Molecular and Thermodynamic Studies on DNA Triplex Formed in the Promoter Region of HMGB1 Gene as a Selective Target for Anticancer Drugs.

Abstract

HMGB1 (High Mobility Group Box-1) is a very versatile highly abundant architectural protein that plays multiple roles in human health and diseases. Under physiological condition it serves as an amazing assortment of roles in different compartments of cell. The reported high expression of HMGB1 in almost all types of human cancers and inflammatory diseases makes it a critical molecular therapeutic target. In the present study, we have mobilized a proximal twenty one base pair nucleotide (21RY) which is in the promoter region (-55 to-75) of hmgb1 gene and targeted it with triplex forming oligonucleotide (TFO) in combination with two widely used chemotherapeutic drugs, actinomycin (ACT) and adriamycin (ADM). The interaction of actinomycin and adriamycin to 21R*R•Y DNA triplex was studied using UV melting profiles, CD spectroscopy, spectrofluorimetry and Isothermal titration calorimetry. The 21R*R•Y formation was confirmed from biphasic thermal melting profiles, continuous variation method, analysis of CD marker band and thermodynamic parameters. The binding of ADM and ACT to 21R*R•Y was characterized by hypochromic and bathochromic shift in their respective absorption spectrum, quenching (ADM) and enhanced fluorescence (ACT) of steady-state fluorescence intensity, perturbation in the circular dichroic spectrum and change in thermal melting temperatures. The ITC profile and Scatchard plot analysis indicate non-cooperative and higher binding affinity of these drugs to 21R*R•Y compared to their corresponding duplexes. Therefore, combining these chemotherapeutic drugs with triplex forming oligonucleotide may offer new diagnostic and therapeutic options in targeting a gene of interest more specifically with fewer side effects. This study shows that ACT and ADM effectively recognize 21R*R•Y triplex DNA formed on the hmgb1 promoter region.