Midazolam induces apoptosis in MA-10 mouse Leydig tumor cells through caspase activation and the involvement of MAPK signaling pathway

Abstract

PurposeThe present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells.MethodsThe apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods.ResultsData showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose) polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase.ConclusionMidazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways.

Midazolam regulated caspase pathway, endoplasmic reticulum stress, autophagy, and cell cycle to induce apoptosis in MA-10 mouse Leydig tumor cells

“…Cells in sub-G1 phase have less DNA contents on cell cycle distribution, which is considered to be DNA fragmentation, and as a result of cell apoptosis. 4 , 5 …”

“…The double-negative cells (viable), annexin V single-positive cells (early apoptotic), PI single positive cells (necrotic), and double positive cells (late apoptotic) could be illustrated in four quadrants. 4 , 5 …”

“…After blocking membranes and incubating it with primary antibodies overnight at 4°C, the membrane was washed and incubated with HRP-conjugated secondary antibodies, and then detected with enhanced chemiluminescence kit (UVP EC3 BioImaging Systems, Upland, CA, USA). 4 , 5 …”

“…2 , 3 In a previous study, we illustrated that midazolam could stimulate steroidogenesis in MA-10 mouse Leydig tumor cells via PKA and PKC pathways along with the expression of peripheral benzodiazepine receptor and StAR proteins, 4 and could induce MA-10 cell apoptosis through caspase and MAPK signaling pathways. 5 In the present study, we further investigated the detailed mechanism of midazolam-induced apoptosis on MA-10 cells.…”

“…In fact, we have found that midazolam could stimulate caspase and MAPK, but inhibit Akt pathways to induce apoptosis in MA-10 cells. 5 In the present study, we would like to further investigate the detailed mechanism activated by midazolam in MA-10 cells, and will focus on the apoptotic pathway, autophagy, ER stress, and cell cycle-related pathway.…”