Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: neuroprotection • neuro-ophthalmology: optic nerve • mitochondria