6-Methoxylated Flavonoids: Jacein, and 3-demethyljacein from Centaurea schmidii with Their Endoplasmic Reticulum Stress and Apoptotic Cell Death in Breast Cancer Cells Along with In-silico Analysis

In phytochemical analysis, Jacein derivatives: 5,7,4''-trihydroxy-3,6,3''-trimethoxyflavone-7(β)-D-glucopyranoside (1), and 3-demethyljacein: 3,5,7,4''-tetrahydroxy-6,3''-dimethoxyflavone-7(β)-D-glucopyranoside (2) were isolated from Campylopus schmidii (C. schmidii) for the first time. The structures were determined by interpretation of NMR, UV, and Mass spectra. To check the roles of ER stress and consequent apoptosis in MCF-7 cell by these compounds, UPR signaling pathway was further examined by analysis of expression of ER stress-related genes. In MTT assay, compounds 1-2 showed cytotoxicity activity against MCF-7 (A) and MDA-MB cells (B) with IC50 values (μM) of 1) 60.04 ± 7.98 (A), and > 200 (B); 2) 42.89 ± 1.91 (A), and 85.31 ± 2.68 (B). The Annexin/PI flow cytometry apoptosis of tested compounds 1-2 was increased significantly in a dose-dependent manner. For example, MCF-7 treatment at the concentration of 100 μM of compounds 1, 2 resulted in total apoptosis (early + late) of 42.04 (18.1 + 24.0), and 66.49 (2.7 + 63.8)%, respectively.  Fluorescence microscopy analysis detected an increased protein aggregation, indicating induced ER stress with a marked increase in XBP-1, sXBP-1, ATF-4, and CHoP compared to untreated cells. In-silico characterization, suggested that Adenosine diphosphate site (A-site) and quercetin site (Q-Site) in IRE1a enzyme are both available interacting sites of a target for the investigated ligands but with different strengths of interactions. The results indicated that the ligand∼A-Site complexes are stronger than the ligand∼Q-Site complexes, but the already available ADP ligand in cells does not allow other ligands to interact with the A-Site and cause them to bond in Q-Site.