Homocysteine induced SH-SY5Y apoptosis through activation of NADPH oxidase in U251MG cells.

Epidemiological studies have indicated a correlation between homocysteinemia and dementia, including Alzheimer's disease. However, the mechanism by which homocysteine (Hcy) induces neuronal cell death remains unknown. We found that micromolar concentrations of Hcy induced neuroblastoma SH-SY5Y cell death only when co-cultured with glioblastoma U251MG cells. In this culture system, cysteine had no effect on SH-SY5Y cell death. There was an increase in TUNEL-positive cells and loss of mitochondrial membrane potential following treatment with 100 µM Hcy. Addition of conditioned medium prepared from U251MG cells in the presence of 100 µM Hcy also reduced SH-SY5Y cell viability, while this effect was prevented when using conditioned medium from U251MG cells exposed to 100 µM Hcy+apocynin, a specific NADPH oxidase inhibitor. Following exposure to 100 µM Hcy in U251MG cells, expression of Rac1, a compartment of NADPH oxidase, was translocated to the plasma membrane, and the active form of Rac1 was increased. There was no change in peroxide concentration in the medium of U251MG cells after addition of Hcy. Overall, these data suggest that Hcy stimulates Rac1 activation and NADPH oxidase, resulting in superoxide anion production that may induce SH-SY5Y cell apoptosis.

Homocysteine and copper induce cellular apoptosis via caspase activation and nuclear translocation of apoptosis-inducing factor in neuronal cell line SH-SY5Y.

Hyperhomocysteinemia has been implicated in dementia and neurodegenerative disease. Physiological homocysteine concentrations did not result in apoptosis in SH-SY5Y cells in the present study. The apoptosis was recognized in millimolar level of homocysteine. However, SH-SY5Y cell death was observed following exposure to micromolar level of homocysteine in combination with copper. Exposure to 250microM homocysteine and 10microM CuCl(2) for one day decreased cell viability by 40%. Homocysteine and copper caused apoptosis, because hallmarks of apoptosis were recognized, such as loss of mitochondrial membrane potential, TUNEL-positive cells, release of cytochrome c from mitochondria, and caspase-3 activation, but not nucleosomal DNA fragmentation. Homocysteine and copper generated the intracellular reactive oxygen species, and homocysteine and copper-induced apoptosis was due to an accumulation of intracellular reactive oxygen species, which was inhibited by catalase. Pan-caspase inhibitor, z-VAD-fmk, could not completely inhibited homocysteine and copper-induced cell death. Homocysteine and copper also caused the nuclear translocation of apoptosis-inducing factor. These results suggested that homocysteine and copper induced not only caspase-dependent apoptosis but also caspase-independent apoptosis-inducing factor related apoptosis.