The Effect of Alpha-Tocopherol on Viability of PC12 Cells during Oxidative Stress and Expression of Genes Encoding Pro- and Anti-Apoptotic Mitochondrial Proteins, SOD2 and Transcription Factors NRF-1, NRF-2 and TFAM

Abstract

Long-term (18 h) preincubation with an antioxidant alpha-tocopherol (alpha-T) both at micromolar and nanomolar concentrations was shown to increase the viability of the PC12 neuronal cell line exposed to oxidative stress, while during short-term preincubation (30 min) alpha-T exhibited a protective effect only at micrimolar concentrations. Using real-time polymerase chain reaction (RT-PCR), it was demonstrated that exposure of PC12 cells to a pro-oxidant hydrogen peroxide increases expression of a proapoptotic mitochondrial protein Bax to a larger extent than that of a antiapoptotic protein Bcl-xL. In contrast, preincubation of PC12 cells with alpha-T before exposure to hydrogen peroxide increased Bcl-xL expression but did not influence Bax expression. Hence, it appears that alpha-T promotes normalization of the Bax/Bcl-xL ratio increased due to the prooxidant’s effect. In control PC12 cells, alpha-T significantly increased expression of the transcription factors NRF-2 and TFAM, but not NRF-1, which perform varied functions and, specifically, can activate mitochondrial biogenesis. In addition, alpha-T decreased the Bax/Bcl-xL ratio in PC12 cells, as shown by immunoblotting. At the same time, both exposure to hydrogen peroxide and preincubation with alpha-T caused no significant changes in NRF-1, NRF-2 and TFAM expression in PC12 cells. Preincubation of PC2 cells with alpha-T before exposure to hydrogen peroxide evoked earlier expression of an antiapoptotic enzyme superoxide dismutase 2 (SOD2) than when the prooxidant acted alone. Thus, it appears that normalization of the Bax/Bcl-xl ratio and a relatively early increase in SOD2 expression contribute to the protective effect of alpha-T towards PC12 cells during oxidative stress. The fact that alpha-T increases NRF-2 and TFAM expression in control PC12 cells suggests that alpha-T can slightly boost mitochondrial biogenesis. However, under oxidative stress conditions, which are characterized by a deficiency of macroergic compounds, no evidence for the alpha-T-induced activation of such an energy-consuming process as mitochondrial biogenesis in PC12 cells was found.