Laryngeal squamous cell carcinoma (LSCC) remains a significant global health challenge, showing minimal improvement in survival rates even with advancements in standard treatments. The growing interest in phytochemicals as potential anticancer agents has highlighted Alpha-tomatine (AT) a steroidal glycoalkaloid derived from tomato plants, because of its strong cytotoxic properties. However, the effects of AT on laryngeal cancer have not been explored previously. This study aims to evaluate the anticancer efficacy of AT in HEp-2 human laryngeal carcinoma cells and to clarify the molecular pathways involved. AT significantly inhibited cell growth in a dose-dependent manner, with an IC₅₀ of 29.6 μM. The DCFH-DA staining results showed a marked increase in intracellular reactive oxygen species (ROS), underscoring oxidative stress as a crucial factor in cytotoxicity. Cells treated with AT exhibited significant mitochondrial membrane depolarization (loss of Δψm) and displayed notable apoptotic morphological changes, as evidenced by AO/EtBr and DAPI staining. Results from Annexin V/PI flow cytometry indicated a concentration-dependent increase in both early and late apoptotic cell populations. The comet assay revealed substantial DNA fragmentation, highlighting the genotoxic effects mediated by reactive oxygen species. The findings indicate that AT induces intrinsic apoptosis in HEp-2 cells through mechanisms related to oxidative stress, mitochondrial dysfunction, and DNA damage. These results suggest that AT may be a valuable phytochemical candidate for further investigation as a treatment for laryngeal cancer.
