13-Oxo-9Z,11E-octadecadienoic Acid Down-regulates c-Myc and Attenuates Breast Cancer Cell Stemness

Abstract

Background/Aim: Breast cancer stem cells (BCSCs) are involved in the development of breast cancer and contribute to therapeutic resistance. This study aimed to investigate the anticancer stem cell (CSC) mechanism of 13-Oxo-9Z,11E-octadecadienoic acid (13-Oxo-ODE) as a potent CSC inhibitor in breast cancer.

Materials and Methods: The effects of 13-Oxo-ODE on BCSCs were evaluated using a mammosphere formation assay, CD44^high/CD24^low analysis, aldehyde dehydrogenase (ALDH) assay, apoptosis assay, quantitative real-time PCR, and western blotting.

Results: We found that 13-Oxo-ODE suppressed cell proliferation, CSC formation, and mammosphere proliferation and increased apoptosis of BCSCs. Additionally, 13-Oxo-ODE reduced the subpopulation of CD44^high/CD24^low cells and ALDH expression. Furthermore, 13-Oxo-ODE decreased c-myc gene expression. These results suggest that 13-Oxo-ODE has potential as a natural inhibitor targeting BCSCs through the degradation of c-Myc.

Conclusion: In summary, 13-Oxo-ODE induced CSC death possibly through reduced c-Myc expression, making it a promising natural inhibitor of BCSCs.

Triple-negative breast cancer cells (TNBCs) are found in 10%-15% of breast cancer patients (1) and lack the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (2). Cancer stem cells (CSCs) exhibit self-renewal and tumorigenic properties (3), and breast CSCs (BCSCs) have distinct properties including aldehyde dehydrogenase (ALDH) 1 and CD44 expression (4,5). Treating CSCs is crucial in cancer therapy (6), and many studies have focused on targeting CSCs to improve cancer treatment outcomes (7).

The transcription factor c-Myc dimerizes with myc-associated factor X and binds to enhancer box sequences (8,9). The transcription of c-myc is initiated at three different sites, and c-Myc1, c-Myc2, or c-MycS are transcribed depending on the site (10). Importantly, c-Myc is associated with the maintenance and metabolism of CSCs (11), and several studies have indicated that c-Myc plays an important role in CSC regulation (12,13). For example, c-Myc is involved in the maintenance of colon CSCs (14), and the down-regulation of c-Myc expression leads to glioma cancer stem cell apoptosis (15) as well as the inhibition of BCSC formation (16). Therefore c-Myc is considered a promising therapeutic target for treating CSCs (17).

Salicornia herbacea L. (glasswort) is a plant found in salt marshes and at coastal locations in Korea, China, and the United States (18). This species contains many components that are used by humans, including saponins and flavonoids (19-22), and exhibits antioxidant, anticancer, and anti-inflammatory activities (23-25). Solvent-extracted samples inhibit the growth of melanoma via the phosphorylated ERK and phosphorylated p38 signaling pathways in mice (23). The oxooctadecadienoic acid 13-Oxo-9Z,11E-octadecadienoic acid (13-Oxo-ODE) has an anti-inflammatory effect (26) and acts as an agonist of peroxisome proliferator-activated receptor-α (27). The effects of 13-Oxo-ODE on BCSCs and the mechanisms underlying these effects have not been studied.

In the present study, 13-Oxo-ODE was investigated as a promising compound for targeting BCSCs. We assessed the anti-BCSC effects of the isolated compound, finding that 13-Oxo-ODE inhibited the gene and protein expression levels of c-myc and c-Myc, respectively, as well as the formation of MDA-MB-231 BCSCs.

Materials and Methods

Materials, kits, and equipment. 60A silica gels, thin-layer chromatography (TLC) plates, and Sephadex LH-20 resin were obtained from Sigma-Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC) was performed using a Shimadzu 20A system (Shimadzu, Kyoto, Japan). Cancer cell proliferation was assayed using a CellTiter 96^® Aqueous Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). A BCSC inhibitor was purified from S. herbacea, and 13-Oxo-ODE was isolated.

Plant specimens. Salicornia herbacea specimens were purchased from Dasarang Ltd. (Sinan, Republic of Korea). This sample (no. 2020_10) was deposited at the Department of Biomaterial, Jeju National University Core-facility Center, Jeju, Republic of Korea.

Isolation of 13-Oxo-ODE. The 13-Oxo-ODE used in this study was prepared as previously reported (26) and isolated as follows. Salicornia herbacea (1,000 g) was incubated with 100% methanol at 32˚C overnight in a shaking incubator. The isolation procedure is shown in Figure 1A. Briefly, methanol-extracted samples were combined with water at a 1:1 ratio. All methanol was evaporated, and the remaining sample was combined with the same volume of ethyl acetate. The ethyl acetate extracts were evaporated, dissolved with methanol, loaded onto a column filled with silica gel (3×30 cm), and isolated with a chloroform: methanol (30:1) solvent mixture. The active fractions were then separated and tested for anti-BCSC effects. Briefly, active fractions were loaded onto a column filled with Sephadex LH-20 gel (2.5×30.0 cm), isolated, and loaded onto a preparatory glass TLC plate (20×20 cm) in a chamber filled with a hexane:ethyl acetate:acetic acid (15.0:5.0:1.0) solvent mixture. The active fraction was analyzed via preparatory HPLC (Shimadzu). An ODS C18 column (10×250 mm) was used with a flow rate of 3 ml/min (acetonitrile proportion: 0%-60% for 20 min; 60%-100% for 10 min; 100% for 10 min; 100%-60% for 10 min, 60%-0% for 10 min; and 0% for 5 min). The peak of the purified sample was detected at a retention time of 32.8 min (Figure 1B).

Figure 1. Procedure for isolating 13-Oxo-ODE from S. herbacea. (A) Flowchart of the purification of the BCSC inhibitor. (B) HPLC analysis of the BCSC inhibitor derived from S. herbacea. (C) Molecular structure of 13-Oxo-ODE, i.e., the CSC inhibitor isolated from S. herbacea.

Breast cancer cell lines and mammosphere culture. Two breast cancer cell lines (MDA-MB-231 and MCF-7 cells) were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific). BCSCs were cultured under humidified conditions (37˚C and 5% CO₂) in MammoCult™ medium (STEMCELL Technologies, Vancouver, BC, Canada) in ultralow-attachment plates at 3×10⁴ and 4×10⁴ cells/well for MDA-MB-231 and MCF-7 cells, respectively. To determine mammosphere formation efficiency (MFE), BCSC formation was assayed using the NICE program (28).

Cell viability assay. Breast cancer cells were seeded in 96-well cell culture plates and incubated with various concentrations (0, 20, 40, 80, 100, 150, 200, 300, and 400 μM) of 13-Oxo-ODE for 24 h. Cell viability was determined using CellTiter 96™ solution according to the manufacturer’s protocol (29). Optical density (OD₄₉₀) was measured using a VersaMax ELISA microplate reader (Molecular Devices, San Jose, CA, USA).

Flow cytometry analysis. After treatment with 13-Oxo-ODE for 24 h, breast cancer cells were treated with trypsin/EDTA (30). The detached cells were incubated with CD44-FITC and CD24-PE antibodies (BD, San Jose, CA, USA) for 20 min at 4˚C. The cells were then centrifuged, washed with 1X FACS buffer, and analyzed using an Accuri C6 Flow Cytometer (BD). An ALDEFLUOR assay was conducted using cultured cells treated with 13-Oxo-ODE for 24 h, and the detached cells were assayed according to the manufacturer’s methods. ALDH1-positive cell populations were assayed using the Accuri C6 Flow Cytometer (31).

Quantitative real-time PCR. We extracted the total RNA from MDA-MB-231-derived mammospheres and performed quantitative real-time PCR using a Real-time One-Step RT-qPCR Kit (Enzynomics, Daejeon, Republic of Korea). The relative mRNA levels of genes were analyzed using the comparative CT method (30). The primers used in this analysis are listed in Table I. β-actin was used as an internal control gene.

Table I. Primer sequences of the target genes used in RT-qPCR.

Western blot analysis. Proteins were extracted from MDA-MB-231-derived mammospheres, subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were incubated with Odyssey Blocking Buffer for 1 h and then incubated with primary antibodies against c-Myc (5605), lamin B (12586, Cell Signaling Technology, Danvers, MA, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) for >4 h at room temperature. The PVDF membranes were then washed twice with PBS-Tween 20 (0.1%, v/v) and incubated with secondary antibodies (IRDye 680RD- and IRDye 800W-labeled) for 1 h at room temperature. Western blot data were analyzed using an Odyssey CLx Imaging System (LI-COR, Lincoln, NE, USA) (32).

Statistical analysis. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). All data are represented as mean±standard deviation (SD). Data were analyzed via one-way ANOVA followed by Dunnett post-hoc test. p-Values of ≤0.05 were considered statistically significant.

Results

BCSC inhibitor purified from Salicornia herbacea. The preparation of 13-Oxo-ODE has been reported previously (26). The compound extracted from S. herbacea inhibited BCSC formation, and was purified based on a CSC inhibition assay (Figure 1A). The 13-Oxo-ODE was isolated using solvent extraction, column chromatography, and preparatory TLC. Finally, the compound was purified using preparatory HPLC (Figure 1B, C).

Breast cancer cell growth and mammosphere formation are inhibited by 13-Oxo-ODE. Breast cancer cells were cultured with a range of 13-Oxo-ODE concentrations (0-400 μM) for 24 h. The antiproliferative effects of 13-Oxo-ODE were tested using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The viability of both breast cancer cell lines was reduced by 13-Oxo-ODE treatment (Figure 2A, B). Additionally, in primary BCSCs treated with 13-Oxo-ODE, the compound reduced the number and size of mammospheres (Figure 2C, D).

Figure 2. Effects of 13-Oxo-ODE on breast cancer cell viability and mammosphere-forming efficiency. MDA-MB-231 (A) and MCF-7 (B) cells were cultured with 13-Oxo-ODE for 24 h. The cytotoxicity of 13-Oxo-ODE was tested using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Treatment with 13-Oxo-ODE suppressed mammosphere formation. To produce mammospheres, 3×10⁴ MDA-MB-231 cells (C) and 4×10⁴ MCF-7 cells (D) per well were plated in ultralow-attachment 6-well plates. The mammospheres were then cultured with 13-Oxo-ODE. Images of representative mammospheres were obtained via inverted light microscopy (scale bar: 100 μm). The mammosphere formation efficiency (MFE) was determined. Data represent the mean±SD from three experiments. *p<0.05, **p<0.01 and ***p<0.001 using t-test or one-way ANOVA followed by Dunnett post-hoc test.

Treatment with 13-Oxo-ODE affects the properties of BCSCs. We examined two BCSC markers: CD44^high/CD24^low expressing and ALDH1-positive subpopulations. MDA-MB-231 cells were treated with 200 μM 13-Oxo-ODE, which reduced the CD44^high/CD24^low-expressing MDA-MB-231 cell subpopulation from 96.5% to 70.7% (Figure 3A) and the ALDH1-positive subpopulation from 5.1% to 0.7% (Figure 3B). Therefore, 13-Oxo-ODE modestly reduced the levels of BCSC markers.

Figure 3. Effects of 13-Oxo-ODE on CD44^high/CD24^low-expressing cells and ALDH1-positive cells. (A) CD44^high/CD24^low subpopulations treated with 13-Oxo-ODE (200 μM) or left untreated were tested via flow cytometry. (B) ALDH1-positive subpopulations were also assayed using flow cytometry. Red lines indicate the binding of an antibody without 13-Oxo-ODE. Data represent the mean±SD from three experiments. *p<0.05, **p<0.01 and ***p<0.001 using t-test or one-way ANOVA followed by Dunnett post-hoc test.

Treatment with 13-Oxo-ODE affects c-Myc regulation. c-Myc has been identified as a promising therapeutic target in breast cancer patients (33). Therefore, we examined the effects of 13-Oxo-ODE on c-myc transcription in BCSCs, finding that the compound decreased c-myc transcription levels in MDA-MB-231 mammospheres (Figure 4A). In addition, 13-Oxo-ODE reduced the total c-Myc protein expression level in MDA-MB-231 cells (Figure 4B). Nuclear proteins were isolated and c-Myc expression was measured. Levels of c-Myc in the cytosolic and nuclear fractions were decreased (Figure 4C). In summary, c-Myc is an important factor in mammosphere formation, and 13-Oxo-ODE inhibits c-Myc expression in MDA-MB-231 BCSCs.

Figure 4. Treatment with 13-Oxo-ODE regulates c-myc mRNA and protein expression. (A) c-myc gene transcript levels in CSCs were measured in mammospheres with or without 13-Oxo-ODE treatment using real-time PCR. β-actin was used as the internal control. (B) Using immunoblotting, total c-Myc protein levels were assayed in MDA-MB-231 cell mammospheres with (150 μM) or without 13-Oxo-ODE treatment for 24 h. Total lysates were used to analyze immunoblots with an anti-c-Myc antibody. β-Actin was used as the internal control. (C) After treatment with 13-Oxo-ODE, c-Myc protein levels in the cytosolic and nuclear fractions were analyzed in mammospheres using western blotting. Nuclear and cytosolic proteins were separated via SDS–PAGE, which was followed by immunoblotting with anti-c-Myc, anti-β-actin, and anti-Lamin B antibodies. Data are presented as the mean±SD of three independent experiments. **p<0.01 and ***p<0.001 vs. the DMSO-treated control group, using t-test or one-way ANOVA followed by Dunnett post-hoc test.

Treatment with 13-Oxo-ODE induces BCSC apoptosis and suppresses CSC marker gene transcription and mammosphere proliferation. BCSCs were treated with 150 μM 13-Oxo-ODE to assess the effects of the compound on apoptosis in mammospheres. Late apoptotic cell subpopulations increased from 6.5% to 23.7% (Figure 5A). Additionally, treatment with 13-Oxo-ODE down-regulated transcription levels of the CSC marker genes Nanog, CD44, and Oct4 (Figure 5B). Furthermore, 13-Oxo-ODE decreased BCSC proliferation (Figure 5C).

Figure 5. Effects of 13-Oxo-ODE on apoptosis, cancer stem cell marker levels, and mammosphere growth. (A) Treatment with 13-Oxo-ODE increased apoptosis in BCSCs. Mammospheres were plated and cultured with 13-Oxo-ODE. Apoptosis was analyzed via Annexin V/propidium oxide (PI) staining after 13-Oxo-ODE treatment. (B) Transcriptional levels of CSC markers, including the Nanog, Oct4, and CD44 genes, were determined in mammospheres with or without 13-Oxo-ODE treatment using real-time PCR. β-actin was used as the internal control. Data represent the mean±SD of three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 vs. the DMSO-treated control using t-test or one-way ANOVA followed by Dunnett post-hoc test. (C) Mammosphere growth was reduced by 13-Oxo-ODE treatment. Mammospheres treated with 13-Oxo-ODE or left untreated were separated into single cells, which were plated in equal numbers in 6 cm dishes. One, two, and three days later, the cells were counted. (D) Proposed model for 13-Oxo-ODE induced CSC death.

Discussion

Salicornia herbacea is a salt-tolerant plant species that grows along the coastline of Korea, China, and the United States. Its extracts have several useful properties, including antioxidant, anti-inflammatory, and anti-CSC properties (34-36). In the present study, we purified the compound 13-Oxo-ODE from S. herbacea using bioassay-based fractionation and determined its anti-BCSC effects. Also known as 13-KODE, 13-Oxo-ODE exerts anti-inflammatory effects by regulating MAPK signaling pathways (26). However, this is the first study to reveal that 13-Oxo-ODE inhibits BCSCs.

BCSCs are found in many breast cancer patients and have distinct properties such as differentiation and self-renewal (37). Biomarkers used to study BCSCs include CD44^high/CD24^low expression and ALDH expression (38). Breast cancer patients with BCSCs typically have poor prognosis, so studying the molecular mechanism of BCSCs may facilitate the development of effective treatment strategies for these patients (39). In the present study, we evaluated the anti-BCSC effects of 13-Oxo-ODE, finding that the compound inhibits BCSC formation and proliferation (Figure 2 and Figure 3). The levels of BCSC biomarkers, i.e., CD44^high/CD24^low-expressing and ALDH1-positive subpopulations, were reduced by 13-Oxo-ODE-treatment (Figure 3). Therefore, we showed that 13-Oxo-ODE exerts anti-BCSC effects.

The transcription factor c-Myc is a proto-oncogene and CSC survival factor that has been associated with the maintenance and self-renewal of CSCs through the regulation of several target genes (40). c-Myc is a short-lived protein owing to its degradation in ubiquitin-dependent or ubiquitin-independent pathways (41,42). It has been associated with the apoptosis of hepatocellular carcinoma cells (43) and maintenance of TNBC-derived CSCs (44). In the present study, 13-Oxo-ODE reduced the transcription level of c-myc as well as the total and nuclear c-Myc protein levels in BCSCs (Figure 4). The compound also inhibited the growth of BCSCs and the transcription levels of CSC-related genes (Figure 5).

Conclusion

The compound 13-Oxo-ODE can be isolated from S. herbacea using bioassay-guided fractionation. In the present study, it inhibited the formation of BCSCs and breast cancer cell proliferation. It also decreased the levels of BCSC markers, namely CD44^high/CD24^low-expressing and ALDH1-positive subpopulations, and some CSC-related genes. Furthermore, 13-Oxo-ODE regulated the transcript and protein levels of c-Myc, which is a known BCSC survival factor. Overall, regulating the c-Myc may be a strategic target for BCSC treatment and the natural compound from S. herbacea may be useful for treating breast cancer.

Conflicts of Interest

The Authors declare that they have no conflicts of interest to report in relation to this study.

Authors’ Contributions

HS Choi and YC Ko designed the experiments and performed all experiments. HS Choi and YC Ko wrote the manuscript. SL Kim helped design and perform the experiments. DS Lee supervised the study.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1A2C1006316, 2022R1I1A1A01068288, and NRF-2016R1A6A1A03012862). It was also supported by a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (grant No. 2020R1A6C101A188).