enhancing the effect of chemotherapy for cervical cancer treated with an anticancer agent, the composition comprising an AP5M1 (adaptor-related protein complex 5, mu 1 subunit) protein or a gene coding therefor as an active ingredient, RTI ID = 0.0 &gt; carboplatin. &Lt; / RTI &gt;
The method according to claim 1,
Wherein said composition is administered simultaneously, separately or sequentially with said anti-cancer agent.
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