WO2022065965A1 - Composition comprising arl6ip5 protein as active ingredient for prevention, alleviation, or treatment of cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising an ADP ribosylation factor like GTPase 6 interacting protein 5 (ARL6IP5) protein or a vector carrying a polynucleotide coding for the ARL6IP5 protein as an active ingredient for prevention or treatment of cancer. ARL6IP5 according to the present invention exhibits an excellent inhibitory effect on growth of cells of ovarian cancer, colorectal cancer, prostate cancer, or uterine cervical cancer, which are difficult to cure, and as such, can be advantageously used in anti-cancer therapy.

Description

A composition for preventing, improving or treating cancer containing ARL6IP5 protein as an active ingredient

The present invention relates to a composition for preventing, improving, or treating cancer containing ARL6IP5 (ADP ribosylation factor like GTPase 6 interacting protein 5) protein as an active ingredient.

Cancer is still one of the incurable diseases despite the ceaseless efforts of mankind over the past decades. Cancer is one of the biggest diseases that threaten human health, and is a disease caused by cells undergoing a series of mutations to proliferate and become immortal in an unrestricted and uncontrolled manner. Various biochemical mechanisms related to cancer have been identified and therapeutic agents have been developed. However, a fundamental treatment method has not yet been proposed.

Accordingly, there is a continuing demand for the identification of various in vivo molecules related to cancer and the development of drugs targeting the molecules, and efforts to enhance the therapeutic effect of cancer by combining some of the drugs are also continuing. Recently, with the remarkable development of various fields such as cancer cell biology, medicine, and chemistry, a number of anticancer drugs such as taxol, rapamycin, and 17-allylaminogeldanamycin (17-AAG) have been developed, and Gleevec ), anticancer drugs with a new mechanism of action are being developed.

On the other hand, Korean Patent Application Laid-Open No. 2016-0114381 discloses 'a pharmaceutical composition for preventing or treating cancer containing CysA5W peptide as an active ingredient', and Korean Patent No. 1540319 includes 'cyb5R3 gene or protein as an active ingredient. A pharmaceutical composition for preventing and treating cancer is disclosed, but there is no description of a composition for preventing, improving or treating cancer containing the ARL6IP5 protein of the present invention as an active ingredient.

The present invention was derived by the above request, and the present inventors treated ARL6IP5 (JWA) recombinant protein in ovarian cancer, colon cancer, prostate cancer and cervical cancer cell lines. As a result, ARL6IP5 compared to the existing anticancer drugs cisplatin and olaparib By confirming that the recombinant protein significantly inhibits the proliferation of cancer cell lines, the present invention was completed.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer containing, as an active ingredient, a vector including an ADP ribosylation factor like GTPase 6 interacting protein 5 (ARL6IP5) protein or a polynucleotide encoding the ARL6IP5 protein. provides

In addition, the present invention provides a health functional food for anticancer adjuvant containing ARL6IP5 protein as an active ingredient.

The ARL6IP5 (JWA) recombinant protein of the present invention shows an excellent cell proliferation inhibitory effect on difficult-to-treat ovarian cancer, colon cancer, prostate cancer or cervical cancer cells, so it will be usefully utilized as an anticancer agent, and in combination with existing anticancer agents It can be expected to increase the anticancer effect through administration.

1 is a result of analyzing the cell proliferation inhibitory effect of ARL6IP5 recombinant protein on SKOV-3 (ovarian cancer cell line).

2 is a result of analyzing the apoptosis effect of ARL6IP5 recombinant protein on SKOV-3. *: p < 0.05, compared with control group; #: p < 0.05, compared with the scramble group.

3 is a result of analyzing the cell death rate of SKOV-3 according to ARL6IP5 recombinant protein, cisplatin and olaparib alone or a combination treatment thereof.

4 is a result of analyzing the cell proliferation inhibitory effect of HT-29 (colon cancer cell line) according to ARL6IP5 recombinant protein, cisplatin, and olaparib alone or a combination thereof.

5 is a result of analyzing the apoptosis rate of HT-29 according to ARL6IP5 recombinant protein, cisplatin and olaparib alone or a combination treatment thereof.

6 is a result of analyzing the cell proliferation inhibitory effect of DU145 (prostate cancer cell line) according to ARL6IP5 recombinant protein, cisplatin and olaparib alone or a combination thereof.

7 is a result of analyzing the apoptosis rate of DU145 according to ARL6IP5 recombinant protein, cisplatin and olaparib alone or a combination treatment thereof.

8 is a result of analyzing the cell proliferation inhibitory effect of HeLa (cervical cancer cell line) according to ARL6IP5 recombinant protein, cisplatin, and olaparib alone or a combination treatment thereof.

9 is a result of analysis of the apoptosis rate of HeLa according to ARL6IP5 recombinant protein, cisplatin and olaparib alone or a combination treatment thereof.

10 is a 3D model of ovarian cancer (SKOV-3), colon cancer (HT-29), prostate cancer (DU145), cervical cancer (HeLa) cells after treatment with cisplatin, olaparib, ARL6IP5 RP cancer cells shows changes in shape and size. control: DMSO treatment, scale bar=100 μm.

11 is a 3D model of ovarian cancer (SKOV-3), colon cancer (HT-29), prostate cancer (DU145), cervical cancer (HeLa) cells formed after treatment with cisplatin, olaparib, ARL6IP5 RP It is the result of measuring the change in the size of the attention. control: DMSO treatment.

12 is a 3D model of ovarian cancer (SKOV-3), colorectal cancer (HT-29), prostate cancer (DU145), cervical cancer (HeLa) cells after treatment with cisplatin, olaparib, ARL6IP5 RP cell viability is the result of the analysis. control: DMSO treatment.

In order to achieve the object of the present invention, the present invention provides a vector comprising an ADP ribosylation factor like GTPase 6 interacting protein 5 (ARL6IP5) protein or a polynucleotide encoding the ARL6IP5 protein as an active ingredient for the prevention or treatment of cancer A pharmaceutical composition is provided.

ARL6IP5 is a protein of the Prenylated rab acceptor (PRA1) family that is encoded by the ARL6IP5 gene in humans. When ARL6IP5 protein is treated with cancer cells, it has the effect of inhibiting the cell proliferation of cancer cells. It was confirmed that the cancer cell proliferation inhibitory effect was superior to that of olaparib.

The scope of the ARL6IP5 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 1 and functional equivalents thereof. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution or deletion of amino acids. It refers to a protein having a sequence homology of 95% or more, and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 1. "Substantially homogenous physiological activity" means an activity for the prevention or treatment of cancer.

In the pharmaceutical composition for preventing or treating cancer of the present invention, the cancer is ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, laryngeal cancer, lung cancer, pancreatic cancer, liver cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, breast cancer, kidney cancer, It may be selected from the group consisting of gallbladder cancer, oral cancer, colon cancer, liver cancer, and bladder cancer, and preferably ovarian cancer, colorectal cancer, prostate cancer or cervical cancer, but is not limited thereto.

In the pharmaceutical composition for preventing or treating cancer of the present invention, the vector containing the polynucleotide encoding the ARL6IP5 protein may be a recombinant viral vector, a plasmid vector, a cosmid vector or a bacteriophage vector, preferably a plasmid vector, but , but is not limited thereto. In addition, the recombinant virus may be any one selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and lentivirus, but is not limited thereto.

The pharmaceutical composition comprising the ARL6IP5 protein of the present invention may further include at least one of cisplatin and olaparib in addition to the active ingredient, but is not limited thereto.

The pharmaceutical composition comprising the ARL6IP5 protein of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.

The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., suppositories and injections, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. can be used.

In addition, the pharmaceutical dosage form of the pharmaceutical composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds as well as suitable combinations. The other pharmaceutically active compounds include, but are not limited to, cisplatin, olaparib, doxorubicin, etoposide, paclitaxel, docetaxel, doxetaxel, fluoropyrimidine ), oxalplatin, campthotecan, belotecan, podophyllotoxin, vinblastine sulfate, cyclophosphamide, actinomycin, vincristine sulfate, methotrexate, bevacuzumab (bevacuzum ab), thalidomide (thalidomide), erlotinib (eriotinib), gefitinib (gefitinib), camptothecin (tamoxifen), Anasterozole, Gleevec, 5-Fluorouracil (5-FU), Fluxuridine, Leuprolide, Flutamide, Zoledronate ( Zoledronate), vincristine, gemcitabine, streptozotocin, carboplatin, topotecan, irinotecan, vinorelbine, hydoxyurea ( hydroxyurea), Valrubicin, Meclorethamine, Chlorambucil, Busulfan, Doxifluridine, Vinblastin, Mitomycin ), prednisone (Prednisone), testosterone (Testosterone), mitoxantrone (Mitoxantron), salicylate (sal) icylates), naproxen, fenoprofen, indomethacin, phenyltazone, mechlorethamine, dexamethasone, prednisolone, celery It may be selected from the group consisting of coxib (celecoxib), valdecoxib (valdecoxib), nimesulide, cortisone and corticosteroids.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art. Therefore, the above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes.

The present invention also provides a health functional food for anticancer supplements containing ARL6IP5 (ADP ribosylation factor like GTPase 6 interacting protein 5) protein as an active ingredient.

In the health functional food for anticancer adjuvant according to an embodiment of the present invention, the ARL6IP5 protein may consist of the amino acid sequence of SEQ ID NO: 1, and the specific range is the same as described above.

In addition, in the health functional food for anticancer supplements according to an embodiment of the present invention, the cancer is ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, laryngeal cancer, lung cancer, pancreatic cancer, liver cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer , may be selected from the group consisting of breast cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, liver cancer and bladder cancer, preferably ovarian cancer, colon cancer, prostate cancer or cervical cancer, but is not limited thereto.

In the health functional food for anticancer adjuvant according to an embodiment of the present invention, there is no particular limitation on the type of the health functional food. Examples of foods to which the ARL6IP5 protein can be added include beverages, tea, drinks and vitamin complexes, and may be used in the form of pills, powders, granules, needles, tablets, capsules, or beverages, and health in the general sense Includes all functional foods.

In addition to the ARL6IP5 protein, the health functional food of the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Preparation of ARL6IP5 overexpressing cell line

ARL6IP5 cDNA ORF clone was purchased from GenScript (#oHu24499, vector-pcDNA3.1+/C-(K)DYK, USA). Dispense SKOV-3 cells at a concentration of 0.5×10 ⁵ cells per well in a 24-well plate and use Lipofectamine 2000 reagent (#11668019, Invitrogent-Thermo Fisher Scientific, USA) when the cell distribution is 80% of the well area. The ARL6IP5 vector was transformed according to the instructions. Briefly, 50 μl serum-free medium containing 1 μg of pcDNA3.1/ALL6IP5 plasmid was prepared and 3 μl Lipofectamine 2000 was separately diluted in 50 μl serum-free medium. After that, the two solutions were mixed and mixed well, and then incubated for 5 minutes to form a fusion. Then, 50 μl of the fusion was added to each well and the plate was gently shaken by hand to ensure that the treatment solution was well dispersed in the wells. Then, the cells were cultured for 48 hours, and the treatment solution was replaced with a normal culture medium and cultured for an additional 12 hours. Thereafter, the cells were harvested, and ARL6IP5 expression was confirmed through western blot analysis.

2. Analysis of ARL6IP5 Recombinant Protein Apoptosis against Cancer Cells

In order to confirm the apoptosis effect of the ARL6IP5 recombinant protein, the cell proliferation rate according to the ARL6IP5 recombinant protein treatment was analyzed using various cancer cells. Specifically, ovarian cancer cell line (SKOV-3), prostate cancer cell line (DU145, ATCC ^® HTB-81 ^TM ), colorectal cancer cell line (HT-29, ATCC ^® HTB-38 ^TM ) and cervical cancer cell line (HeLa, ATCC ^® CCL-2 ^TM ) was dispensed in a 96-well plate at a concentration of 1×10 ⁴ cells per well and cultured for 24 hours, and then human ARL6IP5 recombinant protein (ARL6IP5 RP, #ARLIP5-9863H, Creative BioMart, USA) in the cultured cells ), cisplatin (cis-Dichlorodiammine platinum(II), #P4394, Sigma Aldrich, USA) and olaparib (AZD2281, #A10111-10mM-D, Adooq Bioscience, USA) were treated at different concentrations and for an additional 72 hours. cultured. Then, the cell proliferation rate was measured using the EZ-cytox cell viability assay kit (#DLS-1906, DoGenBioCo., Ltd.). Each experiment was performed in triplicate per treatment group.

In addition, Hoechst 33342 staining was performed to measure apoptosis according to ARL6IP5 recombinant protein treatment. Specifically, cells (2×10 ⁵ cells/well) were transplanted into 6-well plates and cultured overnight in an incubator. The cultured cells were treated with cisplatin (20 μM), olaparib (25 μM) and ARL6IP5 recombinant protein (2.5 μg/ml) individually or in combination, and incubated at 37° C. for 24 hours. After incubation, cells cultured in 6-well plates were washed once with PBS, and then treated with 5 μg/ml of Hoechst 33342 for 15 minutes. Finally, the cells were washed twice with PBS and observed under a reverse fluorescence microscope (Axioskop 2 plus microscope, Carl Zeiss, Germany). Apoptotic nuclei were counted in 5 non-overlapping regions and expressed as a percentage of the total number of nuclei counted.

3. Building 3D Cell Models

3-1. Single cell suspension preparation:

Each cancer cell was trypsinized and cultured at 37° C. until dissociated into single cells. The trypsin activity was stopped by adding 2 ml of medium and the cell fluid was suspended using a 5 ml pipette. Cells were transferred to 15 ml tubes and centrifuged at 2,000 rpm for 4 minutes. The supernatant was discarded and the pellet was washed with 1 ml of medium. The cells were resuspended in 2 ml of medium, and the number of cells was counted to prepare 2×10 ⁶ cells/ml.

3-2. Forming a hanging droplet:

Remove the lid from the 60 mm tissue culture dish and place 5 ml of PBS on the bottom of the dish. It acts as a hydration chamber. Invert the lid and use a pipette to drop 20 μl of the cell suspension onto the bottom of the lid. At this time, place at least 20 drops per plate at an appropriate distance so that each drop does not touch each other. The lid was covered with a bottom chamber filled with PBS, and incubated at 37° C., 5% CO ₂ , 95% humidity conditions and the droplet shape was monitored daily. Cultured until cell sheets or aggregates were formed. Aggregate formation was assessed using stereomicroscopy.

4. Analysis of Cell Viability Using 3D Cell Models

When a cell sheet is formed through the above-described method, transfer the cell sheet to a 1% poly-2-hydroxylethyl methacrylate (poly-HEMA) coated plate containing medium, DMSO (control), Cisplatin, olaparib, and ARL6IP5 recombinant protein (ARL6IP5 RP) were treated, respectively, and cultured at 37° C., 5% CO ₂ , and 95% humidity conditions. Cell viability was measured 72 hours after drug treatment using the EZ-cytox cell viability assay kit. The 3D culture experiment was observed and photographed by inverted contrast microscopy on the 7th day.

Example 1. Analysis of cell proliferation rate of ARL6IP5 overexpressing cells

In order to investigate the effect of ARL6IP5 on the inhibition of cancer cell proliferation, the growth rate was compared after the SKOV-3 cell line overexpressing ARL6IP5 was prepared. As a result, as shown in FIG. 1 , it was confirmed that the SKOV-3 cell line (ARL6IP5++) overexpressing ARL6IP5 had reduced cell proliferation compared to the general SKOV-3 cell line (control). In addition, as a result of treating the SKOV-3 cell line with siRNA to inhibit the expression of ARL6IP5 (siRNA_ARL6IP5), it was found that cell proliferation was increased compared to the normal SKOV-3 cell line (control).

In addition, as a result of treating the ARL6IP5 overexpressing SKOV-3 cell line with the anticancer drug cisplatin, the SKOV-3 cell line (control), the siRNA-treated SKOV-3 cell line (siRNA_ARL6IP5) and siRNA treatment to inhibit the expression of ARL6IP5 It was found that the cell death rate was the highest in the SKOV-3 cell line (ARL6IP5++) overexpressing ARL6IP5 compared to the control SKOV-3 (scramble) ( FIG. 2 ). Through the above results, it was found that ARL6IP5 has an effect of inhibiting the proliferation of cancer cells and increases the sensitivity of cancer cells to anticancer drugs.

Example 2. Analysis of the anticancer effect of ARL6IP5 on various cancer cells

To confirm the anticancer effect of ARL6IP5 on ovarian cancer (SKOV-3), colorectal cancer (HT-29), prostate cancer (DU145), and cervical cancer (HeLa) cells, ARL6IP5 recombinant protein (ARL6IP5 RP) in each cell line , Cell death rates were analyzed by treatment with cisplatin and oliparib alone or in combination. The treatment concentrations of cisplatin, olaparib and ARL6IP5 RP were treated by measuring the 50% inhibitory concentration of the corresponding drug in each cell line (Table 1).

Anticancer drug and ARL6IP5 RP treatment concentration

cell line	Cisplatin (μM)	Olaparib (μM)	ARL6IP5 RP (μg/ml)
SKOV-3	20	25	2.5
HT-29	22	24	3.5
DU145	9	12	2
HeLa	10	14	1.5

As a result, the existing anticancer agents cisplatin and olaparib for all of ovarian cancer (FIG. 3), colorectal cancer (FIGS. 4 and 5), prostate cancer (FIGS. 6 and 7) and cervical cancer (FIG. 8 and 9) cells It was confirmed that ARL6IP5 RP had excellent apoptosis compared to that.

Example 3. Analysis of the anticancer effect of ARL6IP5 using a 3D cell model

The present inventors constructed a 3D cell model to create cellular conditions similar to the in vivo environment. Then, the anticancer effect of ARL6IP5 was analyzed using a 3D cell model.

DMSO (control), cisplatin, olaparib, and ARL6IP5 RP were treated in 3D model-constructed ovarian cancer (SKOV-3), colorectal cancer (HT-29), prostate cancer (DU145), and cervical cancer (HeLa) cells, respectively. , the shape and size of each cancer cell were compared. Table 3 shows the anticancer drug and ARL6IP5 RP treatment concentrations. As a result, in all cancers, it can be confirmed that the size of 3D cells was reduced in the cisplatin, olaparib and ARL6IP5 RP-treated groups compared to the control group (FIG. 10).

In addition, the size of each cancer cell was measured using Image J and compared graphically. In all cancers, it was found that the size of cancer cells was reduced in the cisplatin, olaparib, and ARL6IP5 RP-treated groups compared to the control group (FIG. 11). In particular, it was found that in ovarian cancer (SKOV-3) and prostate cancer (DU145), the size of cancer cells was smaller in the ARL6IP5 RP-treated group compared to the conventional anticancer agents cisplatin and olaparib-treated group.

In addition, as a result of measuring the cell viability, it was confirmed that the cell viability was lower in the cisplatin, olaparib and ARL6IP5 RP-treated groups compared to the control group (FIG. 12). In particular, olaparib and ARL6IP5 RP treatment group showed low cell viability in all cancer cells, and ARL6IP5 RP treatment group had the lowest cell viability in ovarian cancer (SKOV-3) and cervical cancer (HeLa).

Claims (8)

1. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a vector comprising an ADP ribosylation factor like GTPase 6 interacting protein 5 (ARL6IP5) protein or a polynucleotide encoding the ARL6IP5 protein.
2. According to claim 1, wherein the cancer is ovarian cancer, colon cancer, prostate cancer, cervical cancer, laryngeal cancer, lung cancer, pancreatic cancer, liver cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, breast cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, A pharmaceutical composition for preventing or treating cancer, characterized in that it is selected from the group consisting of liver cancer and bladder cancer.
3. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the vector comprising the polynucleotide encoding the ARL6IP5 protein is a recombinant viral vector, a plasmid vector, a cosmid vector, or a bacteriophage vector.
4. The method according to claim 3, wherein the recombinant virus is any one selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, herpes simplex virus and lentivirus. A therapeutic pharmaceutical composition.
5. The pharmaceutical composition for preventing or treating cancer according to claim 1, further comprising at least one of cisplatin and olaparib in addition to the active ingredient.
6. The pharmaceutical composition for preventing or treating cancer according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.
7. A health functional food for anticancer supplements containing ARL6IP5 (ADP ribosylation factor like GTPase 6 interacting protein 5) protein as an active ingredient.
8. The method of claim 7, wherein the cancer is ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, laryngeal cancer, lung cancer, pancreatic cancer, liver cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, breast cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, A health functional food for anticancer supplements, characterized in that it is selected from the group consisting of liver cancer and bladder cancer.

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