Combination of Fish Oil and Selenium Enhances Anticancer Efficacy and Targets Multiple Signaling Pathways in Anti-VEGF Agent Treated-TNBC Tumor-Bearing Mice

Abstract

Fish oil (FO) and selenium (Se) possess antiangiogenic potential in malignant tumors. This study aimed to determine whether combination of FO and Se enhanced treatment efficacy of low-dose antiangiogenic agent Avastin (bevacizumab) in a dose-dependent manner and targeted multiple signaling pathways in triple-negative breast cancer (TNBC)-bearing mice. Randomized into five groups, mice received treatment with either physiological saline (control), Avastin alone, or Avastin in combination with low, medium, and high doses of FO/Se. The target signaling molecules for anticancer were determined either by measuring protein or mRNA expression. Avastin-treated mice receiving FO/Se showed lower tumor growth and metastasis than did mice treated with Avastin alone. Combination-treated mice exhibited lower expressions in multiple proangiogenic (growth) factors and their membrane receptors, and altered cytoplasmic signaling molecules (PI3K-PTEN-AKT-TSC-mTOR-p70S6K-4EBP1, Ras-Raf-MEK-ERK, c-Src-JAK2-STAT3-TMEPAI-Smad, LKB1-AMPK, and GSK3β/β-catenin). Dose-dependent inhibition of down-stream targets including epithelial-to-mesenchymal transition transcription factors, nuclear cyclin and cyclin-dependent kinases, cancer stem cell markers, heat shock protein (HSP-90), hypoxia-inducible factors (HIF-1α/-2α), matrix metalloprotease (MMP-9), and increased apoptosis were observed. These results suggest that combination treatment with FO and Se increases the therapeutic efficacy of Avastin against TNBC in a dose-dependent manner through multiple signaling pathways in membrane, cytoplasmic, and nucleic targets.

Keywords: Avastin; TNBC; antitumor mechanisms; bevacizumab; fish oil; mice; selenium.

Figure 1

Body weight, tumor growth and metastasis, and levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and micronutrient selenium (Se). (a) Increases in actual body weight, tumor size, and tumor growth from day 7 to day 31 during experimental period, (b) distal tumor metastasis, (c) exterior of tumors, and (d) Se and EPA/DHA levels in blood and tumor tissues of 4T1 tumor-bearing mice. Values are expressed as mean ± SEM. Values with different superscripts (a, b, c, and d) significantly different (p < 0.05). 4T1 tumor cells (1 × 10⁵) were injected into the subcutaneous region of the mouse right hind thigh at day 0 of the experiment. Tumor-bearing mice at day 7 were randomized into five weight-matched groups of six mice each as follows: (1) control group injected with saline; (2) Avastin group injected intraperitoneally with 5 mg/kg of the Avastin (once every four days); (3) + low FO/Se, +medium FO/Se, and +high FO/Se groups injected intraperitoneally with 5 mg/kg of the Avastin (once every four days) and supplemented with low, medium, and high concentrations of fish oil/selenium supplements by oral gavage twice a day from day 7 to day 31, respectively. The metastasis for lung was graded as 0/no tumor node; 1: 1–15 tumor nodes; 2: 16–30 nodes; 3: 31–35 nodes; 4: 45–60 nodes. Metastasis for mammary gland was graded as 0: no tumor node; 1: 1–4 nodes; 2: 5–8 nodes; 3: 9–12 nodes; 4: more than 13 nodes. Metastasis for pleural cavity was graded as 1: one to two nodes; 2: three to four nodes; 3: five to six nodes; 4: more than seven tumor nodes. Se = selenium; EPA/DHA = eicosapentaenoic acid/docosahexaenoic acid; FO = fish oil. Increases in actual body weight = (changes in body weight from day 7 to 31)–tumor weight.

Figure 2

Tumor selenoproteins and HSP90-HIFs-SOD-1-COX-2-MMP9 expression. (a) Protein expressions of tumor selenoproteins (SEPN1 and SEPW1), HSP90, HIF-1α/HIF-2α, SOD-1, COX-2, and MMP-9, and (b) the levels of relative HSP90 and MMP9 mRNA in tumor tissues of 4T1 tumor-bearing mice were determined by qRT-PCR. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. SEPN1/SEPW1 = selenoprotein N/W; HSP90 = heat shock protein 90; HIFs = hypoxia-inducible factors (HIF-1α and HIF-2α); SOD-1 = superoxide dismutase-1; COX-2 = cyclo-oxygenase-2; MMP9 = matrix metallopeptidase 9.

Figure 3

Expression of tumor proangiogenic (growth) factors and their receptors. (a) Protein levels of proangiogenic (growth) factors and their receptors, (b) densitometric analysis, and (c) relative CXCL12 mRNA in tumor tissues of 4T1 tumor-bearing mice were determined by qRT-PCR. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. VEGF = vascular endothelial growth factor; EGF=epidermal growth factor; FGF = fibroblast growth factors; PDGF = platelet-derived growth factor; TGFβ = Transforming growth factor beta; GAS6/AXL = growth arrest-specific 6/receptor tyrosine kinase Axl; CXCL12/CXCR4,7 = stromal cell-derived factor-1/C-X-C chemokine receptor type 4,-7; Wnt/FZD7 = WNT/Frizzled-7.

Figure 3

Expression of tumor proangiogenic (growth) factors and their receptors. (a) Protein levels of proangiogenic (growth) factors and their receptors, (b) densitometric analysis, and (c) relative CXCL12 mRNA in tumor tissues of 4T1 tumor-bearing mice were determined by qRT-PCR. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. VEGF = vascular endothelial growth factor; EGF=epidermal growth factor; FGF = fibroblast growth factors; PDGF = platelet-derived growth factor; TGFβ = Transforming growth factor beta; GAS6/AXL = growth arrest-specific 6/receptor tyrosine kinase Axl; CXCL12/CXCR4,7 = stromal cell-derived factor-1/C-X-C chemokine receptor type 4,-7; Wnt/FZD7 = WNT/Frizzled-7.

Figure 4

Combination treatment affects tumor PI3K-Ras-LKB1-AMPK signaling pathways. (a) Protein levels of PI3K-Ras and LKB1-AMPK pathways, and (b) densitometric analysis. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. PI3K = phosphoinositide 3-kinases; PTEN = phosphatase and tensin homolog, a multifunctional tumor suppressor; TSC = tuberous sclerosis complex; mTOR = mechanistic target of rapamycin. PI3K = phosphoinositide 3-kinases; PTEN = phosphatase and tensin homolog; AKT = protein kinase B; TSC = tuberous sclerosis complex; mTOR = mammalian target of rapamycin; MEK = MAPK/ERK kinase; ERK = extracellular signal-regulated kinases; LKB1 = liver kinase B1; AMPK = AMP-activated protein kinase.

Figure 4

Combination treatment affects tumor PI3K-Ras-LKB1-AMPK signaling pathways. (a) Protein levels of PI3K-Ras and LKB1-AMPK pathways, and (b) densitometric analysis. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. PI3K = phosphoinositide 3-kinases; PTEN = phosphatase and tensin homolog, a multifunctional tumor suppressor; TSC = tuberous sclerosis complex; mTOR = mechanistic target of rapamycin. PI3K = phosphoinositide 3-kinases; PTEN = phosphatase and tensin homolog; AKT = protein kinase B; TSC = tuberous sclerosis complex; mTOR = mammalian target of rapamycin; MEK = MAPK/ERK kinase; ERK = extracellular signal-regulated kinases; LKB1 = liver kinase B1; AMPK = AMP-activated protein kinase.

Figure 5

Expression of c-Src-Jak2-STAT3-TMEPAI-Smad and GSK3 β/β-catenin signaling. (a) Protein levels of c-Src-Jak2-STAT3-TMEPAI-Smad and GSK3β/β-catenin, and (b) relative GSK3β and β-catenin mRNA in tumor tissues of 4T1 tumor-bearing mice were determined by qRT-PCR. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. TMEPAI = prostate transmembrane protein androgen induced 1; GSK3β = glycogen synthase kinase 3β.

Figure 6

Analysis of epithelial-to-mesenchymal transition, cancer stem cells, cyclins/CDK, and apoptosis in tumor of 4T1 tumor-bearing mice. (a) epithelial-to-mesenchymal transition (EMT), cancer stem cells, and apoptosis, (b) densitometric analysis, and (c) the levels of relative Cyclin D1 and Cyclin E mRNA in tumor of 4T1 tumor-bearing mice were determined by qRT-PCR. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. Epithelial-to-mesenchymal transition transcription factors (SNAIL, SLUG); Cancer stem cell markers-CD24, CD29, CD44, and CXCR2; PARP = Poly(ADP-ribose) polymerase; Cleaved C3 = cleaved caspase-3/Cleaved C8 = cleaved caspase-8; CFL-1 = cofilin-1; CDK = cyclin-dependent kinases.

Figure 6

Analysis of epithelial-to-mesenchymal transition, cancer stem cells, cyclins/CDK, and apoptosis in tumor of 4T1 tumor-bearing mice. (a) epithelial-to-mesenchymal transition (EMT), cancer stem cells, and apoptosis, (b) densitometric analysis, and (c) the levels of relative Cyclin D1 and Cyclin E mRNA in tumor of 4T1 tumor-bearing mice were determined by qRT-PCR. Values are expressed as relative reading (mean ± SEM) from at least three or four independent observations. Bars with different superscripts (a, b, c, and d) are significantly different (p < 0.05). Details of groups illustrated are as Figure 1. Epithelial-to-mesenchymal transition transcription factors (SNAIL, SLUG); Cancer stem cell markers-CD24, CD29, CD44, and CXCR2; PARP = Poly(ADP-ribose) polymerase; Cleaved C3 = cleaved caspase-3/Cleaved C8 = cleaved caspase-8; CFL-1 = cofilin-1; CDK = cyclin-dependent kinases.

Figure 7

Schematic diagram showing combination treatment with FO and Se increases the therapeutic efficacy of Avastin against TNBC through multiple signaling pathways in the membrane, cytoplasmic, and nucleic targets.