title: Investigating the molecular mechanisms driving NRF2- mediated protection against doxorubicin-induced cardiotoxicity
Creators: name: Roberts, James
Publisher: name: University of Liverpool Repository
Year: 2026

description: Drug-induced cardiotoxicity presents a severe clinical challenge and cause for attrition 
during drug development and post-marketing surveillance. One of the most notorious 
examples of cardiotoxicity arises following treatment with the anthracycline 
chemotherapeutic doxorubicin (DOX), a potent drug for the treatment of many solid 
tumours, haematological cancers and childhood malignancies. The mechanism of 
both anti-cancer activity and cardiotoxic adverse events are highly multifaceted; 
despite clinical utility for &gt; 50 years, they have yet to be completely elucidated. Recent 
evidence has suggested that stimulation of the nuclear factor erythroid 2-related factor 
2 (NRF2) pathway, which drives the expression of a cytoprotective gene battery, can 
alleviate DOX-induced cardiotoxicity in vitro and in vivo.
Within this project, the human cardiomyocyte-like AC16 cell line was utilised to 
investigate the dynamic responses of NRF2 to pharmacological and genetic induction 
and assess the functional consequences for sensitivity to DOX. In agreement with 
previously published data, it was found that upregulated NRF2 activity, notably 
following treatment with the clinically relevant triterpenoid 2-cyano-3,12-dioxoolean 1,9-dien-28-oic acid (CDDO)-Me, herein referred to as CDDO, improved cell viability 
and reduced AC16 cytotoxicity. Moreover, CDDO was not advantageous to cancer cell 
lines of lung or pancreatic lineage which is pertinent given the potential oncological 
roles of NRF2. The mechanisms of both DOX-induced toxicity and CDDO-mediated 
cytoprotection were explored in greater detail primarily through transcriptomic 
profiling. Here, it was found that DOX promoted multiple cell death pathways including 
apoptosis, necrosis and pyroptosis with stark inhibition of cell cycle and topoisomerase 
II, supporting the known canonical pathway aberrations. Treatment of AC16 cells with 
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CDDO decreased interleukin (IL)-1β, IL-6 and VEGFA mRNA expression to ablate 
cardiomyocyte apoptosis and cell death. NRF2 pathway-specificity of CDDO was 
determined at &gt; 73% through analysis of known NRF2 transcriptional targets with 72 
potentially novel targets identified. Finally, AC16 cells cultured in physiologically 
relevant glucose concentrations as opposed to the typically hyperglycaemic conditions 
significantly decreased gene transcription profiles associated with cardiomyocyte cell 
death and apoptosis. Activation of the NRF2 pathway, likely caused by mild stress 
induced by decreased glucose availability, may have primed cells for better adaptation 
to DOX toxicity via hormesis. 
Taken together, data from this project has extensively evaluated the AC16 human cell 
line as a capable in vitro model for DOX-induced cardiotoxicity, characterised NRF2 
dynamics in cardiomyocytes and supported the hypothesis that this transcription factor 
can impact sensitivity to cytotoxic insults. Furthermore, the transcriptomic data will 
serve as a valuable resource for the elucidation of specific cellular responses to DOX 
cardiotoxicity.