Pentadecanoic Acid, an Odd-Chain Fatty Acid, Suppresses the Stemness of MCF-7/SC Human Breast Cancer Stem-Like Cells through JAK2/STAT3 Signaling

Abstract

Saturated fatty acids possess few health benefits compared to unsaturated fatty acids. However, increasing experimental evidence demonstrates the nutritionally beneficial role of odd-chain saturated fatty acids in human health. In this study, the anti-cancer effects of pentadecanoic acid were evaluated in human breast carcinoma MCF-7/stem-like cells (SC), a cell line with greater mobility, invasiveness, and cancer stem cell properties compared to the parental MCF-7 cells. Pentadecanoic acid exerted selective cytotoxic effects in MCF-7/SC compared to in the parental cells. Moreover, pentadecanoic acid reduced the stemness of MCF-7/SC and suppressed the migratory and invasive ability of MCF-7/SC as evidenced by the results of flow cytometry, a mammosphere formation assay, an aldehyde dehydrogenase activity assay, and Western blot experiments conducted to analyze the expression of cancer stem cell markers-CD44, β-catenin, MDR1, and MRP1-and epithelial-mesenchymal transition (EMT) markers-snail, slug, MMP9, and MMP2. In addition, pentadecanoic acid suppressed interleukin-6 (IL-6)-induced JAK2/STAT3 signaling, induced cell cycle arrest at the sub-G1 phase, and promoted caspase-dependent apoptosis in MCF-7/SC. These findings indicate that pentadecanoic acid can serve as a novel JAK2/STAT3 signaling inhibitor in breast cancer cells and suggest the beneficial effects of pentadecanoic acid-rich food intake during breast cancer treatments.

Keywords: JAK2/STAT3 signaling; apoptosis; breast cancer; cancer stem cells; odd-chain fatty acids; pentadecanoic acid.

Figure 1

MCF-7/SC exhibit more prominent cancer stem cell characteristics than the parental MCF-7 cells. (a) Fluorescence-activated cell sorting (FACS) analysis of the CD44⁺/CD24⁻ cell population in MCF-7/SC and MCF-7 cells. (b) Measurement of the ROS levels in MCF-7/SC and MCF-7 cells. (c) Comparison of the mammosphere formation ability of MCF-7/SC and MCF-7 cells cultured in the MammoCult Human Medium for 10 days. Magnification 100×. (d) Analysis of the expression of cancer stem cell markers in MCF-7/SC and MCF-7 cells by Western blotting. GAPDH was used as a loading control. (e) Migratory potential of MCF-7/SC and MCF-7 cells as assessed by the wound healing assay. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 2

Pentadecanoic acid exerts cytotoxicity in MCF-7/SC and MCF-7 cells. Comparison of cytotoxic effects in MCF-7/SC and MCF-7 cells following 48 h of incubation. (a) Unsaturated fatty acids: oleic acid (C18:1) and linoleic acid (C18:2). (b) Odd-chain fatty acids: pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). (c) Effects of pentadecanoic acid and heptadecanoic acid on MCF-10A cell proliferation after 48 h of incubation. (d) Time-dependent (24 and 48 h) cytotoxic effects of pentadecanoic acid in MCF-7/SC. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 3

Pentadecanoic acid inhibits the migration and invasion of MCF-7/SC. (a) Cell migration was determined by the wound healing assay following 48 h of exposure. (b) Invasive cells were stained with crystal violet after treatment with pentadecanoic acid for 48 h (magnification 100×). (c) Western blot analysis of epithelial–mesenchymal transition (EMT) markers in MCF-7/SC was performed after pentadecanoic acid treatment for 48 h. GAPDH was used as a loading control. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 4

Pentadecanoic acid suppresses the stem like-cell characteristics of MCF-7/SC. (a) Effects of pentadecanoic acid on MCF-7/SC mammosphere formation (magnification 100×). (b,b’) The effects of pentadecanoic acid on the expression of cell surface markers in MCF-7/SC were analyzed by FACS analysis following pentadecanoic acid exposure. (c) Flow cytometry analysis of the ALDH⁺ population using the ALDEFLUOR assay kit. Cells were treated with pentadecanoic acid for 48 h prior to the assay. (d) The levels of cancer stem cell (CSC) markers were assessed by Western blot experiments following pentadecanoic acid treatment for 48 h. (e) The levels of CSC markers as assessed by Western blot experiments following 100 μM pentadecanoic acid exposure. GAPDH was used as a loading control. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 4

Pentadecanoic acid suppresses the stem like-cell characteristics of MCF-7/SC. (a) Effects of pentadecanoic acid on MCF-7/SC mammosphere formation (magnification 100×). (b,b’) The effects of pentadecanoic acid on the expression of cell surface markers in MCF-7/SC were analyzed by FACS analysis following pentadecanoic acid exposure. (c) Flow cytometry analysis of the ALDH⁺ population using the ALDEFLUOR assay kit. Cells were treated with pentadecanoic acid for 48 h prior to the assay. (d) The levels of cancer stem cell (CSC) markers were assessed by Western blot experiments following pentadecanoic acid treatment for 48 h. (e) The levels of CSC markers as assessed by Western blot experiments following 100 μM pentadecanoic acid exposure. GAPDH was used as a loading control. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 5

Pentadecanoic acid suppressed JAK2/STAT3 signaling in MCF-7/SC. (a) Representative Western blot analysis showing the dose-dependent effects of pentadecanoic acid on the expression of STAT3, pSTAT3, JAK2, and pJAK2 in MCF-7/SC following 48 h of incubation. (b) Representative Western blot analysis showing the time-dependent effects of 100 μM pentadecanoic acid on the expression of STAT3, pSTAT3, JAK2, and pJAK2 in MCF-7/SC. (c) Effects of pentadecanoic acid on IL-6-inducible JAK2/STAT3 signaling. MCF-7/SC were pre-treated with 150 μM pentadecanoic acid for 48 h and stimulated with 20 ng/mL of IL-6 for 15 min. In Figure 5c, PTDCN indicates pentadecanoic acid. GAPDH was used as a loading control. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 5

Pentadecanoic acid suppressed JAK2/STAT3 signaling in MCF-7/SC. (a) Representative Western blot analysis showing the dose-dependent effects of pentadecanoic acid on the expression of STAT3, pSTAT3, JAK2, and pJAK2 in MCF-7/SC following 48 h of incubation. (b) Representative Western blot analysis showing the time-dependent effects of 100 μM pentadecanoic acid on the expression of STAT3, pSTAT3, JAK2, and pJAK2 in MCF-7/SC. (c) Effects of pentadecanoic acid on IL-6-inducible JAK2/STAT3 signaling. MCF-7/SC were pre-treated with 150 μM pentadecanoic acid for 48 h and stimulated with 20 ng/mL of IL-6 for 15 min. In Figure 5c, PTDCN indicates pentadecanoic acid. GAPDH was used as a loading control. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.

Figure 6

Pentadecanoic acid induced apoptosis in MCF-7/SC. (a,a’) Fluorescein-conjugated annexin V (annexin V-FITC) vs. propidium iodide (PI) staining analysis showing apoptosis induction following treatment with pentadecanoic acid for 48 h. (b) Cell cycle analysis of MCF-7/SC exposed to pentadecanoic acid for 48 h. (c) The levels of apoptosis markers were assessed by Western blot experiments following pentadecanoic acid treatment for 48 h. GAPDH was used as a loading control. Data are shown as the mean ± standard deviation of three biologically independent experiments. * p < 0.05 vs. control.