KR20240072366A - Anti-cancer adjuvant comprising pentadecanoic acid as effective component and uses thereof - Google Patents

Abstract

The present invention relates to an anticancer adjuvant containing pentadecanoic acid as an active ingredient and its use, and specifically, to a composition for enhancing anticancer drug susceptibility of anticancer drug resistant cancer containing pentadecanoic acid as an active ingredient, pentadecanoic acid. It relates to a pharmaceutical composition for preventing or treating tamoxifen-resistant breast cancer, comprising a mixture of tamoxifen as an active ingredient, and a method for treating tamoxifen-resistant cancer in a mammalian subject using the pharmaceutical composition.

Description

Anti-cancer adjuvant comprising pentadecanoic acid as effective component and uses thereof}

The present invention relates to an anticancer adjuvant containing pentadecanoic acid as an active ingredient and its use. More specifically, when tamoxifen-resistant breast cancer cells are treated with pentadecanoic acid in combination with tamoxifen, tamoxifen is treated alone. It was confirmed that it can significantly enhance apoptosis of tamoxifen-resistant breast cancer cells.

Breast cancer is the most commonly diagnosed cancer in women, and its heterogeneity and various subtypes reduce the effectiveness of breast cancer treatment. Identifying the breast cancer subtype in a patient is one of the basic steps in selecting an appropriate treatment strategy. In addition to histopathological characteristics, breast cancer is classified based on the presence of estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor-2 positivity (ERBB2/HER2+). Among the types of breast cancer, ER-α positive breast cancer is the most frequently discovered, accounting for nearly 75% of all breast cancers. ER expression has a significant impact on the success of hormonal therapy, the mainstay of treatment for ER-positive breast cancer patients. In fact, loss of ER expression in ER-negative breast cancer patients results in failure to respond to conventional hormonal treatment.

Tamoxifen, an antineoplastic nonsteroidal selective estrogen receptor modulator, is one of the most popular chemotherapeutics for treating ER-positive breast cancer. However, the development of resistance to tamoxifen has emerged as a major clinical problem, and loss of ER-α expression has been reported to be associated with tamoxifen resistance. Therefore, re-expression of ER-α in ER-α-negative breast cancer tumors may help overcome resistance to tamoxifen and other hormonal treatments. In addition, combination therapy using ligustilide or epigallocatechin-3-gallate (EGCG) has been reported as a promising strategy to overcome tamoxifen resistance (Ma, H. et al., Oncotarget (2017) 8 :29328; Sulaiman, A. et al., Cell Death Dis (2018) 9:815; Li, Y. et al., 7:9345.

Meanwhile, Korean Patent Publication No. 1995-0000642 discloses 'pentanoic acid derivatives' for preventing and/or treating neurodegenerative diseases and neurological dysfunction caused by seizures or trauma, but 'pentadecanoic acid' of the present invention is disclosed. There is no description regarding ‘anti-cancer adjuvants included as active ingredients and their uses’.

The present invention was developed in response to the above-mentioned needs, and the present invention sought to provide an anticancer adjuvant that can improve the effect of endocrine therapy for tamoxifen-resistant breast cancer cells.

The present inventor investigated whether anticancer drug-resistant breast cancer cells could be re-sensitized to an anticancer drug (tamoxifen) by treating pentadecanoic acid, which has an effect of suppressing breast cancer metastasis, in combination with tamoxifen, and found that the combined treatment of tamoxifen and pentadecanoic acid resulted in apoptosis. And the effect on epithelial-mesenchymal transition was also investigated.

As a result of the analysis, ER-α expression and apoptosis levels of tamoxifen-resistant breast cancer cells were significantly increased under the conditions of combined treatment with tamoxifen or pentadecanoic acid compared to treatment alone, and the effect of inhibiting epithelial-mesenchymal transition was also excellent, confirming the present invention. Completed.

In order to solve the above problems, the present invention provides a composition for enhancing anticancer drug susceptibility of anticancer drug-resistant cancer, containing pentadecanoic acid as an active ingredient.

Additionally, the present invention provides an anticancer adjuvant for anticancer drug-resistant cancer containing pentadecanoic acid as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating tamoxifen-resistant breast cancer, comprising a mixture of pentadecanoic acid and tamoxifen as an active ingredient.

Additionally, the present invention provides a method of treating tamoxifen-resistant cancer in a mammalian subject, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a mammalian subject with cancer resistant to tamoxifen.

Pentadecanoic acid of the present invention can improve the anticancer drug sensitivity of tamoxifen-resistant breast cancer due to insufficient expression of estrogen receptors, and thus can improve the effect of endocrine therapy in the treatment of tamoxifen-resistant breast cancer cells. In addition, it is expected that the side effects of the anticancer drug itself can be reduced by reducing the dose of tamoxifen used in combination.

Figure 1 shows the characteristics of drug-resistant human breast cancer cells MCF-7/SC. (A) is the cell image of MCF-7 and MCF-7/SC, (B) and (C) are the results of comparing the colony formation levels of MCF-7 and MCF-7/SC cells, (D) and (E) is the result of comparing the invasion ability of MCF-7 and MCF-7/SC cells, (F) is the Western blot result for drug resistance marker analysis, and (G) is the Western blot result of (F). This is a quantified graph. * means p<0.05 compared to the control group.
Figure 2 relates to tamoxifen resistance of MCF-7/SC cells, (A) and (B) show cell survival rates 24 hours and 48 hours after tamoxifen treatment, respectively, (C) and (D) show These are the results of comparing the level of colony formation according to tamoxifen treatment, (E) and (F) are the results of comparing cell migration ability according to tamoxifen treatment, and (G) and (H) are the results of comparing cell invasion ability according to tamoxifen treatment. This is the result of comparison. * means p<0.05 compared to the control group.
Figure 3 shows the tamoxifen resistance of MCF-7/SC cells. (A) and (B) are flow cytometry results detecting apoptotic populations after tamoxifen treatment, and (C) and (D) are cells after tamoxifen exposure. This is the result of analyzing the expression level of death markers by Western blot. * means p<0.05 compared to the control group.
Figure 4 confirms the additive effect of the combined treatment of pentadecanoic acid (PDCN) and tamoxifen (TAM). (A) and (B) are the effects of pentadecanoic acid alone or pentadecanoic acid and tamoxifen, respectively. Cell viability after 24 and 48 hours of combination treatment, (C) and (D) are the results of analyzing CI (combination index) values after 24 and 48 hours of combination treatment, respectively, and (E) and (F) are 5. This is the result of analyzing the level of colony formation 24 hours after treatment with 10 μM tamoxifen and various concentrations of pentadecanoic acid. (G) and (H) are results of treatment with 10 μM tamoxifen and various concentrations of pentadecanoic acid. This is the result of analyzing the level of colony formation after 24 hours. * means p<0.05 compared to the control group, and # means that the combination treatment group is significant at the p<0.05 level compared to the single treatment group.
Figure 5 confirms apoptosis following the combined treatment of pentadecanoic acid (PDCN) and tamoxifen (Tam), and (A) and (B) show flow cytometric analysis of the apoptotic population using Annexin V/PI staining. This is the result confirmed by , and (C) and (D) are the results of confirming the expression level of the apoptosis marker by Western blot. * means p<0.05 compared to the control group, and # means that the combination treatment group is significant at the p<0.05 level compared to the single treatment group.
Figure 6 shows that the combined treatment of pentadecanoic acid (PDCN) and tamoxifen has an effect of suppressing epithelial-mesenchymal transition, and (A) and (B) show cell migration by performing a wound healing assay in MCF-7/SC. (C) and (D) are the results of analyzing cell invasion ability 48 hours after combined treatment, and (E) and (F) are the results of epithelial-mesenchymal transition (EMT) markers confirmed by Western blot. . * means p<0.05 compared to the control group, and # means that the combination treatment group is significant at the p<0.05 level compared to the single treatment group.
Figure 7 confirms that the combined treatment of pentadecanoic acid (PDCN) and tamoxifen (Tam) can induce the expression of estrogen receptor (ER-α) in MCF-7/SC. (A) shows 48 hours of combined treatment. (B) and (C) are the results of confirming the protein level of ER-α by Western blot, and (D) is the result of confirming the gene expression level of ER-α after 48 hours of combined treatment. This is the result of confirming the expression level.

In order to achieve the object of the present invention, the present invention provides a composition for enhancing anticancer drug susceptibility of anticancer drug resistant cancer containing pentadecanoic acid as an active ingredient.

In the present invention, the term "anticancer drug sensitivity" refers to the sensitivity of response to the use of an anticancer drug, and refers to a characteristic that can maximize the effect on an individual when an anticancer drug is administered. Increasing anticancer drug sensitivity can increase the therapeutic effect of anticancer drugs by ensuring that the drug effects work smoothly.

In the composition for enhancing anticancer drug sensitivity according to the present invention, the anticancer agent may be tamoxifen, but is not limited thereto, and any anticancer agent that can inhibit the action of estrogen by competitively binding to the estrogen receptor can be used without limitation. .

In addition, in the present invention, the anticancer drug-resistant cancer may preferably be breast cancer that is resistant to anticancer drugs, and more specifically, may be breast cancer that is resistant to tamoxifen, but is not limited thereto. Breast cancer that is resistant to tamoxifen is characterized by low expression of estrogen receptors.

In addition, in the present invention, the composition for enhancing sensitivity to anticancer drugs can significantly improve the sensitivity enhancing effect by further including other types of anticancer agents. Here, the type of anticancer agent that may be further included is not particularly limited, but for example, cisplatin, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, and vandetanib. , nirotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cetuximab, Viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamycin, ibritumomab tuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtu Zumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, Cytarabine, fluorouracil, fludagabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topo Tecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pyra Rubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chloram. Busil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, navelvin, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, boro It may include, but is not limited to, one or more selected from the group consisting of sol, bicalutamide, lomustine, and carmustine.

In one embodiment of the present invention, the composition for enhancing anticancer drug sensitivity targets breast cancer with loss or reduced estrogen receptor expression that is resistant to tamoxifen, an anticancer drug.

The present invention also provides an anticancer adjuvant for anticancer drug-resistant cancer containing pentadecanoic acid as an active ingredient.

In the present invention, the term “anticancer adjuvant” refers to an agent that can improve, enhance, or increase the anticancer effect of an anticancer agent.

In the present invention, the anticancer adjuvant can be used as an anticancer agent or an anticancer adjuvant depending on the treatment concentration, and can improve sensitivity to the anticancer agent.

In the present invention, the anti-cancer adjuvant can be administered in combination with a known compound that has the effect of preventing, improving, or treating cancer.

The anti-cancer adjuvant according to the present invention may be used in combination with tamoxifen for breast cancer that has lost or reduced expression of estrogen receptors that is resistant to the anti-cancer drug tamoxifen, but is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating tamoxifen-resistant breast cancer, comprising a mixture of pentadecanoic acid and tamoxifen as an active ingredient.

In the pharmaceutical composition according to the present invention, the mixture of pentadecanoic acid and tamoxifen increases the expression level of estrogen receptors in breast cancer cells, increases apoptosis, and inhibits epithelial-mesenchymal transition. It is characterized by

In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects. The pharmaceutical composition is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods.

In the pharmaceutical composition according to one embodiment of the present invention, the pentadecanoic acid may be formulated by mixing with tamoxifen in advance, or may be formulated separately.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration. For injections, buffers, preservatives, and analgesics can be used. Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, Includes sublingual or rectal areas. Oral or parenteral administration is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention varies depending on several factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art.

The present invention also provides a method of treating tamoxifen-resistant cancer in a mammalian subject, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a mammalian subject with cancer resistant to tamoxifen.

In the method for treating anticancer drug-resistant cancer according to the present invention, the mammalian subject may preferably be a mammalian subject other than a human, but is not limited thereto.

Additionally, the tamoxifen-resistant cancer may preferably be tamoxifen-resistant breast cancer, but is not limited thereto.

In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and activity of the drug. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention can be administered at a dosage of 0.1 mg/kg to 1 g/kg, and more preferably at a dosage of 1 mg/kg to 500 mg/kg. Meanwhile, the dosage can be appropriately adjusted depending on the patient's age, gender, and condition.

Hereinafter, the present invention will be described in detail by 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. Cell lines and culture methods

In the present invention, human estrogen receptor positive breast cancer cells (MCF-7) and breast cancer stem cell-like cells (MCF-7/SC) were used. MCF-7 cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin according to instructions from ATCC. MCF-7/SC cells were generated from parental MCF-7 by sorting the CD44+/CD24-/dim cell population (To, N.B. et al., Nutrients (2020) 12:1663), 10% FBS, 100 U/mL penicillin. and RPMI 1640 supplemented with 100 g/mL streptomycin. Proliferation of MCF-7 and MCF-7/SC is approximately 29 and 25 hours, respectively.

2. Cell viability analysis

For the MTT test, cells (4 × 10 ³ cells/well) were seeded in 96-well plates using the indicated culture medium and cultured at 37°C. After incubation, they were treated with various concentrations of pentadecanoic acid, tamoxifen, or their combination for 48 hours. After 24 or 48 hours of treatment, MTT assay was performed to determine cell viability (To, NB et al., 2020).

3. Colony formation assay

MCF-7 and MCF-7/SC cells (4×10 ² cells/culture dish) were cultured for 24 hours and then treated with pentadecanoic acid, tamoxifen, or a combination thereof. After culturing for 10 days, each colony was fixed with 4% paraformaldehyde and stained with 2% crystal violet. Stained colonies were manually counted and expressed as percentage compared to untreated controls for each concentration of treated substance.

4. Wound healing analysis

MCF-7 and MCF-7/SC cells (1× ¹⁰⁵ cells/well) were cultured in 6-well plates until they formed a monolayer. The tightly grown cell monolayer was scratched uniformly using a sterile pipette tip and washed with PBS. Afterwards, each cell was treated with pentadecanoic acid, tamoxifen, or a combination thereof and reacted for 24 and 48 hours, and the width of the scratch was measured using a phase contrast microscope (×100).

5. Cell invasion assay

The cell invasion assay used a 24-well transwell system (pore size 0.2 μm, Corning, USA) as previously reported (Nobili, S. et al., Pharmacol. Res. (2009) 59:365-378). . Pentadecanoic acid and tamoxifen, alone or in combination, were added to the upper chamber under serum-free medium conditions, and medium supplemented with 10% FBS was added to the lower chamber. After 48 hours of culture, cells were fixed with 4% paraformaldehyde and stained with 2% crystal violet. Infiltrating cells were observed under phase contrast microscopy (×100).

6. Flow cytometry

The effects of treatment with pentadecanoic acid, tamoxifen, or their combination on cell cycle and apoptosis were confirmed by flow cytometry according to a previous report (Nobili, S. et al., 2009). Cells (1 × 10 ⁵ cells/culture dish) were treated with pentadecanoic acid and tamoxifen alone or in combination for 48 hours. Apoptosis populations were analyzed using BD FACSDiva™ software. Annexin V-FITC apoptosis detection kit (BD Biosciences) was used to detect apoptosis, cell cycle analysis was performed by an ACSCalibur flow cytometer (Becton Dickinson), and the proportion of each population in the cell cycle was calculated using GraphPad Prism 7. It was analyzed and graphed using .

7. Western blotting

RIPA (Radioimunoopination assay) buffer was used to prepare cell lysates from MCF-7 or MCF-7/S cells (3 × 10 ⁵ cells/culture dish) after treatment with pentadecanoic acid, tamoxifen, or their combination, and protein A BCA assay (Thermo Fisher Scientific) was performed to determine the amount. After sample preparation, proteins were separated using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Membrane transfer, blocking, primary and secondary antibody reactions, and protein detection procedures were performed in the same manner as in a previous study (Nobili, S. et al., 2009). Antibody dilution was performed according to the manufacturer's instructions, and information on the antibodies used in Western blot is shown in Table 1.

8. qRT-PCR (quantitative reverse transcription PCR)

MCF-7/SC cells (1×10 ⁶ cells/culture dish) were cultured overnight and then treated with pentadecanoic acid and tamoxifen alone or in combination for 48 hours. After drug treatment, total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific), and the extracted total RNA was used for cDNA synthesis. Then, real-time PCR was performed with a 20 μl reaction mixture consisting of 1 μl of cDNA, 2 μl of primers (1 μl of each primer), 10 μl of master mix (Takara), and 7 μl of RNA-free water. Real-time PCR consisted of initial denaturation at 95°C for 15 minutes, 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C, and then generated a dissociation curve through 15 seconds at 95°C and 30 seconds at 60°C. The temperature was gradually increased for 15 seconds. Primer sequences of target genes are shown in Table 2, and gene expression was quantified using the ^{2 -ΔΔCq} method (Livak, KJ Schmittgen, TD Methods (2001) 25:402-408).

Primer information used in the present invention Gene Sequence information (5'→3') sequence number BCL2 F TCCCTCGCTGCACAAATACTC One R ACGACCCGATGGCCATAGA 2 CA12 F TGGCATTCTTGGCATCTGTA 3 R TTGGTGGCTGGCTTGTAAAAT 4 XBP1 F GGCGCTCCACGCACCTG 5 R GCTGCTACTCTGTTTTTCAGTTTCC 6 GREB1 F CAAAGAATAACCTTGTTGGCCCTGC 7 R GACATGCCTGCGCCTCTCATACTTA 8 FOS F CGTCTTCCTTCGTCTTCACC 9 R GTCAGAGGAAGGCTCATTGC 10 NRIP1 F TCGCACTCACCACAGAAAAC 11 R AGCCAAGCTCTTCTCCATGT 12 RARA F CGCTGCTGGAGGCGCTAAAG 13 R GCGCTGATGCTTCGCAGGTC 14 TFF1 F GTGTCAGCCCTCCCAGT 15 R GGACCCCACGAACGGTG 16

9. Calculate CI (combination index) value

The effects of pentadecanoic acid and tamoxifen on drug interactions were calculated and expressed as CI. The following formula was used to calculate the CI value based on the median-effect principle.

CI = D1/(Dx)1 + D2/(Dx)2

((Dx)1 and (Dx)2 in the denominator represent the concentrations that can inhibit x% of the first drug (Drug1) and the second drug (Drug2) alone, respectively. D1 and D2 in the numerator correspond to the corresponding values in the denominator, respectively. It represents the portion of the first drug and the second drug in the combination (D1+D2) that has the same effect (x% inhibition) as the concentration of the drug.)

CI values according to the Chou-Talalalay model were calculated using CompuSyn software (Chou, T.-C. Cancer Res. (2010) 70:440-446). Indicators of additive impact, synergism, and antagonism are CI = 1, 1, and >1, respectively.

10. Statistical analysis

Statistical analysis was performed using GraphPad Prism 7. The mean and standard deviation of three independent experiments were used to represent the results. One-way analysis of variance with Dunnett's post hoc test was used for group comparisons, and p < 0.05 was considered statistically significant.

Example 1. Characterization of drug-resistant human breast cancer MCF-7/SC

In a recent study, the present inventors performed RNA sequencing in MCF-7 and MCF-7/SC cells and identified differentially expressed genes (DEGs) between MCF-7 and MCF-7/SC cells. According to the transcriptome analysis results, a distinct gene profile and significant increase in DEGs related to drug resistance were confirmed in MCF-7/SC cells.

To confirm the transcriptome analysis results, the characteristics of MCF-7/SC cells were further characterized. Microscopic analysis revealed clear morphological differences between MCF-7 and MCF-7/SC cells. MCF-7 cells showed a round or irregular epithelial-like shape, but MCF-7/SC cells had a more luminal and elongated shape and increased intercellular distance (Figure 1A). In addition, the clonogenic ability of MCF-7/SC cells was confirmed to be higher than that of parental MCF-7 cells (Figure 1B, C). In addition, the results of transwell analysis showed that the invasion ability of MCF-7/SC cells was improved (Figures 1D, E). The present inventors performed Western blot to analyze the expression levels of several proteins involved in tumor drug resistance, and the results showed that MCF-7/SC cells expressed snail, Bcl-2, STAT3, and p-STAT3 compared to parental MCF-7 cells. It was expressed at a high level, and the expression level of tumor suppressor proteins such as Rb, p-Rb, E-cad, and p21 was confirmed to be reduced (Figure 1F, G). In particular, we observed that the mRNA and protein expression levels of ER-α in MCF-7/SC cells were significantly lower than those in MCF-7 cells (Figures 1F, G). These results implied that loss of ER-α may play an important role in drug resistance of MCF-7/SC cells.

Example 2. Tamoxifen resistance analysis of MCF-7/SC cells

Because the therapeutic effects of tamoxifen are primarily ER-mediated, loss of ER expression in breast cancer cells is highly associated with tamoxifen resistance. MTT assay was performed to determine whether MCF-7/SC cells were resistant to tamoxifen. As a result, tamoxifen showed lower cytotoxicity in MCF-7/SC compared to MCF-7 (Figures 2A, B). In addition, tamoxifen showed stronger colony formation inhibitory activity in MCF-7 cells than in MCF-7/SC cells. Under 10 μM tamoxifen treatment conditions, the number of colonies in MCF-7 cells was reduced by more than 90% compared to the untreated control group. , in the case of MCF-7/SC cells, the number of colonies was reduced by about 40% compared to the untreated control group, showing that MCF-7/SC cells showed resistance to tamoxifen (Figure 2 C,D). The cell migration and invasion inhibition effects of tamoxifen were also significantly greater in the parental MCF-7 cells (Figures 2E to H). In addition, it was observed that apoptosis increased in a tamoxifen concentration-dependent manner in parental cells, MCF-7, but in MCF-7/SC cells, it was confirmed that apoptosis did not increase in the tamoxifen-treated group compared to the untreated control group. (Figure 3A, B). Western blot results analyzing the expression levels of apoptosis marker proteins also showed that the protein levels of full-length caspase-7 and caspase-9 in MCF-7 cells were decreased by tamoxifen treatment, but in MCF-7/SC cells, In this case, it was confirmed that there was no change in protein level (Figure 3C, D).

Example 3. Analysis of the effect of combined treatment of pentadecanoic acid and tamoxifen

The present inventors analyzed the effect of combination therapy of pentadecanoic acid and tamoxifen on tamoxifen-resistant MCF-7/SC cells. As a result of analyzing the survival rate of MCF-7/SC cells using the MTT assay after treatment with a combination of pentadecanoic acid and tamoxifen, the survival rate of MCF-7/SC cells was higher in the group treated with the combination of pentadecanoic acid and tamoxifen compared to treatment with pentadecanoic acid alone. It was confirmed that it was falling (Figure 4A, B). In addition, as a result of calculating the CI values according to various treatment concentration combinations of pentadecanoic acid and tamoxifen, the conditions of pentadecanoic acid/tamoxifen (μM) 25/5 and 50/5 showed an additive effect (CI) at both 24 and 48 hours. < 1), and the conditions of pentadecanoic acid/tamoxifen 100/5 were additive at 24 hours, and the conditions of pentadecanoic acid/tamoxifen 50/10, 75/10, and 100/10 were additive at 48 hours. It was found to be effective (Figure 4C, D). In the colony formation assay, the combined treatment of pentadecanoic acid and tamoxifen showed a significantly superior inhibitory effect compared to the individual treatment of pentadecanoic acid and tamoxifen (Figures 4E to H). The above results showed that pentadecanoic acid effectively re-sensitized MCF-7/SC cells to tamoxifen.

Example 4. Effect of combined treatment of pentadecanoic acid and tamoxifen on apoptosis of MCF-7/SC cells

The present inventors analyzed the effect of combined treatment of pentadecanoic acid and tamoxifen on apoptosis of MCF-7/SC cells. As confirmed through Annexin V/PI staining, treatment with 10 μM tamoxifen alone did not appear to affect apoptosis of MCF-7/SC cells, but combining 10 μM tamoxifen with 50 or 100 μM pentadecanoic acid It was found that apoptosis was significantly increased in treated cells (Figure 5A, B). Western blot results confirming the expression levels of apoptosis marker proteins also showed that combined treatment with pentadecanoic acid and tamoxifen significantly reduced the expression of caspase-3, -7, and -9 proteins compared to individual treatment with pentadecanoic acid or tamoxifen ( Figure 5C,D).

Example 5. Effect of combined treatment of pentadecanoic acid and tamoxifen on epithelial-mesenchymal transition (EMT)

Previous studies have reported the important role of EMT in cancer cell metastasis and drug resistance. Therefore, the effect of pentadecanoic acid and tamoxifen combination on EMT was evaluated using cell migration assay, invasion ability assay, and Western blot. It was confirmed that when 50 μM pentadecanoic acid and 10 μM tamoxifen were treated together, the migration and invasion abilities of MCF-7/SC cells were significantly inhibited (Figures 6A to D). These facts were further confirmed through Western blot results for EMT-related markers such as Snail, Slug, matrix metalloproteinase 9 (MMP9), and vimentin. No significant inhibitory effect was observed on the expression of these proteins under conditions of individual treatment with tamoxifen or pentadecanoic acid (Figure 6E-F), but combined treatment significantly reduced the expression of EMT-related proteins in MCF-7/SC cells. The above results showed that the combined treatment of pentadecanoic acid and tamoxifen could effectively inhibit cell migration and invasion ability and reduce the expression of EMT-related markers in MCF-7/SC cells.

Example 6. Changes in estrogen receptor expression by combined treatment of pentadecanoic acid and tamoxifen

We hypothesized that the combination of pentadecanoic acid and tamoxifen could induce ER-α expression in MCF-7/SC cells. To investigate this, MCF-7/SC cells were first treated with pentadecanoic acid (50 or 100 μM) alone or together with 10 μM tamoxifen, and ER-α expression was examined. As a result, pentadecanoic acid increased both the mRNA and protein expression levels of ER-a in all conditions of treatment alone at 100 μM and in combination with tamoxifen (Figures 7A to C). We further investigated whether the combined treatment of pentadecanoic acid and tamoxifen affects ER-α-related genes. As a result, it was confirmed that treatment with high concentration of pentadecanoic acid alone or in combination with tamoxifen can induce the expression of ER-α-related genes including CA12, XBP1, GREB1, FOS, NRIP1, RARA, BCL2 , and TFF1 ( Figure 7D). The above results indicate that re-expression of ER-α by pentadecanoic acid alone or in combination with tamoxifen contributes to restoration of sensitivity to tamoxifen in MCF-7/SC breast cancer cells.

Sequence list electronic file attached

Claims (8)

A composition for enhancing anticancer drug susceptibility in anticancer drug-resistant cancer, comprising pentadecanoic acid as an active ingredient. The composition for enhancing anticancer drug susceptibility according to claim 1, wherein the anticancer drug-resistant cancer is tamoxifen-resistant breast cancer. An anticancer supplement for anticancer drug-resistant cancer containing pentadecanoic acid as an active ingredient. The anti-cancer adjuvant according to claim 3, wherein the anti-cancer adjuvant is used in combination with an anti-cancer drug. The anti-cancer adjuvant according to claim 3, wherein the anti-cancer drug-resistant cancer is tamoxifen-resistant breast cancer. A pharmaceutical composition for preventing or treating tamoxifen-resistant breast cancer, comprising a mixture of pentadecanoic acid and tamoxifen as active ingredients. The pharmaceutical composition of claim 6, wherein the mixture of pentadecanoic acid and tamoxifen increases the expression level of estrogen receptors in cancer cells, increases apoptosis, and inhibits epithelial-mesenchymal transition. Composition. A method of treating tamoxifen-resistant cancer in a mammalian subject, comprising administering a pharmaceutically effective amount of the pharmaceutical composition of claim 6 to a mammalian subject with cancer resistant to tamoxifen.