Extraction of pH-Dependent DNA-Binding Anti-Tumoral Peptides from Saccharomyces cerevisiae

Abstract

Cancer remains a significant challenge in the field of medicine, primarily due to its inherent plasticity and the development of resistance to conventional therapeutic interventions. Genomic mutations and the activation of oncogenes enable cancer cells to resist senescence and apoptosis, leading to uncontrolled growth with harmful consequences. Small peptides are molecules with interesting anti-tumour properties and represent a valid alternative to conventional treatments. Our group has previously identified a class of small peptides bound to the DNA that can be extracted from the chromatin of various tissues, including wheat germ and trout. These peptide pools have been shown to possess interesting antiproliferative and apoptotic properties, and they are associated with cell cycle regulation. However, given the complexity of the extraction process, it is necessary to identify a substrate that will enable a more efficient extraction of these peptides, while also ensuring a composition that is simple to investigate. The present study developed a method for the extraction of this group of peptides from yeast, and the extract was then tested on cancer cells in order to confirm its anti-tumoral properties. The peptides were obtained from chromatin extracted from Saccharomyces cerevisiae cells through alkalisation and purification by gel filtration chromatography. The extract was tested on HeLa cells to verify its effects on vitality and the cell cycle. The data demonstrate that the chromatographic profile of this peptide extract indicates a more basic composition than the pool extracted from other tissues and exhibits comparable antiproliferative properties. The ability to rapidly obtain a biologically active, analytically accessible, and adequately purified fraction from the widely available substrate Saccharomyces cerevisiae represents a significant advance in the study of these DNA-binding peptides.

Keywords: DNA damage; anti-tumoral peptide; cancer; cell cycle; drug discovery; peptide extraction; small peptide.

Figure 1

Chromatography profile of YCPE (a) and wheat germ extract (b) on Sephadex G25; the fraction corresponding to Ve/V0 = 2 corresponds to 120–180 mL for YCPE and 90–120 for wheat germ extract. (c) TLC chromatography of the YCPE. Spots 1 and 2 indicate YCPE spots (2.5 and 1 μg/mL), and 3 to 5 indicate the reference standard amino acids (3-serine, 4-threonine, 5-phenylalanine) used as a positive control for ninhydrin staining. Red arrows indicate the spots used for calculate the Rf for sample 1 and 2. Rf for the samples was 0.74 and 0.77 for YCPE 2.5 (1) and 1 μg/mL (2), 0.75 for serine (3), 0.71 for threonine (4), and 0.57 for phenylalanine (5).

Figure 2

(a) Bar plot of MTT viability assay of HeLa cells treated with different concentrations of YCPE. The bars represent the mean values of at least three experiments plus SD. **** p < 0.0001 indicates the significance obtained with Dunnett’s multiple comparison test of treated cells (red) vs. control (dark gray). (b) Representative images of control cells (upper) and cells treated with 6 μg/mL of YCPE (lower). Scale bar: 100 μm.

Figure 3

Bar graph representing the percentage of cells in the various phases of the cell cycle following 24 h of treatment with 1 and 6 μg/mL of YCPE. Each bar represents the percentage change in the number of treated cells compared to the number of cells in the corresponding phase of the cell cycle of the untreated control group. The red dashed line indicates the value 100% as a reference point to facilitate the visualization of the results.