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The membrane efflux transporter P-glycoprotein (P-gp, [ABCB1, MDR1]) exports a wide range of xenobiotic compounds, resulting in a continuous first line of defense against toxicant accumulation at basal expression levels, and contributing to the multixenobiotic resistance (MXR) phenotype at elevated expression levels. Relatively little information exists on P-gp inhibition in fish by chemosensitizers, compounds which lower toxicity thresholds for harmful P-gp substrates in complex mixtures. The effects of four known mammalian chemosensitizers (cyclosporin A [CsA], quinidine, valspodar [PSC833], and verapamil) on the P-gp-mediated transport of rhodamine 123 (R123) and cortisol in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes were examined. Competitive accumulation assays using 25 µM R123 or cortisol and varying concentrations of chemosensitizers (0-500 µM) were used. CsA, quinidine, and verapamil inhibited R123 export (IC50 values ± SE: 132 ± 60, 83.3 ± 27.2, and 43.2 ± 13.6 µM, respectively). CsA and valspodar inhibited cortisol export (IC50 values: 294 ± 106 and 92.2 ± 34.9 µM, respectively). In an ATP depletion assay, hepatocytes incubated with all four chemosensitizers resulted in lower free ATP concentrations, suggesting that they act via competitive inhibition. Chemosensitizers that inhibit MXR transporters are an important class of environmental pollutant, and these results show that rainbow trout transporters are inhibited by similar chemosensitizers (and mostly at similar concentrations) as seen in mammals and other fish species., Competing Interests: Declarations Ethics approval Research was approved by the SFU University Animal Care Committee, Protocol numbers 1138B-14 and 1308B-20. Consent to participate All authors consent to participate. Consent for publication All authors consent to publication. Conflicts of interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Investigating blood-brain barrier (BBB) dysfunction has become a pre-clinical and clinical research focus as it accompanies many neurological disorders. Nevertheless, knowledge of how diagnostic BBB tracers cross the endothelium from blood-to-brain or vice versa often remains incomplete. In particular, brain-to-blood transport (efflux) may reduce tracer extravasation of intravascularly (i.v.) applied tracers. Conversely, impaired efflux could mimic phenotypic extravasation. Both processes would affect conclusions on BBB properties primarily attributed to blood-to-brain leakage. Here, we specifically investigated efflux of fluorescent BBB tracers, focusing on the most common non-toxic marker, sodium fluorescein, which is applicable in patients. We used acute neocortical slices from mice and applied fluorescein, sulforhodamine-B, rhodamine-123, FITC dextran to the artificial cerebrospinal fluid. Anionic low molecular weight (MW) fluorescein and sulforhodamine-B, but not ~ 10-fold larger FITC-dextran and cationic low MW rhodamine-123, showed efflux into the lumen of blood vessels. Our data suggest that fluorescein efflux depends on organic anion transporter polypeptides (Oatp) rather than P-glycoprotein. Furthermore, sodium-potassium ATPase inhibition and incomplete oxygen-glucose deprivation (OGD, 20% O 2 ) reduced fluorescein efflux, while complete OGD (0% O 2 ) abolished efflux. We provide evidence for active efflux of fluorescein in vitro. Impaired efflux of fluorescein could thus contribute to the frequently observed BBB dysfunction in neuropathologies in addition to blood-to-brain leakage., (© 2024. The Author(s).)

The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell ® -based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

There are several in vitro systems that enable evaluation of the absorption direction, but there are few quantitative systems that enable easy evaluation of the excretion direction. Enteroids, organoids derived from intestine, have been frozen and passaged for various research. But it is not clear how the freezing and passaging affect the expression and function of transporters. We investigated the effects of passage and cryopreservation of enteroids. We focused on P-gp (P-glycoprotein) and compared the transfer rates of rhodamine 123 (Rh123) into the lumen of enteroids with and without a P-gp inhibitor. mRNA expression levels did not change significantly before and after passage and cryopreservation. Accumulation of Rh123 in the lumen of enteroids was observed. With some P-gp inhibitors, excretion of Rh123 into the lumen of enteroids was inhibited and the nonexcreted Rh123 accumulated in enteroids epithelial cells. The transfer rate of Rh123 into the lumen of enteroids with a P-gp inhibitor was significantly decreased compared to that of without a P-gp inhibitor. Before and after passage and cryopreservation, the transfer rate was almost the same as that of primary cultured enteroids. We succeeded in easily evaluating whether a component is a substrate of P-gp using enteroids., Competing Interests: Conflicts of interest The authors report no conflicts of interest in this work., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Background: Individuals with Alzheimer's disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model., Methods: This study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 - 10 µg/mL over 24 - 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms., Results: P-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively., Conclusions: Different apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner., (© 2024. The Author(s).)

P-glycoprotein (P-gp), a multidrug efflux pump encoded by the ABCB1 (formerly MDR1 ) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored.We generated canine colonoid-derived 2D monolayers to investigate ABCB1 gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture.A significant increase in gene expression of P-gp encoded by the ABCB1 was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model.Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research.

The inhibition of P-glycoprotein (P-gp) has emerged as an intriguing strategy for circumventing multidrug resistance (MDR) in anticancer chemotherapy. In this study, we have designed and synthesized 30 indole-selenides as a new class of P-gp inhibitors based on the scaffold hopping strategy. Among them, the preferred compound H27 showed slightly stronger reversal activity (reversal fold: 271.7 vs 261.6) but weaker cytotoxicity (inhibition ratio: 33.7% vs 45.1%) than the third-generation P-gp inhibitor tariquidar on the tested MCF-7/ADR cells. Rh123 accumulation experiments and Western blot analysis demonstrated that H27 displayed excellent MDR reversal activity by dose-dependently inhibiting the efflux function of P-gp rather than its expression. Besides, UIC-2 reactivity shift assay revealed that H27 could bind to P-gp directly and induced a conformation change of P-gp. Moreover, docking study revealed that H27 matched well in the active pockets of P-gp by forming some key H-bonding interactions, arene-H interactions and hydrophobic contacts. These results suggested that H27 is worth to be a starting point for the development of novel Se-containing P-gp inhibitors for clinic use., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The ATP-dependent membrane transporter P-glycoprotein (P-gp) is associated with resistance to a wide variety of chemical substrates, as well as the multi-drug resistance (MDR) phenotype in mammals. Less is known regarding P-gp's function and relevance in teleosts; this study expanded the range of known substrates and the inhibitory effects of a model chemosensitizer verapamil. The P-gp-mediated uptake and efflux dynamics of 5 known mammalian substrates (berberine, cortisol, doxorubicin, rhodamine 123 [R123], and vinorelbine) were examined in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes with and without co-exposure to varying doses of verapamil. Initial substrate uptake rates (pmol/10 6 cells/min) varied widely and were in order: berberine (482 ± 94) > R123 (364 ± 67) > doxorubicin (158 ± 41) > cortisol (20.3 ± 5.9) > vinorelbine (15.3 ± 3.5). Initial efflux rates (pmol/10 6 cells/min) were highest in berberine (464 ± 110) > doxorubicin (341 ± 57) > R123 (106 ± 33) > cortisol (26.6 ± 6.1) > vinorelbine (9.0 ± 2.4). Transport of vinorelbine and R123 is verapamil sensitive, but verapamil had no effect on transport of berberine, cortisol, or doxorubicin. Cortisol and doxorubicin showed evidence of high P-gp affinity, thus displacing verapamil from their shared P-gp binding site. Cortisol, doxorubicin, R123, and vinorelbine transport by rainbow trout P-gp was confirmed, while berberine could not be confirmed or excluded as a substrate. Binding sites and affinities were similar between mammalian and trout P-gp for doxorubicin, R123, and vinorelbine, while fish P-gp had a higher affinity for cortisol than mammalian P-gp. This study demonstrated that the range of substrates, as well as binding sites and affinities, of fish P-gp are well-aligned with those in mammals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp., (© 2023. The Author(s).)

A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp; the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

Forsythiaside, one of the main bioactive components of Chinese medicine Lian Qiao, exerts antioxidant, anti-bacterial, and anti-inflammatory effects. To date, the mechanism of Forsythiaside in cardiomyocyte injury remains unclear. However, the antioxidant effects of Forsythiaside on cardiac cells are currently unknown. This study investigated the effect and mechanism of Forsythiaside on oxidative stress in H9c2 cardiomyocytes. H9c2 cells were treated with H 2 O 2 and Forsythiaside and then transfected with small-interfering RNA against nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability, apoptosis, accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential were measured using methyl thiazolyl tetrazolium (MTT), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123, respectively. The levels of oxidative stress-related markers were determined using their respective detection kits. Furthermore, the levels of apoptosis- and Nrf2 pathway-related molecules were determined via Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Forsythiaside had no obvious toxicity on H9c2 cells. H 2 O 2 suppressed the viability, and reduced the levels of mitochondrial membrane potential, B-cell lymphoma-2 (Bcl-2), glutathione peroxidase (GSH-Px) and catalase (CAT) and superoxide dismutase (SOD), while promoted apoptosis, ROS accumulation, and elevated the levels of cleaved caspase 3, BCL2-Associated X (Bax) and malondialdehyde (MDA) in H9c2 cells. Contrarily, Forsythiaside reversed the aforementioned effects. H 2 O 2 advanced the levels of cytoplasm Nrf2, heme oxygenase-1 (HO-1), and nucleus Nrf2 in H9c2 cells, whereas Forsythiaside enhanced these effects. SiNrf2 reversed the functions of H 2 O 2 or Forsythiaside in cell viability, MDA, SOD, GSH-Px, CAT, Nrf2, and HO-1 in H9c2 cells, whereas Forsythiaside reversed the aforementioned effects of siNrf2. In sum, Forsythiaside protected H9c2 cells from oxidative stress and apoptosis induced by H 2 O 2 by activating the Nrf2/HO-1 pathway.

Objectives: Epithelial-mesenchymal transition (EMT) plays a role in cancer metastasis as well as in drug resistance through various mechanisms, including increased drug efflux mediated by P-glycoprotein (P-gp). In this study, we investigated the activation mechanism of P-gp, including its regulatory factors, during EMT in hepatoblastoma-derived HepG2 cells., Methods: HepG2 cells were transfected with SNAI1 using human adenovirus serotype 5 vector. We quantified mRNA and protein expression levels using qRT-PCR and western blot analysis, respectively. P-gp activity was evaluated by uptake assay, and cell viability was assessed by an MTT assay., Key Findings: P-gp protein expression on plasma membrane was higher in SNAI1-transfected cells than in Mock cells, although there was no difference in P-gp protein level in whole cells. Among the scaffold proteins such as ezrin, radixin and moesin (ERM), only radixin was increased in SNAI1-transfected cells. Uptake of both Rho123 and paclitaxel was decreased in SNAI1-transfected cells, and this decrease was blocked by verapamil, a P-gp inhibitor. The reduced susceptibility of SNAI1-transfected cells to paclitaxel was reversed by elacridar, another P-gp inhibitor., Conclusions: Increased expression of radixin during SNAI1-induced EMT leads to increased P-gp membrane expression in HepG2 cells, enhancing P-gp function and thereby increasing drug resistance., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Semi-synthetic A-cycle modified triterpenic derivatives with A-cycle condensed with a heterocyclic fragment (compound 1) and fragmented A-ring (compound 2) were tested for cytotoxicity against several tumor cell cultures and doxorubicin (Dox)-resistant cell lines. The equal cytotoxicity of the tested compounds to the parental tumor cell lines (HBL-100, K562) and their resistant subclones (HBL-100/Dox, K562/i-S9) was revealed. The overexpression of ABCB1 (MDR1) gene and P-glycoprotein (P-gp) was confirmed for both resistant subclones of tumor cells. Compounds 1 and 2 were shown to inhibit the ABC-transporter gene expression (MDR1, MRP, MVP, and BCRP) and the transport of well-known P-gp substrate Rhodamine 123 from resistant cells. The docking of triterpenoids 1 and 2 into the drug binding site of P-gp revealed a similarity between the conformation of the tested triterpenoids and that of classical inhibitor verapamil, thus assuming these compounds to be more likely the inhibitors than the substrates of P-gp. Any tested triterpenic derivatives, when combined at non-toxic concentrations with doxorubicin, improved cytotoxic effect of the therapeutic drug against resistant subclones of tumor cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study was aimed to investigate the inhibitory activity of flavonoids on P-glycoprotein (P-gp). Effects of 39 flavonoids on the cellular uptake (CU) of rhodamine123 (Rho) and daunomycin (DNR) were investigated in both parental KB and P-gp overexpressed KB/MDR cells. The inhibition mechanism of selected flavonoids was further investigated by measuring the ATPase activity and expression level of P-gp. Twelve flavonoids improved the uptake of Rho (↑Rho F ) and nineteen flavonoids increased the uptake of DNR (↑DNR F ) in KB/MDR cells with nine flavonoids overlapped. Structure-activity relationship (SAR) indicated that 8-OCH 3 , and 2'-OH have a negative effect on Rho and DNR transport. Whereas 5-OH, 5-OCH 3 , 6-OH, 7-OCH 3 , 3'-OH, and 4'-OH, are essential for inhibition of flavonoids on P-gp and reversing the resistance of Rho and DNR. Eleven selected flavonoids significantly induced the basal P-gp-ATPase activity but much lower than that induced by verapamil. Tangeretin, galangin, kaempferol, quercetin, and morin significantly reversed the ATPase activity stimulated by verapamil. Six of eleven flavonoids significantly decreased P-gp expression, whereas three flavonoids slightly increased P-gp expression. These results provide valuable information that flavonoids can effectively reverse multidrug resistance of P-gp-mediated transport of nutraceutical and drugs by co-administration., (Copyright © 2021. Published by Elsevier Ltd.)

P-gp-associated multidrug resistance is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure-activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone to the moiety bearing the pyrimidine favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4-7 and 12 is required for inhibition., (© 2021. The Author(s).)

Background: P-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems., Purpose and Study Design: The aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well., Methods: Calcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses., Results: 5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively., Conclusion: The present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Overexpression of P-glycoprotein (P-gp), which is linked to multidrug resistance (MDR), is one of the underlying obstacles to the success of chemotherapy as it reduces the efficacy of anticancer drugs and the side effects of these increase as a result of any increased dose to achieve the therapeutic effect. To identify agents with P-gp inhibitory properties, ethanol extracts from 80 plants were screened for their ability to increase intracellular doxorubicin-associated fluorescence, and the extract of Ligaria cuneifolia was found to be the most effective. Its bioassay-guided isolation yielded the pentacyclic triterpene betulin as active agent. This efficiently inhibited P-gp mediated efflux, as demonstrated by the enhancement of the intracellular accumulation of doxorubicin and rhodamine 123 from 1.56 μM in the P-gp overexpressing MDR leukemia cell, Lucena 1. Betulin was also able to render Lucena 1 sensitive to Dox from 0.39 μM. The docking studies revealed that betulin tightly binds to a key region of the TMDs, with a binding mode overlapping one main site of doxorubicin and, more interestingly, emulating the same contacts as tariquidar, as revealed by the per-residue energetic analysis from molecular dynamics simulations. MTT assay using peripheral blood mononuclear cells and hemolysis assay showed that betulin is devoid of toxicity. These findings provide important evidence that betulin may be a safe and promising entity to be further investigated to develop agents able to overcome P-gp-mediated MDR, resulting in a more effective and less toxic chemotherapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Our previous study demonstrated that the total saponins from Paris forristii (PCT3) had obvious inhibitory effect on the proliferation of adriamycin-resistant human breast adenocarcinoma cells (MCF-7/ADM), and this effect was significantly stronger than that in parental cells (MCF-7). This study was designed to test the reversal effect of PCT3 on MCF-7/ADM cells and to understand its mechanism of action. Results demonstrated that low cytotoxic concentrations of PCT3 (0.3, 1 and 3 µg/mL) reversed resistance of MCF-7/ADM cells to ADM, cisplatin (DDP) and 5-fluorouracil (5-FU), with reversal fold of 16.4, 19.5 and 31.7 for ADM, 1.6, 1.4 and 1.4 for DDP, 1.7, 1.8 and 5.6 for 5-FU, respectively. Moreover, PCT3 significantly increased the accumulation of ADM and Rhodamine 123 (Rh123) in MCF-7/ADM cells, suggesting that PCT3 may act by affecting the function of drug efflux pump P-glycoprotein (P-gp), which is encoded by MDR1 gene. Both MDR1 gene and P-gp protein expression was downregulated by PCT3 treatment. Further results demonstrated that p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathway was remarkably activated in MCF-7/ADM cells, inhibition of p38 or ERK attenuated P-gp expression. While, only the phosphorylation level of ERK was downregulated by PCT3, indicating that PCT3 sensitized P-gp overexpressed MCF-7/ADM cells to ADM via inhibition of ERK signaling pathway.

Context: Shenmai Injection (SMI) is usually used to treat atherosclerotic coronary heart disease and viral myocarditis in China. However, the effect of SMI on multidrug resistance has not been reported. Objective: To investigate the reversal effect of SMI in adriamycin (ADR) resistant breast cancer cell line (MCF-7/ADR) and explore the related molecular mechanisms. Materials and methods: The effect of SMI (0.25, 0.5, 1 mg/mL) to reverse chemoresistance in MCF-7/ADR cells was elucidated by MTT, HPLC-FLD, DAPI staining, flow cytometric analysis, western blotting. At the same time, in vivo test was conducted to probe into the effect of SMI on reversing ADR resistance, and verapamil (10 μM) was used as a positive control. Results: The results showed that the toxicity of ADR to MCF-7/ADR cells was strengthened significantly after treated with SMI (0.25, 0.5, 1 mg/mL), the IC 50 of ADR was decreased 54.4-fold. The intracellular concentrations of ADR were increased 2.2-fold ( p  < 0.05) and ADR accumulation was enhanced in the nuclei ( p  < 0.05). SMI could strongly enhance the ADR-induced apoptosis and increase intracellular rhodamine 123 accumulation in MCF-7/ADR cells. Additionally, a combination of ADR and SMI (5 mg/kg) could dramatically reduce the weight and volume of tumour ( p  < 0.05). Furthermore, the results revealed that SMI might reverse MDR via inhibiting ADR-induced activation of the mitogen-activated protein kinase/nuclear factor (NF)-κB pathway to down-regulated the expression of P-glycoprotein (P-gp). Discussion and conclusions: SMI could potentially be used to treat ADR-resistance. This suggests possibilities for future clinical research.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14 ) were able to resensitize CEM Vbl100 and LoVo Doxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 μM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.

Multidrug resistance (MDR) is a severe problem in the treatment of cancer with overexpression of glycoprotein P (Pgp, ABCB1) as a reason for chemotherapy failure. A series of 14 novel 5-arylideneimidazolone derivatives containing the morpholine moiety, with respect to two different topologies (groups A and B), were designed and obtained in a three- or four-step synthesis, involving the Dimroth rearrangement. The new compounds were tested for their inhibition of the ABCB1 efflux pump in both sensitive (parental (PAR)) and ABCB1-overexpressing (MDR) T-lymphoma cancer cells in a rhodamine 123 accumulation assay. Their cytotoxic and antiproliferative effects were investigated by a thiazolyl blue tetrazolium bromide (MTT) assay. For active compounds, an insight into the mechanisms of action using either the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp was performed. The safety profile in vitro was examined. Structure-activity relationship (SAR) analysis was discussed. The most active compounds, representing both 2-substituted- ( 11 ) and Dimroth-rearranged 3-substituted ( 18 ) imidazolone topologies, displayed 1.38-1.46 fold stronger efflux pump inhibiting effects than reference verapamil and were significantly safer than doxorubicin in cell-based toxicity assays in the HEK-293 cell line. Results of mechanistic studies indicate that active imidazolones are substrates with increasing Pgp ATPase activity, and their dye-efflux inhibition via competitive action on the Pgp verapamil binding site was predicted in silico.

PLGA (poly(lactic-co-glycolic acid))-based nanoparticles (NPs) are promising drug carrier systems because of their excellent biocompatibility and ability for sustained drug release. However, it is not well understood how the kinetics of such drug delivery system perform in the retinal blood circulation as imaged in vivo and in real time. To answer this question, PLGA NPs were loaded either with lipophilic carbocyanine perchlorate (DiI) or hydrophilic Rhodamine 123 (Rho123) and coated with poloxamer 188 (P188): PLGA-DiI/P188 and PLGA-Rho123/P188. All particles had narrow size distributions around 130 nm, spherical shape and negative potential. Subsequently, we performed in vivo real-time imaging of retinal blood vessels, combined with ex vivo microscopy to monitor the kinetics and to detect location of those two fluorescent markers. We found that DiI signals were long lasting, detectable >90 min in blood vessels after intravenous injection as visible by homogeneous labelling of the vessel wall as well as by spots in the lumen of blood vessels. In contrast, Rho123 signals mostly disappeared after 15 min post intravenous injection in such compartment. To explore how PLGA NP-loaded cargoes are released in the retina in vivo, we thereafter monitored the Cyanine5.5 amine (Cy5.5) covalently linked PLGA polymer (Cy5.5-PLGA) in parallel to DiI and Rho123. The Cy5.5 signal from PLGA polymer was detectable in the retina vessels >90 min for both, the Cy5.5-PLGA-DiI/P188 and Cy5.5-PLGA-Rho123/P188 groups. Microscopy of the ex vivo retina tissue revealed partial level of colocalization of PLGA with DiI but no colocalization between PLGA and Rho123 at 2 h post injection. This indicates that at least a fraction of the lipophilic DiI was preserved within NPs, whereas no hydrophilic Rho123 was associated with NPs at that time point. In conclusion, the properties of PLGA carrier-cargo system in the blood circulation of the retina might be strongly influenced by the combination of factors, including the individual properties of loaded compounds and blood milieu. Thus, it is unlikely that a single nanoparticle formulation will be identified that is universally effective for the delivery of different compounds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study was devised to investigate if P-glycoprotein (P-gp) mediated the drug-drug interaction (DDI) between genistein and repaglinide. When genistein was added, the plasma concentrations of repaglinide in rats were increased. The maximum plasma concentration (Cmax) of repaglinide increased from 70.80 ± 7.98 ng/mL to 124.71 ± 9.02 ng/mL and the area under the plasma concentration-time curve (AUC) increased from 134.89 ± 13.65 μg·h/L to 245.95 ± 7.24 μg·h/L. Intestinal absorption of repaglinide was markedly enhanced by genistein or P-gp inhibitor verapamil (Ver), both in situ rat jejunal perfusion studies and in vitro transport assays using everted rat intestinal sac preparations. Furthermore, the accumulation of repaglinide in both Caco-2 cells and IEC-6 cells also increased significantly in the presence of genistein and Ver. The transepithelial transport rate of repaglinide from basolateral-to-apical in MDR1-MDCK cells was 3.6-fold higher than the apical-to-basolateral rate with a net efflux ratio of 1.92 compared with mock-MDCK cells, which was significantly decreased following co-administration with genistein or Ver. In an intracellular accumulation experiment using Rhodamine 123 as a P-gp substrate, genistein significantly increased the intracellular fluorescence of Rhodamine 123. These results indicated that genistein had an inhibitory effect on the efflux function of P-gp. Through molecular docking assays we further found that genistein could bind to the nucleotide-binding domains (NBD) in the cytoplasm of P-gp, thus affecting the functions of P-gp. In conclusion, genistein inhibited the efflux of repaglinide mediated by P-gp in rats and in vitro. The findings suggested that the DDI between genistein and repaglinide is mediated by P-gp, and a dosage adjustment may be needed when they are co-administered in a clinical setting., Competing Interests: Declaration of Competing Interest The authors declare that we have no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: Ezrin (Ezr), radixin (Rdx) and moesin (Msn) (ERM) proteins anchor other proteins to the cell membrane, serving to regulate their localization and function. Here, we examined whether ERM proteins functionally regulate breast cancer resistance protein (BCRP) and P-glycoprotein in cell lines derived from lung, intestinal and renal cancers., Methods: ERM proteins were each silenced with appropriate siRNA. BCRP and P-gp functions were evaluated by means of efflux and uptake assays using 7-ethyl-10-hydroxycamptothecin (SN-38) and rhodamine123 (Rho123) as specific substrates, respectively, in non-small cell lung cancer HCC827 cells, intestinal cancer Caco-2 cells and renal cancer Caki-1 cells., Key Findings: In HCC827 cells, the efflux rates of SN-38 and Rho123 were significantly decreased by knockdown of Ezr or Msn, but not Rdx. However, BCRP function was unaffected by Ezr or Rdx knockdown in Caco-2 cells, which do not express Msn. In Caki-1 cells, Rdx knockdown increased the intracellular SN-38 concentration, while knockdown of Ezr or Msn had no effect., Conclusions: Our findings indicate that regulation of BCRP and P-gp functions by ERM proteins is organ-specific. Thus, if the appropriate ERM protein(s) are functionally suppressed, accumulation of BCRP or P-gp substrates in lung, intestine or kidney cancer tissue might be specifically increased., (© 2020 Royal Pharmaceutical Society.)

Human P-glycoprotein (P-gp) is a multispecific drug-efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo-electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry-related outer gates. Experiments were aided by availability of the X-ray structure of a homodimeric eukaryotic homolog of P-gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation. We mutated analogous tyrosine residues in each half of the human full-length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry-related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry-related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P-gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

The development of multidrug resistance is often associated with the over-expression of P-glycoprotein (P-gp). This protein prevents drug accumulation and extrudes them out of the cell before they reach the intended target. The aim of this study was to develop an in vitro MCF-7 cell line with increased expression of P-gp and test the phototoxicity of a novel photoactivated zinc phthalocyanine tetrasulfonic acid (ZnPcS4) on these cells. The over-expressed P-gp MCF-7 cells (MCF-7/DOX) were developed from wildtype (WT) MCF-7 cells by a stepwise continuous exposure of the WT cells to different concentrations of Doxorubicin (DOX) (0.1 - 1 μM) over a period of 4 months. The P-gp expression was measured using flow cytometry, immunofluorescence and enzyme immunoassay. To verify whether zinc phthalocyanine-mediated photodynamic therapy (ZnPcS4 - PDT) is effective in MCF-7/DOX, we studied the subcellular localization, phototoxicity and nuclear damage. The flow cytometry result showed two distinct peaks of P-gp positive and negative expression in MCF-7/DOX cell population, which correlates with the ELISA-based assay (p˂0.001). The ME16C (Normal breast cells) was used as control. The localization studies showed that ZnPcS4 have greater affinity for lysosome than mitochondria. Phototoxicity results indicated that photoactivated zinc phthalocyanine decreased the cell proliferation and viability as the drug and laser light dosages increased to 16 μM and 20 J/cm 2 respectively. PDT-induced cytotoxicity using lactose dehydrogenase (LDH) enzyme leakage as measure did not increase likewise. The ZnPcS4-induced PDT was less effective for MCF-7/DOX cells which could be attributed to decreased retention of ZnPcS4 in major cellular organelles due to the presence of increased drug efflux P-gp. The current findings suggest that, increased P-gp expression, a characteristic of multidrug resistance together with other related intrinsic mechanisms might contribute to render MCF-7/DOX cells less sensitive to ZnPcS4-induced phototoxicity., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Assessment of the mitochondrial membrane potential (Δψ) in living cells, although not trivial, can be used to estimate mitochondrial bioenergetic state. For this purpose, fluorescent lipophilic cations are broadly applied. These cations enter cells and accumulate primarily in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of the cations tetramethylrhodamine methyl ester (TMRM) and rhodamine 123 (Rhod123) for semi-quantitative Δψ analysis in living mammalian cells. Two different strategies are presented: (1) steady-state measurements that are suited to compare Δψ between different conditions (i.e., for comparing disease states or treatments) and (2) dynamic measurements allowing temporal monitoring of Δψ changes (i.e., to assess the effect of acute perturbations). We discuss the rationale for the use of TMRM and Rhod123 in their non-quenching/redistribution and quenching mode, how these modes are associated with different fluorescence responses, and how data can be interpreted. Practically, three experimental protocols are provided describing the use of TMRM and/or Rhod123 to assess Δψ in primary human skin fibroblasts (PHSFs) and neuron/astrocyte co-cultures by live-cell fluorescence microscopy., (© 2020 Elsevier Inc. All rights reserved.)

Glucosamine (GlcN) is a natural amino monosaccharide in the human body, and evidence of its anticancer effects is growing. In this study, we aimed to evaluate the effects of GlcN for its cytotoxicity, MDR reversal effects and inhibitory effects on function and expression of P-glycoprotein (P-gp) transporter in the daunorubicin-resistant human gastric cancer cells. Cell viability was measured by MTT assay to evaluate the cytotoxicity and multidrug resistance (MDR) reversal effects of GlcN. The effects of GlcN on function and mRNA expression level of P-gp transporter were assessed by flow cytometry and real-time RT-qPCR, respectively. Our results indicated that GlcN reduced the proliferation of human gastric cancer cell line EPG85-257 and its drug-resistant variant EPG85-257RD in a dose-dependent manner. GlcN (at the concentrations of 0.5 and 1 mM) also enhanced the sensitivity of EPG85-257RDB cells to daunorubicin. The cellular accumulation studies showed that GlcN inhibited efflux activity of P-gp and enhanced the mean fluorescent intensity of Rho123 while ˙ it had no effects on P-gp gene expression in these cells. This study suggested that the inhibition of P-gp activity is a novel mechanism of action by which GlcN could reverse MDR in EPG85-257RDB cells.

Oral drug delivery is a preferred administration route due to its low cost, high patient compliance and fewer adverse events compared to intravenous administration. However, many pharmaceuticals suffer from poor solubility and low oral bioavailability. One major factor that contributes to low bioavailability are efflux transporters which prevent drug absorption through intestinal epithelial cells. P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two important efflux transporters in the intestine functioning to prevent toxic materials from entering systemic circulation. However, due to its broad substrate specificity, P-gp limits the absorption of many therapeutics, including chemotherapeutics and antibacterial agents. Methods to inhibit P-gp with competitive inhibitors have not been clinically successful. Here, we show that micron scale devices (microdevices) made from a commonly used biomaterial, polyethylene glycol (PEG), inhibit P-gp through a biosimilar mucus in Caco-2 cells and that transporter function is restored when the microdevices are removed. Microdevices were shown to inhibit P-gp mediated transport of calcein AM, doxorubicin, and rhodamine 123 (R123) and BCRP mediated transport of BODIPY-FL-prazosin. When in contact with Caco-2 cells, microdevices decrease the cell surface amount of P-gp without affecting the passive transport. Moreover, there was an increase in mucosal to serosal transport of R123 with microdevices in an ex-vivo mouse model and increased absorption in vivo. This biomaterial-based approach to inhibit efflux transporters can be applied to a range of drug delivery systems and allows for a nonpharmacologic method to increase intestinal drug absorption while limiting toxic effects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Characterizing interaction of newly synthetized molecules with efflux pumps remains essential to improve their efficacy and safety. Caco-2 cell line cultivated on inserts is widely used for measuring apparent permeability of drugs across biological barriers, and for estimating their interaction with efflux pumps such as P-gp, BCRP and MRPs. However, this method remains time consuming and expensive. In addition, detection method is required for measuring molecule passage across cell monolayer and false results can be generated if drugs concentrations used are too high as demonstrated with quinidine. For this reason, we developed a new protocol based on the use of Caco-2 cell directly seeded on 96- or 384-well plates and the use of fluorescent substrates for efflux pumps. We clearly observed that the new method reduces costs for molecule screening and leads to higher throughput compared to traditional use of Caco-2 cell model. This accelerated model could provide quick feedback regarding the molecule design during the early stage of drug discovery and therefore reduce the number of compounds to be further evaluated using the traditional Caco-2 insert method.

Objective: Methotrexate (MTX) is a commonly used drug for the treatment of rheumatoid arthritis (RA) and it has been studied in RA resistance recently. P-glycoprotein (P-gp) is one of the important transporters that mediate MTX resistance. This study investigated the effect of Paeoniflorin-6'-O-benzene sulfonate (code: CP-25) in the resistance of P-gp-mediated MTX to RA., Methods: Adjuvant arthritis (AA) was induced in rats via complete Freund's adjuvant. The experimental groups were divided into normal group; AA model group; monotherapy groups, including CP-25, MTX and dexamethasone; and CP-25 combined with MTX group. The expression of P-gp in synovial tissue was measured by western blot and histochemistry. Besides, P-gp high expression of human hepatoma cell line Bel7402/5-FU and Bel7402 were chose to study in MTX resistance and the function of P-gp was detected by Flow cytometry., Results: CP-25 had a good therapeutic effect on AA rats, significantly improved manifestations and reduced the expression of P-gp in synovial tissue, spleen medulla and small intestinal epithelial cells in the apical tissues of AA rats. In addition, CP-25 significantly inhibited the up-regulation of P-gp induced by TNF-α stimulation in synoviocytes. Furthermore, according to the accumulation and efflux of rhodamine 123 in Bel7402/5-FU resistant cells and Bel7402 sensitive cells, CP-25 could reverse the resistance of MTX in Bel7402/5-FU cells compared with Bel7402 cells, which was reflected by the reduced IC50 values of MTX. Further study indicated that CP-25 could decrease P-gp expression and inhibit P-gp function in Bel7402/5-FU cells., Conclusion: CP-25 regulates the expression of P-gp and inhibits the function of P-gp, thereby improving the resistance of MTX., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Overexpression of p-glycoprotein (p-gp) is the main cause of multidrug resistance and chemotherapy failure in leukemia. Sodium azulene sulfonate (SAS) was used to reverse the multidrug resistance of human leukemia adriamycin-resistant strain K562/A02, and the underlying mechanism was investigated. Human leukemia cell line K562 and drug-resistant cell line K562/A02 in logarithmic phase were used in this study. After 48 hours of treatment of K562/A02 cells with SAS, the intrinsic cytotoxicity of chlorogenic acid and its sensitivity to adriamycin (ADM) were determined with MTT assay. The degree of reversal was calculated. Using ADM accumulation and rhodamine 123 efflux experiments, the average fluorescence intensity of ADM and rhodamine 123 (Rh123) in chlorogenic acid-treated K562/A02 cells was determined flow cytometrically. The expressions of p-gp, t-Akt and p-Akt in K562/A02 cells were assayed using Western blotting. SAS had almost no cytotoxic effect, and the degree of inhibition was only about 20% at the highest concentration of 100 mu-M. The EC50 of MDR reversal by SAS was in the nano range (539±37nM), and it had a high selectivity index for normal cells (&gt;185). The accumulation of ADM in drug-resistant cells was increased significantly after treatment with 1 and 5 mu-M SAS, while the efflux of Rh123 was significantly inhibited, suggesting that SAS reversed MDR by inhibiting p-gp function. Western blotting experiments showed that SAS downregulated the expression of p-gp by inhibiting PI3K/Akt signaling pathway. This contributed to the reversal of drug resistance.SAS effectively reverses multidrug resistance in vitro by inhibiting the function of p-gp in K562/A02 cells, through a mechanism involving downregulation of the P13K/Akt signaling pathway. Therefore, SAS may be a potential candidate drug for reversal of MDR.

Background: Neutrophils ex vivo in whole blood specimens are widely understood to decay rapidly when compared to other leukocytes, requiring assessment of neutrophil activity to be performed shortly after blood collection. There is a disparity in evidence for decay rates in measurements and recommended time-frames for assaying neutrophil parameters in particular assays following blood collection. We, therefore, evaluated the decline in the neutrophil respiratory burst, typically screened for assessing congenital NADPH oxidase defects, over a shorter time-course than previously published experiments., Methods: The neutrophil respiratory burst was assessed by flow cytometric detection of DHR-123 oxidation to rhodamine-123 (Rho123), following stimulation of neutrophils by phorbol myristate acetate (PMA), in heparinized healthy donor blood specimens immediately following venipuncture, and then at 3 and 5 h later with ambient temperature or refrigerated specimen storage., Results: A consistent time-dependent decline in the Rho123 fluorescence of PMA-stimulated neutrophils was detected in the healthy donor specimens, indicating a decay in respiratory burst activity. Neutrophil oxidative indexes calculated for half of the specimens at 3 and 5 h of age, fell below our normal laboratory lower limit. We also found that Rho123 histograms of PMA-stimulated neutrophils from stored healthy donor specimens have a risk of misinterpretation due to mimicking the appearance of histograms from carriers of CGD and other NADPH oxidase defects. Refrigeration of specimens did not significantly minimize decay., Conclusions: DHR assay of the neutrophil respiratory burst from blood specimens at 3 h post-venipuncture and beyond can generate unreliable clinical measurements due to decay. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Herein, the purpose of this study is to evaluate the effects of kaempferol on bioavailability and pharmacokinetics of nifedipine and its metabolite dehydronifedipine in rats. The experimental design is based on with or without kaempferol in the oral and intravenous administration of nifedipine in rats. Moreover, the pharmacokinetic parameters including nifedipine and dehydronifedipine were evaluated in rats.The in vitro studies ofkaempferol were investigated on P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 activity. Kaempferol reduced a 50% inhibitory concentration (IC 50 ) of 8.6 μmol·L -1 on CYP3A4 enzyme activity. Moreover, kaempferol clearly improved the cell internalization of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. Depending on increased concentrations of kaempferol, the areas under the plasma concentration-time curve (AUC 0-∞ ) and the peak concentration (C max ) of nifedipine were increased after oral and intravenous administration. Moreover, the absolute bioavailability (AB) and relative bioavailability (RB) of nifedipine in the presence of kaempferol was significantly higher than those of the control group after oral and intravenous administration. Improvement of bioavailability of nifedipine by kaempferol may be mainly because of the inhibition of the P-gp-mediated efflux transporter in the small intestine and CYP3A4-mediated metabolism in the small intestine or liver, or both., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Objective: In this study, we develop and apply a high-throughput screening protocol to investigate the activity of non-ionic surfactants, with a broad range of hydrophilic-lipophilic balance values, against ABCB1-mediated efflux transport and ABCC2-mediated efflux transport., Methods: Caco-2 cells were grown for 7 days in 96-well plates, then washed and incubated with the test materials for 2 h in the presence of 2.5 μm of either rhodamine 123 (R-123) or 5(6)-Carboxy-2',7' dichlorofluorescein diacetate as probes of ABCB1 and ABCC2, respectively., Key Findings: Of the surfactants tested, no activity against ABCC2 was detected and all surfactants showing efficacy against ABCB1 had a HLB value of 22 or below. Inhibition of ABCB1 was seen in the order of efficacy to be poloxamer 335 > poloxamer 40 > Crovol A-70 > Myrj S-40 > poloxamer 184 > poloxamer 182 > Etocas 40 > Tween 20 > Etocas 29 > Tween 80 > Acconon C-44 > Span 20. With regard to this inhibition, the distribution of hydrophilic regions is more important than the HLB value., Conclusion: This work demonstrates a high-throughput protocol for detecting materials that can modulate ABCB1-mediated efflux. These surfactants could be exploited to improve oral delivery of drugs prone to efflux., (© 2019 The Authors. Journal of Pharmacy and Pharmacology published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society.)

This study was aimed to determine the mechanism for flavonoid poor absorption related to P-glycoprotein (P-gp). The cellular uptake (CU) of 40 flavonoids was investigated in P-gp overexpressing KB/multidrug-resistant (MDR) cells. A total of 9 flavonoids, including 5,7,3',4'-tetramethoxyflavone, with a significant ( p < 0.05) CU KBE (2.90 ± 0.146 μmol/g) higher than CU KBP (1.57 ± 0.129 μmol/g) were identified as P-gp substrates. Besides, 8 substrates, including tangeretin, showed a significant ( p < 0.05) CU KB (9.72 ± 1.09 μmol/g) higher than its CU KBP (7.36 ± 0.692 μmol/g). A total of 7 of 17 flavonoid substrates stimulated the P-gp efflux of rhodamine 123, and most substrates increased P-gp expression in KB/MDR cells. Docking analyses showed a good correlation ( R = 0.764; p < 0.01) between efflux fold and S&#95;scoring of flavonoids to the P-gp model, indicating consistency between in silico and in vitro results. A structure-affinity relationship exhibited that 3-OH, 5-OH, 3'-OCH 3 , and 4'-OCH 3 are crucial for flavonoids binding to P-gp. These results provide valuable information for finding a solution to improve the absorption of flavonoids.

Objectives: This study investigated the absorptive potential of phyllanthin across the polarized Caco-2 monolayers and the potential role of phyllanthin in P-glycoprotein (P-gp)-mediated drug interaction., Methods: The absorptive potential of phyllanthin was predicted from its apparent permeability (P app ) across the Caco-2 monolayers under the pH gradient condition (pH 6.5 AP -7.4 BL ) at 37°C. Integrity of paracellular transport was assessed by monitoring transepithelial electrical resistance (TEER) and lucifer yellow (LY) leakage. P-gp-mediated interaction was evaluated by transport studies of phyllanthin and rhodamine-123., Key Findings: The absorptive P app of phyllanthin (34.90 ± 1.18 × 10 -6 cm/s) was in the same rank order as the high permeable theophylline and antipyrine. Phyllanthin transport in the absorptive and secretive directions was comparable (the efflux ratio (ER) of 1.19 ± 0.01). Phyllanthin caused no changes in TEER nor LY leakage in the monolayers. However, phyllanthin increased rhodamine-123 ER in a concentration-dependent manner, suggesting its inhibition on P-gp function. In addition, phyllanthin aqueous solubility was <5 μg/ml at 37°C., Conclusions: Phyllanthin is a highly permeable compound that could passively diffuse through the absorptive barrier via transcellular pathway with little hindrance from P-gp. Phyllanthin could interfere with transport of P-gp drug substrates, when concomitantly administered. In addition, aqueous solubility could be a limiting factor in phyllanthin absorption., (© 2018 Royal Pharmaceutical Society.)

Overexpression of P-gp is the main cause of multidrug resistance (MDR) and chemotherapeutic failure in leukemia. In this study, the multidrug resistance reverse effect of Schizandrol A (SchA) was demonstrated with P-gp overexpressed drug-resistant K562/A02 cells. SchA had almost no cytotoxic activity, the EC50 value reversed to DOX was in the nanomole range of (707 ± 29nM) and had a high selectivity index (&gt; 113) to normal cells. DOX accumulation and Rh123 efflux tests demonstrated that the MDR reversal activity of SchA was induced by inhibiting P-gp function. Western blotting assay showed that SchA down-regulated the expression of P-gp by inhibiting the PI3K / Akt signaling pathway, which was also a key factor in reversal activity. Therefore, SchA may be a potential candidate for natural MDR reversal agents.

Multidrug resistance (MDR) poses a great impediment to cancer treatment. Excessive expression of ATP-binding cassette transport protein AC-1 (P-glycoprotein, P-GLP) is usually involved in MDR. In this study, ailanthone (AIL), a natural compound extracted from the whole seedlings of Ailanthus altissima (Simaroubaceae) was shown to mediate the reversal of P-GLP-induced MDR and restore the susceptibility of K562/A02 cells to doxorubicin (DOX). Further mechanistic studies revealed that AIL increased intracellular DOX accumulation and interrupted Rh123 efflux through suppression of P-GLP, and also suppressed P-GLP ATPase activity. At the same time, it markedly inhibited MDR1 gene expression and P-GLP protein to sensitize the cytotoxic effect of DOX. Furthermore, AIL down-regulated P-GLP expression by inhibiting the PI3K/Akt pathway. Thus, AIL could be a potential therapeutic compound for reversing P-GLP-mediated drug resistant cancer.

Regarding post-complication of convenient therapies against breast cancer, the emergence of effective approaches is essential. Photodynamic therapy is touted as a novel invasive therapeutic approach by the application of a photosensitizer promoted by laser irradiation. This study aimed to investigate the combined regime of low-level laser irradiation with zinc phthalocyanine in human breast cancer ZR-75-1 cell line. Cells were treated with 0.01 and 5 μg/ml of ZnPc for 24 h and exposed to radiation (70 mW) for 60 s. Cell viability was evaluated by MTT and flow cytometry. Cell migration capacity was monitored by scratch test, Transwell migration insert, and gelatin zymography. The function of MDR in treated cells was examined by Rhodamine 123 exclusion test. The level of GALNT11 was measured by ELISA. The expression of Bax and Bcl-2 genes was evaluated by real-time PCR. Laser irradiation and zinc phthalocyanine induced cell cytotoxicity in a dose-dependent manner. Flow cytometry analysis showed the induction of apoptotic and necrotic changes in treated cells. We found a reduction in migration rate and MMP-9 activity in cells undergoing the experimental procedure (p < 0.05). Immunofluorescence imaging revealed the intracellular accumulation of Rhodamine 123 coincided with a reduction in the level of GALNT11 in treated cells, showing the reduction of MDR activity and tumor cell resistance. Similar to flow cytometry assay, the reduction of Bcl-2 (approximately twofold) and upregulation of Bax genes were found in treated cells. Photodynamic therapy could be as an effective and alternative method for the treatment of breast cancer in a human.

Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability ( P app ) of <5 × 10 -7 cm s -1 , while propranolol demonstrated a P app of 9.69 × 10 -6 cm s -1 . Rh123 was transported in a luminal direction ( P app,efflux / P app,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo.

Background: Multidrug resistance (MDR) is the major reason for the failure of chemotherapy in colon cancer. Bufalin (BU) is one of the most effective antitumor active constituents in Chansu. Our previous study found that BU can effectively reverse P-glycoprotein (P-gp)-mediated MDR in colon cancer. However, the clinical application of BU is limited due to its low solubility in water and high toxicity. In the present study, a multifunctional delivery system based on vitamin-E- succinate grafted chitosan oligosaccharide (VES-CSO) and cyclic (arginine-glycine-aspartic acid peptide) (RGD)-modified d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) was prepared by emulsion solvent evaporation method for targeted delivery of BU to improve the efficacy of drug-resistant colon cancer therapy., Methods: The cytotoxicity of BU-loaded micelles against drug-resistant colon cancer LoVo/ADR and HCT116/LOHP cells was measured by CCK-8 assay. The cellular uptake, Rho123 accumulation, and cell apoptosis were determined by flow cytometry. The expression of apoptosis-related protein and P-gp was measured by Western blot assay. The antitumor activity of BU-loaded micelles was evaluated in LoVo/ADR-bearing nude mice., Results: BU-loaded VES-CSO/TPGS-RGD mixed micelles (BU@VeC/T-RGD MM) were 140.3 nm in diameter with zeta potential of 8.66 mV. The BU@VeC/T-RGD MM exhibited good stability, sustained-release pattern, higher intracellular uptake, and greater cytotoxicity in LoVo/ADR cells. Furthermore, the mechanisms of the BU@VeC/T-RGD MM to overcome MDR might be due to enhanced apoptosis rate and P-gp efflux inhibition. Subsequently, in vivo studies confirmed an enhanced therapeutic efficiency and reduced side effects associated with BU@VeC/T-RGD MM compared with free BU, owing to the enhanced permeation and retention effect, improved pharmacokinetic behavior, and tumor targeting, which lead to MDR-inhibiting effect in LoVo/ADR-bearing nude mice., Conclusion: Our results demonstrated that VeC/T-RGD MM could be developed as a potential delivery system for BU to improve its antitumor activity against drug-resistant colon cancer., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

P-glycoprotein affects the transport of numerous drugs including chemotherapeutic drugs vincristine sulfate (VCR) and docetaxel (DTX), and is one of the main causes for multidrug resistance. Our previous studies have shown that oxypeucedanin (OPD) can enhance the intestinal transit of puerarin and VCR. However, the underlying mechanism is unclear. This study investigated the potential mechanism by which OPD improves P-gp-mediated drug transport. Molecular docking was performed to predict the binding force between OPD and P-gp and the contribution of OPD on P-gp activity. We observed the effect of OPD on the transport of VCR in MDCK-MDR1 cell monolayer and also measured the plasma pharmacokinetic parameters of DTX in the presence and absence of OPD by LC-MS/MS. Moreover, we further investigated the reversal mechanism of OPD on P-gp-mediated drug transport by determining the intracellular accumulation of Rhodamine-123 (Rh123) and P-gp ATPase activity as well as protein expression and mRNA level of P-gp. Our molecular docking results revealed that the binding force between OPD and P-gp was much lower than that between P-gp and verapamil (a P-gp substrate). The transport study in vitro indicated that OPD increased the flux of VCR across MDCK-MDR1 cell monolayer. The in vivo pharmacokinetic parameters data showed OPD increased the absorption of DTX. OPD activated P-gp ATPase activity and enhanced intracellular accumulation of Rh123 in MDCK-MDR1 cells. Western blotting and qRT-PCR outcomes indicated that OPD suppressed P-gp protein expression as well as downregulated P-gp mRNA level. Thus, OPD reverse P-gp-mediated drug transport via inhibition of P-gp activity and P-gp protein expression as well as downregulation of P-gp mRNA level. Our results suggest that OPD could reverse P-gp-mediated drug resistance in tumor cells.

Background/aim: Malignant diseases present a significant public health burden worldwide and their treatment is further complicated by the phenomenon of multidrug resistance. Derivatives of imidazopyridine exhibit several remarkable pharmacological activities and they could reverse the multidrug resistance of cancer cells due to overexpressing P-glycoprotein., Materials and Methods: A series of novel imidazo[4,5-b]pyridine derivatives were synthesized and their biological activities were evaluated in vitro using parental (PAR) and multidrug resistant (MDR; ABCB1-overexpressing) mouse T-lymphoma cells. The cytotoxic activity and selectivity of the tested compounds were assessed by the thiazolyl blue tetrazolium bromide (MTT) assay, the ABCB1 modulating activity was measured by rhodamine 123 accumulation assay using flow cytometry., Results: Six compounds ( b, c, d, f, h and i ) showed moderate-to-high cytotoxic activity on the tested cell lines, while derivative i presented with promising selectivity towards the MDR cell line. Derivatives a, d, f, g and i were proven to be effective modulators of the ABCB1 multidrug efflux pump, with two compounds showing efflux pump modulatory activity at 2 μM concentration., Conclusion: Based on our experimental results, compounds that showed potent activity are those with a short carbon side chain; a methoxy group on the benzene ring; a heterocyclic (triazole) side chain and the presence of an alkylated N-atom at position 4., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Multidrug resistance (MDR) is one of the main obstacles of clinical chemotherapy. A great deal of research shows that the occurrence of drug resistance in various malignant tumors is closely related to the expression of P-glycoprotein (P-gp) on the surface of the cell membrane. In this paper, based on the structure-activity relationship of phenylethyl tetrahydroisoquinoline, we choose tariquidar as the lead compound for the design and synthesis of 17 novel tetrahydroisoquinoline P-gp inhibitors. Additionally, in vitro and in vivo cytotoxicity assays and reversed MDR activity assays were evaluated. Among them, compound 3 had a good reversal of MDR activity and the reversal mechanism study of it was carried out. All of these results demonstrated that compound 3 was considered to be a promising P-gp-mediated MDR reversal candidate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recently, a new target Ca 2+ -binding protein SORCIN was reported to participate in multidrug resistance (MDR) in cancer. Here we aim to investigate whether dihydromyricetin (DMY), a dihydroflavonol compound with anti-inflamatory, anti-oxidant, anti-bacterial and anti-tumor actions, reverses MDR in MCF-7/ADR and K562/ADR and to elucidate its potential molecular mechanism. DMY enhanced cytotoxicity of adriamycin (ADR) by downregulating MDR1 mRNA and P-gp expression through MAPK/ERK pathway and also inhibiting the function of P-gp significantly. Meanwhile, DMY decreased mRNA and protein expression of SORCIN, which resulted in elevating intracellular free Ca 2+ . Finally, we investigated co-administration ADR with DMY remarkably increased ADR-induced apoptosis. Further study showed DMY elevated ROS levels and caspase-12 protein expression, which signal apoptosis in endoplasmic reticulum. At the same time, proteins related to mitochondrial apoptosis were also changed such as Bcl-2, Bax, caspase-3, caspase-9, and PARP. Finally, nude mice model also demonstrated that DMY strengthened anti-tumor activity of ADR in vivo. In conclusion, DMY reverses MDR by downregulating P-gp, SORCIN expression and increasing free Ca 2+ , as well as, inducing apoptosis in MCF-7/ADR and K562/ADR. These fundamental findings provide evidence for further clinical research in application of DMY as an assistant agent in the treatment of cancer., (© 2017 Wiley Periodicals, Inc.)

Recently, thioxanthone derivatives were found to protect cells against toxic P-glycoprotein (P-gp) substrates, acting as potent inducers/activators of this efflux pump. The study of new P-gp chiral modulators produced from thioxanthone derivatives could clarify the enantioselectivity of this ABC transporter towards this new class of modulators. The aim of this study was to evaluate the P-gp modulatory ability of four enantiomeric pairs of new synthesized chiral aminated thioxanthones (ATxs) 1 - 8 , studying the influence of the stereochemistry on P-gp induction/ activation in cultured Caco-2 cells. The data displayed that all the tested compounds (at 20 μM) significantly decreased the intracellular accumulation of a P-gp fluorescent substrate (rhodamine 123) when incubated simultaneously for 60 min, demonstrating an increased activity of the efflux, when compared to control cells. Additionally, all of them except ATx 3 (+), caused similar results when the accumulation of the P-gp fluorescent substrate was evaluated after pre-incubating cells with the test compounds for 24 h, significantly reducing the rhodamine 123 intracellular accumulation as a result of a significant increase in P-gp activity. However, ATx 2 (-) was the only derivative that, after 24 h of incubation, significantly increased P-gp expression. These results demonstrated a significantly increased P-gp activity, even without an increase in P-gp expression. Therefore, ATxs 1 - 8 were shown to behave as P-gp activators. Furthermore, no significant differences were detected in the activity of the protein when comparing the enantiomeric pairs. Nevertheless, ATx 2 (-) modulates P-gp expression differently from its enantiomer, ATx 1 (+). These results disclosed new activators and inducers of P-gp and highlight the existence of enantioselectivity in the induction mechanism., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.