NF-kappaB activation is associated with homocysteine-induced injury in Neuro2a cells

Background: Perinatal exposure to hyperhomocysteinemia might disturb neurogenesis during brain development and growth. Also, high levels of homocysteine trigger neurodegeneration in several experimental models. However, the putative mechanisms of homocysteine-induced toxicity in the developing nervous system have poorly been elucidated. This study was aimed to investigate homocysteine effects in undifferentiated neuroblastoma cells, Neuro2a. Results: A 4 h exposure to homocysteine in a concentration range of 10-100 mu M did not affect cell viability and ROS production in Neuro2a cell cultures. Instead, ROS levels were increased by two-three folds in cells treated with 250 mu M and 500 mu M homocysteine, respectively, in comparison with control cells. Also, the highest homocysteine dose significantly reduced the viable cell number by 40%. Notably, the treatment with homocysteine (250 mu M-500 mu M) in the presence of antioxidants, such as N-acetylcysteine and IRFI 016, a synthetic alpha-tocopherol analogue, recovered cell viability and significantly reduced homocysteine-evoked increases in ROS production. Moreover, antioxidants, particularly IRFI 016, were able to counteract NF-kappa B activation induced by 250 mu M homocysteine. Cell treatment with 250 mu M homocysteine also triggered the onset of apoptosis, as demonstrated by the increased expression of early apoptotic markers such as Bax, caspase-3 and p53. In contrast, Bcl2 expression was not affected by homocysteine exposure. Interestingly, the specific inhibition of NF-kappa B nuclear translocation by the synthetic peptide SN50 was able to almost completely suppress the homocysteine-evoked rises in pro- apoptotic protein expression as well as in caspase-3 activity. Further, also IRFI 016 and N-acetylcysteine were able to significantly reduce caspase-3 activation induced by homocysteine treatment. Conclusion: These observations suggest an involvement of redox state alterations and activated NF-kappa B in apoptosis onset triggered by homocysteine in neuroblastoma cells Neuro2a. However, further investigations are needed to characterize molecular events involved in the NF-kappa B activation induced by homocysteine.