In-cell western assay: a new approach to study the hypocholesterolemic effects of food components at HepG2 cell line

Cardiovascular disease, such as atherosclerosis and coronary heart disease, is a leading death cause in the developed world. Significant research efforts focusing on the prevention and treatment of this disease have identified elevated plasma cholesterol as primary risk factor for cardiovascular disease. In particular, the low density lipoprotein (LDL) receptor (LDLR) is a transmembrane glycoprotein that plays a pivotal role in the binding and endocytosis of circulating LDL increasing its plasma clearance. In literature, it is widely recognized that the LDLR pathway could be implicated in the hypocholesterolemic activity of several food components, such as plant proteins (soy [1,2], lupin [3,4]) and polyphenols (resveratrol [5]) via the activation of transcription factors and the expression of lipogenic enzymes. In particular, the transcription factor Sterol Regulatory Element Binding Protein (SREBP) Pathway is directly involved in the expression regulation of some lipogenic enzymes, such as LDLR and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase). The aim of the present work is the development of the In-Cell Western (ICW), a novel quantitative colorimetric cell-based technique, in order to screen and to evaluate in a throughput way the effects of lupin bioactive peptides on the LDLR-SREBP2 pathway. In particular, the ICW has been optimized and validated at HepG2 cell line using lovastatin as known reference compound. The ICW optimization is a multi step process in which different parameters and conditions have been standardized at HepG2 cells. Experiments have been performed in 96-well plate format using fixed cells in order to establish the right conditions in which lovastatin is able to properly stimulate LDLR. This optimized method will be used for the studying the effects of food components. Results demonstrated that the ICW optimization and validation have provided a robust, reproducible, and easy to use assay that will permit to study the functionality of plant peptides, such as lupin bioactive peptides through the detection and quantification of LDLR protein level directly in the HepG2 cells. References: 1) Lovati MR, Manzoni C, Gianazza E, Arnoldi A, Kurowska E, Carroll KK, and. Sirtori CR. Soy Protein Peptides Regulate Cholesterol Homeostasis in Hep G2 Cells. J Nutr. 2000;130:2543-2549. 2) Torres N, Torre-Villalvazo I, Tovar AR. Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders. J Nutr Biochem. 2006;17:365-73 3) Sirtori CR, Lovati MR, Manzoni C, Castiglioni S, Duranti M, Magni C, Morandi S, D'Agostina A, Arnoldi A. Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells. J Nutr. 2004;134:18-23. 4) Bettzieche A, Brandsch C, Weisse K, Hirche F, Eder K, Stangl GI. Lupin protein influences the expression of hepatic genes involved in fatty acid synthesis and triacylglycerol hydrolysis of adult rats. Br J Nutr. 2008;99:952-62. 5) Takuya Y., Manami N., Makoto S., Jun I., Ryuichiro S. Resveratrol increases the expression and activity of the low density lipoprotein receptor in hepatocytes by the proteolytic activation of the sterol regulatory element-binding proteins. Atherosclerosis 2012 ; 220:369-384