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

Małgorzata Zakłos-Szyda,Grażyna Budryn

doi:10.3390/nu12010257

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.

Highlights

Cancer cells’ uncontrolled growth, and their migration and invasion abilities, are commonly found in malignant tumors
Extracts obtained from fermented sprouts at IC0 dosages were able to inhibit migration of breast cancer cells through their influence on intracellular Reactive Oxygen Species (ROS) generation; membrane stiffening; adhesion; regulation of membrane potential (MMP)-9, N-cadherin and
The phenolic composition of red clover (Trifolium pratense L.) sprouts was described in detail previously in [10,12,13]

Summary

Introduction

Cancer cells’ uncontrolled growth, and their migration and invasion abilities, are commonly found in malignant tumors. Estrogen receptor β (ERβ), as a member of the nuclear receptor superfamily, shows potent tumor suppressive activities in many cancers [1]. Estrogens such as 17 β-estradiol work as natural ligands for estrogen receptors (ER)—proteins belonging to the nuclear receptor superfamily. Despite the classical or genomic action of steroid receptors, they possess non-genomic rapid activity via upstream interactions with signaling or scaffold proteins controlling cell cycle, proliferation, migration or nuclear exclusion of steroid receptors [3,4]

Methods

Results

Conclusion