Fish Oil and Selenium with Doxorubicin Modulates Expression of Fatty Acid Receptors and Selenoproteins, and Targets Multiple Anti-Cancer Signaling in Triple-negative Breast Cancer Tumors

Abstract

Omega-3 fatty acids from fish oil (FO) and selenium (Se) potentiate some conventional therapies and have anticancer immune potential. This study aims to determine whether FO/Se modulates G-protein-coupled polyunsaturated fatty acid receptors (GPR-40 and GPR-120) and selenoproteins (Sel-H, Sel-W, and GPx4), and increases the therapeutic effect of doxorubicin in a dose-dependent manner on triple-negative breast cancer (TNBC) mouse. Mice were randomized into 5 groups (n = 7/group) and treated with physiological saline (control), low-dose doxorubicin, and doxorubicin in combination with low, medium, or high doses of FO/Se. The expression of signaling molecules in tumors was determined by measuring either mRNA or protein expression. Compared with doxorubicin alone, combination treatment resulted in lower tumor sizes and fewer overall metastasis, lower GPR-40 mRNA levels, and higher expression of all selenoproteins. Doxorubicin-FO/Se combination treatment decreased expression of membrane EGFR and FGFR, down-regulated downstream PI3K/AKT/mTOR, MAPK/ERK, and JAK2/c-Src/STAT3 signaling, increased tumor suppressor PTEN/TSC1/TSC2 expression and P53 activation, and suppressed oncogenic transcription factor expression. Dose-dependent inhibition of proliferation index Ki-67, cell cycle, and stem-cell-related markers were observed. Decreased immune check-points PD-L1/CTLA-4/Foxp3/CD86 and increased PD-1/CD28/IL-2 expression was also found. These observations suggest that the nutritional supplements FO/Se increase the chemotherapeutic efficacy of doxorubicin against TNBC by modulating GPR-40 and selenoprotein and targeting multiple signaling pathways in tumor tissues.

Keywords: Anticancer signaling; Doxorubicin; Fish oil and selenium; GPR-40; Immune microenvironment; Selenoproteins; TNBC mouse tumor.

Figure 1

(A) Tumor growth and tumor and body weights. (B) Internal organ weights. (C) Number of metastases in different organs. (D) Gross appearance of primary tumors in TNBC-bearing mice. Values are expressed as the mean (standard error). *p < 0.05 doxorubicin vs control; doxorubicin vs + low, medium, and high FO/Se. 4T1 tumor cells (1 × 10⁵) were subcutaneously injected into the mouse right hind thigh on day 0. Tumor-bearing mice on day 7 were randomized into 5 weight-matched groups of 7 mice each as follows: 1) Control group, saline injected; 2) doxorubicin group, injected intraperitoneally with 5 mg/kg doxorubicin (once every 4 days); 3) +low FO/Se, +medium FO/Se, and +high FO/Se groups were injected intraperitoneally with 5 mg/kg doxorubicin (once every 4 days) together with a low, medium, or high dose of fish oil/selenium supplement p.o. twice a day from day 7 to day 31. Healthy mice were randomly assigned to Healthy (H, n=6) and H+high FO/Se (n=6) groups and treated with or without 0.4 g of high dose-FO/Se by oral gavage twice a day from day 7 to day 31. Doxo, doxorubicin; FO, fish oil; Se, selenium. Carcass weight = total body weight minus tumor weight. Superscript ^{a, b, c, d}: Bars sharing the same superscript are not significantly different from each other; Bars with different superscript are significantly different from each other (p < 0.05).

Figure 2

(A) Selenoprotein (Sel-H and GPx4) and G-protein-coupled polyunsaturated fatty acid receptors (GPR-40/120) mRNA levels in TNBC mouse tumor. (B) Sel-H/Sel-W and EGFR/FGFR protein expression. (C) EGFR mRNA levels. (D) Densitometric analysis of three separate western blots. Values are expressed as relative readings (mean ± standard error) from 3-4 mice in each group. Further details of the groups illustrated are described in Figure 1. Superscript ^{a, b, c, d}: Bars sharing the same superscript are not significantly different from each other; Bars with different superscript are significantly different from each other (p < 0.05).

Figure 3

(A) PTEN mRNA levels in TNBC mouse tumor. (B) Expression of PI3K/PTEN/AKT/mTOR/4EBP1/p70S6K, Ras/Raf/MEK/ERK, and c-Src/JAK2/STAT3 signaling markers. (C) Densitometric analysis of three separate western blots. Values are expressed as relative readings (mean ± standard error) from 3-4 mice in each group. Further details of the groups illustrated are described in Figure 1. Superscript ^{a, b, c, d}: Bars sharing the same superscript are not significantly different from each other; Bars with different superscript are significantly different from each other (p < 0.05).

Figure 4

(A) c-Jun, c-Fos, and TP53 mRNA levels in TNBC mouse tumor. (B) Expression of c-Myc, NF-κB p65, HIFs, and P53 proteins. (C) Densitometric analysis of three separate western blots. Values are expressed as relative readings (mean ± standard error) from 3-4 mice in each group. Details of groups illustrated above are described in Figure 1. Superscript ^{a, b, c, d}: Bars sharing the same superscript are not significantly different from each other; Bars with different superscript are significantly different from each other (p < 0.05).

Figure 5

(A) mRNA levels of PD-1/PD-L1, CTLA-4, Foxp3, and IL-2 in TNBC mouse tumor. (B) PD-1/PD-L1/PD-L2, and CTLA-4, Foxp3, Nkp46, CD28, CD80/86, and IL-2 protein levels. (C) Densitometric analysis of three separate western blots. Values are expressed as relative readings (mean ± standard error) from 3-4 mice in each group. Details of groups illustrated are given in Figure 1. Superscript ^{a, b, c, d}: Bars sharing the same superscript are not significantly different from each other; Bars with different superscript are significantly different from each other (p < 0.05).

Figure 6

(A) mRNA levels of Ki-67 and cyclin D1/E in TNBC mouse tumor. (B) Expression of CDK2/4/6 and cyclin D1/E proteins. (C) Expression of cancer stemness markers. (D) Densito-metric analysis of three separate western blots. Values are expressed as relative readings (mean ± standard error) from 3-4 mice in each group. Details of groups illustrated are listed in Figure 1. Superscript ^{a, b, c, d}: Bars sharing the same superscript are not significantly different from each other; Bars with different superscript are significantly different from each other (p < 0.05).

Figure 7

Schematic diagram showing combination treatment with FO and Se increases the therapeutic efficacy of doxorubicin against TNBC.