The Effects of Trifolium pratense L. Sprouts' Phenolic Compounds on Cell Growth and Migration of MDA-MB-231, MCF-7 and HUVEC Cells

Abstract

Uncontrolled growth and migration and invasion abilities are common for cancer cells in malignant tumors with low therapeutic effectiveness and high mortality and morbidity. Estrogen receptor β (ERβ), as a member of the nuclear receptor superfamily, shows potent tumor suppressive activities in many cancers. Phytoestrogens' structural resemblance to 17 β-estradiol allows their binding to ERβ isoform predominantly, and therefore, expression of genes connected with elevated proliferation, motility and invasiveness of cancer cells may be downregulated. Among polyphenolic compounds with phytoestrogenic activity, there are isoflavones from Trifolium pratense L. (red clover) sprouts, containing high amounts of formononetin and biochanin A and their glycosides. To determine the source of the most biologically active isoflavones, we obtained four extracts from sprouts before and after their lactic fermentation and/or β-glucosidase treatment. Our previous results of ITC (isothermal titration calorimetry) modelling and a docking simulation showed clover isoflavones' affinity to ERβ binding, which may downregulate cancer cell proliferation and migration. Thus, the biological activity of T. pratense sprouts' extracts was checked under in vitro conditions against highly invasive human breast cancer cell line MDA-MB-231 and non-invasive human breast cancer cell line MCF-7 cells. To compare extracts' activities acquired for cancer cells with those activities against normal cells, as a third model we choose human umbilical vein endothelial cells (HUVEC), which, due to their migration abilities, are involved in blood vessel formation. Extracts obtained from fermented sprouts at IC₀ dosages were able to inhibit migration of breast cancer cells through their influence on intracellular ROS generation; membrane stiffening; adhesion; regulation of MMP-9, N-cadherin and E-cadherin at transcriptional level; or VEGF secretion. Simultaneously, isolated phenolics revealed no toxicity against normal HUVEC cells. In the manuscript, we proposed a preliminary mechanism accounting for the in vitro activity of Trifolium pratense L. isoflavones. In this manner, T. pratense sprouts, especially after their lactic fermentation, can be considered a potent source of biological active phytoestrogens and a dietary supplement with anti-cancer and anti-invasion properties.

Keywords: Trifolium pratense L.; breast cancer; estrogen receptors; isoflavones; migration.

Figure 1

The influence of T. pratense extracts on MDA-MB-231 (A), MCF-7 (B) and HUVEC (C) cells’ metabolic activity determined by PrestoBlue assay after 48 h exposure to CU (sprouts polyphenolic extract), CUH (sprouts polyphenolic extract after hydrolysis), CUF (fermented sprouts polyphenolic extract) and CUFH (fermented sprouts polyphenolic extract after hydrolysis). Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations from at least three independent experiments (n ≥ 16).

Figure 2

The influence of 48 h of exposure of T. pratense extracts on phosphatidylserine externalization on the outer membrane leaflet of apoptotic cells and membrane permeabilization detected with Annexin-V in MDA-MB-231 (A), MCF-7 (B) and HUVEC (C) cells. Values are means ± standard deviations, n ≥ 6; statistical significance was calculated versus control cells (untreated); * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

The effect of T. pratense extracts on intracellular ROS generation analyzed by DCFH-DA assay after 48 h incubation with MDA-MB-231 (A), MCF-7 (B) and HUVEC (C) cells. Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations from at least three independent experiments; statistical significance was calculated versus control cells (untreated); *** p ≤ 0.001.

Figure 4

Changes in cell membrane fluidity expressed as values of generalized polarization (GP) for Laurdan probe after incubation of cells with T. pratense at IC₀ (A). The effect of T. pratense extracts on mitochondrial membrane potential was determined with JC-1 probe. As a positive control for depolarization, carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (50 μM) was used (B). control cells were not exposed to any compound but the vehicle; values are means ± standard deviations from at least three independent experiments; statistical significance was calculated versus control cells (untreated); * p ≤ 0.05, *** p ≤ 0.001.

Figure 5

The influence of 48 h of exposure of T. pratense extracts on the migration (A) and wound healing (B) of MDA-MB-231 cells. Migration rates of MDA-MB-231 cells incubated with extracts at IC₀ dosages into the free detection zones were photographed (×8) (C). Cell adhesion to the substrate was measured after staining with crystal violet; randomly chosen fields were photographed at ×200 (D). Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations from at least three independent experiments; statistical significance was calculated versus control cells (untreated); ** p ≤ 0.01, *** p ≤ 0.001.

Figure 6

The influence of 48 h of exposure of T. pratense extracts on the wound healing (A) and migration (B) of MCF-7 cells. Migration rates of MCF-7 cells incubated with extracts at IC₀ dosages into the free detection zones were photographed (×8) (C). Cell adhesion to the substrate was measured after staining with crystal violet; randomly chosen fields were photographed at ×200 (D). Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations from at least three independent experiments; statistical significance was calculated versus control cells (untreated); ** p ≤ 0.01, *** p ≤ 0.001.

Figure 7

The influence of 48 h of exposure of T. pratense extracts on wound healing (A) and migration (B) of HUVEC cells. Migration rates of HUVEC cells incubated with extracts at IC₀ dosages into the free detection zones were photographed (×8) (C). Cells adhesion to the substrate was measured after staining with crystal violet; randomly chosen fields were photographed at ×200 (D). Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations from at least three independent experiments; statistical significance was calculated versus control cells (untreated); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 8

The influence of 48 h of exposure to T. pratense extracts on the expression of genes in MDA-MB-231 (A), MCF-7 (B) and HUVEC (C) cells. The expression levels of MMP-9, MMM-2, ERα, ERβ, E-cadherin, and N-cadherin were quantified by real-time PCR and normalized using hypoxanthine phosphoribosyltransferase 1 (HPRT1) as a reference gene. Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations, n ≥ 3; statistical significance was calculated versus control cells (untreated) * p ≤ 0.05, ** p ≤ 0.01.

Figure 9

Analysis of relative ERα and ERβ expressions at the mRNA (A) and protein levels (B) in MDA-MB-231 and MCF-7 cells (samples were calibrated by MDA-MB-231 ERβ). The influence of 48 h of exposure to T. pratense extracts on the expression of estrogen receptor proteins in MDA-MB-231 (C) and MCF-7 (D) cells—bands of western blot representative experiment. The expression levels of ERα and ERβ were quantified by western blot and normalized using β actin as a reference protein. Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations, n = 3. (E) Relative proliferative effects (RPE) of extracts and 17 β-estradiol on proliferative estrogenic activity (RPE) properties according to E-screen assay (n ≥ 8).

Figure 10

The influence of 48 h of exposure of T. pratense extracts at IC₀ dosages on VEGF secretion and VEGF mRNA level in MDA-MB-231 (A), MCF-7 (B) and HUVEC cells (C). Control cells were not exposed to any compound but the vehicle; values are means ± standard deviations, n = 3; statistical significance was calculated versus control cells (untreated) * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 11

Trifolium pratense isoflavones as modulators of cell migration—the proposed mechanism of action. They possess cytoprotective activity against ROS generation, stiffen cellular membrane, induce apoptotic type cell death and influence expression of MMP9 and VEGF. ERα/β—estrogen receptor α/β; ERE—estrogen responsive element; MMP9—matrix metalloproteinase 9; VEGF—vascular endothelial growth factor.