Cell experiment:

After cytometry, 1×3 cells are implanted in a 96-well plate in 100 μL DMEM supplemented with 10 % FBS. Ten microliter (10 % of the total volume) WST-1 (Water Soluble Tetrazolium) is added to cells and incubated at 37℃ for 30 min before colorimetric assay with 450 nm excitation and 630 nm emission at 24 h intervals up to 96 h. The mean fluorescence value is counted, and the cell number is determined using the standard curve.

Animal experiment:

U87MG cells are injected into both flanks of the nude mice subcutaneously at a dose of 2.0×3/200 μL per side. Eight days after injection, neoplasm growth is observed macroscopically on both sides of the mice. Then, 20 nM phenoxybenzamine hydrochloride is injected into the right side subcutaneously at a 2-day interval, and the dissolvent DMSO is used as control. The tumor volume (V) is determined by measuring the length (a) and the width (b) and calculated using the equation: V=(ab)2/2.

Phenoxybenzamine hydrochloride is a selective antagonist of both α-adrenoceptor and calmodulin that is commonly used for the treatment of hypertension, specifically caused by pheochromocytoma.
The IC50 (100 nM) derived from the blockade of [3H]yohimbine binding by Phenoxybenzamine hydrochloride is significantly less than the IC50 (550 nM) for the corresponding reversal by Phenoxybenzamine hydrochloride of the effects of norepinephrine on cyclic AMP accumulation[1]. Phenoxybenzamine hydrochloride (50 nM) in conbination with Phenoxybenzamine hydrochloridetolamine (1000 nM) enhances Phenoxybenzamine hydrochlorideylephrine-induced contraction compared with pretreatment with Phenoxybenzamine hydrochloride (50 nM) alone in endothelium-intact aortae. Combined treatment with either dexmedetomidine (300 or 1000 nM) and Phenoxybenzamine hydrochloride (50 nM) or Phenoxybenzamine hydrochloridetolamine (1000 nM) and Phenoxybenzamine hydrochloride (50 nM) enhance Phenoxybenzamine hydrochlorideylephrine-induced contraction compared with Phenoxybenzamine hydrochloride alone (50 nM). In addition, combined treatment with Phenoxybenzamine hydrochloridetolamine and Phenoxybenzamine hydrochloride enhances Phenoxybenzamine hydrochlorideylephrine-induced contraction compared with dexmedetomidine (1000 nM) and Phenoxybenzamine hydrochloride combined treatment. Combined treatment with high concentrations of dexmedetomidine (1000 nM) and Phenoxybenzamine hydrochloride enhances Phenoxybenzamine hydrochlorideylephrine-induced contraction compared with combined treatment with low concentrations of dexmedetomidine (300 nM) and Phenoxybenzamine hydrochloride[2]. Phenoxybenzamine hydrochloride (0.1-100 μM) inhibits glioma proliferation, migration, and invasion and suppresses the tumorigenesis capacity. Phenoxybenzamine hydrochloride also inhibits self-renewal of glioma stem-like cells. Phenoxybenzamine hydrochloride activates LINGO-1 and inhibits the TrkB-Akt pathway[3]. Phenoxybenzamine hydrochloride (0.1 μM-1 mM) preserves primary neurons within the CA1, CA3 and dentate gyrus and produces a robust neuroprotective effect, and prevents neuronal death from OGD in all regions of the hippocampus when delivered at 2, 4, and 8 h post-OGD at 100 μM[4].
Phenoxybenzamine hydrochloride (20 nM, s.c.) effectively suppresses the tumorigenesis of glioma cells in mice and the cell density in Phenoxybenzamine hydrochloride-U87MG xenografts decreases significantly[3]. Phenoxybenzamine hydrochloride (1 mg/kg, i.v.) treated rats shows significant improvements in NSS and foot fault scoring[4].
Reference:
[1]. Lenox, R.H., et al, Alpha 2-adrenergic receptor-mediated regulation of adenylate cyclase in the intact human platelet. Evidence for a receptor reserve. Mol Pharmacol, 1985. 27(1): p. 1-9.
[2]. Byon HJ, et al. Dexmedetomidine Inhibits Phenylephrine-induced Contractions via Alpha-1 Adrenoceptor Blockade and Nitric Oxide Release in Isolated Rat Aortae. Int J Med Sci. 2017 Feb 7;14(2):143-149.
[3]. Lin XB, et al. Anti-tumor activity of phenoxybenzamine hydrochloride on malignant glioma cells. Tumour Biol. 2016 Mar;37(3):2901-8.
[4]. Rau TF, et al. Phenoxybenzamine is neuroprotective in a rat model of severe traumatic brain injury. Int J Mol Sci. 2014 Jan 20;15(1):1402-17.