Cellular, genomic and transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

Abstract

Purpose of the study. To evaluate the cellular, genomic (gene copy number) and transcriptomic (gene expression) effects of P.hybridus (L.) secondary metabolites when they affect the HeLa cell line.Materials and methods. The isolation of secondary metabolites from plant material and its identification were carried out by preparative chromatography. The composition was determined using mass spectrometric analysis, and the final verification of structural formulas was carried out by nuclear magnetic resonance at the Department of Natural Compounds, the Faculty of Chemistry of the Southern Federal University. The subsequent phase of the study was conducted using both cultural and molecular methods. HeLa cells were cultivated under standard conditions in a MEM medium. Once the confluence level was reached 75–80 %, the nutrient medium was replaced with the introduction of the studied compounds (at a concentration of 4 micrograms/ml) and cultivated for 72 hours. Cell mortality was determined using a NanoEnTek JuliFl counter (Korea) in the presence of 0.4 % trypan blue. The assessment of apoptosis following secondary metabolite exposure was conducted on a BD FACSCanto II flow cytometer using the FITC Annexin V Apoptosis Detection Kit I. The level of replication and expression of the genes responsible for apoptosis was assessed by digital droplet PCR (ddPCR).Results. The following compounds were isolated and verified, and were assigned the following sequence numbers to facilitate their use in the experiment: No. 2 – 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one, No. 3 – 5-(hydroxymethyl) furan-2-carbaldehyde, No. 5.3 – 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, P. hybridus (L.) At the stage of cell death assessment, it was found that the greatest effect was achieved in the compound under ordinal No. 2. However, the evaluation of the copy number and expression of the CASP8, CASP9, CASP3, BAX, BCL2, TP53, MDM2, CDKN1B, CDK1, CCND1, CCND3, and RB1 genes by DD-PCR revealed the presence of apoptosis initiation in tumor cells at the molecular level under the action of compounds No. 2 and No. 5.3 obtained from P. hybridus (L.).Conclusion. The outcomes were multifeatured. Only compound 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one exhibited a pronounced cytostatic effect out of all compounds utilized in the experiment. Concurrently, the compound 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde was found to induce an increase in the expression of the CASP3, CASP8, TP53, and BAX genes.

Keywords

• secondary plant metabolites
• apoptosis
• gene expression
• copy number variation
• hela cell line
• digital droplet pcr