We investigated the mechanisms of P-glycoprotein (P-gp) transporter regulation in Caco-2 cells under exogenous and endogenous oxidative stress (OS). Exogenous OS was modeled by exposure of the growth medium to hydrogen peroxide at concentrations of 0.1, 0.5, and 1 M for 24 h or 10 M for 72 h. Endogenous OS was modeled by incubating cells with DL-buthionine sulfoximine (BSO, gamma-glutamylcysteine synthetase inhibitor) at a concentration of 10, 50, and 100 M for 24 h. The levels of intracellular reactive oxygen species (ROS) were assessed using MitoTracker Red CM-H2XRos fluorescent probes. Relative P-gp contents were analyzed using Western blot. Exogenous and endogenous OS was shown to increase relative to P-gp contents. An important role played by the Nrf2-Keap1 signaling pathway in increasing the P-gp contents under H2O2-induced exogenous OS was revealed using specific inhibitors. The transcription factor HIF1 is involved in the regulation of the P-gp levels under 24-hour exogenous OS, and the transcription factor CAR is involved in the regulation of transporter levels under 72-hour OS. All tested transcription factors and signaling pathways are involved in P-gp induction under endogenous OS. Most likely, this is associated with the bimodal effect of BSO on Pgp. On the one hand, BSO induces the development of OS; on the other, BSO, as a xenobiotic, is able to stimulate PXR and CAR, which, in turn, increase the P-gp contents.
Background. P-glycoprotein (Pgp) is a multidrug resistance protein 1 with broad substrate specificity. Its functioning can change under the influence of various substances, so the search for endogenous and exogenous compounds that modulate the activity of the transporter protein is an important area of research.
Aim. To evaluate the effect of DL-butionine sulfoximine on the activity and amount of the Pgp transporter protein in Caco-2 cells.
Material and methods. The study was performed on a human colon adenocarcinoma cell line (Caco-2). Cells were incubated with DL-butionine sulfoximine and quinidine (a classical Pgp inhibitor) at concentrations of 1, 5, 10, 50, 100, and 500 M for 3 hours. Pgp activity was evaluated by the transport of its substrate, fexofenadine, which concentration was determined by high performance liquid chromatography with ultraviolet detection (Stayer, Russia). The results were analyzed using StatSoft Statistica 13.0 (ANOVA), IC50 was calculated using GraphPad Prism 8 software. Differences were considered statistically significant at p 0.05.
Results. Incubation with DL-butionine sulfoximine and quinidine at concentrations of 1500 M for 3 hours did not affect the amount of Pgp in Caco-2 cells. Pgp activity decreased when using DL-butionine sulfoximine at concentrations of 50500 M by a maximum of 47.7% (p=0.040). Quinidine at concentrations of 5500 M reduced Pgp activity by a maximum of 79.1% (p=0.0002) at a concentration of 500 M. Quinidine inhibited Pgp activity at lower concentrations compared to DL-butionine sulfoximine: the IC50 of fexofenadine with quinidine was 5.160.59 mol/l, for DL-butionine sulfoximine it was 17.212.46 mol/l (p= 0.001).
Conclusion. DL-butionine sulfoximine has a direct inhibitory effect on the activity of the Pgp transporter protein on the Caco-2 cell line.
A bioanalytical technique for quantitative determination of MDA by HPLC-MS/MS. The proposed method for determining MDA includes the release stage of bound MDA and excludes the derivatization reaction. The lower limit of quantitative detection was 600 nmol/l, the volume of the required sample was 10 µl, the analysis time was 7 min. The range of concentrations obtained during the study makes it possible to use this bioanalytical technique to determine the concentration of MDA in biological material when assessing physiological and pathological conditions.
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