Abstract
BackgroundTemozolomide (TMZ) induces a G2/M cell cycle arrest and is used for treatment of paediatric tumours, especially neuroblastomas. Patients treated with TMZ frequently receive midazolam for sedation prior to surgery and other interventions. Previous studies suggested both cytoprotective and apoptosis-inducing properties of midazolam. Therefore, the impact of midazolam on TMZ-induced cytotoxicity was investigated in vitro.MethodsHuman neuroblastoma cells were incubated with midazolam alone, as a pretreatment prior to incubation with TMZ or a coincubation of both. Cell viability and proliferation was analysed (XTT and BrdU assay) after 24 h and flowcytometric cell cycle analysis was performed after 24 and 48 h.ResultsMidazolam alone increased cell viability at lower concentrations (2, 4, 8, 16 μM), whereas higher concentrations (128, 256, 512 μM) reduced cell viability. Pretreatment with midazolam 6 h prior to TMZ incubation reduced cytotoxic effects (IC25 1005 ± 197 μM; IC50 1676 ± 557 μM; P < 0.05) compared to incubation with TMZ alone (IC25 449 ± 304 μM; IC50 925 ± 196 μM) and reduced the antiproliferative effect of TMZ (1000 μM) by 43.9 % (P < 0.05). In contrast, cytotoxic effects of TMZ were increased (IC75 1175 ± 221 μM vs. 2764 ± 307 μM; P < 0.05) when midazolam pretreatment was followed by coincubation of midazolam and TMZ. Cell cycle analysis revealed increased fractions of cells in G2/M phase after TMZ treatment (100 μM; 48 h), irrespective of midazolam pretreatment.ConclusionMidazolam causes a hormetic dose–response relationship in human neuroblastoma cells. Pretreatment with midazolam reduces the cytotoxic and antiproliferative effects of TMZ without interfering with G2/M cell cycle arrest. In contrast, subsequent midazolam coincubation increases overall cytotoxicity.
Highlights
Temozolomide (TMZ) induces a G2/M cell cycle arrest and is used for treatment of paediatric tumours, especially neuroblastomas
Cell viability after incubation with different concentrations of midazolam To evaluate the impact of midazolam on the viability of neuroblastoma cells, XTT assay was performed after incubation for 24 h with increasing concentrations of midazolam
Midazolam pretreatment followed by coincubation with TMZ did not change the IC25 and IC50 of TMZ, while the IC75 of TMZ was decreased significantly (IC75 1175 ± 221 μM vs. 2764 ± 307 μM; P < 0.05), indicating an amplification of the TMZ-induced cytotoxicity (Fig. 3a-c)
Summary
Temozolomide (TMZ) induces a G2/M cell cycle arrest and is used for treatment of paediatric tumours, especially neuroblastomas. Patients treated with TMZ frequently receive midazolam for sedation prior to surgery and other interventions. Previous studies suggested both cytoprotective and apoptosis-inducing properties of midazolam. The relevance of temozolomide (TMZ), a DNA-methylating agent, for the treatment of paediatric solid tumours, especially neuroblastomas, has been elucidated [1,2,3,4,5,6]. Midazolam has been shown to exert apoptosis-inducing properties, which are mediated by the mitochondrial pathway in neuroblastoma cells and primary rat neurons [12], while other modes of cytotoxicity were reported to be predominant in other human cell types [13]. Whereas high concentrations of midazolam (>50 μM) induce apoptosis in a large fraction of neuroblastoma cells and primary rat neurons, low
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