Efflux Transporter Activity Research Articles

Changes in Disposition of Ezetimibe and Its Active Metabolites Induced by Impaired Hepatic Function: The Influence of Enzyme and Transporter Activities.

Ezetimibe (EZE) is a selective cholesterol absorption inhibitor. Hepatic impairment significantly increases the systemic exposure of EZE and its main active phenolic glucuronide, EZE-Ph. Although changes in efflux transporter activity partly explain the changes in EZE-Ph pharmacokinetics, the causes of the changes to EZE and the effects of the administration route on EZE-Ph remain unclear. A carbon tetrachloride (CCl4)-induced hepatic failure rat model was combined with in vitro experiments to explore altered EZE and EZE-Ph disposition caused by hepatic impairment. The plasma exposure of EZE and EZE-Ph increased by 11.1- and 4.4-fold in CCl4-induced rats following an oral administration of 10 mg/kg EZE, and by 2.1- and 16.4-fold after an intravenous injection. The conversion of EZE to EZE-Ph decreased concentration-dependently in CCl4-induced rat liver S9 fractions, but no change was observed in the intestinal metabolism. EZE-Ph was a substrate for multiple efflux and uptake transporters, unlike EZE. In contrast to efflux transporters, no difference was seen in the hepatic uptake of EZE-Ph between control and CCl4-induced rats. However, bile acids that accumulated due to liver injury inhibited the uptake of EZE-Ph by organic anion transporting polypeptides (OATPs) (glycochenodeoxycholic acid and taurochenodeoxycholic acid had IC50 values of 15.1 and 7.94 μM in OATP1B3-overexpressed cells). In conclusion, the increased plasma exposure of the parent drug EZE during hepatic dysfunction was attributed to decreased hepatic glucuronide conjugation, whereas the increased exposure of the metabolite EZE-Ph was mainly related to transporter activity, particularly the inhibitory effects of bile acids on OATPs after oral administration.

Anticancer and chemosensitizing activities of stilbenoids from three orchid species.

Recently, we have isolated and identified several bioactive flavonoids and stilbenoids with potential anticancer activity from Thai orchids. In this study, we further investigated the cytotoxic and chemosensitizing activities of these phytochemicals (namely, pinocembrin, cardamonin, isalpinin, galangin, pinosylvin monomethyl ether, 2,3'-dihydroxy-5'-methoxystilbene, (E)-2,5'-dihydroxy-2'-(4-hydroxybenzyl)-3'-methoxystilbene, 2,3-dihydroxy-3',5'-dimethoxystilbene, 2,3'-dihydroxy-5,5'-dimethoxystilbene, 3,4'-dihydroxy-5-methoxystilbene and batatasin III) against breast cancer MCF7 cells and its two multidrug resistant (MDR) sublines (MCF7/DOX and MCF7/MX). Cytotoxicity was determined with MTT assay for the estimation of the half maximal cytotoxic concentrations (IC50). Effects of the test compounds on activities of efflux transporters (BCRP, P-gp, MRP1, and MRP2) were evaluated with substrate accumulation assays using fluorometry and flow cytometry analysis. Out of these 11 test compounds, the stilbene pinosylvin monomethyl ether displayed its cytotoxicity specifically toward MCF7 cells (IC50 = 6.2 ± 1.2μM, 72-h incubation) with 4.96 folds higher than normal fibroblast. Its potency decreased in MCF7/DOX and MCF7/MX cells by 3.94 and 7.38 folds, respectively. Our transporter assay indicated that this stilbene significantly reduced the activities of P-gp, MRP1, and MRP2, but not BCRP. After 48-h co-incubation, this stilbene (at 2μM) synergistically increased doxorubicin- and mitoxantrone-mediated cytotoxicity in MCF7, MCF7/DOX, and MCF7/MX cells potentially by increasing the intracellular level of cytotoxic drug. Pinosylvin monomethyl ether could sensitize breast cancer cells to chemotherapy and overcome MDR, in part, via the inhibition of drug efflux transporters.

ATP Mimetic Attack on the Nucleotide-Binding Domain to Overcome ABC Transporter Mediated Chemoresistance.

Since the problem of transporter-mediated multidrug resistance of tumor cells is becoming increasingly important in cancer therapy, it is necessary to modulate the activity of efflux transporters of the ABC family, among which P-glycoprotein is the best known. We consider the nucleotide binding domain, a universal fragment of these transporters, as a target for the rational design of small molecule compounds capable of preventing ATP-dependent drug efflux. Using various ATP mimetics, we showed that they suppress the efflux of fluorescent substrates and paclitaxel from the cells due to suppressing the ATPase activity of the transporters. The combined use of paclitaxel and ATP mimetics significantly increases its antitumor efficacy, including in cells with the multidrug resistance phenotype. The considered compounds are promising agents for the development of therapeutic efflux modulators, since they are not toxic at the given concentrations and do not induce the transporter overexpression. Moreover, the compounds overcome not only P-gp-mediated but also BCRP-mediated resistance of tumor cells.

Glutathione Transferase P1: Potential Therapeutic Target in Ovarian Cancer.

Chemotherapy resistance of ovarian cancer, regarded as the most lethal malignant gynecological disease, can be explained by several mechanisms, including increased activity of efflux transporters leading to decreased intracellular drug accumulation, increased efflux of the therapeutic agents from the cell by multidrug-resistance-associated protein (MRP1), enhanced DNA repair, altered apoptotic pathways, silencing of a number of genes, as well as drug inactivation, especially by glutathione transferase P1 (GSTP1). Indeed, GSTP1 has been recognized as the major enzyme responsible for the conversion of drugs most commonly used to treat metastatic ovarian cancer into less effective forms. Furthermore, GSTP1 may even be responsible for chemoresistance of non-GST substrate drugs by mechanisms such as interaction with efflux transporters or different signaling molecules involved in regulation of apoptosis. Recently, microRNAs (miRNAs) have been identified as important gene regulators in ovarian cancer, which are able to target GST-mediated drug metabolism in order to regulate drug resistance. So far, miR-186 and miR-133b have been associated with reduced ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GSTP1 and MDR1. Unfortunately, sometimes miRNAs might even enhance the drug resistance in ovarian cancer, as shown for miR-130b. Therefore, chemoresistance in ovarian cancer treatment represents a very complex process, but strategies that influence GSTP1 expression in ovarian cancer as a therapeutic target, as well as miRNAs affecting GSTP1 expression, seem to represent promising predictors of chemotherapeutic response in ovarian cancer, while at the same time represent potential targets to overcome chemoresistance in the future.

Are active efflux transporters contributing to infant drug exposure via breastmilk? A longitudinal study.

Although most drugs are considered safe and compatible with breastfeeding, cases of toxic drug exposure have been reported. Active efflux transporters have been implicated as a mechanism in the transfer of drugs from mother to baby via breastmilk. Using breastmilk as a source of human mammary epithelial cells, this novel longitudinal study investigated the expression of four active transporters, namely, MDR1, MRP1, MRP2 and BCRP in the lactating human breast. BCRP gene was found to be strongly overexpressed with levels peaking at 5 months postpartum, potentially indicating a time where a breastfed infant may be at risk of inadvertent exposure to BCRP substrates. Serum albumin, a major component of human breastmilk was increasingly downregulated as lactation progresses. Xanthine oxidase/dehydrogenase, an enzyme in breastmilk attributed to a reduced risk of gastroenteritis caused by Escherichia coli and Salmonella enteritides, was downregulated. Lysozyme and fatty acid synthase are progressively upregulated. This study also shows that breastmilk-derived epithelial cells, when propagated in culture, exhibit characteristics significantly different to those derived directly from breastmilk. This serves to warn that in vitro studies are not a true representation of in vivo processes in the lactating breast; hence, application of in vitro data should be conducted with caution.

Generation of a VeroE6 Pgp gene knock out cell line and its use in SARS-CoV-2 antiviral study

Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.

Sodium arsenite but not aluminum chloride stimulates ABC transporter activity in renal proximal tubules of killifish (Fundulus heteroclitus)

ABC export proteins including Multidrug resistance-related protein 2 (Mrp2) serve as detoxification mechanism in renal proximal tubules due to active transport of xenobiotics and metabolic waste products into primary urine. The environmental pollutants aluminum and arsenic interfere with a multitude of regulatory mechanisms in the body and here their impact on ABC transporter function was studied. NaAsO2 but not AlCl3 rapidly stimulated Mrp2-mediated Texas Red (TR) transport in isolated renal proximal tubules from killifish, a well-established laboratory model for the determination of efflux transporter activity by utilizing fluorescent substrates for the ABC transporters of interest and confocal microscopy followed by image analysis. This observed stimulation remained unaffected by the translation inhibitor cycloheximide (CHX), but it was abrogated by antagonists and inhibitors of the endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) signaling pathway. NaAsO2-triggered effects were abolished as a consequence of PKCα inhibition through Gö6976 and PKCα inhibitor peptide C2–4. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294,002 as well as the mammalian target of rapamycin (mTOR) inhibitor rapamycin suppressed NaAsO2-triggered stimulation of luminal TR transport. In addition, the stimulatory effect of NaAsO2 was abolished by GSK650394, an inhibitor of serum- and glucocorticoid-inducible kinase 1 (SGK1), which is an important downstream target. Environmentally relevant concentrations of NaAsO2 further stimulated transport function of P-glycoprotein (P-gp), Multidrug resistance-related protein 4 (Mrp4) and Breast cancer resistance protein (Bcrp) while AlCl3 was ineffective. To our knowledge, this is the first report engaging in the impact of NaAsO2 on efflux transporter signaling and it may contribute to the understanding of defense mechanisms versus this worrying pollutant.

Trivariate Linear Regression and Machine Learning Prediction of Possible Roles of Efflux Transporters in Estimated Intestinal Permeability Values of 301 Disparate Chemicals.

A system for predicting apparent bidirectional permeability (Papp) across Caco-2 cells of diverse chemicals has been reported. The present study aimed to investigate the relationship between in silico-generated Papp (from apical to basal side, Papp A to B) for 301 substances with diverse structures and a binary classification of the reported roles of efflux P-glycoprotein or breast cancer resistant protein. The in silico log(Papp A to B/Papp B to A) values of 70 substances with reported active efflux and 231 substances with no reported active efflux were significantly different (p < 0.01). The probabilities of active efflux transport estimated by trivariate analysis with log MW, log DpH 6.0, and log DpH 7.4 for the 70 active-efflux-positive compounds were higher than those of the other 231 substances (p < 0.01); the area under the corresponding receiver operating characteristic (ROC) curve was 0.81. Further probability values estimated using a machine learning algorithm with 30 chemical descriptors as inputs yielded an area under the ROC curve of 0.79. Using a secondary set of 52 efflux-positive and 48 efflux-negative medicines, the final trivariate-generated probabilities resulted in no significant differences between these binary groups (p = 0.09); however, the final machine learning model demonstrated a good area under the ROC curve of 0.79. Consequently, a combination of the previously established system for generating the permeability coefficients across intestinal monolayers (a continuous variable) and the currently proposed system for predicting the roles of additional active efflux (a binary classification) could prove useful; high accuracy was achieved by applying machine learning using in silico-generated chemical descriptors.

P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT

Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells. Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles. Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used. Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells. Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters.

Penetration of Organophosphate Triesters and Diesters across the Blood–Cerebrospinal Fluid Barrier: Efficiencies, Impact Factors, and Mechanisms

The penetration of organophosphate triesters (tri-OPEs) and diesters (di-OPEs) across the blood-brain barrier and their influencing factors remain unclear in humans. In this study, 21 tri-OPEs and 8 di-OPEs were measured in 288 paired serum and cerebrospinal fluid (CSF) samples collected in Jinan, China. Six tri-OPEs were frequently detected in both serum and CSF, with median concentrations ranging from 0.062 to 1.62 and 0.042-1.11 ng/mL, respectively. Their penetration efficiencies across the blood-CSF barrier (BCSFB) (RCSF/serum, CCSF/Cserum) were calculated at 0.667-2.80, and these efficiencies first increased and then decreased with their log Kow values. The reduced penetration efficiencies of triphenyl phosphate (TPHP) and 2-ethylhexyl diphenyl phosphate (EHDPP) may be attributed to their strong binding affinities for human serum albumin and p-glycoprotein due to their high hydrophobicity and aryl structure, as indicated by molecular docking. This suggests that active efflux transport may be involved in the penetration of TPHP and EHDPP in addition to passive diffusion similar to the other four tri-OPEs. Di-OPEs were found in few serum samples and even fewer CSF samples, indicating their limited BCSFB permeability. This may be due to their high polarity, low hydrophobicity, and ionic state in blood. This study has important implications for understanding the neurotoxicity of tri-OPEs and di-OPEs and the underlying mechanisms.

The Effects of Propofol on a Human in vitro Blood-Brain Barrier Model.

BackgroundRecently, the safety of repeated and lengthy anesthesia administration has been called into question, a subset of these animal studies demonstrated that anesthetics induced blood-brain barrier (BBB) dysfunction. The BBB is critical in protecting the brain parenchyma from the surrounding micro-vasculature. BBB breakdown and dysfunction has been observed in several neurodegenerative diseases and may contribute to both the initiation and the progression of the disease. In this study we utilize a human induced pluripotent stem cell (iPSC) derived-BBB model, exhibiting near in vivo properties, to evaluate the effects of anesthetics on critical barrier properties.MethodsiPSC-derived brain microvascular endothelial cells (BMECs) expressed near in vivo barrier tightness assessed by trans-endothelial electrical resistance and para-cellular permeability. Efflux transporter activity was determined by substrate transport in the presence of specific inhibitors. Trans-cellular transport was measured utilizing large fluorescently tagged dextran. Tight junction localization in BMECs was evaluated with fluorescent microscopy. The anesthetic, propofol was exposed to BMECs at varying durations and concentrations and BBB properties were monitored post-exposure.ResultsFollowing propofol exposure, BMECs displayed reduced resistance and increased permeability indicative of a leaky barrier. Reduced barrier tightness and the dysregulation of occludin, a tight junction protein, were partly the result of an elevation in matrix metalloproteinase (MMP) levels. Efflux transporter activity and trans-cellular transport were unaffected by propofol exposure. Propofol induced barrier dysfunction was partially restored following matrix metalloproteinase inhibition.ConclusionFor the first time, we have demonstrated that propofol alters BBB integrity utilizing a human in vitro BBB model that displays key in vivo characteristics. A leaky BBB enables otherwise impermeable molecules such as pathogens and toxins the ability to reach vulnerable cell types of the brain parenchyma. A robust human in vitro BBB model will allow for the evaluation of several anesthetics at fluctuating clinical scenarios and to elucidate mechanisms with the goal of ultimately improving anesthesia safety.

Establishment of ion channel and ABC transporter assays in 3D-cultured ReNcell VM on a 384-pillar plate for neurotoxicity potential

Neurotoxicity potential of compounds by inhibition of ion channels and efflux transporters has been studied traditionally using two-dimensionally (2D) cultured cell lines such as CHO and HEK-293 overexpressing the protein of interest. However, these approaches are time consuming and do not recapitulate the activity of ion channels and efflux transporters indigenously expressed in neural stem cells (NSCs) in vivo. To overcome these issues, we established ion channel and transporter assays on a 384-pillar plate with three-dimensionally (3D) cultured ReNcell VM and demonstrated high-throughput measurement of ion channel and transporter activity. RNA sequencing analysis identified major ion channels and efflux transporters expressed in ReNcell VM, followed by validating 3D ReNcell-based ion channel and transporter assays with model compounds. Major ion channel activities were measured by specifically inhibiting potassium channels Kv 7.2 with XE-991 and Kv 4.3 with fluoxetine, and a calcium channel with 2-APB. Activities of major efflux transporters, MDR1, MRP1, and BCRP, were assessed using their respective blockers, verapamil, probenecid, and novobiocin. From this study, we demonstrated that 3D-cultured ReNcell VM on the 384-pillar plate could be a good alternative to rapidly identify environmental chemicals and therapeutic compounds for their role in modulating the activity of ion channels and efflux transporters, potentially leading to neurotoxicity.

442 Repurposing FDA-approved PI3K/Akt Inhibitors to Improve Anti-Cancer Drug Brain Uptake in Glioblastoma Resection Models

OBJECTIVES/GOALS: We have shown that glioblastoma upregulates blood-brain barrier drug efflux transporters via a mechanism that likely involves TNFα and PI3K/Akt. Our goal is to repurpose FDA-approved PI3K/Akt inhibitors to increase anticancer drug brain concentrations, which holds the potential for translation into the neuro-oncology clinic. METHODS/STUDY POPULATION: GL261 Red-FLuc and MBR525-1 Red-FLuc cells (2μl; 2.5K cells/μl; 1μl/min) were injected into the right hemisphere of 8-week old female J:NU mice (coordinates relative to bregma: AP -2 mm, ML -2 mm, DV -3 mm). Tumor burden was assessed weekly with IVIS® Spectrum in vivo imaging; tumor volume and invasiveness were measured by MRI and histopathology, respectively. On day 14 post-injection, mice received 5-ALA (200 mg/kg ip), and tumors were resected with a 2 mm punch biopsy tool and surgical fluorescence microscope (ex/em: 405/635nm). Drug efflux transporter expression and activity in isolated brain capillaries were determined by Western blot and substrate fluorescence assays, respectively. Cytotoxicity was assessed after 48-hour drug incubation using CyQuant MTT Cell Proliferation Assay kits. RESULTS/ANTICIPATED RESULTS: IC50 values of temozolomide, lapatinib, alpelisib, and miltefosine were N/A, 32, 20, and 190 μM for GL261 Red-FLuc cells and N/A, 49, 36, and 148 μM for MBR525-1 Red-FLuc cells, respectively. Median survival of GL261 Red-FLuc mice was 26.5d and significantly increased to 34d with resection (p=0.116). In GBM mice, drug efflux transporter expression and activity levels in brain capillaries isolated from the contralateral hemisphere were significantly upregulated compared to sham controls. Furthermore, treatment with FDA-approved PI3K/Akt inhibitors, alpelisib and miltefosine, significantly reduced drug efflux transporter expression and activity to control levels. In PK and survival studies, we expect that PI3K/Akt inhibition will increase brain uptake of anticancer drugs and prolong GBM mouse survival. DISCUSSION/SIGNIFICANCE: We have previously shown that PI3K/Akt inhibition reduces P-gp/BCRP levels in brain capillaries. Here, we vertically extend this strategy by repurposing the FDA-approved PI3K/Akt inhibitors alpelisib/miltefosine to improve brain uptake of anticancer drugs in GBM resection models.

The Bradykinin B2 Receptor Agonist (NG291) Causes Rapid Onset of Transient Blood-Brain Barrier Disruption Without Evidence of Early Brain Injury.

Background: The blood–brain barrier (BBB) describes the brain’s highly specialized capillaries, which form a dynamic interface that maintains central nervous system (CNS) homeostasis. The BBB supports the CNS, in part, by preventing the entry of potentially harmful circulating molecules into the brain. However, this specialized function is challenging for the development of CNS therapeutics. Several strategies to facilitate drug delivery into the brain parenchyma via disruption of the BBB have been proposed. Bradykinin has proven effective in disrupting mechanisms across the blood–tumor barrier. Unfortunately, bradykinin has limited therapeutic value because of its short half-life and the undesirable biological activity elicited by its active metabolites.Objective: To evaluate NG291, a stable bradykinin analog, with selective agonist activity on the bradykinin-B2 receptor and its ability to disrupt the BBB transiently.Methods: Sprague Dawley rats and CD-1 mice were subjected to NG291 treatment (either 50 or 100 μg/kg, intravenously). Time and dose-dependent BBB disruption were evaluated by histological analysis of Evans blue (EB) extravasation. Transcellular and paracellular BBB leakage were assessed by infiltration of 99mTc-albumin (66.5 KDa) and 14C-sucrose (340 Da) radiolabeled probes into the brains of CD-1 mice treated with NG291. NG291 influence on P-glycoprotein (P-gp) efflux pump activity was evaluated by quantifying the brain accumulation of 3H-verapamil, a known P-gp substrate, in CD-1 mice.Results: NG291-mediated BBB disruption was localized, dose-dependent, and reversible as measured by EB extravasation. 99mTc-albumin leakage was significantly increased by 50 μg/kg of NG291, whereas 100 μg/kg of NG291 significantly augmented both 14C-sucrose and 99mTc-albumin leakage. NG291 enhanced P-gp efflux transporter activity and was unable to increase brain uptake of the P-gp substrate pralidoxime. NG291 did not evoke significant short-term neurotoxicity, as it did not increase brain water content, the number of Fluoro-Jade C positive cells, or astrocyte activation.Conclusion: Our findings strongly suggest that NG291 increases BBB permeability by two different mechanisms in a dose-dependent manner and increases P-gp efflux transport. This increased permeability may facilitate the penetration into the brain of therapeutic candidates that are not P-gp substrates.

Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC–MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22–C26). Inhibitors of endoplasmic reticulum–associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.

Expression and Function of ABC Proteins in Fish Intestine.

In fish, the intestine is fundamental for digestion, nutrient absorption, and other functions like osmoregulation, acid-base balance, and excretion of some metabolic products. These functions require a large exchange surface area, which, in turn, favors the absorption of natural and anthropogenic foreign substances (xenobiotics) either dissolved in water or contained in the food. According to their chemical nature, nutrients, ions, and water may cross the intestine epithelium cells’ apical and basolateral membranes by passive diffusion or through a wide array of transport proteins and also through endocytosis and exocytosis. In the same way, xenobiotics can cross this barrier by passive diffusion or taking advantage of proteins that transport physiological substrates. The entry of toxic substances is counterbalanced by an active efflux transport mediated by diverse membrane proteins, including the ATP binding cassette (ABC) proteins. Recent advances in structure, molecular properties, and functional studies have shed light on the importance of these proteins in cellular and organismal homeostasis. There is abundant literature on mammalian ABC proteins, while the studies on ABC functions in fish have mainly focused on the liver and, to a minor degree, on the kidney and other organs. Despite their critical importance in normal physiology and as a barrier to prevent xenobiotics incorporation, fish intestine’s ABC transporters have received much less attention. All the ABC subfamilies are present in the fish intestine, although their functionality is still scarcely studied. For example, there are few studies of ABC-mediated transport made with polarized intestinal preparations. Thus, only a few works discriminate apical from basolateral transport activity. We briefly describe the main functions of each ABC subfamily reported for mammals and other fish organs to help understand their roles in the fish intestine. Our study considers immunohistochemical, histological, biochemical, molecular, physiological, and toxicological aspects of fish intestinal ABC proteins. We focus on the most extensively studied fish ABC proteins (subfamilies ABCB, ABCC, and ABCG), considering their apical or basolateral location and distribution along the intestine. We also discuss the implication of fish intestinal ABC proteins in the transport of physiological substrates and aquatic pollutants, such as pesticides, cyanotoxins, metals, hydrocarbons, and pharmaceutical products.

Factors Influencing Luciferase-Based Bioluminescent Imaging in Preclinical Models of Brain Tumor.

Bioluminescent imaging (BLI) is a powerful tool in biomedical research to measure gene expression and tumor growth. The current study examined factors that influence the BLI signal, specifically focusing on the tissue distribution of two luciferase substrates, D-luciferin and CycLuc1. D-luciferin, a natural substrate of firefly luciferase, has been reported to have limited brain distribution, possibly due to the efflux transporter, breast cancer resistance protein (Bcrp), at the blood-brain barrier. CycLuc1, a synthetic analog of D-luciferin, has a greater BLI signal at lower doses than D-luciferin, especially in the brain. Our results indicate that limited brain distribution of D-luciferin and CycLuc1 is predominantly dictated by their low intrinsic permeability across the cell membrane, where the efflux transporter, Bcrp, plays a relatively minor role. Both genetic ablation and pharmacological inhibition of Bcrp decreased the systemic clearance of both luciferase substrates, significantly increasing exposure in the blood and, hence, in organs and tissues. These data also indicate that the biodistribution of luciferase substrates can be differentially influenced in luciferase-bearing tissues, leading to a "tissue-dependent" BLI signal. The results of this study point to the need to consider multiple mechanisms that influence the distribution of luciferase substrates. SIGNIFICANCE STATEMENT: Bioluminescence is used to monitor many biological processes, including tumor growth. This study examined the pharmacokinetics, brain distribution, and the role of active efflux transporters on the luciferase substrates D-luciferin and CycLuc1. CycLuc1 has a more sustained systemic circulation time (longer half-life) that can provide an advantage for the superior imaging outcome of CycLuc1 over D-luciferin. The disparity in imaging intensities between brain and peripheral sites is due to low intrinsic permeability of these luciferase substrates across the blood-brain barrier.

Modelling the role of APOE4 in blood-brain barrier dysfunctions with isogenic IPSC-derived brain microvascular endothelial cells and pericytes.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer's Disease, and is known to affect the function of multiple neuronal and glial cell types. Recent research has suggested that blood-brain barrier (BBB) dysfunction is commonly observed among AD patients. However, it remains unclear whether and how APOE4 directly contributes to blood-brain barrier dysfunction. Isogenic iPSC lines with different APOE isoforms were differentiated into brain microvascular endothelial cell-like cells (BMECs) and neural crest-derived pericyte-like cells (PCs). Isogenic BMECs with different APOE isoforms were compared for their tight junction integrity, efflux transporter activity and amyloid clearance capabilities. Isogenic PCs with different APOE isoforms were compared for their APOE secretion levels and amyloid uptake capabilities. APOE4, APOE3 and APOE2 BMECs exhibited similar levels of tight junction protein expression, efflux transporter activities and trans-endothelial electrical resistance. However, we found that the presence of APOE ε4 protein, when compared with APOE ε2 and APOE ε3, led to reduced BMEC clearance of amyloid in a Transwell model. Compared to APOE3 and APOE2 PCs, APOE4 PCs demonstrated similar levels of APOE secretion, but had lower amyloid uptake capabilities. Our findings reveal that although APOE4 did not directly affect BMEC barrier properties in our hPSC-derived model, it plays a potentially important role in amyloid clearance by both BMECs and PCs.

Evaluation of the Influence of Zhenwu Tang on the Pharmacokinetics of Digoxin in Rats Using HPLC-MS/MS.

Digoxin (DIG) is a positive inotropic drug with a narrow therapeutic window that is used in the clinic for heart failure. The active efflux transporter of DIG, P-glycoprotein (P-gp), mediates DIG absorption and excretion in rats and humans. Up to date, several studies have shown that the ginger and Poria extracts in Zhenwu Tang (ZWT) affect P-gp transport activity. This study aimed to explore the effects of ZWT on the tissue distribution and pharmacokinetics of DIG in rats. The deionized water or ZWT (18.75 g/kg) was orally administered to male Sprague–Dawley rats once a day for 14 days as a pretreatment. On day 15, 1 hour after receiving deionized water or ZWT, the rats were given the solution of DIG at 0.045 mg/kg dose, and the collection of blood samples was carried out from the fundus vein or excised tissues at various time points. HPLC-MS/MS was used for the determination of the DIG concentrations in the plasma and the tissues under investigation. The pharmacokinetic interactions between DIG and ZWT after oral coadministration in rats revealed significant reductions in DIG Cmax and AUC0-∞, as well as significant increases in T1/2 and MRT0-∞. When coadministered with ZWT, the DIG concentration in four of the investigated tissues statistically decreased at different time points except for the stomach. This study found that combining DIG with ZWT reduced not only DIG plasma exposure but also DIG accumulation in tissues (heart, liver, lungs, and kidneys). The findings of our study could help to improve the drug's validity and safety in clinical applications and provide a pharmacological basis for the combined use of DIG and ZWT.

Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling.

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-β signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.