The Apoptotic Mechanism of Midazolam on MA-10 Mouse Leydig Tumor Cells.

Abstract

Midazolam is widely used as a sedative anesthetic induction agent, and it is a derivative from benzodiazepine drug. In previous study, the midazolam could modulate GABAA receptor in central nervous system to achieve sedative effect. Moreover, midazolam could rapidly onset the action with highly metabolic clearance when compared with other benzodiazepine drug. In fact, it has been reported that benzodiazepine could regulate adrenocortical steroidogenesis, indicating that midazolam might influence the steroidogenesis when it is used as a sedative drug. In our previous data we found that midazolam induced StAR-dependent steroidogenesis via PKC and PKA signaling transduction pathways in MA-10 mouse Leydig tumor cells and primary Leydig cells. Our previous data also showed that midazolam at higher dosages would induce MA-10 Leydig tumor cell rounding-up, membrane blebbing and then cell death. But the cell apoptotic phenomenon was not found in primary mouse Leydig cells. Thus, we further examined the cell death effect of midazolam on MA-10 cells. First, we exploited MTT assay and flow cytometry to investigate apoptotic effect and results showed that midazolam decreased cells viability and induced cell cycle accumulation in sub-G1 phase in time- and dose-dependent manners. We then examined the expression of apoptotic proteins by immunoblotting. We found that only the expressions of caspase-8, -9, -3 and poly (ADP-ribose) polymerase (PARP) were increased in higher dosages and later time points of midazolam treatments. In addition, we detected that midazolam could inhibit Akt protein expression. In conclusion, we found that midazolam induced MA-10 mouse Leydig tumor cell apoptosis through activating the caspase-8, -9, -3 and PARP pathway. (poster)