Evaluation of the Role of P-Glycoprotein in Ivermectin Uptake by Primary Cultures of Bovine Brain Microvessel Endothelial Cells

The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.     

Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

P-glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging

P-glycoprotein (P-gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over-expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P-gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P-gp in human lymphocytes, comparing two different methodologies to assess both parameters. P-gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30-years old; group 2: above 60-years old). P-gp expression was assessed using the anti-P-gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P-gp activity was evaluated measuring the efflux rate of the fluorescent P-gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, P-gp expression and each of the P-gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age-dependent increase in mean P-gp expression (p = 0.029), which was not reflected in the transporter's activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P-gp activity. Despite the significant correlation between P-gp expression and P-gp activity found in lymphocytes (Gp1(group 1)-r = 0.609, p < 0.001; Gp2-r = 0.461, p = 0.012), using UIC2 shift assay, these data reinforce the need for P-gp activity assessment, rather than P-gp expression determination alone, when starting new therapeutic regimens with P-gp substrates, especially in men older than 60 years of age.     

Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells.

The objective of this study was to examine effects of interleukin-6 (IL-6) on the expression and activity of the drug resistance transporters (MDR1 and MRP) in human hepatoma cell lines. Expression and activity of MDR1 and MRP transporters were examined in IL-6-treated and control HuH 7 and HepG2 cells using semi-quantitative RT-PCR analysis and by rhodamine 123 and 5-carboxyfluorescin efflux assays. Results from RT-PCR demonstrated expression of MRP3, MRP6, and MDR1 in HuH 7 cells and expression of MRP1, MRP2, MRP3, MRP6, and MDR1 in HepG2 cells. Compared with controls, treatment of HuH 7 cells with IL-6 (10 ng/mL, 24 h) resulted in a 1.8-fold increase in MRP-mediated efflux of 5-CF with a corresponding 1.5-fold induction of MRP3 mRNA levels (p < 0.05). Similarly, in HepG2 cells, a 2-fold increase in MRP functional activity and a 1.8-fold induction of MRP1 mRNA levels were seen in the IL-6 treated cells (p < 0.05). Treatment of cells with IL-6 was also found to cause significant reductions in the expression and activity of MDR1 in HuH 7 cells, but not in HepG2 cells. Our data suggest that IL-6 induces MRP expression and activity in human hepatoma cell lines. Suppressive effects of IL-6 on MDR1 expression and activity were also observed in HuH 7 cells. This underscores the importance of examining the regulation of multiple drug resistance proteins as these proteins may have opposing regulatory mechanisms in malignant cells.     

Bone marrow stromal cell interaction reduces Syndecan-1 expression and induces kinomic changes in myeloma cells

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.     

Proliferation kinetics of plasma cells and of normal haemopoietic cells in multiple myeloma

DNA synthesis time (Ts) and 3H thymidine (TdR) labelling index (LI) of bone marrow (BM) myelomatous plasma cells (PC) and of the residual haemopoietic cell population (RHCP) were measured by in vitro quantitative 14C-TdR autoradiography in five patients with multiple myeloma (MM) in different phases of disease (three at presentation and two at relapse) and in one patient with solitary extra-osseous myeloma. One other patient with plasma cell leukaemia (PCL) was studied during an initial relapse phase and later during the leukaemic terminal phase. PC Ts was 18.8 +/- 3.7 (from 13.3 to 25.0) hr and PC LI was 2.5 +/- 1.8% (from 1.0 to 6.3%). In the case of PCL, circulating PC had a Ts of 14.4 hr and a LI of 3.1. From these experimental measurements, the fractional turnover rate (FTR-percentage of cells produced per unit time) and the potential doubling time (Td) of BMPC were calculated assuming that all BMPC were in a steady-state at the time of the study. BMPC FTR was 3.53 +/- 2.3% cells per day (from 1.2 to 6.72) and BMPC Td was 46.8 +/- 27.5 days (from 15.0 to 75.4). Comparison with results obtained in BM blasts of children with acute lymphoblastic leukaemia (ALL) indicated that BMPC had a lower proliferative activity (P less than 0.001), although BMPC Ts was not significantly different. In two patients a tumour doubling time of 6 and 13 months was determined by clinical follow up. Comparison of this parameter with Td showed a cell loss factor of more than 90% in both patients. Kinetic data relative to RHCP showed slight variations with respect to those found in normal subjects, with a general tendency towards a prolongation of Ts and a reduction of LI.     

Swelling-activated chloride channels in multidrug-sensitive and - resistant cells

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug- efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug- sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively. gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV. Cl- channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells. gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM). None of these blockers inhibited R123 efflux. NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered. Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling- activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

The membrane transport system responsible for multidrug resistance is operating in nonresistant cells

Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium. Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release. We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells. The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR). By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium. It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.     

Assessment of P-glycoprotein-dependent drug transport in isolated rat hepatocytes using rhodamine 123

To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.Abbreviations AAF acetaminofluorene - MDR multidrug resistance - Pgp P-glycoprotein - R-123 rhodamine 123     

Microtubule-targeting anticancer drug eribulin induces drug efflux transporter P-glycoprotein

This study examined the effects of microtubule-targeting anticancer drugs (paclitaxel, cabazitaxel, and eribulin) on the expression of drug efflux transporter P-glycoprotein, which is encoded by MDR1. Paclitaxel and eribulin induced MDR1 promoter activity in a concentration-dependent manner, while cabazitaxel had little effect in human intestinal epithelial LS174T cells. Overexpression of the nuclear receptor pregnane X receptor (PXR) gene (NR1I2) enhanced paclitaxel- and eribulin-induced MDR1 activation, but expression of the nuclear receptor co-repressor silencing mediator for retinoid and thyroid receptors (SMRT) gene (NCOR2) repressed MDR1 activation. Eribulin increased the mRNA and protein expression of P-glycoprotein in LS174T cells. Cellular uptake of rhodamine 123 and calcein-acetoxymethyl ester (calcein-AM), P-glycoprotein substrates, decreased in paclitaxel- or eribulin-treated LS174T cells. Eribulin also increased MDR1 promoter activity in human breast cancer MCF7 cells. The results suggest that the microtubule-targeting anticancer drug eribulin can induce the drug efflux transporter P-glycoprotein via PXR in human intestinal and breast cancer cells and thus influence the efficacy of anticancer drugs.     

穿心莲内酯联合氟康唑抗耐药白假丝酵母菌作用机制研究

目的探讨穿心莲内酯联合氟康唑抗耐药白假丝酵母菌作用及其机制。方法采用微量稀释法检测穿心莲内酯（AG）及联合氟康唑（FLC）对耐药白假丝酵母菌的MIC;采用罗丹明6G（Rh6G）检测AG对耐药白假丝酵母菌CDR外排功能的影响;利用罗丹明123评估AG对耐药白假丝酵母菌MDR外排功能的影响：采用二氢罗丹明检测AG单用及联合FLC对耐药白假丝酵母菌活性氧（ROS）的影响;采用实时荧光定量PCR（qRT—PCR）检测AG联合FLC对耐药白假丝酵母菌外排泵相关基因CDR1、CDR2和MDR1表达的影响。结果AG联合FLC抗耐药白假丝酵母菌呈相加作用;AG对CDR外排功能无影响;AG可抑制MDR外排功能;AG联合FLC能显著提高耐药白假丝酵母菌细胞ROS水平;AG与FLC联合作用于耐药白假丝酵母菌可下调CDR1和MDR1的表达量,上调CDR2的表达量。结论AG联合FLC抗耐药白假丝酵母菌具有相加作用,其机制可能与抑制外排泵及相关基因表达,提高胞内ROS水平有关。     

Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.     

Proliferation kinetics of plasma cells and of normal haemopoietic cells in multiple myeloma

Abstract. DNA synthesis time ( T _s) and ³H thymidine (TdR) labelling index (LI) of bone marrow (BM) myelomatous plasma cells (PC) and of the residual haemopoietic cell population (RHCP) were measured by in vitro quantitative ¹⁴C-TdR autoradiography in five patients with multiple myeloma (MM) in different phases of disease (three at presentation and two at relapse) and in one patient with solitary extra-osseous myeloma. One other patient with plasma cell leukaemia (PCL) was studied during an initial relapse phase and later during the leukaemic terminal phase. PC T _s was 18.8 ± 3.7 (from 13.3 to 25.0) hr and PC LI was 2.5 ± 1.8%; (from 1.0 to 6.3%). In the case of PCL, circulating PC had a T _s of 14.4 hr and a LI of 3.1. From these experimental measurements, the fractional turnover rate (FTR—percentage of cells produced per unit time) and the potential doubling time ( T _d) of BMPC were calculated assuming that all BMPC were in a steady-state at the time of the study. BMPC FTR was 3.53 ± 2.3% cells per day (from 1.2 to 6.72) and BMPC T _d was 46.8 ± 27.5 days (from 15.0 to 75.4). Comparison with results obtained in BM blasts of children with acute lymphoblastic leukaemia (ALL) indicated that BMPC had a lower proliferative activity ( P < 0.001), although BMPC T _s was not significantly different. In two patients a tumour doubling time of 6 and 13 months was determined by clinical follow up. Comparison of this parameter with T _d showed a cell loss factor of more than 90% in both patients.
Kinetic data relative to RHCP showed slight variations with respect to those found in normal subjects, with a general tendency towards a prolongation of T _s and a reduction of LI.     

Non-Invasive Imaging Provides Spatiotemporal Information on Disease Progression and Response to Therapy in a Murine Model of Multiple Myeloma

Background

Multiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.

Material and Methods

A mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.

Results

Strong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase⁺ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase⁺ cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.

Conclusions

This study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.     

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.     

Reversal of multidrug resistance by 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide through the reversible inhibition of P-glycoprotein

Overexpression of P-glycoprotein (P-gp) is one of the major obstacles to successful cancer chemotherapy. In this study, we examined the ability of 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide (C-4) to reverse multidrug resistance (MDR) in P-gp expressing KBV20C cells. Treatment of KBV20C cells with C-4 led to a dramatic increase in paclitaxel- or vincristine-induced cytotoxicity without any cytotoxicity by itself. In parallel, C-4 treatment caused an increase in apoptotic cell death by paclitaxel or vincristine. Furthermore, C-4 treatment significantly increases in intracellular accumulation of fluorescent P-gp substrate rhodamine 123, indicating that C-4 treatment leads to reversal of the MDR phenotype resulting from an increased accumulation of anticancer drugs by inhibiting drug efflux function of P-gp. This notion is further supported by the observation that C-4 treatment potentiates paclitaxel-induced G(2)/M arrest of the cell cycle. In addition, the drug efflux function of P-gp was reversibly inhibited by C-4 treatment, while the expression level of P-gp was not affected. Collectively, our results describe the potential of C-4 to reverse the P-gp-mediated MDR phenotype through reversible inhibition of P-gp function, which may make it an attractive new agent for the chemosensitization of cancer cells.