Synergistic Action of Thymol-citral is Associated with Cell Cycle Arrest and Intracellular ROS Generation in A549 Cells

Objective: NSCLC is the predominant form of lung cancer, often exhibiting resistance to chemotherapy. Thymol and citral have shown promise as anticancer agents in different cancer cell lines but have not been evaluated in combination against NSCLC. Hence, we planned to investigate the anticancer effect of thymol-citral combination and explore its mechanisms of action against A549 cells. Methods: A549 cells were exposed to varying concentrations of thymol and citral, alone and in combination. Cell proliferation, plasma membrane integrity, apoptotic markers, reactive oxygen species (ROS) levels, cell cycle distribution, senescence induction, and migration potential were assessed. Additionally, in vitro safety was evaluated in human bronchial epithelial cells (HBECs) and human red blood cells (RBCs). Results: Thymol and citral showed synergistic action against A549 cells, with a CI value of 0.75. After 24 h, they induced apoptosis, caused G0/G1 phase arrest, and increased ROS levels, suggesting oxidative stress as the mechanism. This combination also induced cell senescence, significantly inhibited A549 cell migration, and was non-toxic to human RBCs and HBECs. Conclusion: Overall, the thymol-citral synergistic combination was found to be a safe and effective therapy option for non-small cell lung cancer.