Molecular and cellular aspects of the impact of secondary metabolites from common barberry and hybrid goat’s rue on the HeLa cell line

Abstract

Purpose of the study. To isolate and verify pure fractions of secondary plant metabolites contained in B. vulgaris (L.) and P. hibridus (L.), as well as to conduct a model experiment and molecular genetic study to evaluate their cytotoxic effect in vitro on the HeLa cell line.Materials and methods. The isolation and verification of all compounds used in the experiment were carried out using column chromatography and nuclear magnetic resonance methods at the Department of Natural Compounds, Faculty of Chemistry, Southern Federal University. Subsequently, an experiment was conducted using cultural and molecular methods on the HeLa cell line in three repetitions for each test compound; after incubation with them, the numbers of dead cells were counted on the automated NanoEnTek JuliFl counter, and the numbers of cells in apoptosis were measured by flow cytometry on the BD analyzer FACS Canto II. The level of copy number variation and expression of genes responsible for apoptosis were assessed by real-time PCR (RT-PCR). In total, three substances were studied, with two concentrations (4 and 12 μg/ml) and two exposures (24 and 72 hours) for each of them.Results. In the first stage of the study, we isolated and verified the berberine alkaloid extracted from the roots of B. vulgaris (L.), as well as 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde from P. hibridus (L.). The subsequent stage of the study demonstrated the maximal cellular death under the action of berberine at a 72-hour exposure. However, the RT-PCR assessment of the copy number variation and expression of the CASP8, CASP9, CASP3, BAX, BCL2, TP53 and MDM2 genes revealed the presence of apoptosis initiation in tumor cells at the molecular level under the action of all the studied compounds: both berberine and furan and azulene derivatives derived from P. hibridus (L.).Conclusion. All compounds used in the experiment exhibited a cytotoxic effect on the HeLa cell line. Berberine alkaloid showed the most pronounced cytotoxic effect on the HeLa line as recorded by all methods used in the study. Terpenoids 4-dihydroxy-2,5-dimethylfuran-3(2H)-one and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, when exposed to the HeLa line, caused an increase in the copy number variation and expression of the CASP9, CASP3 loci, which are among the main activators of apoptosis. They also influenced the expression of TP53 and MDM2 loci.

Keywords

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