KR20230072029A - Composition for preventing or treating ovarian cancer comprising atorvastatin and carboplatin - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an ovarian cancer containing atorvastatin and carboplatin as active ingredients. More specifically, the atorvastatin was checked to reduce the cancer stem cell characteristics of ovarian cancer cells through NANOG-miR-424/503-WEE1 signaling axis control and the combined administration of the atorvastatin and the carboplatin suppresses tumor growth and peritoneal dissemination in an animal model with a peritoneal ovarian cancer. It was found that the carboplatin alone significantly has suppressed tumor growth, but when administered in combination with the atorvastatin, the inhibitory effect on tumor growth was significantly higher. Therefore, the combined administration of the atorvastatin and the carboplatin can be effectively used in the treatment of an ovarian cancer.

Description

Composition for preventing or treating ovarian cancer comprising atorvastatin and carboplatin as active ingredients

The present invention relates to a composition for preventing or treating ovarian cancer containing atorvastatin and carboplatin as active ingredients.

Epithelial ovarian cancer is the most lethal cancer in women and has a high mortality rate. The causes of this high mortality are late diagnosis due to the absence of early symptoms and high recurrence rates due to the specific metastatic behavior of ovarian cancer. Ovarian cancer cells in the late stage detach from the primary cancer, form spheroids, spread into the abdominal cavity, and then metastasize to nearby organs and peritoneum to aggressively form secondary lesions. Ovarian cancer cells that form spheroids exhibit self-renewal and tumor formation, which are characteristics of cancer stem cells, and resistance to anticancer drugs. Therefore, development of treatments that can reduce metastasis and recurrence of cancer by targeting these cancer stem cells need is emerging.

Oncotarget. 2020; 11:3660-3674. (2020.10.06. Public)

An object of the present invention is to provide a pharmaceutical composition or health functional food composition for preventing or treating ovarian cancer, which contains atorvastatin and carboplatin as active ingredients.

Another object of the present invention is to provide a pharmaceutical composition for enhancing the anti-cancer effect of carboplatin against ovarian cancer, containing atorvastatin as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating ovarian cancer comprising atorvastatin and carboplatin as active ingredients.

In addition, the present invention provides a health functional food composition for preventing or improving ovarian cancer comprising atorvastatin and carboplatin as active ingredients.

In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of carboplatin against ovarian cancer, containing atorvastatin as an active ingredient.

The present invention relates to a composition for preventing or treating ovarian cancer containing atorvastatin and carboplatin as active ingredients, and more specifically, atorvastatin is a NANOG-miR-424/503-WEE1 signaling axis It was confirmed that cancer stem cell characteristics of ovarian cancer cells were reduced through control, and that the combined administration of atorvastatin and carboplatin inhibited tumor growth and peritoneal dissemination in an animal model of peritoneal ovarian cancer. Although carboplatin alone significantly inhibited tumor growth, it was found that the inhibitory effect on tumor growth was remarkably high when administered in combination with atorvastatin. Therefore, the combined administration of atorvastatin and carboplatin can be effectively used for the treatment of ovarian cancer.

1 shows the results that atorvastatin alleviates cancer stem cell characteristics of ovarian cancer cells through the NANOG-miR-424/503-WEE1 axis.
Figure 2 shows the results that the combined administration of atorvastatin and carboplatin inhibits tumor growth and peritoneal dissemination in peritoneal ovarian cancer animal models.

The present invention provides a pharmaceutical composition for preventing or treating ovarian cancer comprising atorvastatin and carboplatin as active ingredients.

Preferably, the composition may include 10mg/kg atorvastatin and 40mg/kg carboplatin, but is not limited thereto.

Preferably, the composition can reduce cancer stem cell characteristics of ovarian cancer cells by regulating the NANOG-miR-424/503-WEE1 axis.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the active ingredient of the present invention, and such a pharmaceutically acceptable carrier is commonly used in drug preparation. , Lactose, Dextrose, Sucrose, Sorbitol, Mannitol, Starch, Gum Acacia, Calcium Phosphate, Alginate, Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Cellulose, Water, Syrup, Methyl Cellulose, Methyl It may include hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. In addition, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like as additives.

The method of administration of the pharmaceutical composition is determined according to the degree of symptoms, and a topical administration method is usually preferred. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the patient's age, sex, weight, etc., and may be administered once or several times a day.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration can be envisaged, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebroventricular injection.

The pharmaceutical composition may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or prepared by putting it into a multi-dose container. At this time, the dosage form may be in the form of a solution, suspension or emulsion, or in the form of an elixir agent, an extract agent, a powder agent, a granule agent, a tablet agent, a warning agent, a lotion agent, an ointment agent, and the like.

In addition, the present invention provides a health functional food composition for preventing or improving ovarian cancer comprising atorvastatin and carboplatin as active ingredients.

When the composition of the present invention is a health functional food composition, the health functional food composition may be provided in the form of a powder, granule, tablet, capsule, syrup, drink or pill, and the health functional food composition may be provided in addition to the active ingredient of the present invention. It can be used together with other foods or food additives, and can be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition may be used according to the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. It is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.

There is no particular limitation on the type of health functional food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes.

In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of carboplatin against ovarian cancer, containing atorvastatin as an active ingredient.

Preferably, the composition can reduce cancer stem cell characteristics of ovarian cancer cells by regulating the NANOG-miR-424/503-WEE1 axis.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell culture and transfection

The ovarian cancer cell line was cultured in a 37 degree, 5% CO2 incubator, and RPMI-1640 (HyClone) supplemented with 10% FBS (HyClone) and 1% penicillin-streptomycin was used as the medium. Lipofectamine RNAimax products were used for miRNA and siRNA transfection.

2. Human specimens

This study was approved by the Pusan National University Bioethics Committee (IRB-2001-010-087), and all patients enrolled in the study signed informed consent before the study. Ovarian cancer tissues were collected during surgery, confirmed by the pathology department, and moved to the cell culture laboratory. Tissues were separated into single cells by enzymatic and mechanical methods, and RNA and protein were extracted after culturing in stem cell conditions.

3. Animal Testing

Female 7-week-old Balb/C nude mice (Orient) were bred in a facility controlled by a 12-hour light/dark cycle, and all experiments were conducted in accordance with the Pusan National University Hospital and Pusan National University Animal Research Ethics Committee (PNUH-2017-110) regulations. For the SKOV3 spheroid ovarian cancer model, isolated 1×10 ⁷ SKOV3 spheroids were intraperitoneally administered to mice. After 3 days, they were randomly divided into 4 groups (8 animals per group), and carboplatin (50 mg/kg) in 100 μl PBS was intraperitoneally administered once a week, and atorvastatin (10 mg/kg) was intraperitoneally administered daily. For the combined administration of carboplatin and atorvastatin, carboplatin (40 mg/kg) was intraperitoneally administered once a week and atorvastatin (10 mg/kg) three times a week. Ovarian carcinogenesis was determined by weekly checks for ascites formation and palpable tumors. After 28 days of administration, they were sacrificed by cervical dislocation and abdominal cavity photographs were taken to confirm ascites and tumor formation. Tumors formed in the abdominal cavity were separated, measured for weight and size, and fixed in 10% formalin for histological analysis.

4. Immunohistochemical staining

Immunohistochemical staining was performed using ImmPRESS HRP REAGENT kit (MP-7402) and WEE1 antibody (Santa Cruz, SC-5285) for tissues embedded in paraffin after fixation. Counterstaining was performed with hematoxylin, and images were observed at 400-fold magnification using Aperio ImageScope software.

5. RNA extraction and real-time polymerase chain reaction ( qRT - PCR )

Total RNA was extracted using the miRNeasy RNA isolation kit (QIAGEN) and reverse transcribed using the Taqman miRNA Reverse Transcription kit (Applied Biosystems). Real-time polymerase chain reaction was performed using TaqMan Universal Master Mix II, no UNG (Applied Biosystems), and expression of miR-424 and miR-503 was detected with Taqman probe. RNAU6B was used as a control. mRNA was reverse transcribed with qPCRBIO cDNA Synthesis Kit (PCR Biosystmes) and 18S rRNA was used as a control.

The sequences of the primers are as follows.

WEE1, forward: 5'-GATGTGCGACAGACTCCTCAAG-3', reverse: 5'-CTGGCTTCCATGTCTTCACCAC-3',

18S, forward: 5'-CTGGCTTCCATGTCTTCACCAC-3', reverse: 5'-ACCCGTTGAACCCCATTCGTGA-3'

6. Western blot

Cells were lysed with RIPA buffer (Biosesang) containing protease inhibitors (Gendepot), and proteins were extracted by centrifugation at 13000 rpm and 4 degrees for 15 minutes. For protein quantification, Pierce BCA Protein assay kit (Thermo Scientific) was used. The same amount of protein was electrophoresed on an SDS-polyacrylamide gel, and the protein was transferred to a polyvinyl-difluoride (PVDF) membrane and reacted with an antibody. Primary antibodies used were WEE1 (1:1000, Cell Signaling), FLAG (1:3000, Sigma-Aldrich), NANOG (1:1000, Cell Signaling), GAPDH (1:4000, Cell Signaling), and HRP was bound The amount of protein was detected using a secondary antibody and chemiluminescence detection method (Thermo Scientific).

7. spheroid Formation

SKOV3 was seeded in a 6-well plate coated with poly 2-hydroxylethyl methacrylate (poly-HEMA; Sigma-Aldrich) at a density of 1 × 10 ⁵ cells/ml, and 20 ng/mL epidermal growth factor (Invitrogen), 10 ng/mL basic They were cultured in serum-free DMEM/F12 (Welgene) medium containing fibroblast growth factor (Invitrogen), 0.4% bovine serum albumin (Gendepot), and 5 mg/mL insulin (Sigma-Aldrich). Spheroid formation was observed using a microscope at 100x magnification on the 5th day after seeding. The size of spheroids was measured using ImageJ software.

8. Cell proliferation assay

Cells were seeded in a 96-well plate at 1×10 ⁴ cells/well and treated with atorvastatin for 24 hours in a monolayer state or treated with atorvastatin for 3 days in a spheroid state in a poly-HEMA-coated 96-well plate. Cytotoxicity was measured using the EZ-Cytox WST kit (Daeil Lab). After adding 10ul of WST-1 reagent and reacting for 30 minutes, absorbance was measured at 450nm.

9. limiting dilution method ( in vitro Limiting dilution assay)

Cells were seeded in various numbers (1-128 cells/well) in poly-HEMA-coated 96-well plates and simultaneously treated with DMSO or 10 µM atorvastatin. After 7 days, the number of sphere-free wells was measured for each seeding density, and a graph was drawn for the number of cells per well.

10. flow cell Analysis (Flow cytometry analysis)

For single cell analysis, spheroids were separated using cell dissociated buffer (Thermo Scientific). The cell population with aldehyde dehydrogenase (ALDH) enzyme activity was measured using the Aldefluor kit (Stem Cell Technologies). 1×10 ⁶ cells were suspended in 1 ml of ALDEFLOR assay buffer containing ALDH substrate (BAAA), and as a negative control, a portion of each sample was treated with diethylaminobenzaldehyde (DEAB), an ALDH inhibitor, and used. Samples were analyzed after reaction at 37 degrees for 45 minutes. To identify cancer stem cell markers CD44 and CD133, CD133-PE antibody (1:300, Miltenyi Biotec) and CD44-FITC (1:300, BD Science) were reacted in PBS containing 0.1% BSA for 1 hour and FACS Detection was performed using a CantoII flow cytometer. Analysis was done using FlowJo software.

11. Statistical Analysis

All experiments were performed at least three times, and analysis was performed using GraphPad Prism 7.0 software. Statistical significance was analyzed by unpaired two-tailed Student's t-test and ANOVA method. Statistically significant when P<0.05 (*P < 0.05; **P < 0.01; and ***P < 0.001).

< Example 1> NANOG - miR -424/503- WEE1 of ovarian cancer cells through the axis cancer stem tax Atorvastatin to alleviate blistering properties

In order to confirm the role of the NANOG-miR-424/503-WEE1 axis in ovarian cancer cells in regulating cancer stem cell properties, we restored the altered NANOG-miR-424/503-WEE1 axis in ovarian cancer cell spheroids. strategy was used. MiR-424 and miR-503 are closely regulated by apelin/APJ signaling, and previous studies have shown that statins increase apelin/APJ signaling. This means that statins can regulate miR-424/503 expression. In addition, previous studies have shown that statins inhibit tumor growth and metastasis of various carcinomas and reduce pluripotency markers such as NANOG and OCT-4 in cancer cells. Accordingly, it was confirmed whether atorvastatin could restore miR-424 and miR-503 expressions, NANOG, and WEE1 expressions in ovarian cancer cell spheroids. As a result, the expression of miR-424 and miR-503 increased by atorvastatin treatment (FIG. 1A), and mRNA and protein expressions of NANOG and WEE1 decreased (FIGS. 1B and 1C). As a result of confirming whether atorvastatin can regulate the characteristics of ovarian cancer stem cells, atorvastatin significantly reduced the viability of ovarian cancer spheroids and reduced the formation of spheroids of primary ovarian tumor cells and SKOV3 (Fig. 1D and Figure 1E). In addition, to confirm whether atorvastatin can regulate cancer stem cell characteristics, self-renewal activity and expression of cancer stem cell markers were confirmed. By using an in vitro limiting dilution assay, it was confirmed that atorvastatin treatment suppressed self-replication ability (FIG. 1F) and significantly lowered the ALDH1-positive population of primary ovarian cancer cells and SKOV3 (FIG. 1G). Therefore, these results indicate that atorvastatin can reduce cancer stem cell properties of ovarian cancer cells through the regulation of the NANOG-miR-424/503-WEE1 signaling axis.

< Example 2> Inhibiting tumor growth and peritoneal dissemination in peritoneal ovarian cancer animal models ah Concomitant administration of torvastatin and carboplatin

To examine the effects of atorvastatin and carboplatin co-administration on tumor growth and peritoneal dissemination, an peritoneal ovarian cancer mouse model was used. The degree of ascites formation was significantly reduced when carboplatin was administered alone, and was further reduced in the group administered in combination with atorvastatin (FIGS. 2A and 2B). In addition, the combined administration of atorvastatin and carboplatin significantly reduced the degree of peritoneal dissemination compared to the control group (FIG. 2C). Carboplatin alone significantly inhibited tumor growth, but when administered in combination with atorvastatin, the inhibitory effect on tumor growth was remarkably high (FIGS. 2D and 2E). In addition, it was confirmed that the expression of WEE1 and NANOG in cancer tissues was not changed in the carboplatin monotherapy, but decreased in the atorvastatin monotherapy group and the atorvastatin/carboplatin combination administration group (FIGS. 2F and 2G). Therefore, it was confirmed that atorvastatin inhibits tumor metastasis and increases carboplatin sensitivity in the ovarian cancer peritoneal metastasis model using spheroids by inhibiting cancer stem cells by regulating miR-424/503 expression.

Claims (6)

A pharmaceutical composition for preventing or treating ovarian cancer, comprising atorvastatin and carboplatin as active ingredients. The pharmaceutical composition for preventing or treating ovarian cancer according to claim 1, wherein the composition comprises 10 mg/kg atorvastatin and 40 mg/kg carboplatin. The pharmaceutical composition for preventing or treating ovarian cancer according to claim 1, wherein the composition regulates the NANOG-miR-424/503-WEE1 axis to reduce cancer stem cell characteristics of ovarian cancer cells. A health functional food composition for preventing or improving ovarian cancer, comprising atorvastatin and carboplatin as active ingredients. A pharmaceutical composition for enhancing the anticancer effect of carboplatin against ovarian cancer, comprising atorvastatin as an active ingredient. The pharmaceutical composition for enhancing the anticancer effect of carboplatin against ovarian cancer according to claim 5, wherein the composition reduces cancer stem cell characteristics of ovarian cancer cells by regulating the NANOG-miR-424/503-WEE1 axis. .

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