Yu.V. Abalenikhina1, A.V. Shculkin2, A.A. Seidkulieva3,  M.O. Poroshina4, E.A. Sudakova5, E.N. Yakusheva6

1–6 Ryazan State Medical University (Ryazan, Russia)

P-glycoprotein (Pgp, ABC-transporter) is of great clinical importance, since it is involved in the removal of endogenous and exogenous substances from the cell. The regulation of its expression and activity has been extensively studied under the influence of drugs, but under conditions of oxidative stress has not been sufficiently described. Oxidative stress is a typical pathological process; therefore, it seems relevant to study the mechanisms of Pgp regulation in order to increase the effectiveness of pharmacotherapy.

Aim – to evaluate changing of Pgp amount and to study regulatory role of NF-E2-related factor 2 (Nrf2) in the process of Pgp expression under glutathione synthesis inhibition in vitro.

The research was done on Caco-2 cells (Human colon adenocarcinoma cells). Caco-2 cells were cultured for 21 days because it was the time interval required for their spontaneous differentiation into small intestine enterocyte-like cells overexpressing Pgp. Glutathione synthesis inhibition was modeled by adding of DL-buthionine sulfoximine (BSO, Sigma-Aldrich, Germany) to the culture medium in final concentrations 1, 5, 10, 50, 100 µM and incubation for 24 hours. Pgp and Nrf2 level in cells lysate was evaluated via ELISA test (ELISA kit, Blue gene, China). The concentration of protein and non-proteinn thiol groups was determined using the Ellman method with 5,5′-dithiobis(2-nitro)-benzoic acid (SERVA, Germany) under non-denaturing conditions.

Buthionine sulfoximine in concentrations 1 and 5 µM didn`t influence on the level of reduced thiols, Nrf2 and Pgp. Influence of buthionine sulfoximine in concentrations 10-100 µM in Caco-2 cells led to decrease of protein and non-proteinn thiol groups level. Buthionine sulfoximine in concentrations 10, 50 and 100 µM stimulated growth of Nrf2 level what correlated with concentration of reduced protein thiols in a negative way. Pgp amount increased under the influence of buthionine sulfoximine in concentrations 10 and 50 µM what correlated with Nrf2 level in a positive way.

That way, in the conditions of glutathione synthesis deficit modeling under influence of buthionine sulfoximine in concentration of 100 µM Pgp level growth is the adaptive mechanism aiming at maintenance of Pgp level up to the standard. Under the mild oxidative stress (buthionine sulfoximine in concentration 10 and 50 µM) increase of Pgp level is of defensive aim due to transport protein can participate in the efflux of peroxidation products from the cells. But, an increase in the amount of Pgp with a decrease in glutathione content may explain the increased level of the transporter protein in tumor cells, the formation of which is associated with the development of oxidative stress. Proteins oxidation on thiol residues activates Nrf2 transcription factor which in its turn increases Pgp amount in the conditions of glutathione synthesis inhibition.

Abalenikhina Yu.V., Shculkin A.V., Seidkulieva A.A., Poroshina M.O., Sudakova E.A., Yakusheva E.N. Multi-drug resistance  P-glycoprotein in the conditions of glutathione synthesis inhibition in vitro. Technologies of Living Systems. 2021. V. 18. № 3. Р. 43−51. DOI: https://doi.org/10.18127/j20700997-202103-04 (in Russian).

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