In-Cell Western Assay: a tool to study the hypocholesterolemic effects of potentially bioactive food components at HepG2 cell line

Since reducing time and saving money are very important issues while developing innovative functional foods and nutraceutics, it is crucial to use specific and highly throughput assays for the fast screening of potentially bioactive ingredients. The In-Cell Western (ICW) assay, a quantitative colorimetric cell-based technique, has all these characteristics. Having the final goal of sorting out novel hypocholesterolemic food components, the aim of the present investigation was the development of the In-Cell Western (ICW) at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds at the low density lipoprotein (LDL) receptor (LDLR). This receptor plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimized and validated by using monacolin K, the main hypocholesterolemic component of red yeast rice. Experiments were performed using fixed cells in order to establish the best conditions for a proper stimulation of the LDLR protein level. Subsequently, the ICW was used to investigate the activity of peptide mixtures obtained by pepsin or trypsin hydrolysis of lupin protein. The ICW optimization and validation provided a robust and reproducible assay useful for characterizing the cholesterol-lowering properties of bioactive food components. It is important to underline that this is the first application of the ICW technique in the field of functional foods and nutraceutics.