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

So EC, Lin YX, Tseng CH, Pan BS, Cheng KS, Wong KL, Hao LJ, Wang YK, Huang BM - Onco Targets Ther (2014)

Bottom Line: Data 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia, Tainan Municipal An Nan Hospital, China Medical University, Tainan, Taiwan ; Department of Anesthesia, China Medical University, Taichung, Taiwan.

ABSTRACT

Purpose: The 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.

Methods: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods.

Results: Data 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.

Conclusion: Midazolam 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.

No MeSH data available.


Related in: MedlinePlus

Midazolam activated caspase cascade in MA-10 mouse Leydig tumor cells. MA-10 cells were treated without or with different concentrations of midazolam (6 μM, 30 μM, and 150 μM) for 3 hours, 6 hours, 12 hours, and 24 hours, respectively. The levels of procaspase-8 protein (57 KDa), cleaved caspase-8 (47 KDa), cleaved caspase-9 (17 KDa), cleaved caspase-3 (19 KDa), and cleaved poly (ADP-ribose) polymerase (PARP) (90 KDa) were analyzed by Western blot. Immunoblots represent the observations from one single experiment repeated three times (A). The integrated optical densities of procaspase-8 (B), cleaved caspase-8 (C), cleaved caspase-9 (D), cleaved caspase-3 (E), and cleaved PARP (F) proteins were analyzed after normalization with β-actin (43 kDa) in each lane. Data in (B–F) represent the mean ± standard error of the mean of three separate experiments.Note: *Indicates significant difference between control and midazolam-treated groups at the same time (P<0.05).
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f2-ott-7-211: Midazolam activated caspase cascade in MA-10 mouse Leydig tumor cells. MA-10 cells were treated without or with different concentrations of midazolam (6 μM, 30 μM, and 150 μM) for 3 hours, 6 hours, 12 hours, and 24 hours, respectively. The levels of procaspase-8 protein (57 KDa), cleaved caspase-8 (47 KDa), cleaved caspase-9 (17 KDa), cleaved caspase-3 (19 KDa), and cleaved poly (ADP-ribose) polymerase (PARP) (90 KDa) were analyzed by Western blot. Immunoblots represent the observations from one single experiment repeated three times (A). The integrated optical densities of procaspase-8 (B), cleaved caspase-8 (C), cleaved caspase-9 (D), cleaved caspase-3 (E), and cleaved PARP (F) proteins were analyzed after normalization with β-actin (43 kDa) in each lane. Data in (B–F) represent the mean ± standard error of the mean of three separate experiments.Note: *Indicates significant difference between control and midazolam-treated groups at the same time (P<0.05).

Mentions: To further elucidate the mechanism of apoptosis induced by midazolam in MA-10 cells, caspase cascade was investigated by Western blotting. Results showed that procaspase-8 significantly decreased with 150 μM midazolam after 12-hour and 24-hour treatments (Figure 2A and B, P<0.05), whereas cleaved caspase-8 significantly increased after 12-hour treatment with 150 μM midazolam (Figure 2A and C, P<0.05). In addition, midazolam significantly induced caspase-9 cleavage at a dosage of 150 μM after 24-hour treatment (Figure 2A and D, P<0.05). Moreover, cleaved caspase-3 significantly increased after 12-hour treatment with 150 μM midazolam (Figure 2A and E, P<0.01). It is well known that PARP is one of the downstream substrates of activated caspase-3, which could be cleaved into 85 KDa degraded product of PARP.8 We also examined the cleavage of PARP upon midazolam treatment, and the results showed that cleavage of PARP activated by caspase-3 significantly increased after 6-hour treatment with 150 μM midazolam, and this phenomenon was sustained for 24 hours (Figure 2A, P<0.05). These results suggested that midazolam could stimulate the cleavage of caspase-8, -9, and -3 and PARP to induce apoptosis in MA-10 mouse Leydig tumor cells.


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

So EC, Lin YX, Tseng CH, Pan BS, Cheng KS, Wong KL, Hao LJ, Wang YK, Huang BM - Onco Targets Ther (2014)

Midazolam activated caspase cascade in MA-10 mouse Leydig tumor cells. MA-10 cells were treated without or with different concentrations of midazolam (6 μM, 30 μM, and 150 μM) for 3 hours, 6 hours, 12 hours, and 24 hours, respectively. The levels of procaspase-8 protein (57 KDa), cleaved caspase-8 (47 KDa), cleaved caspase-9 (17 KDa), cleaved caspase-3 (19 KDa), and cleaved poly (ADP-ribose) polymerase (PARP) (90 KDa) were analyzed by Western blot. Immunoblots represent the observations from one single experiment repeated three times (A). The integrated optical densities of procaspase-8 (B), cleaved caspase-8 (C), cleaved caspase-9 (D), cleaved caspase-3 (E), and cleaved PARP (F) proteins were analyzed after normalization with β-actin (43 kDa) in each lane. Data in (B–F) represent the mean ± standard error of the mean of three separate experiments.Note: *Indicates significant difference between control and midazolam-treated groups at the same time (P<0.05).
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Related In: Results  -  Collection

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f2-ott-7-211: Midazolam activated caspase cascade in MA-10 mouse Leydig tumor cells. MA-10 cells were treated without or with different concentrations of midazolam (6 μM, 30 μM, and 150 μM) for 3 hours, 6 hours, 12 hours, and 24 hours, respectively. The levels of procaspase-8 protein (57 KDa), cleaved caspase-8 (47 KDa), cleaved caspase-9 (17 KDa), cleaved caspase-3 (19 KDa), and cleaved poly (ADP-ribose) polymerase (PARP) (90 KDa) were analyzed by Western blot. Immunoblots represent the observations from one single experiment repeated three times (A). The integrated optical densities of procaspase-8 (B), cleaved caspase-8 (C), cleaved caspase-9 (D), cleaved caspase-3 (E), and cleaved PARP (F) proteins were analyzed after normalization with β-actin (43 kDa) in each lane. Data in (B–F) represent the mean ± standard error of the mean of three separate experiments.Note: *Indicates significant difference between control and midazolam-treated groups at the same time (P<0.05).
Mentions: To further elucidate the mechanism of apoptosis induced by midazolam in MA-10 cells, caspase cascade was investigated by Western blotting. Results showed that procaspase-8 significantly decreased with 150 μM midazolam after 12-hour and 24-hour treatments (Figure 2A and B, P<0.05), whereas cleaved caspase-8 significantly increased after 12-hour treatment with 150 μM midazolam (Figure 2A and C, P<0.05). In addition, midazolam significantly induced caspase-9 cleavage at a dosage of 150 μM after 24-hour treatment (Figure 2A and D, P<0.05). Moreover, cleaved caspase-3 significantly increased after 12-hour treatment with 150 μM midazolam (Figure 2A and E, P<0.01). It is well known that PARP is one of the downstream substrates of activated caspase-3, which could be cleaved into 85 KDa degraded product of PARP.8 We also examined the cleavage of PARP upon midazolam treatment, and the results showed that cleavage of PARP activated by caspase-3 significantly increased after 6-hour treatment with 150 μM midazolam, and this phenomenon was sustained for 24 hours (Figure 2A, P<0.05). These results suggested that midazolam could stimulate the cleavage of caspase-8, -9, and -3 and PARP to induce apoptosis in MA-10 mouse Leydig tumor cells.

Bottom Line: Data 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia, Tainan Municipal An Nan Hospital, China Medical University, Tainan, Taiwan ; Department of Anesthesia, China Medical University, Taichung, Taiwan.

ABSTRACT

Purpose: The 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.

Methods: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods.

Results: Data 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.

Conclusion: Midazolam 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.

No MeSH data available.


Related in: MedlinePlus