Co-medication effects of Doxorubicin and Bendamustine on DNA molecules ananlyized with a variety of single-molecule...

Combination drug therapy is an important strategy to reduce the side effects and enhance the positive effects of chemotherapy drugs. Doxorubicin (DOX) and Bendamustine (BENDA) are two widely used and effective chemotherapeutic agents for treating tumors by targeting DNA molecules, and recent clinical data have displayed that their combination therapy is efficacious; However, no systematic study has clarified the synergistic or competitive binding mechanism of these two drugs to DNA at the molecular level. In this study, cooperated with the surface-enhanced Raman spectroscopy, UV-Vis spectroscopy and molecular docking simulation, we systematically investigated the combined effects of DOX and BENDA on ctDNA at the single-molecule level. The results show that DOX primarily binds to the N7 site of adenine via hydrogen bonding, while BENDA interacts with the N3 site of the same adenine molecule, demonstrating a synergistic effect. In contrast, both DOX and BENDA compete for binding to the N3 and NH₂ sites of guanine, showing a competitive relationship. Moreover, the binding behavior is significantly influenced by the sequence of drug administration: when DOX is added before BENDA, BENDA can partially replace the previously bound DOX at its binding sites, and vice versa. Gel electrophoresis experiments showed that BENDA can induce DNA strand breaks, and this effect can be partially inhibited by DOX pre-bound to DNA. This study, for the first time, reveals the synergistic and competitive interaction mechanisms of DOX and BENDA on DNA at the single-molecule level, providing theoretical support and experimental evidence for optimizing combination therapies and designing low-toxicity, high-efficacy anticancer drugs.