Abstract Carnosic acid (CA), a phenolic diterpene found in rosemary (Rosmarinus officinalis), has demonstrated anticancer activity in multiple tumor models; however, its mechanisms in non-small cell lung cancer (NSCLC) remain poorly defined. Many polyphenols exert biological effects through reactive oxygen species (ROS) generation and activation of apoptosis signal-regulating kinase 1 (ASK1)-mediated signalling, prompting this investigation into the role of ROS and ASK1 in the actions of CA. Cell viability was measured in NSCLC cells (H1299, A549, H460), and normal lung fibroblasts (MRC-5) following 48-hour CA treatment using the MTT assay. Migration of H1299 cells was assessed by scratch assay. ROS generation was quantified using dichlorodihydrofluorescin diacetate (DCFDA) fluorescence, and protein expression and activation were evaluated by western blotting and immunocytochemistry. CA decreased cell viability in a dose-dependent manner across NSCLC cell lines with IC50 in the micromolar range; H1299 showed the greatest sensitivity. In MRC-5 fibroblasts, maximal inhibition reached only ∼50% of control compared with complete inhibition of growth observed in the NSCLC cell lines. In H1299 cells, CA significantly impaired wound closure after 24 hours, comparable to inhibition by paclitaxel. CA increased intracellular ROS levels and activated the antioxidant response pathway, evidenced by nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulation of heme oxygenase-1 (HO-1). Western blotting demonstrated activation of ASK1 and downstream mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Pretreatment with N-acetylcysteine (NAC) attenuated CA-induced cytotoxicity, supporting a role for ROS in mediating these effects. In summary, carnosic acid inhibits viability and migration of NSCLC cells while partially sparing normal lung fibroblasts. Its effects involve ROS generation and activation of ASK1-dependent MAPK signaling. These findings provide mechanistic insight into the anticancer potential of CA in lung cancer. Future work will evaluate CA using an in vivo tumor xenograft model. Citation Format: Eric J. O'Neill, Evangelia Tsiani, . Carnosic acid induces ROS and activates ASK1 signalling in NSCLC cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3651.
O'Neill et al. (Fri,) studied this question.