In-vitro evaluation of apoptotic effect of OEO and thymol in 2D and 3D cell cultures and the study of their interaction mode with DNA

Oliveria decumbens is an Iranian endemic plant used extensively in traditional medicine. Recently, some studies have been performed on biological effects of Oliveria essential oil (OEO). However, to our knowledge, the anticancer activity of OEO has not been reported. Based on our GC/MS analysis, the basic ingredients of OEO are thymol, carvacrol, p-cymene and γ-terpinene. Therefore, we used OEO and its main component, thymol, to explore their effects on cell growth inhibition and anticancer activity. Despite having a limited effect on L929 normal cells, OEO/thymol induced cytotoxicity in MDA-MB231 breast cancer monolayers (2D) and to a lesser extent in MDA-MB231 spheroids (3D). Flow cytometry, caspase-3 activity assay in treated monolayers/spheroids and also fluorescence staining and DNA fragmentation in treated monolayers demonstrated apoptotic death mode. Indeed, OEO/thymol increased the Reactive Oxygen Species (ROS) level leading to mitochondrial membrane potential (MMP, ΔΨm) loss, caspase-3 activation and DNA damage caused S-phase cell cycle arrest. Furthermore, immunoblotting studies revealed the activation of intrinsic and maybe extrinsic apoptosis pathways by OEO/thymol. Additionally, in-vitro experiments, indicated that OEO/thymol interacts with DNA via minor grooves confirmed by docking method. Altogether, our reports underlined the potential of OEO to be considered as a new candidate for cancer therapy.