BIOLOGICAL INVESTIGATION OF H2O2-ACTIVATED DNA CROSS-LINKING AGENTS: SAFETY, EFFICACY, MECHANISM, AND COMBINATION THERAPIES

Abstract

Developing targeted anticancer treatments with minimal side effects remains a significant challenge in oncology. One promising strategy is to exploit the unique biochemical changes, such as oxidative stress, present in cancer cells. Reactive oxygen species (ROS)-responsive prodrugs activated by hydrogen peroxide (H₂O₂) have demonstrated potential; however, the variability in ROS levels among different cancer cells can limit the effectiveness of these prodrugs. To address this issue, we propose a novel approach that combines ROS-activated prodrugs with prooxidants designed to increase oxidative stress specifically in cancer cells. Our research focused on the combination of vitamin C (vitC) and H₂O₂-activated alkylating agents. Through extensive screening of various prooxidants, we discovered that vitC, when combined with an H₂O₂-activated alkylating agent, selectively targets cancer cells while sparing normal cells. VitC induces high levels of H₂O₂ within cancer cells, thereby activating the alkylating agents, which in turn amplify ROS levels and induce DNA damage. This cascade effect triggers extensive cancer cell death via a p53-mediated apoptosis pathway.VitC effectively sensitizes cancer cells to the H₂O₂-activated prodrug, allowing for lower therapeutic doses and enhanced anticancer effects. This selectivity is attributed to the differential catalase activity between cancer and normal cells: cancer cells exhibit low catalase activity, leading to H₂O₂ accumulation and subsequent prodrug activation, whereas normal cells possess high catalase activity, protecting them from oxidative damage. In our in vitro experiments, vitC demonstrated high selectivity towards cancer cells, with IC50 values ranging from 1.0 to 2.3 mM, compared to IC50 values of 2.7 to 4.2 mM for normal cells, indicating its potent cytotoxic effects were reserved for cancer cells. Specifically, VitC’s remarkable selectivity towards cancer cells reduced the prodrug's IC50 value by 6-fold, 4-fold, 3-fold, and 2-fold for MDA-MB-468, MDA-MB-231, MCF7, and U87, respectively. In vivo studies demonstrated the synergistic anticancer effects and selectivity of this combination therapy. Toxicity tests revealed that high doses of vitC (up to 4 g/kg) and the prodrug (up to 10 mg/kg) were well-tolerated in mice. Importantly, safe doses of vitC (500 mg/kg) and the prodrug (3 mg/kg) significantly reduced tumor size with no observable side effects. The efficacy of this therapy was closely correlated with the increased ROS levels in tumors. This novel combination therapy has shown remarkable efficacy, particularly against cancers with poor prognosis and limited treatment options, such as triple-negative breast cancers and glioblastoma. These findings underscore the potential of combining vitC with H₂O₂-activated prodrugs for selective, effective cancer treatments with reduced adverse effects, paving the way for new therapeutic strategies in oncology. The second part of this thesis focuses on the cytotoxicity and cellular DNA damage screening of H₂O₂-responsive theranostic prodrugs. It also explores the UV-induced cytotoxicity and selectivity of 4,4’-dibromobinaphthalene analogues, as well as anthryl trimethyl ammonium and triphenylphosphonium salts.