SYNERGIC EFFECT OF EXOGENOUS P53 AND SODIUM BUTYRATE ON TUMOR CELL SURVIVAL



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Abstract

Relevance: The P53 protein is a transcription factor that regulates the expression of genes associated with a variety of cellular functions, including cell cycle arrest, apoptosis, cell proliferation, DNA repair, etc. The status of a “guardian of the genome” and a multifunctional tumor suppressor makes P53 an attractive and promising target for cancer therapy.

Aim: The aim of the study was to analyze the effect of the combined effects of transfection of a plasmid encoding the p53 gene (genetic method), the histone deacetylase inhibitor sodium butyrate (epigenetic regulation) and co-cultivation with exosomes secreted by cells with wild type P53 (modeling of intercellular communication) on the survival of cell lines of various human tumors.

Materials and methods: The study was conducted on four continuous cell lines: wild type P53 is expressed in cells of the HeLa (epithelioid carcinoma of the cervix) and HT-1080 (fibrosarcoma) lines, cells of two other lines K562 (chronic myelogenous leukemia) and Gl-V (primary culture of glioma cells) it is not contained (P53-/-).

Cells were transfected with a P53-GFP plasmid created in our laboratory, encoding the P53 protein carrying green fluorescent protein (GFP) at the N-terminus. Successful transfection was monitored by P53-GFP protein expression using a confocal microscope. The level of P53 protein in cells was determined by immunoblotting. To quantify proliferation and cell cycle parameters under deacetylase inhibition conditions, sodium butyrate (NaBu) was added to the culture medium to a final concentration of 2.5 mM. Analysis was performed using a manual automatic cell counter, flow cytometry technique, or the ability of cells to form colonies. Exosomes were isolated from the accumulated conditioned medium by ultracentrifugation. The concentration of exosomes was determined using a nanoparticle size and concentration analyzer Malvern Panalytical NanoSight LM10.

Results: The result was highly dependent on the P53 status in the cancer cells analyzed using only one of the above approaches. The combination of correction of the epigenetic profile by inhibiting the activity of deacetylases with genetic regulation or with exosomes from cells with wild type P53 demonstrated a significant synergistic effect and increased the efficiency of suppressing tumor cell growth several times compared to the use of monotherapy.

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About the authors

Roman Kovalev

National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute

Email: kovalev_ra@pnpi.nrcki.ru
ORCID iD: 0000-0003-2214-0269
SPIN-code: 1386-2357
Scopus Author ID: 55634543500

Младший научный сотрудник

Russian Federation, Gatchina, Russia, 188300

Elena Semenova

National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute

Email: semenova_el.spb@mail.ru
ORCID iD: 0000-0003-0852-6595
SPIN-code: 2758-6825
Scopus Author ID: 7102695886

Научный сотрудник

Russian Federation, Gatchina, Russia, 188300

Tatyana Shtam

National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute

Email: Shtam_ta@pnpi.nrcki.ru
ORCID iD: 0000-0003-0651-4785
SPIN-code: 3738-8187
Scopus Author ID: 6508065266

Старший научный сотрудник

Russian Federation, Gatchina, Russia, 188300

Vladimir Burdakov

National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute

Email: Burdakov_vs@pnpi.nrcki.ru
ORCID iD: 0000-0001-6025-7367
SPIN-code: 8832-9047
Scopus Author ID: 55347037600

Младший научный сотрудник

Russian Federation, Gatchina, Russia, 188300

Elena Varfolomeeva

National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute

Author for correspondence.
Email: Varfolomeeva_EY@pnpi.nrcki.ru
ORCID iD: 0000-0003-3287-4709
SPIN-code: 9426-1667
Scopus Author ID: 6701723593

Старший научный сотрудник

Gatchina, Russia, 188300

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