P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Drug transporter,P-glycoprotein (MDR1), is an integrated component of the mammalian blood–testis barrier

Throughout spermatogenesis, leptotene spermatocytes traverse the blood–testis barrier (BTB) to enter the adluminal compartment of the seminiferous epithelium for continued development. At the same time, the integrity of the BTB, which is constituted by co-existing tight junctions (TJ), basal ectoplasmic specializations (basal ES) and desmosome-like junctions, must be maintained since a breach in barrier function can result in spermatogenic arrest and even infertility. There is evidence to suggest that drug transporters may function at the BTB, but little is known about how they contribute to spermatogenesis. In this study, we investigate the role of P-glycoprotein (P-gp), a drug efflux pump, in BTB dynamics. A survey by RT-PCR revealed several transporter genes to be expressed by the testis, including Mdr1 (gene symbol for P-gp), Mrp1, Abcc5 and Slc15a1. It was also demonstrated that P-gp localizes to the BTB in all stages of the seminiferous epithelial cycle in the adult rat testis, as well as to the Sertoli cell–elongated spermatid interface in stages VII and VIII. We continued our study by examining the levels of several transporters in the testis following oral administration of Adjudin, a compound known to affect Sertoli–germ cell adhesion. In this experiment, the steady-state levels of P-gp, MRP1, ABCG1 and SLC15A1 were all found to increase by several-fold within hours of Adjudin treatment during junction restructuring. More importantly, an increase in P-gp association with TJ proteins (e.g., occludin, claudin-11 and JAM-A) was noted when testis lysates from Adjudin-treated rats were used for co-immunoprecipitation experiments, suggesting that P-gp may enhance BTB function during Sertoli–germ cell junction restructuring.     

Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells

We have investigated the involvement of intracellular pH (pH_i) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na⁺/H⁺ exchanger1 (NHE1) inhibitor cariporide and the “high K⁺” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH_i than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH_i and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH_i recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH_i and P-gp in K562/DOX cells.     

A novel in vivo regulatory role of P-glycoprotein in alloimmunity

P-glycoprotein (P-gp) is required for adaptive immunity through defined functions in T cell activation and antigen presenting cell (APC) maturation. The potential role of P-gp as an in vivo regulator of alloimmunity is currently unknown. Here we show that P-gp blockade prolongs graft survival in a murine heterotopic cardiac allotransplantation model through in vivo inhibition of the T helper 1 (Th1) cytokine IFN-γ and the Th2 product IL-4, and via downregulation of the APC-expressed positive costimulatory molecule CD80. In vitro, the P-gp antagonist PSC833, a non-calcineurin-inhibitory cyclosporine A analogue, specifically inhibited cellular efflux of the P-gp substrate rhodamine-123 in wild-type CD3⁺ T cells and MHC class II⁺ APCs but not their P-gp knockout counterparts that lacked rhodamine-123 efflux capacity. Additionally, P-gp blockade significantly inhibited murine alloimmune T cell activation in a dose-dependent fashion. In vivo, P-gp blockade significantly prolonged graft survival in Balb/c recipients of C57BL/6 cardiac allografts from 8.5 ± 0.5 to 11.7 ± 0.5 days (P < 0.01), similar in magnitude to the effects of monotherapy with cyclosporine A. Moreover, P-gp blockade, compared to controls, attenuated intragraft expression of CD3 and CD80, but not CD86, and inhibited IFN-γ and IL-4 production (P < 0.05). In the setting of systemic CD86 inhibition, P-gp blockade suppressed IFN-γ and IL-4 production significantly further (to 98% and 89% inhibition, respectively) compared to either P-gp or anti-CD86 blockade alone, and markedly prolonged allograft survival compared to anti-CD86 blockade alone (40.5 ± 4.6 versus 22.5 ± 2.6 days, respectively, P < 0.01). Our findings define a novel in vivo regulatory role of P-gp in alloimmunity and identify P-gp as a potential therapeutic target in allotransplantation.     

Lobeline,a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells

Multidrug resistance (MDR) can limit efficacy of chemotherapy. The best studied mechanism involves P-gp (P-glycoprotein) mediated drug efflux. This study focuses on MDR reversal agents from medicinal plants, which can interfere with P-gp. Rhodamine 123 accumulation assay and flow cytometry analysis were employed to screen for P-gp dependant efflux inhibitors. Lobeline, a piperidine alkaloid from Lobelia inflata and several other Lobelia species, inhibited P-gp activity. MDR reversal potential of lobeline could be demonstrated in cells treated with doxorubicin in that lobeline can sensitize resistant tumor cells at non-toxic concentrations. However, lobeline cannot block BCRP (Breast Cancer Resistance Protein) dependent mitoxantrone efflux. Lobeline could be a good candidate for the development of new MDR reversal agents.     

The contribution of peroxynitrite generation in HIV replication in human primary macrophages

Background

The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability.

Results

The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells.

Conclusion

To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.     

The Interactions of P-Glycoprotein with Antimalarial Drugs,Including Substrate Affinity,Inhibition and Regulation

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10⁻⁶ cm/sec, followed by amodiaquine around 20 x 10⁻⁶ cm/sec; both mefloquine and artesunate were around 10 x 10⁻⁶ cm/sec. Methylene blue was between 2 and 6 x 10⁻⁶ cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.     

MDR1 Gene Polymorphisms and Clinical Relevance

In vivo and in vitro studies have demonstrated that P-glycoprotein (P-gp) plays a very significant role in the ADME processes (absorption, distribution, metabolism, excretion) and drug-drug interaction (DDI) of drugs in humans. P-gp is the product of multidrug resistance gene (MDR1/ABCB1). Pharmacogenomics and pharmacogenetics studies have revealed that genetic polymorphisms of MDR1 are associated with alteration in P-gp expression and function in different ethnicities and subjects. By now, 50 single nucleotide polymorphisms (SNPs) and 3 insertion/deletion polymorphisms have been found in the MDR1 gene. Some of them, such as C3435T, have been identified to be a risk factor for numerous diseases. It is believed that further understanding of the physiology and biochemistry of P-gp with respect to its genetic variations may be important for individualized pharmacotherapy. Therefore, based on the latest public information and our studies, this review focuses on the following four aspects: 1) the impact of P-gp on pharmacokinetics; 2) MDR1 genetic polymorphisms and their impacts on pharmacogenetics; 3) relationship between altered P-gp expression and function and the MDR1^C3435T SNP, and 4) relevance of MDR1 polymorphisms to certain human diseases.     

A novel flavonoid from Fissistigma cupreonitens, 5‑hydroxy‑7,8‑dimethoxyflavanone,competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance

BackgroundP-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 designThe 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.MethodsCalcein-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.Results5‑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.ConclusionThe 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.     

HZ08 Reverse P-Glycoprotein Mediated Multidrug Resistance In Vitro and In Vivo

BackgroundMultidrug efflux transporter P-glycoprotein (P-gp) is highly expressed on membrane of tumor cells and is implicated in resistance to tumor chemotherapy. HZ08 is synthesized and studied in order to find a novel P-gp inhibitor.MethodsMDCK-MDR1 monolayer transport, calcein-AM P-gp inhibition and P-gp ATPase assays were used to confirm the P-gp inhibition capability of HZ08. Furthermore, KB-WT and KB-VCR cells were used to evaluate the P-gp inhibitory activity of HZ08 both in vitro and in vivo.ResultsResults showed that HZ08 was more potent than verapamil in MDCK-MDR1 monolayer transportation model. Meanwhile, P-gp ATPase assay and calcein-AM P-gp inhibition assay confirmed that HZ08 inhibited P-gp ATPase with a calcein-AM IC50 of 2.44±0.31μM. In addition, significantly greater in vitro multidrug resistance reversing effects were observed when vincristine or paclitaxel was used in combination with 10μM HZ08 compared with 10μM verapamil. Moreover, HZ08 could significantly enhance the sensitivity of vincristine with a similar effect like verapamil in both KB-WT and KB-VCR tumor xenograft models.ConclusionsThe novel structure HZ08 could be a potent P-gp inhibitor.     

Kuguacin J isolated from Momordica charantia leaves inhibits P-glycoprotein (ABCB1)-mediated multidrug resistance

Multidrug resistance (MDR) is a major factor in the failure of chemotherapy in cancer patients. Resistance to chemotherapy has been correlated to the overexpression of ABC drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. Our previous study showed that bitter melon (Momordica charantia) leaf extract (BMLE) was able to reverse the MDR phenotype by increasing the intracellular accumulation of chemotherapeutic drugs. In the present study, bioguided fractionation was used to identify the active component(s) of BMLE that is able to modulate the function of P-gp and the MDR phenotype in a human cervical carcinoma cell line (KB-V1). We found that kuguacin J, one of the active components in BMLE, increased sensitivity to vinblastine and paclitaxel in KB-V1 cells. A flow cytometry assay indicated that kuguacin J inhibits the transport function of P-gp and thereby significantly increases the accumulation of rhodamine 123 and calcein AM in the cells. These results were confirmed by [³H]-vinblastine transport assay. Kuguacin J significantly increases intracellular [³H]-vinblastine accumulation and decreased the [³H]-vinblastine efflux in the cells. Kuguacin J also inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin into P-gp in a concentration-dependent manner, indicating that kuguacin J directly interacts with the drug-substrate-binding site on P-gp. These results indicate that kuguacin J modulates the function of P-gp by directly interacting at the drug-substrate-binding site, and it appears to be an effective inhibitor of P-gp activity in vitro and thus could be developed as an effective chemosensitizer to treat multidrug-resistant cancers.     

Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia

BackgroundKidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs.MethodsHuman conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm² or low peak FSS of 0.5 dyne/cm². Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined.ResultsA primary cilium knock-out cell model, ciPTEC-KIF3α^−/−, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α^−/−. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity.ConclusionsFSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane.General significanceFSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism.     

Isopetasin and S-isopetasin as novel P-glycoprotein inhibitors against multidrug-resistant cancer cells

BackgroundA major problem of cancer treatment is the development of multidrug resistance (MDR) to chemotherapy. MDR is caused by different mechanisms such as the expression of the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These transporters efflux xenobiotic toxins, including chemotherapeutics, and they were found to be overexpressed in different cancer types.PurposeIdentification of novel molecules that overcome MDR by targeting ABC-transporters.MethodsResazurin reduction assay was used for cytotoxicity test. AutoDock 4.2. was used for molecular docking. The function of P-gp and BCRP was tested using a doxorubicin uptake assay and an ATPase assay. ROS generation was detected using flow cytometry for the measurement of H₂DCFH-DA fluorescence. Annexin/PI staining was applied for the detection of apoptosis. Bioinformatic analyses were performed using LigandScout 3.12. software and DataWarrior software.ResultsIn our search for new molecules that selectively act against resistant phenotypes, we identified isopetasin and S-isopetasin, which are bioactive natural products from Petasites formosanus. They exerted collateral sensitivity towards leukemia cells with high P-gp expression in CEM/ADR5000 cells, compared to sensitive wild-type CCRF-CEM leukemia cells. Also, they revealed considerable activity towards breast cancer cells overexpressing breast cancer resistance protein, MDA-MB-231-BCRP clone 23. This motivated us to investigate whether the function of P-gp was inhibited. In-silico results showed the compounds bound with high affinity and interacted with key amino acid residues in P-gp . Then, we found that the two compounds increased doxorubicin accumulation in P-gp overexpressing CEM/ADR5000 by three-fold compared to cells without inhibitor. P-gp-mediated drug efflux was ATP-dependent. Isopetasin and S-isopetasin increased the ATPase activity of human P-gp in a comparable fashion as verapamil used as control P-gp inhibitor. As isopetasin and S-isopetasin exerted dual roles, first as cytotoxic compounds and then as P-gp inhibitors, we suggested that their P-gp inhibition is part of a larger complex of mechanisms to induce cell death in cancer patients. P-gp dysfunction induces mitochondrial stress to generate ATP. Upon continuing stress by P-gp inhibition, the mitochondria generate reactive oxygen species (ROS). Initially established for verapamil, this theory was validated in the present study for isopetasin and S-isopetasin, as treatment with the two candidates increased ROS levels in CEM/ADR5000 cells followed by apoptosis.ConclusionOur study highlights the importance of isopetasin and S-isopetasin as novel ROS-generating and apoptosis-inducing P-gp inhibitors.     

The P-glycoprotein Inhibitor GF120918 Modulates Ca2+-Dependent Processes and Lipid Metabolism in Toxoplasma Gondii

Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca²⁺ signaling in the parasite. In support of a potential role of P-gp in Ca²⁺-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca²⁺ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.     

P-Glycoprotein Mediated Efflux Limits the Transport of the Novel Anti-Parkinson's Disease Candidate Drug FLZ across the Physiological and PD Pathological In Vitro BBB Models

FLZ, a novel anti-Parkinson''s disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson''s conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.     

In vitro concentration dependent transport of phenytoin and phenobarbital,but not ethosuximide,by human P-glycoprotein

AimsOne possible mechanism for epilepsy drug resistance is overexpression of P-glycoprotein in the blood–brain barrier, but whether (or which) antiepileptic drugs (AEDs) are transported by P-gp remains unclear. We evaluated AEDs as P-gp substrates using cell monolayers.Main methodsBi-directional transport assays and concentration equilibrium transport assays (CETAs) were performed for phenytoin (PHT), phenobarbital (PB), and ethosuximide (ESM) using wildtype Madin–Darby Canine Kidney II cell line MDCKII and porcine renal endothelial cell line LLC–PK1 cells and these cells transfected with human MDR1 cDNA to express P-gp.Key findingsWildtype cells demonstrated no efflux transport of PHT, PB, or ESM. In CETAs, both MDR1-transfected cell lines transported PHT from basolateral to apical when PHT loading concentrations were 5 or 10, but not 20 µg/ml. MDCK–MDR1 cells transported PB when initial concentrations were 10 or 20, but not 5 µg/ml. LLC–MDR1 did not transport PB. P-gp inhibitor verapamil blocked efflux transport. MDR1-transfected cells did not transport ESM at 5.6 or 56 µg/ml. Bi-directional transport assays demonstrated weak transport for PHT but not PB or ESM.SignificanceHuman P-gp transports PHT and PB, but not ESM, in a concentration dependent manner. CETA may be more sensitive than bi-directional assays to detect transport of drugs with high passive diffusion. Potential P-gp substrates should be tested at clinically relevant concentration ranges.     

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein

Sorcin, a 22 kDa Ca²⁺ binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between −716 and −709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.