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A. F. Thompson, C. J. Lieven, L. A. Levin; Synthesis of Novel Borane-Protected Phosphines That are Neuroprotective Against Superoxide-Dependent Apoptosis in RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):131.
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We previously synthesized and studied borane-protected phosphines bis (3-propionic acid methyl ester) phenylphosphine borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine borane complex (PB2). These reducing agents were shown to be neuroprotective in cultured primary retinal ganglion cells (Schlieve et al., Exp Eye Res 83:1252, 2006) and RGC-5 cells (Seidler et al, unpublished data). We have now synthesized three new borane-protected phosphines, PB3, PB4, and PB6, which are designed for improved cell permeability and mitochondrial targeting. We examined the ability of these drugs to protect RGC-5 cells from apoptosis resulting from menadione-induced mitochondrial superoxide.
RGC-5 cells suspended in growth medium were plated in 96-well plates at a density of 2,000 cells per well. After 24 hours in culture, cells were treated with 17.8 µM menadione. For treatment conditions, PB3, PB4, or PB6 was added to wells directly before menadione treatment. At 24 hours after menadione treatment, cells were stained with calcein-AM and photographed. Live (calcein-positive) cells were counted using ImageJ software.
Treatment with PB3, PB4, or PB6 significantly rescued cells from menadione-induced death. PB3 was significantly protective at all concentrations tested between 100 pM and 10 µM. Treatment with 100 pM, 1 µM, and 10 µM PB3 resulted in rescue of 30.9 ± 4.3% (p = 0.000032), 46.4 ± 16.8% (p = 0.024), and 38.3 ± 12.2% (p = 0.014) of cells respectively. PB4 and PB6 were less effective neuroprotectants than PB3. A concentration of 1 µM was most effective for PB4, and resulted in rescue of 19.0 ± 5.5% of cells (p = 0.031). PB6 was most effective at 10 pM, and resulted in rescue of 25.6 ± 8.4% of cells (p = 0.016).
Borane-protected phosphines PB3, PB4, and PB6 are neuroprotective against menadione-induced death in RGC-5 cells. Differences in the effectiveness of these compounds may be due to differences in cell permeability or intracellular targeting.
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