Nutr Res Pract.  2023 Oct;17(5):917-933. 10.4162/nrp.2023.17.5.917.

Peanut sprout tea extract inhibits lung metastasis of 4T1 murine mammary carcinoma cells by suppressing the crosstalk between cancer cells and macrophages in BALB/c mice

• Jung JI
• Lee HS
• Lee J
• Kim EJ

• KMID: 2546948
• DOI: http://doi.org/10.4162/nrp.2023.17.5.917

Abstract

BACKGROUND/OBJECTIVES
As peanuts germinate, the content of the components beneficial to health, such as resveratrol, increases within the peanut sprout. This study examined whether the ethanol extract of peanut sprout tea (PSTE) inhibits breast cancer growth and metastasis.
MATERIALS/METHODS
After orthotopically injecting 4T1 cells into BALB/c mice to induce breast cancer, 0, 30, or 60 mg/kg body weight/day of PSTE was administered orally. Angiogenesis-related protein expression in the tumors and the degree of metastasis were analyzed. 4T1 and RAW 264.7 cells were co-cultured, and reverse transcription polymerase chain reaction was performed to measure the crosstalk between breast cancer cells and macrophages.
RESULTS
PSTE reduced tumor growth and lung metastasis. In particular, PSTE decreased matrix metalloproteinase-9, platelet endothelial cell adhesion molecule-1, vascular endothelial growth factor-A, F4/80, CD11c, macrophage mannose receptor, macrophage colony-stimulating factor, and monocyte chemoattractant protein 1 expression in the tumors. Moreover, PSTE prevented 4T1 cell migration, invasion, and macrophage activity in RAW 264.7 cells. PSTE inhibited the crosstalk between 4T1 cells and RAW 264.7 cells and promoted the macrophage M1 subtype while inhibiting the M2 subtype.
CONCLUSIONS
These results suggest that PSTE blocks breast cancer growth and metastasis to the lungs. This may be because the PSTE treatment inhibits the crosstalk between mammary cancer cells and macrophages and inhibits the differentiation of macrophages into the M2 subtype.

Keyword

Peanuts; breast; tumor; metastasis; macrophage

Figure

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  Fig. 1 High-performance liquid chromatography chromatograms of PSTE. (A) Standard, 0.02 mg/mL p-coumaric acid, 0.02 mg/mL resveratrol. (B) The 20 mg/mL PSTE.PSTE, peanut sprout tea extract.

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  Fig. 2 Effects of PSTE on solid tumor growth and lung metastasis in 4T1 tumor-bearing BALB/c mice. 4T1 cells (1 × 105 cells/mouse) were injected into the mammary fat pads of 6-week-old female BALB/c mice. After the tumor reached 50 mm3 (7 days after 4T1 cell inoculation), PSTE was administered by oral gavage for 18 days. Subsequently, the mice were euthanized, and the tumors and lungs were removed. (A) Tumor volume. (B) Tumor weights. (C) Photographs of the lungs, representative of 10 animals, are shown. (D) Metastatic tumor nodules in the lungs were counted. The bars represent the mean ± SE of the mean (n = 10).PSTE, peanut sprout tea extract; CON, vehicle (saline)-treated control group; PSTE30, 30 mg/kg body weight/day peanut sprout tea extract-treated group; PSTE60, 60 mg/kg body weight/day peanut sprout tea extract-treated group.The means without a common letter are different, P < 0.05.

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  Fig. 3 Effects of PSTE on MMP-9, PECAM-1, VEGF, and F4/80 expression in 4T1 tumors in BALB/c mice. The mice were injected with 4T1 cells and administered PSTE, as described in Fig. 2. (A) Tumor sections were stained with MMP-9, PECAM-1, VEGF-A, and F4/80 antibodies. Representative immunofluorescence staining images are shown. Scale bar, 50 µm. (B) Staining intensities were quantified. The bars represent the mean ± SE of the mean (n = 4). (C) Relative Mmp-9, Pecam-1, Vegf, and F4/80 mRNA expression levels in the tumors were analyzed, and the results were normalized to glyceraldehyde 3-phosphate dehydrogenase. The bars represent the mean ± SE of the mean (n = 4).PSTE, peanut sprout tea extract; Mmp-9, matrix metalloproteinase-9; Pecam-1, platelet endothelial cell adhesion molecule-1; Vegf, vascular endothelial growth factor; CON, vehicle (saline)-treated control group; PSTE30, 30 mg/kg body weight/day peanut sprout tea extract-treated group; PSTE60, 60 mg/kg body weight/day peanut sprout tea extract-treated group.The means without a common letter are different, P < 0.05.

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  Fig. 4 Effects of PSTE on the migration and invasion of 4T1 cells and inflammatory response in LPS-stimulated RAW 264.7 cells. (A) Serum-deprived 4T1 cells and (B) Serum-deprived RAW 264.7 cells were incubated in a serum-deprivation medium with or without 500 µg/mL PSTE for 24 h. The cell viability was measured using an MTT assay. (C) Serum-deprived 4T1 cells were plated in the upper inserts at 5 × 104 cells/insert and treated with 500 µg/mL PSTE. The lower chamber was filled with DMEM supplemented with 100 mL/L hormone-free, gelatinase-free FBS as a chemoattractant. After 16 h of incubation, the ability of 4T1 cells to migrate was quantified. (Left panel) Representative images of the migrated 4T1 cells stained with hematoxylin and eosin are shown. (Right panel) Quantitative analyses of the migrated cells. The bars represent the mean ± SE of the mean (n = 3). (D) Serum-deprived 4T1 cells were plated at 5 × 104 cells/insert in the upper inserts and treated with 500 µg/mL PSTE. The lower chamber was filled with DMEM supplemented with 100 mL/L hormone-free, gelatinase-free FBS as a chemoattractant. After 16 h of incubation, 4T1 cell invasion was quantified. The bars represent the mean ± SE of the mean (n = 3). (E) Serum-deprived 4T1 cells were incubated in a serum-deprivation medium with or without 500 µg/mL PSTE for 24 h. The relative Mmp-2, Mmp-9, Timp-1, Timp-2, upa, Pai-1, Vegf, M-csf, Mcp-1, and Cox-2 mRNA expression levels in 4T1 cells were analyzed, and the results were normalized to Gapdh and presented relative to the PSTE (−) group. The bars represent the mean ± SE of the mean (n = 4). (F) Serum-deprived RAW 264.7 cells were incubated in a serum-deprivation medium with or without 500 µg/mL PSTE in the absence or presence of 1 µg/mL LPS. The 24-h conditioned media were collected and the nitric oxide content was measured. Relative Cox-2, Tnf-α, and Il-6 mRNA expression levels in RAW 264.7 cells were analyzed, and the results were normalized to Gapdh and presented relative to the LPS (−)/PSTE (−) group. The bars represent the mean ± SE of the mean (n = 4).PSTE, peanut sprout tea extract; LPS, lipopolysaccharide; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; Mmp, matrix metalloproteinase; Timp, tissue inhibitor of metalloproteinase; upa, urokinase plasminogen activator; Pai-1, plasminogen activator inhibitor-1; Vegf, vascular endothelial growth factor; M-csf, macrophage-colony stimulating factor; Mcp-1, monocyte chemoattractant protein-1; Cox-2, cyclooxygenase-2, Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Tnf-α, tumor necrosis factor-α; Il-6, interleukin-6.*P < 0.05, **P < 0.01, ***P < 0.001 significantly different from the PSTE (−) group.†††P < 0.001 significantly different from the LPS (−)/PSTE (−) group.‡P < 0.05, ‡‡P < 0.01, ‡‡‡P < 0.001 significantly different from the LPS (+)/PSTE (−) group.

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  Fig. 5 Effects of PSTE on the crosstalk between 4T1 and RAW 264.7 cells. 4T1 and RAW 264.7 cells were plated separately on the upper inserts and lower transwell system chambers, respectively, and serum-starved for 4 h in Dulbecco’s Modified Eagle’s Medium. The upper insert containing 4T1 cells was inserted into the lower transwell chamber containing RAW 264.7 cells. The co-culture was treated with 0 or 500 µg/mL PSTE for 36 h. (A) (Left panel) Representative images of migrated 4T1 cells stained with hematoxylin and eosin are shown. (Right panel) Quantitation of the migrated cells. The bars represent the mean ± SE of the mean (n = 8). (B) Relative Mcp-1, M-csf, and Vegf mRNA expression levels in 4T1 cells were analyzed, and the results were normalized to Gapdh and presented relative to the 4T1-only group. The bars represent the mean ± SE of the mean (n = 4). (C) Relative Cd11c, Nos, Arg1, and Mrc1 mRNA expression levels in RAW 264.7 cells were analyzed. The results were normalized to Gapdh and presented relative to the RAW 264.7-only group. The bars represent the mean ± SE of the mean (n = 4).PSTE, peanut sprout tea extract; Mcp-1, monocyte chemoattractant protein-1; M-csf, macrophage-colony stimulating factor; Vegf, vascular endothelial growth factor; Nos, nitric oxide synthase; Arg1, arginase-1; Mrc1, mannose receptor 1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase.*P < 0.05, **P < 0.01, ***P < 0.001 significantly different from the 4T1 only group.†P < 0.05, ††P < 0.01, †††P < 0.001 significantly different from the co-culture group.‡P < 0.05, ‡‡P < 0.01, ‡‡‡P < 0.001 significantly different from the RAW 264.7 only group.

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  Fig. 6 Effects of PSTE on CD11c, MMR, M-CSF, and MCP-1 expression in 4T1 tumors in BALB/c mice. The mice were injected with 4T1 cells and treated with PSTE, as described in Fig. 2. The total RNA in the tumors was extracted, reverse-transcribed, and real-time PCR was conducted. Relative Cd11c, Mmr, M-csf, and Mcp-1 mRNA expression levels in the tumors were analyzed, and the results were normalized to that of Gapdh. The bars represent the mean ± SE of the mean (n = 5).PSTE, peanut sprout tea extract; Mmr, macrophage mannose receptor; M-csf, macrophage-colony stimulating factor; Mcp-1, monocyte chemoattractant protein-1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase, CON, vehicle (saline)-treated control group; PSTE30, 30 mg/kg body weight/day peanut sprout tea extract-treated group; PSTE60, 60 mg/kg body weight/day peanut sprout tea extract-treated group.The means without a common letter are different, P < 0.05.

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