Barakol-induced apoptosis in P19 cells through generation of reactive oxygen species and activation of caspase-9

Ethnopharmacological relevance: Barakol, an anxiolytic agent isolated from Senna siamea leaves which has been traditionally used for producing natural sleep, has been described as toxic to patients.

Aim of the study: The aim of current study was to investigate the molecular mechanism of barakol-induced toxicity in mouse embryonal carcinoma P19 cell model.

Materials and methods: XTT assay was used to determine cell viability in P19 cells treated with barakol. Apoptotic cells were detected by Hoechst 33342 staining. Intracellular Reactive Oxygen Species (ROS) generation was analyzed by flow cytometry using a Fluorescent Dye, DCFH-DA. Detection of apoptotic protein expression in P19 cells was performed by Western blot analysis. Caspase-9 activity was measured using a fluorescent immunosorbent Enzyme assay kit.

Results: Treatment with barakol decreased cell viability in a concentration- and time-dependent manner with an IC(50) value of 1.5mM in 24-h treated cells. A Hoechst 33342 assay revealed that barakol cytotoxicity was due to a significant increase in the number of apoptotic cells. Different scavengers to characterize ROS were utilized and revealed that hydroxyl radicals played a major role in ROS-induced Apoptosis in barakol-treated cells. Western blot analysis demonstrated that barakol-induced Apoptosis was mediated by the increase in expression ratio of Bax/Bcl-2. Furthermore, increase in caspase-9 activity after exposure to barakol for 24h was also observed. Pretreatment of cells with N-acetyl-l-cysteine (NAC) attenuated intracellular ROS generation, the Bax/Bcl-2 protein expression, and Apoptosis.

Conclusions: The mechanism of barakol-mediated toxicity in P19 cells is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-9 activation leading to apoptotic cell death. Pretreatment of cells with NAC could antagonize the toxicity produced by barakol.