Thymol-rich Ajwain essential oil attenuates EMT-related features associated with the PTEN–AKT2 axis in MDA-MB-231 breast cancer cells

Epithelial-to-mesenchymal transition (EMT) has a fundamental role in carcinoma metastasis. The PI3K/AKT signaling pathway makes an imperative contribution to EMT progression, with PTEN and AKT being its main inhibitor and effector, respectively. AKT and PTEN function can be regulated by phosphorylation. Ajwain essential oil (AEO) is rich in thymol. Thymol is a natural compound with well-studied anticancer effects; however, data on its anti-metastatic potential remain limited. This study investigated the potential of AEO in reversing EMT linked to modulation of the PTEN–AKT2 axis, along with its anti-proliferative properties, in MDA-MB-231 cells. Chemical profiling of AEO by GC/MS was provided, followed by an evaluation of its anticancer features using MTT, colony formation, and flow cytometry. Moreover, attenuation of EMT properties and its effect on PTEN and AKT2 were assessed using wound-healing, Transwell/invasion, and Western blot assays. Phytochemical profiling of AEO identified thymol as the major constituent (≈ 43%). Treatment led to a dose-dependent decrease in proliferation and clonogenic potential. Aside from apoptosis induction, AEO also led to accumulation of cells at the G2/M phase and reduced their migration and invasion potential. In response to treatment, the protein abundance of p-PTEN (Thr366) and p-AKT2 (Ser474) was decreased without significant changes in total PTEN and AKT2. Moreover, N-cadherin and Snail were downregulated; conversely, E-cadherin was upregulated. Collectively, these findings suggest that thymol-rich AEO could attenuate EMT properties, associated with a decrease in p-PTEN (Thr366) and p-AKT2 (Ser474) in MDA-MB-231 cells. Therefore, thymol-rich AEO merits further studies on its anti-metastatic properties.