Dammarolic acid improves tyrosine kinase inhibitor activity in lung cancer cells.

Lung cancer is a leading global health concern, ranking as the second-highest cause of cancer-related deaths. Tyrosine kinase inhibitors (TKIs) such sorafenib or regorafenib are considered the second-line drugs to treat advanced lung cancer. At advanced stages of lung cancer, the efficacy of TKIs gets reduced and does not produce prolonged effects. Thus, there is a need to discover new ways to increase the efficacy of TKIs for better outcomes. In the present study, synergistic approaches are utilized to improve the anticancer activity of TKIs. We investigated the synergisitic potential of dammarolic acid (DA) with sorafenib in lung cancer cell lines. DA, a triterpenoid derived from Centella asiatica, has demonstrated various medicinal properties. This study explores the combined effects of DA and sorafenib on lung cancer cells focusing on their synergistic mechanisms. To assess this synergy, the half-maximal inhibitory concentration (IC) of both agents was measured using the MTT assay on lung cancer cell lines. The Chou-Talalay method was applied to calculate synergy parameters and identify effective minimum concentrations. Following this, minimally effective doses of DA and sorafenib were tested to further evaluate cytotoxicity and synergistic potential. Flow cytometric  analyses were conducted to measure reactive oxygen species (ROS), mitochondrial membrane potential (MMP), annexin V binding, and cell cycle distribution. Western blotting evaluated autophagy markers LC3-II and p62. Findings indicate that increasing DA concentrations significantly reduced the IC and Dose Reduction Index (DRI) values of sorafenib. The co-treatment elevated ROS, disrupted MMP, and increased lung cancer cell cytotoxicity. Overexpression of p62 and LC3-II suggested autophagic inhibition and late apoptosis, confirmed through ATG5 knockdown experiments. Notably, this combination effectively targeted metastatic lung cancer cells by arresting them in the G0/G1 phase, promoting late-stage apoptosis and inhibiting autophagy in metastatic-prone NSCLC cells.