Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms. 0.05, **< 0.01, ***< 0.001 for AGS cells treated with oxaliplatin vs. controls). (E) AGS cells were treated with or without oxaliplatin and cell growth was dynamically monitored using impedance technology. Normalized cell index values measured over 70 hours are shown. The tNOX-mediated modulation of the NAD+ concentration and SIRT1 are involved in oxaliplatin-induced apoptosis Next, we explored whether tNOX could be involved in the molecular mechanisms that govern oxaliplatin-mediated growth inhibition and apoptosis. As tNOX catalyzes the oxidation of reduced NADH to oxidized NAD+ [6, 7], we questioned whether oxaliplatin affects the intracellular levels of NAD+ and NADH. Indeed, we found that exposure of AGS cells to oxaliplatin at doses of 1 1 M and above effectively decreased the intracellular NAD+/NADH ratio compared to that in control cells (Figure ?(Figure2A),2A), possibly due to the inhibition of tNOX activity. The NAD+/NADH ratio is an indicator of cellular redox balance, which affects many cellular pathways. The availability of NAD+ also modulates the activity of SIRT1, which acts as a deacetylase for both histone and non-histone proteins (including the tumor suppressor, p53) and affects many cellular responses . We examined the effect of oxaliplatin on SIRT1 activity, and found that the activity of recombinant SIRT1 was significantly inhibited by 20 M oxaliplatin, making its inhibitory power comparable to that of the well-defined SIRT1 inhibitor, sirtinol (25 M) (Figure ?(Figure2B).2B). Interestingly, we observed an increase in p53 acetylation in cells treated with as little as 1 M of oxaliplatin (Figure ?(Figure2C).2C). The SIRT1 activity requires NAD+ as a cofactor and we speculate that the tNOX-mediated decrease of NAD+ concentrations and oxaliplatin-induced down-regulation of tNOX together reduce SIRT1deacetylase activity SIRT1 deacetylase activity was determined using a direct fluorescence assay kit. Sirtinol was used as a control. Values (means SEs) were obtained from three independent experiments performed in at least triplicate (**< 0.01 and ***< 0.001 for samples treated with oxaliplatin or sirtinol vs. controls). (C) AGS cells were treated with oxaliplatin or ddH2O for 24 hours, and cell lysates were separated by SDS-PAGE and analyzed by Western blotting. -Actin was used as an internal control. Representative images are shown. We next Formoterol hemifumarate used RNA interference (RNAi) to down-regulate tNOX expression in AGS cells. RNA interference targeting tNOX reduced expression of SIRT1 and this SIRT1 suppression was accompanied by enhanced p53 acetylation (Figure ?(Figure3A).3A). As expected, the intracellular NAD+/NADH ratio was decreased in these tNOX-knockdown AGS cells (Figure ?(Figure3B).3B). More importantly, apoptotic populations were significantly higher in tNOX-knockdown AGS control cells and tNOX-depletion also enhanced sensitivity of AGS cells to oxaliplatin-induced apoptosis at 0.1 M compared to the controls (Figure ?(Figure3C).3C). Rabbit polyclonal to TP53BP1 On the other hand, overexpression of tNOX in AGS cells has reversed the oxaliplatin-mediated apoptosis, suggesting a pro-survival role of tNOX (Figure ?(Figure3D3D). Open in a separate window Figure 3 The effects of siRNA-mediated tNOX knockdown or tNOX overexpression on oxaliplatin-induced apoptosis in AGS cellstNOX was knockdown by RNA interference in AGS cells (A, B) and then these cells were treated with ddH2O or oxaliplatin for 24 hours, and the percentage of apoptotic cells was determined by flow cytometry (C). A, Cell lysates were separated by SDS-PAGE and analyzed by Western blotting. -Actin was used Formoterol hemifumarate as an internal control. Representative images are shown. B, NAD+ and NADH were quantified in extracts of control and tNOX-knockdown cells. Formoterol hemifumarate The optical density at 450.