Search Results (11)

In the human ABCG2 (ATP Binding Casette transporter G2/BCRP/MXR) multidrug transporter, a so-called “leucin plug/valve” (a.a. L554/L555) has been suggested to facilitate substrate exit and the coupling of drug transport to ATPase activity. In this work, we analyzed the effects of selected variants in this region by expressing these variants, both in mammalian and Sf9 insect cells. We found that, in mammalian cells, the L554A, L554F, L555F, and a combination of L554F/L555F variants of ABCG2 were functional, were processed to the plasma membrane, and exhibited substrate transport activity similar to the wild-type ABCG2, while the L555A and L554A/L555A mutants were poorly expressed and processed in mammalian cells. In Sf9 cells, all the variants were expressed at similar levels; still, the L555A and L554A/L555A variants lost all transport-related functions, while the L554F and L555F variants had reduced dye transport and altered substrate-stimulated ATPase activity. In molecular dynamics simulations, the mutant variants exhibited highly rearranged contacts in the central transmembrane helices; thus, alterations in folding, trafficking, and function can be expected to occur. Our current studies reinforce the importance of L554/L555 in ABCG2 folding and function, while they do not support the specific role of this region in selective substrate handling and show a general reduction in the coupling of drug transport to ATPase activity in the mutant versions. Full article

Acquired resistance to chemotherapeutic drugs is the primary cause of treatment failure in the clinic. While multiple factors contribute to this resistance, increased expression of ABC transporters—such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins—play significant roles in the development of resistance to various chemotherapeutics. We found that Erastin, a ferroptosis inducer, was significantly cytotoxic to NCI/ADR-RES, a P-gp-expressing human ovarian cancer cell line. Here, we examined the effects of both Erastin and RSL3 (Ras-Selected Ligand 3) on reversing Adriamycin resistance in these cell lines. Our results show that Erastin significantly enhanced Adriamycin uptake in NCI/ADR-RES cells without affecting sensitive cells. Furthermore, we observed that Erastin enhanced Adriamycin cytotoxicity in a time-dependent manner. The selective iNOS inhibitor, 1400W, reduced both uptake and cytotoxicity of Adriamycin in P-gp-expressing NCI/ADR-RES cells only. These findings were also confirmed in a BCRP-expressing human breast cancer cell line (MCF-7/MXR), which was selected for resistance to Mitoxantrone. Both Erastin and RSL3 were found to be cytotoxic to MCF-7/MXR cells. Erastin significantly enhanced the uptake of Hoechst dye, a well-characterized BCRP substrate, sensitizing MCF-7/MXR cells to Topotecan. The effect of Erastin was inhibited by 1400W, indicating that iNOS is involved in Erastin-mediated enhancement of Topotecan cytotoxicity. RSL3 also significantly increased Topotecan cytotoxicity. Our findings—demonstrating increased cytotoxicity of Adriamycin and Topotecan in P-gp- and BCRP-expressing cells—suggest that ferroptosis inducers may be highly valuable in combination with other chemotherapeutics to manage patients’ cancer burden in the clinical setting. Full article

Mixed reality (MxR) enables the projection of virtual three-dimensional objects into the user’s field of view via a head-mounted display (HMD). This phantom model study investigated three different workflows for navigated common femoral arterial (CFA) access and compared it to a conventional sonography-guided technique as a control. A total of 160 punctures were performed by 10 operators (5 experts and 5 non-experts). A successful CFA puncture was defined as puncture at the mid-level of the femoral head with the needle tip at the central lumen line in a 0° coronary insertion angle and a 45° sagittal insertion angle. Positional errors were quantified using cone-beam computed tomography following each attempt. Mixed effect modeling revealed that the distance from the needle entry site to the mid-level of the femoral head is significantly shorter for navigated techniques than for the control group. This highlights that three-dimensional visualization could increase the safety of CFA access. However, the navigated workflows are infrastructurally complex with limited usability and are associated with relevant cost. While navigated techniques appear as a potentially beneficial adjunct for safe CFA access, future developments should aim to reduce workflow complexity, avoid optical tracking systems, and offer more pragmatic methods of registration and instrument tracking. Full article

The teleost ATP Binding Cassette (ABC) transporter P-glycoprotein (P-gp) is an active transmembrane transporter that plays a pivotal role in facilitating the movement of both endogenous and xenobiotic substrates (moderately hydrophobic and amphipathic compounds) across cell membranes. P-gp exhibits substrate specificity often shared with other ABC transporters and solute carrier proteins, thereby ensuring the maintenance of chemical homeostasis within cells. These transporters are integral to chemical defense systems in fish, as they actively expel a wide range of substrates, primarily unmodified compounds, from cells. This transport process assists in preventing chemical absorption (e.g., intestine), safeguarding sensitive tissues (e.g., brain and gonads), and effectively excreting substances (e.g., liver and kidney). Upregulated P-gp export activity in aquatic animals results in the multi-xenobiotic resistance (MXR) phenotype that plays an essential protective role in survival in contaminated environments. Pollutants inhibiting P-gp are termed chemosensitizers and heighten fish sensitivity to toxic P-gp substrates. While the known intrinsic functions of P-gp in fish encompass steroid hormone and bile acid processing, relatively little attention has been given to endogenous substrates and inhibitors. Fish P-glycoprotein regulation is orchestrated by pivotal nuclear transcription factors, including pregnane X receptor (PXR) and nuclear factor erythroid 2-related factor 2 (Nrf2). This comprehensive review provides profound insights into P-gp’s significance across diverse fish species, contributing to an enhanced understanding of fish physiology, evolution, and toxicology, and provides information with potential applications, such as environmental monitoring. Full article

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354–367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357–360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter. Full article

ATP-binding cassette transporters, including ABCB1 (P-glycoprotein) and ABCG2 (BCRP/MXR/ABCP), are pivotal in multidrug resistance (MDR) development in cancer patients undergoing conventional chemotherapy. The absence of approved therapeutic agents for multidrug-resistant cancers presents a significant challenge in effectively treating cancer. Researchers propose repurposing existing drugs to sensitize multidrug-resistant cancer cells, which overexpress ABCB1 or ABCG2, to conventional anticancer drugs. The goal of this study is to assess whether furmonertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor overcomes drug resistance mediated by ABCB1 and ABCG2 transporters. Furmonertinib stands out due to its ability to inhibit drug transport without affecting protein expression. The discovery of this characteristic was validated through ATPase assays, which revealed interactions between furmonertinib and ABCB1/ABCG2. Additionally, in silico docking of furmonertinib offered insights into potential interaction sites within the drug-binding pockets of ABCB1 and ABCG2, providing a better understanding of the underlying mechanisms responsible for the reversal of MDR by this repurposed drug. Given the encouraging results, we propose that furmonertinib should be explored as a potential candidate for combination therapy in patients with tumors that have high levels of ABCB1 and/or ABCG2. This combination therapy holds the potential to enhance the effectiveness of conventional anticancer drugs and presents a promising strategy for overcoming MDR in cancer treatment. Full article

Prions are self-propagating, misfolded forms of proteins associated with various neurodegenerative diseases in mammals and heritable traits in yeast. How prions form spontaneously into infectious amyloid-like structures without underlying genetic changes is poorly understood. Previous studies have suggested that methionine oxidation may underlie the switch from a soluble protein to the prion form. In this current study, we have examined the role of methionine sulfoxide reductases (MXRs) in protecting against de novo formation of the yeast [PSI⁺] prion, which is the amyloid form of the Sup35 translation termination factor. We show that [PSI⁺] formation is increased during normal and oxidative stress conditions in mutants lacking either one of the yeast MXRs (Mxr1, Mxr2), which protect against methionine oxidation by reducing the two epimers of methionine-S-sulfoxide. We have identified a methionine residue (Met124) in Sup35 that is important for prion formation, confirming that direct Sup35 oxidation causes [PSI⁺] prion formation. [PSI⁺] formation was less pronounced in mutants simultaneously lacking both MXR isoenzymes, and we show that the morphology and biophysical properties of protein aggregates are altered in this mutant. Taken together, our data indicate that methionine oxidation triggers spontaneous [PSI⁺] prion formation, which can be alleviated by methionine sulfoxide reductases. Full article

Earthworms are key organisms of the soil ecosystem and bioindicators for soil quality. While pesticides are used for the improvement of crop yields, they also present a burden for soil organisms. To understand the complex effects of pesticides on soil organisms, it is important to test these effects in soil exposures to include influences of the soil matrix on the toxicity. Therefore, the aim of this study was the assessment of the effects pesticides on earthworm Eisenia andrei. In an initial screening, active ingredients and commercial preparations were tested for comparison. Since the commercial preparations showed a higher toxicity, all further investigations (biomarkers, multixenobiotic resistance (MXR) activity, and avoidance behavior) were performed using the commercial pesticide formulations only: Sumialfa (esfenvalerate), Calypso (thiacloprid), Frontier (dimethenamid-p), and Filon (prosulfocarb). Significant differences in avoidance behavior were observed for Filon and Frontier. All pesticides inhibited the MXR activity and affected oxidative stress-related markers. Frontier was the only pesticide that did not affect enzymatic biomarkers related to neurotransmission. The results show the potential hazards associated with the usage of the tested pesticides and the importance of evaluating the effects of commercial pesticide preparations for a more realistic insight into the adverse effects on the environment. Full article

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and βO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18βH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and β-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC₅₀^(24h) = 0.7–4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and βO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and βO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates. Full article

The mussel Mytilus galloprovincialis is one of the most important aquaculture species in Europe. Its main production problem is the accumulation of toxins during coastal blooms, which prevents mussel commercialization. P-glycoprotein (ABCB1/MDR1/P-gp) is part of the multixenobiotic resistance system in aquatic organisms, and okadaic acid, the main DSP toxin, is probably a substrate of the P-gp-mediated efflux. In this study, the presence and possible role of P-gp in the okadaic acid detoxification process was studied in M. galloprovincialis. We identified, cloned, and characterized two complete cDNAs of mdr1 and mdr2 genes. MgMDR1 and MgMDR2 predicted proteins had the structure organization of ABCB full transporters, and were identified as P-gp/MDR/ABCB proteins. Furthermore, the expression of mdr genes was monitored in gills, digestive gland, and mantle during a cycle of accumulation-elimination of okadaic acid. Mdr1 significantly increased its expression in the digestive gland and gills, supporting the idea of an important role of the MDR1 protein in okadaic acid efflux out of cells in these tissues. The expression of M. galloprovincialismrp2, a multidrug associated protein (MRP/ABCC), was also monitored. As in the case of mdr1, there was a significant induction in the expression of mrp2 in the digestive gland, as the content of okadaic acid increased. Thus, P-gp and MRP might constitute a functional defense network against xenobiotics, and might be involved in the resistance mechanisms to DSP toxins. Full article

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts. Full article