A New Method To Assay Estrogen Receptor: Probing Into Its Expression Related To Phosphatidylinositol3-Kinase Pathway In Breast Cancer Cells

Objective：Emerging evidence implies that estrogen receptor (ER) and phosphatidylinositol3-kinase (PI3K) pathway play central roles in both breast cancer progression andresponse to therapy. The measurement of ER and the investigation of the link betweenER level and PI3K pathway will be of great help to fully understand the underlyingmechanisms and evaluate the effect of therapeutic interventions. Here, we present anovel electrochemical method to assay ER, and explore the relationship between ERand PI3K pathway in ER positive breast cancer.Methods:We present a novel electrochemical method to assay ER by using anExonuclease III protection-based strategy. First, two complementary DNA (P1/P2)strands incorporating the EREs are firstly prepared. Among them, P1is thiolated toensure self-assembly on the gold electrode surface, while P2is modified with MB toserve as an electroactive signaling probe. Hybridization of P1and P2grafts the P2strand to be immobilized on the electrode surface, forming a stable duplex with a3’-blunt end at the signaling strand. If there is ER in the test system, the digestion ofthe exonuclease will be blocked by the formation of ER-DNA complex due to thesignificant steric hindrance. Since more ER will protect more P2from digesting,resulting in an increased electrochemical signal, relationship between the ERconcentration and the obtained electrochemical signal can be established. To furtherexplore the relationship between the ER expression and PI3K pathway inhibition, we have interrogated the ER levels in nuclear extracts from BT474cells treated withdifferent concentrations of PI3K inhibitor BEZ-235.Results：1. It can be observed that a signal increase with increasing ER concentration. Alinear correlation of the peak currents versus the ER concentrations can be obtained inthe range from0.5to100nM with a detection limit of0.38nM.2. MDA-MB-231cell extracts produce only a small peak; while BT474andMCF-7cell extracts produce significant signals.3. The SWV peaks increased along with the number of MCF-7cells4. Treatment of BT474breast cancer cells with PI3K inhibitor BEZ-235for6hours can cause a significant increased signal, compared with that of the controlexperiments by the treatment with DMSO. A dose-dependent increased signal can beobserved.Conclusions：1. We have designed an electrochemical biosensor so as to propose a newmethod for ER detection. The sensitivity of the proposed method had improved by65-fold as compared with the previously reported metal nanoparticle-basedcolorimetric assay.2. The method to be directly used in cell nuclear extracts, disclosing that themethod can be used to monitor the expression level of ER in cancer cells.3. PI3K inhibition may raise total nuclear ER concentration, here is an intimatelink between ER and PI3K pathways.