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.

Plants are an important source of pharmacologically active compounds. In the present work, we characterize the impact of black cumin (Nigella sativa L.) aqueous extracts on a yeast model of p53-dependent apoptosis. To this end, the Saccharomyces cerevisiae recombinant strain over-expressing p53 was used. The over-expression of p53 triggers the expression of apoptotic markers: the externalization of phosphatidylserine, mitochondrial defect associated with cytochrome-c release and the induction of DNA strand breaks. These different effects were attenuated by Nigella sativa L. aqueous extracts, whereas these extracts have no effect on the level of p53 expression. Thus, we focus on the anti-apoptotic molecules present in the aqueous extract of Nigella sativa L. These extracts were purified and characterized by complementary chromatographic methods. Specific fluorescent probes were used to determine the effect of the extracts on yeast apoptosis. Yeast cells over-expressing p53 decrease in relative size and have lower mitochondrial content. The decrease in cell size was proportional to the decrease in mitochondrial content and of mitochondrial membrane potential (ΔΨm). These effects were prevented by the purified aqueous fraction obtained by fractionation with different columns, named C4 fraction. Yeast cell death was also characterized by reactive oxygen species (ROS) overproduction. In the presence of the C4 fraction, ROS overproduction was strongly reduced. We also noted that the C4 fraction promotes the cell growth of control yeast cells, which do not express p53, supporting the fact that this purified extract acts on cellular mediators activating cell proliferation independently of p53. Altogether, our data obtained on yeast cells over-expressing p53 demonstrate that anti-apoptotic molecules targeting p53-induced apoptosis associated with mitochondrial dysfunction and ROS overproduction are present in the aqueous extracts of Nigella seeds and in the purified aqueous C4 fraction.

Since its discovery in 1979, the tumor suppressor p53 has been widely studied and expressed in various organisms, including yeast. Yeast has proven to be a very informative model and an effective system for studying the roles and functions of this protein and gene. This review is a compilation of our team's studies involving p53 expression in yeast. These researches investigated certain aspects, essentially the apoptotic function of p53. Our main contribution to the study and understanding of the p53 gene in the yeast context is the confirmation of a negative effect of p53 on cell growth in both Saccharomyces cerevisiae and Pichia pastoris strains, which ultimately led to apoptotic cell death. This involves a high dose of p53 and the NLS signal, which enables p53 to target both the mitochondria and the nucleus. Prior to that, obtaining the whole protein required a cDNA without its UTR. Thus, a yeast model was developed, allowing verification of p53 activity. Cancer mutants and their revertants could thereby be assessed. This has evolved into a real antioxidant/anti-apoptotic molecular screening mechanism. Two primary applications were achieved: testing the co-expression with the thioredoxin 2 gene (TRX2) and assessing the impact of Nigella sativa seed extracts. Furthermore, the high yield of yeast P53 production allowed its use in serological cancer diagnosis.