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  • Effect of secondary metabol...
    Enin, Yaroslav S.; Burov, Oleg N.; Ryadinskaya, Ludmila A.; Abramova, Natalia A.; Agieva, Aza A.; Alieva, Faina V.; Sergeeva, Marina M.; Tikhanovskaya, Natalia M.; Teplyakova, Maria A.; Novoselova, Kristina A.; Myagkova, Valeriya S; Popova, Irina L.; Zlatnik, Elena Yu; Vladimirova, Liubov Yu

    Journal of clinical oncology, 05/2021, Letnik: 39, Številka: 15_suppl
    Journal Article

    Abstract only e15063 Background: The study of the possible antitumor activity of secondary plant metabolites either in the form of individual agents or in combination with clinically used drugs is considered as a promising direction in the therapy of malignant tumors. The aim of this study was an estimation of antitumor activity of secondary plant metabolites in the in vitro experiment on the HeLa cell line. Methods: Secondary metabolites were extracted from plant raw materials and were isolated by preparative chromatography. Determination of their composition was carried out using HPLC; obtained compound structures were identified by NMR. We selected 4 secondary metabolites from Petasites hybridus for testing: (2,4-dihydroxy-2,5-dimethylfuran-3 (2H), 5-(hydroxymethyl) furan-2-carbaldehyde, 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, Corynan), and 1 secondary metabolite from Berberis vulgaris : a berberine chloride (BBR, C 20 H 18 NO 4 + Cl - is a derivative of 5,6-dihydrobenzoa,g isoquinolinium). HeLa CCL2 cultivation was carried out under standard conditions in the MEM medium. When reaching the 75-80% confluence level we replaced nutrient medium with the introduction of secondary metabolites (concentration 4 and 12 μg/ml) and cultured for 24 and 72 hours. Cell survival was determined on a NanoEnTek JuliFl counter (Korea) in the presence of 0.4% trypan blue. Apoptosis was assessed on a flow cytometer BD FACSCanto II using FITC Annexin V Apoptosis Detection Kit I. Post-exposure copy number and expression were assessed by Real-time PCR with a panel of genes CASP9, CASP8, CASP3, TP53, MDM2, BAX, BCL2, CDK1, BRCA1, BRCA 2, RB1. Results: All obtained data were normalized by negative control. When we used 4 μg/ml berberine solution with 72-hour exposition, the proapoptogenic effect was maximal, causing the death of 67.2% of HeLa cells (29.3% early apoptosis, 37.2% late apoptosis, 0.7% necrosis). Within 24 hours, berberine at the same concentration caused a 2-fold increase in TP53 expression relative to MDM2. An increase in its concentration to 12 μg/ml and exposure for up to 72 hours led to a 31-fold increase in TP53/MDM2. The terpenoid 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde at a concentration of 12 μg/ml after 72 hours of cultivation caused a 6.5-fold increase in the TP53/MDM2 ratio. The corynan alkaloid (12 μg/ml) at an exposure of 72 hours increased the BAX/BCL ratio by 2.4 times. There were no statistically significant differences in the expression and copy number of the remaining genes studied. Conclusions: Berberine, corynan, 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde showed high promise in the HeLa cell line in vitro, since they surpassed the antitumor activity of other metabolites of Berberis vulgaris and Petasites hybridus.