Abstract
BackgroundBoth human and veterinary cancer chemotherapy are undergoing a paradigm shift from a “one size fits all” approach to more personalized, patient-oriented treatment strategies. Personalized chemotherapy is dependent on the identification and validation of biomarkers that can predict treatment outcome and/or risk of toxicity. Many cytotoxic chemotherapy agents, including doxorubicin, base their mechanism of action by interaction with DNA and disruption of normal cellular processes. We developed a high-resolution/accurate-mass liquid chromatography-mass spectrometry DNA screening approach for monitoring doxorubicin-induced DNA modifications (adducts) in vitro and in vivo. We used, for the first time, a new strategy involving the use of isotope-labeled DNA, which greatly facilitates adduct discovery. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology.ResultsWe used our novel mass spectrometry approach to screen for adducts in purified DNA exposed to doxorubicin. This initial in vitro screening identified nine potential doxorubicin-DNA adduct masses, as well as an intense signal corresponding to DNA-intercalated doxorubicin. Two of the adduct masses, together with doxorubicin and its metabolite doxorubicinol, were subsequently detected in vivo in liver DNA extracted from mice exposed to doxorubicin. Finally, the presence of these adducts and analytes was explored in the DNA isolated from dogs undergoing treatment with doxorubicin. The previously identified nine DOX-DNA adducts were not detected in these preliminary three samples collected seven days post-treatment, however intercalated doxorubicin and doxorubicinol were detected.ConclusionsThis work sets the stage for future evaluation of doxorubicin-DNA adducts and doxorubicin-related molecules as candidate biomarkers to personalize chemotherapy protocols for canine cancer patients. It demonstrates our ability to combine in one method the analysis of DNA adducts and DNA-intercalated doxorubicin and doxorubicinol. The last two analytes interestingly, were persistent in samples from canine patients undergoing doxorubicin chemotherapy seven days after treatment. The presence of doxorubicin in all samples suggests a role for it as a promising biomarker for use in veterinary chemotherapy. Future studies will involve the analysis of more samples from canine cancer patients to elucidate optimal timepoints for monitoring intercalated doxorubicin and doxorubicin-DNA adducts and the correlation of these markers with therapy outcome.
Highlights
Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a “one size fits all” approach to more personalized, patient-oriented treatment strategies
We previously developed a nanoLC-MS3 DNA adductomics approach that allows for the screening of potentially every adduct in a hydrolyzed DNA sample
Screening for DOX‐DNA adducts in vitro Initial screening for DOX-DNA adducts was performed by reacting DOX in the presence of formaldehyde with DNA from calf thymus (CT-DNA) and with DNA extracted from E.coli bacteria
Summary
Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a “one size fits all” approach to more personalized, patient-oriented treatment strategies. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology. Genetic variations in patients can result in different responses to therapy and varying degrees of toxicity, despite phenotypically similar diseases [2, 3]. For these reasons, cancer chemotherapy is currently shifting from the concept of “one size fits all” to more personalized, patient-oriented approaches, with the goal of optimizing individual therapeutic protocols to increase treatment success and/or decrease undesired side effects [1]. A drug dose or a combination drug protocol may be adapted as a result of biomarker measurement to allow for less unwanted side effects without compromising treatment success
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