Plumbagin promotes mitochondrial mediated apoptosis in gefitinib sensitive and resistant A549 lung cancer cell line through enhancing reactive oxygen species generation

Plumbagin (PL) is a natural naphthoquinone compound, isolated from Plumbago zeylanica that has cytotoxic and antimigratory potential in many cancer. However, the cytotoxic mechanism of plumbagin in drug resistant lung cancer is poorly understood. To reveal the mechanism, we studied the anticancer effect of plumbagin in both gefitinib-sensitive and resistant A549 lung cancer cells. The anticancer potential of PL was demonstrated by MTT assay and the result suggested that PL showed cytotoxicity in both gefitinib-sensitive (A549) and gefitinib-resistant (A549GR) lung cancer cells. IC₅₀ values of PL in A549 and A549GR were 3.2 μM and 4.5 μM, respectively. Morphological changes were also observed after treatment with PL. Furthermore, PL decreased cell survival by inhibiting colony formation ability, and inhibited cell migration at very low concentrations. From Annexin V-FITC/PI, AO/EtBr, and DAPI staining, we found that increasing concentration of PL leads to increase in apoptosis of lung cancer cells. Furthermore, western blotting results suggested that Bax and Caspase 3 levels were upregulated after PL treatment. In addition, treatment of PL caused DNA damage in a dose-dependent manner. PL arrested the cell cycle at S-G_2/M phase, and enhanced reactive oxygen species (ROS) generation. Excess ROS generated by PL disrupted mitochondrial membrane resulted in depletion of mitochondrial membrane potential (MMP). These results conclude that PL decreases lung cancer cell viability by arresting cells at S-G_2/M phase, and induces apoptosis by activation of mitochondrial-mediated apoptotic pathway through excess ROS generation. Overall findings suggest that plumbagin shows cytotoxic and therapeutic potential against both A549 and A549GR cell lines.

Keywords: Apoptosis; Drug resistance; Lung cancer; Mitochondrial membrane potential; Plumbagin; Reactive oxygen species.