FTIR spectroscopy of whole cells for the monitoring of yeast apoptosis mediated by p53 over-expression and its suppression by Nigella sativa extracts.

Abstract

p53 over expression in yeast results in cell death with typical markers of apoptosis such as DNA fragmentation and phosphatidylserine externalization. We aimed to substitute/supplement classical fluorescent techniques (TUNEL, Annexin V, ROS detection) usually used to detect biochemical changes occurring during yeast apoptosis mediated by p53 over expression and the effect of anti-apoptotic purified molecules from Nigel (Nigella sativa) extracts on these same yeasts by the label free technique of FTIR spectroscopy. The comparison of the entire IR spectra highlighted clear modifications between apoptotic p53-expressing yeasts and normal ones. More precisely, DNA damage was detected by the decrease of band intensities at 1079 and 1048 cm-1. While phosphatidylserine exposure was followed by the increase of νsCH2 and νasCH2 bands of unsaturated fatty acids that were exhibited at 2855 and 2926 cm-1, and the appearance of the C = O ester functional group band at 1740 cm-1. In a second step, this FTIR approach was used to estimate the effect of a purified fraction of the Nigel extract. The modulation of band intensities specific to DNA and membrane status was in agreement with apoptosis supression in presence of the Nigel extracts. FTIR spectroscopy is thus proven to be a very reliable technique to monitor the apoptotic cell death in yeast and to be used as a means of evaluating the biomolecules effect on yeast survival.