Interaction of xanthoxyletin with protein, molecular docking and antitumor activity in human chronic myeloid leukemia K562 cells

Xanthoxyletin (C₁₅H₁₄O₄), a natural linear pyranocoumarin, is known to trigger anti-cancer activities, although its anticancer mechanisms are largely unknown. Human hemoglobin (HHb) as a major plasma protein could play a key role in the transportation and biodistribution of therapeutic molecules In this study, the interaction of xanthoxyletin with HHb was measured under physiological conditions using multi-spectroscopic techniques along with molecular docking simulation. Also, the interaction of xanthoxyletin with human chronic myeloid leukemia K562 cells was assessed by cellular assays. It was deduced that each xanthoxyletin molecule shows a thermodynamically favorable interaction with one molecule of HHb with a binding constant (K_b) in the orders of 10^3-4 M⁻¹. These interactions did not significantly result in the loss of α-helical content of HHb. In silico studies supported the experimental findings and demonstrated that non-covalent forces including hydrogen bonds (Ala88 and Arg92), pi-pi stacking (Trp37), and hydrophobic interactions (and Pro95 and Pro36) are the main forces contributing to the xanthoxyletin-HHb complex formation. Cellular and molecular assays showed that xanthoxyletin mitigated the proliferation of K562 cells through apoptosis induction mediated by overexpression of p53, caspase-3, and PARP cleavage. This data may hold great promise for the development of anticancer compounds with potential blood protein binding stemming from xanthoxyletin derivatives/hybrids.