Cellular effects of barakol-induced apoptosis in P19 cells

Abstract

Barakol is an active constituent extracted from Cassia siamea Lam., which has been used in folk medicine for the treatment of insomnia, however, its toxicity was reported and became an important limitation of using this herbal drug. Since, the mechanism by which barakol-induced toxicity has not been elucidated, this study thus investigated the cytotoxicity of barakol and its underlying mechanism in P19 embryonic stem cells. The extraction of barakol was obtained with a 0.1% yield. The barakol stability in culture medium was clarified and its stability was still stable until 24 h. Treament with barakol decreased cell viability in a time-dependent and a concentration-dependent manner measured by XTT assay with the IC[subscript 50] value of 1.5 mM. Thus the type of cell death was evaluated. Hoechst 33342 assay showed a significant increase in apoptosis after 24 h incubation of barakol. While the pretreatment with antioxidants such as N-acetyl-L-cysteine (NAC) or glutathione (GSH) showed the significant decrease in apoptotic cells, suggesting that reactive oxygen species (ROS) may play a role in barakol-mediated apoptosis. Whereas the flow cytometric analysis using propidium iodide staining showed that the distribution G[subscript 0]/ G[subscript 1] cell cycle phase was different from control. The molecular mechanism of barakol-induced apoptosis via the generation of ROS was further elucidated. Treatment with barakol showed a significant increase in the ROS level, peaking at 2h , detected by flow cytometry using 2' , 7' -dichlorodihydrofluorescein diacetate (DCFH-DA). Pretreatment of cells with NAC or GSH significantly inhibited ROS generation as well as apoptosis induced by barakol. To characterize ROS, The scavenging effects unsing manganese (III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP, a superoxide dismutase mimetic), catalase (a hydrogen peroxide scavenger), and sodium formate (a hydroxyl radical scavenger) were performed. Pretreatment with MnTBAP or catalase had no significant change in ROS signal comparing to the barakol treated control, indicating the absence of the involvement of superoxide anion or hydrogen peroxide in this system. Pretreatment with sodium formate completely abolished the effect of barakol-induced ROS generation. The result indicated that the predominant ROS in barakol-induced apoptosis was hydroxyl radical. Furthermore, the activity of caspase-9 significantly increased after treatment of the cells with barakol. Taken together, induction of apoptosis in P19 cells by barakol was mainly associated with the ROS generation, mitochondrial dysfunction, and caspase-9 activation.