Evaluation of Isoflavones as Bone Resorption Inhibitors upon Interactions with Receptor Activator of Nuclear Factor-κB Ligand (RANKL)

Abstract

Receptor activator of nuclear factor-κB ligand (RANKL) is a cytokine responsible for bone resorption. It binds its receptor RANK, which activates osteoporosis. High levels of osteoprotegerin (OPG) competitively binding RANKL limit formation of ligand-receptor complexes and enable bone mass maintenance. The new approach to prevent osteoporosis is searching for therapeutics that can bind RANKL and support OPG function. The aim of the study was to verify the hypothesis that isoflavones can form complexes with RANKL limiting binding of the cytokine to its receptor. Interactions of five isoflavones with RANKL were investigated by isothermal titration calorimetry (ITC), by in silico docking simulation and on Saos-2 cells. Daidzein and biochanin A showed the highest affinity for RANKL. Among studied isoflavones coumestrol, formononetin and biochanin A showed the highest potential for Saos-2 mineralization and were able to regulate the expression of RANKL and OPG at the mRNA levels, as well as osteogenic differentiation markers: alkaline phosphatase (ALP), collagen type 1, and Runt-related transcription factor 2 (Runx2). Comparison of the osteogenic activities of isoflavones showed that the use of physicochemical techniques such as ITC or in silico docking are good tools for the initial selection of substances showing a specific bioactivity.

Viburnum opulus L. Juice Phenolic Compounds Influence Osteogenic Differentiation in Human Osteosarcoma Saos-2 Cells

“…Thus, our effective dose of purified juice (25 mg/L) contained 16 mg/L chlorogenic acid. Additionally, V. opulus phenolics' binding affinity to human serum albumin may effectively regulate their bioavailability and cellular metabolism [22,30]. Data indicate that phenolic compounds, as well as chlorogenic acids, are metabolized by gut microbiota, and then their aglycons and other metabolites are absorbed in the small intestine by passive diffusion or with transporters [62].…”

“…After 8 days of incubation with V. opulus samples, the Saos-2 cells were washed with PBS and fixed with 5% formaldehyde solution for 30 min at room temperature [22,76]. After cell rinsing with water-staining, a solution of 1% alizarin red S in 2% ethanol (pH 4.0) was added for 30 min.…”

Cicer arietinum L. Sprouts’ Influence on Mineralization of Saos-2 and Migration of MCF-7 Cells

“…Based on the obtained results, it could be hypothesized that the high biological activity of the extracts obtained from fermented sprouts was connected with the presence of large amounts of coumestrol, formononetin and biochanin A. The pro-osteogenic potential of these isoflavonoids was associated with the activation of ALP, as well as the upregulation of RUNX2 and COL1A mRNA levels in Saos-2 cells [ 29 ].…”

“…Bone and breast tissues rely on estrogen, which stimulates bone mineralization but, at the same time, may induce breast malignancy in excess [ 19 ]. MCF-7 human breast-cancer cells are known to express both isoforms of estrogen receptors (ERα and ERβ) [ 29 ], and were thus used in a proliferation experiment to evaluate the estrogenic potency of C. arietinum extracts. Taking into account that bone is one of the most common metastatic sites for breast cancer [ 20 ], the effect of the C. arietinum isoflavones on MCF-7 cell migration was assessed.…”

“…Furthermore, a diet rich in phytoestrogens (especially soy isoflavones) has been documented as preventive for postmenopausal osteoporosis and breast cancer [ 17 , 47 ]. In this regard, it was shown that biochanin A, one of the components of C. arietinum sprouts, may decrease osteoclast activation, osteoporosis and mammary carcinogenesis via interaction with RANKL [ 29 , 44 , 48 ].…”