Osteogenic bioactivity and oestrogen growth factor response induced by extracts from Ceratonia siliqua

Abstract

The main aim of the study was the production of an extract from a local indigenous plant that induces the differentiation of the osteoblast cell line MC3T3-El without inducing an estrogen growth factor response in the breast cancer cell line MCF-7, therefore being a suitable candidate for the substitution of the currently utilized hormone replacement therapy. A wide range of different methodologies have been used. Methanol extraction has been utilized for the extraction process. Titrations were performed so as to obtain the working concentrations for 17 β-oestradiol, the extracts, and a commercialized product (Lignan, Brevail®). To determine the cytotoxicity, XTT assays were performed. Alizarin Red staining measurements were utilized to directly measure the osteogenesis rate. Real time-PCR was utilized for expression studies of identified oestradiol response genes.

The working concentrations of the test compounds have been identified and XTT assays indicated us that none of the test compounds tested (17β-oestradiol 10-4 M, lignan 5000 ppm, and carob pod extract 32750 ppm, carob flower extract 6000 ppm) was cytotoxic in the highest concentrations tested. The osteogenesis rate has been quantified and after 16 days, a 30-fold increase in osteogenesis has been obtained with lignan and a 20-fold increase in osteogenesis has been obtained with carob flower extract, when compared with osteogenesis induction medium.

For the investigation of oestrogen growth factor response on the MCF-7 breast cancer cell line, 241 direct ERα target genes were selected utilizing a published dataset and utilizing another published dataset, 11 genes from the previous 241 were selected that contain estrogen response elements (EREs). Utilizing current literature and the involvement in breast cancer, a total of 3 out of 11 genes were selected; gene regulated by estrogen in breast cancer protein (GREBl), nuclear receptor interacting protein 1 (NRIPl), and insulin-like growth factor binding protein 4 (Igfbp4). Whilst ERα expression is downregulated in MCF-7 cells at both time points in all the test compounds utilized, expression of NRIPl and Igfbp4 is transient with the carob flower extract. Thus we concluded that the carob flower extract, is a potential candidate for the substitution of the currently utilized hormone replacement therapy.