title: Midazolam regulated caspase pathway, endoplasmic reticulum stress, autophagy, and cell cycle to induce apoptosis in MA-10 mouse Leydig tumor cells.
abstract: OBJECTIVE
  Midazolam is widely used as a sedative and anesthetic induction agent by modulating the different GABA receptors in the central nervous system. Studies have also shown that midazolam has an anticancer effect on various tumors. In a previous study, we found that midazolam could induce MA-10 mouse Leydig tumor cell apoptosis by activating caspase cascade. However, the detailed mechanism related to the upstream and downstream pathways of the caspase cascade, such as endoplasmic reticulum (ER) stress, autophagy, and p53 pathways plus cell cycle regulation in MA-10 mouse Leydig tumor cells, remains elusive.
  
  
  METHODS
  Flow cytometry assay and Western blot analyses were exploited.
  
  
  RESULTS
  Midazolam significantly decreased cell viability but increased sub-G1 phase cell numbers in MA-10 cells (P<0.05). Annexin V/propidium iodide double staining further confirmed that midazolam induced apoptosis. In addition, expressions of Fas and Fas ligand could be detected in MA-10 cells with midazolam treatments, and Bax translocation and cytochrome c release were also involved in midazolam-induced MA-10 cell apoptosis. Moreover, the staining and expression of LC3-II proteins could be observed with midazolam treatment, implying midazolam could induce autophagy to control MA-10 cell apoptosis. Furthermore, the expressions of p-EIF2α, ATF4, ATF3, and CHOP could be induced by midazolam, indicating that midazolam could stimulate apoptosis through ER stress in MA-10 cells. Additionally, the expressions of cyclin A, cyclin B, and CDK1 could be inhibited by midazolam, and the phosphorylation of p53, P27, and P21 could be adjusted by midazolam, suggesting that midazolam could manage cell cycle through the regulation of p53 pathway to induce apoptosis in MA-10 cells.
  
  
  CONCLUSIONS
  Midazolam could induce cell apoptosis through the activation of ER stress and the regulation of cell cycle through p53 pathway with the involvement of autophagy in MA-10 mouse Leydig tumor cells.
authors.affiliation: Department of Anesthesia, An Nan Hospital, China Medical University, Tainan, Taiwan, Republic of China; Department of Anesthesia, School of Medicine, China Medical University, Taichung, Taiwan; Republic of China.
authors.forename: Edmund Cheung
authors.initials: EC
authors.lastname: So
authors.affiliation: Department of Anatomy, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.
authors.forename: Yung-Chia
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
authors.forename: Shu-Chun
authors.initials: SC
authors.lastname: Wang
authors.affiliation: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
authors.forename: Chia-Ching
authors.initials: CC
authors.lastname: Wu
authors.affiliation: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
authors.forename: Man-Chi
authors.initials: MC
authors.lastname: Huang
authors.affiliation: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
authors.forename: Meng-Shao
authors.initials: MS
authors.lastname: Lai
authors.affiliation: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China; Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, NC, USA.
authors.forename: Bo-Syong
authors.initials: BS
authors.lastname: Pan
authors.affiliation: Department of Anesthesia, Chi Mei Medical Center, Chiali, Tainan, Taiwan, Republic of China.
authors.forename: Fu-Chi
authors.initials: FC
authors.lastname: Kang
authors.affiliation: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
authors.forename: Bu-Miin
authors.initials: BM
authors.lastname: Huang
keywords: ER stress; MA-10 cells; apoptosis; autophagy; caspase; cell cycle; midazolam; tumor