Evaluating binding behavior of quercetin to human serum albumin and calf thymus DNA: Insights from molecular dynamics, spectroscopy, and apoptotic pathway regulation

Abstract

In this work, we sought to apprehend quercetin binding affinity and its interaction behavior in complex with human serum albumin (HSA) and calf thymus DNA (ctDNA) through multi spectroscopy and molecular dynamics and also evaluated its effects on colorectal cancer. The binding constants of ctDNA-quercetin and HSA-quercetin complexes at 298 K, which were calculated to be (2.67 ± 0.04) × 10³ M^-1 and (4.77 ± 0.05) × 10⁴ M^-1 respectively, denoted the strong binding of quercetin with ctDNA and HSA. The K_sv and K_b values decrease with increasing temperature, indicating that the quenching of HSA and ctDNA in the presence of quercetin is caused by the combined dynamic and static effects. The obtained thermodynamic parameters for the ctDNA-quercetin interaction represented the existence of electrostatic forces (ΔH⁰ < 0 and ΔS⁰ > 0), and the thermodynamic parameters of HSA-quercetin complex disclose the dominance of hydrogen bonds and van der Waals interactions (ΔH⁰ < 0 and ΔS⁰ < 0). Moreover, the interactions were exothermic, as evidenced by the negative ΔH⁰ value for both interactions. According to molecular docking and MD simulation data, quercetin was capable of placing into the site 1 of HSA and forming stable interaction plus this ligand tended to unwind DNA's strands as an intercalator ligand, which was confirmed by experimental results. The fluorescence competition studies between the two intercalator probes of ethidium bromide (EB) and acridine orange (AO), as well as the effect of ionic strength, proposed the strong tendency of quercetin to exist between the two strands of ctDNA as a sign of its intercalative property. Consequently, quercetin can be assumed as an efficient intercalator ligand carried by HSA with an anticancer property. We also conducted cell viability experiments on HT-29 and SW620 cell lines to validate the anticancer ability of quercetin, and observed its decreasing impact on the cell viability of these two cell lines. Additionally, the outcomes of Real-time qPCR proved its capability to reduce the CXCR4 expression and increase the NKD2 expression in Wnt signaling pathway. Therefore, these facts confirm the inhibiting ability of quercetin towards colorectal cancer growth via the prevention of Wnt pathway and approve its functionality as a potential anticancer agent for this cancer.

Keywords: HSA; MTT assay; Molecular dynamics; Quercetin; RT-qPCR; Spectroscopy; ctDNA.