Purchase this article with an account.
Ian H. Chan, Dan-Ning Hu, Min Chen, Joseph Walsh; Rescue Of Cybids Containing Leber Hereditary Optic Neuropathy (lhon) Mutation Using Necrostatin-1 And Pan-caspase Inhibitor Combination. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3933.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the combination use of necrostatin-1(N) and pan-caspase inhibitors (Z-VMD-FMK(Z) and B-D-FMK(B)) to block apoptosis and necroptosis in cybrids containing LHON mutation.
Homeoplasmic 143B osteosarcoma cybrids containing mtDNA (G11778A) mutation were grown in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose supplemented with 10% fetal bovine serum. Cells were seeded into 96 well plates and cultured with DMEM, glucose-free galactose medium (GFGM) and GFGM with combinations of N at 20 µmol, Z at 20 and 50 µmol and B at 20 and 50 µmol concentrations. After 48 hrs, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 50 µl of 1 mg/ml) was added, incubated for 4 hrs and replaced by DMSO. A microplate spectrophotometer at 540 nm was used to evaluate for % cell survival.
Compared to cybrids in DMEM medium, cell viability from MTT assay showed that cybrids survival % dropped to 58% after incubation in GFGM after 48 hours. Adding various agents(in µmol) to GFGM improved survival after 48 hrs: N20=62%(p=0.76) , Z20=78%(p=0.13), B20=79%(p=0.12), N20+Z20=90%(p=0.02), N20+Z50=79%(p=0.11), N20+B20=76%(p=0.17), and N20+B50=81%(p=0.09). P-values were calculated from 2-tailed t-test comparing cell survival of GFGM with different agents to GFGM alone.
Combination use of necrostatin-1 with pan-caspase inhibitor Z-VAD-FMK each at 20 µmol concentrations demonstrated statistically significant protective effects in LHON cybrid model.
This PDF is available to Subscribers Only