Table 1.

Characteristics of included studies.

First author	Year	Area	Study design	Cell lines/animal	Concentrations of midazolam	Pathway	Assays	Results	Conclusion
Stevens [12]	2011	Netherlands	In vitro	Human lymphoma and neuroblastoma cell lines	0, 50, 70, 100, and 150 μM for Jurkat T-lymphoma cells 0, 100, 200, 300, and 400 μM neuroblastoma cells	Mitochondrial pathway	XTT assay	Midazolam induced apoptosis in all investigated cell types in a concentration-dependent manner. Bcl2 overexpression and caspase-9 deficiency protected against toxicity, whereas caspase-8 or FADD deficiency had no effect.	Midazolam induces apoptosis via activation of the mitochondrial pathway in a concentration-dependent manner. The mechanism of midazolam toxicity switches from caspase-dependent apoptosis to necrosis with increasing concentrations of midazolam.
Mak [28]	1997	Hong Kong	In vitro	Murine myeloid leukemia WEHI 3B (JCS) and M1 cells	0, 10, 20, 30, and 40 μg/ml for both JCS and M1 cells for 18, 48, and 72 hours	NA	Flow cytometry; mRNA phenotyping; phagocytosis; cell morphology; Southern-blot	Midazolam inhibits proliferation of both M1 and JCS cells in a dose-dependent manner. mRNA phenotyping also indicated that the expression of tumor necrosis factor and the neutrophil-specific J11d differentiation marker was significantly upregulated in midazolam-treated JCS cells. In addition, the phagocytic activity of midazolam-treated JCS cells was increased towards opsonized yeast cells.	Midazolam inhibits proliferation of both M1 and JCS cells.
So [29]	2014	Taiwan	In vitro	MA-10 mouse Leydig tumor cell line	0, 6, 30, and 150 μM for 3, 6, 12, and 24 hours	pAkt pathway and p38 and c-Jun NH2-terminal kinase pathways	Flow cytometry and immunoblotting analysis	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. It induced the activation of caspase-8, caspase-9, and caspase-3 and poly(ADP-ribose) polymerase, decreased both pAkt and Akt expression, and stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase.	Midazolam induced MA-10 cell apoptosis via activation of the caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways.
Ohno [30]	2012	Japan	In vitro	Human OSCC cell lines; HL-60 cells; glioblastoma; keratinocytes; oral normal cells	0–1000 μM for 3, 6, 9, 12, 24, and 48 hours	NA	Cytotoxic activity by MTT; DNA fragmentation by ultraviolet irradiation; caspase activation; autophagy; electron microscopy	Midazolam showed the highest cytotoxicity. In HL-60 cells, it induced the appearance of many vacuoles, mitochondrial swelling, and cell membrane rupture in HSC-2 and HSC-4 cells.	Midazolam may induce necrotic cell death, rather than apoptosis or autophagy, in OSCC cell lines.
So [31]	2016	Taiwan	In vitro	MA-10 mouse Leydig tumor cells	0, 6, 30, and 150 μM for 1, 3, 6, 12, and 24 hours	ER stress and p53 pathway	Flow cytometry assay and Western blot analyses	Midazolam significantly decreased cell viability but increased sub-G1 phase cell numbers and apoptosis. Expression of Fas, Fas ligand, p-EIF2α, ATF4, ATF3, CHOP, and LC3-II proteins was detected. Midazolam was able to regulate the cell cycle via regulation of the p53 pathway.	Midazolam induced cell apoptosis in MA-10 mouse Leydig tumor cells through activation of ER stress and regulation of the cell cycle via the p53 pathway, with the involvement of autophagy.
Braun [32]	2015	Germany	In vitro	Human neuroblastoma cells	2, 4, 8, 16, 128, 256, and 512 μM for 24 and 48 hours	NA	Cell proliferation; cell cycle; cell viability	Midazolam increased cell viability at lower concentrations, whereas higher concentrations reduced cell viability.	Midazolam causes a hormetic dose-response relationship in human neuroblastoma cells.
Dou [33]	2014	China	In vitro	Hypopharyngeal squamous carcinoma cells	0, 6.25, 12.5, 25, 50, and 100 μM for 24 and 48 hours	CDKI/RB pathway	MTT and BrdU incorporation RT-PCR and Western blotting	Midazolam inhibited the expression of p300 and the proliferation of FaDu cells. Additionally, knockdown of p300 resulted in increased expression of p21 and p27 and decreased expression of p-Rb, while inhibiting the proliferation of FaDu cells.	Midazolam inhibits the proliferation of human head and neck squamous carcinoma cells by downregulating p300.
Dou [34]	2013	China	In vitro	FaDu human hypopharyngeal squamous cell carcinoma cells	0, 6.25, 12.5, 25, 50, and 100 μM for 24 and 48 hours	TRPM7 inhibition	Cell death assay; cell cycle analysis; Western blot analysis; quantitative real-time PCR	Midazolam inhibits the growth and proliferation of FaDu cells. Midazolam triggers G0/G1 cell cycle arrest by regulating cell cycle regulators. The inhibitory effect of midazolam on proliferation is benzodiazepine receptor- (BR-) independent but TRPM7-dependent.	The inhibitory activity of midazolam on cancer cell growth and proliferation, combined with the TRPM-dependent mechanism, reveals the anticancer potential of midazolam as a TRPM7 inhibitor.
Mishra [35]	2013	Korea	In vitro; in vivo	BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts	0, 10, 30, 100, and 200 μM for 24 and 48 hours (in vitro), 0.83 mg/kg body weight (in vivo)	Mitochondrial intrinsic pathway	Cell viability; flow cytometric analysis; cell cycle analysis; measurement of mitochondrial membrane potential; DNA fragmentation; Western blot; intracellular superoxide generation; in vivo mouse xenograft	Midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, caspase-3 and PARP, lowered mitochondrial membrane potential, increased apoptotic DNA fragmentation, and exhibited ROS scavenging activity through the inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. It also resulted in inhibition of pERK1/2 signaling which led to inhibition of the antiapoptotic proteins Bcl-XL and XIAP and phosphorylation activation of the proapoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice.	Midazolam inhibited the growth of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis. It also inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects may be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins.
Chen [36]	2016	China	In vitro	Malignant glioblastoma cell line T98-MG cells	0, 25, 50, and 100 μM for 24 and 48 hours	Inhibited the TRPM7 expression	Immunofluorescence; cell proliferation and cell viability; caspase-3 activity and LDH release assay; reverse transcription-PCR; electrophysiology; cell cycle analysis; Western blot analysis; calcium imaging	Brief midazolam treatment (seconds) suppressed TRPM7 channels and calcium influx, while treatment for 48 h inhibited the TRPM7 expression. The inhibitory effect on TRPM7 accounts for the decrease in proliferation and G0/G1 phase cell cycle arrest induced by midazolam.	Midazolam represses proliferation of human malignant glioma cells via the inhibition of TRPM7 channels.
Hong [37]	2013	China	In vitro	Mantle cell; lymphoma JeKo-1 cell line	10, 20, 40, and 80 μmol/L for 24, 48, 72 hours	Mitochondrial pathway	CCK8; flow cytometry; Western blot	Midazolam inhibited the growth of JeKo-1 cells; it induced apoptosis and reduction of Bcl-2, procaspase-9, and procaspase-3 protein expression and an increase in cyto-C protein expression in a concentration-dependent manner.	Midazolam may initiate the mitochondrial pathway by reducing the expression of Bcl-2, leading in turn to the release of Cyto-C in the mitochondria. This leads to the activation of caspase-9 and caspase-3 protein and triggers the caspase cascade, ultimately inducing apoptosis of the JeKo-1 cells.
Dou [38]	2012	China	In vitro	Human colon cancer SW480 cells	0, 6.25, 12.5, 25, 50, and 100 μM for 24 and 48 hours	CDKI/Rb pathway	MTT and BrdU incorporation RT-PCR and Western blotting	Midazolam inhibited the growth and proliferation of SW480 cells in a time- and dose-dependent manner. It downregulated USP22 expression. When USP22 expression was silenced by siRNA, proliferation of the SW480 cells was inhibited. P21 and P27 expression was upregulated, while pRB was downregulated.	Midazolam inhibits the proliferation of human colon cancer SW480 cells. The mechanism may be through mediation of the CDKI/RB pathway via downregulation of USP22.

GABAA, γ-aminobutyric acid A; CBM, Chinese Biomedical Literature; Bci-2, B cell lymphoma 2; FADD, Fas-associating protein with a novel death domain; ER stress, endoplasmic reticulum stress; CHOP, C/EBP-homologous protein; ATF-4, activating transcription factor 4; p-eIF2α, phosphorylated α subunit of eukaryotic initiation factor 2; Bax, Bcl-2 associated X; OSCC, oral squamous cell carcinoma; LC3-II, LC3-phosphatidylethanolamine conjugate; CKD1, cyclin-dependent kinase 1; TRPM7, transient receptor potential melastatin 7; USP22, ubiquitin-specific protease 22; CDKI/RB, cyclin-dependent kinase inhibitor/retinoblastoma protein; TNF, tumor necrosis factor; ROS, reactive oxygen species; NA: not applicable.