Disarming the Heat-Shock Response: Rosmarinic Acid Potentiates Mild Hyperthermia-Induced Apoptosis in A549 Non-Small Cell Lung Cancer Cells

This study investigated the biochemical mechanisms by which rosmarinic acid (RA), a natural polyphenol, sensitizes A549 non-small cell lung cancer (NSCLC) cells to mild hyperthermia (43°C). A549 cells were treated with RA (100 or 200 μM) and exposed to mild hyperthermia (43°C, 30 min), after which we performed a comprehensive analysis of cell viability, clonogenic potential, and migratory recovery. The molecular underpinnings were explored through flow cytometry for reactive oxygen species (ROS) and cell cycle progression, together with western blotting for MAPK signaling (JNK, ERK), apoptosis-related proteins (Bcl-xL, survivin, caspases), and the heat-shock response (HSP27, 70, 90, and HSF1). Our results show that the combination of RA and hyperthermia impaired cell proliferation and migration while promoting S-phase accumulation and apoptosis. At the biochemical level, combined RA and hyperthermia increased intracellular ROS beyond the level produced by either treatment alone. This rise in ROS occurred alongside an altered p-JNK/total JNK profile and caspase activation-associated apoptosis. Importantly, RA suppressed the canonical heat-shock response, lowering the p-HSF1/total HSF1 ratio and reducing the expression of the molecular chaperones HSP27, 70, and 90. Pretreatment with N-acetylcysteine (NAC) attenuated the observed ROS, Annexin V, and immunoblot changes, consistent with a contribution of redox imbalance to this sensitization process. In conclusion, RA enhanced hyperthermia-induced oxidative stress and suppressed the HSF1-HSP axis in A549 cells, providing a rationale for its further preclinical evaluation as a biochemical adjuvant in hyperthermic lung cancer therapy.