Agent Verapamil Research Articles

Dictyostelium discoideum Expresses a Malaria ChloroquineResistance Mechanism upon Transfection with Mutant, but Not Wild-type,Plasmodium falciparum TransporterPfCRT

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.

Effects of Arsenic Trioxide on Protein Kinase C and Cytosolic Calcium in Ventriclar Myocardium of Guinea Pig.

AIM To clarify the mechanisms of the cardiac side effects of arsenic trioxide (As2O3) in leukemia treatment, the effects of As2O3 on protein kinase C(PKC) and cytosolic calcium ([Ca2+]i) in ventriclar myocardium of guinea pig were investigated.METHODS Fluo-3/AM fluorescent probe tagged [Ca2+]i of guinea pig's ventriclar myocardium, detected the [Ca2+]i, by laser confocal microscopy before and after exposed to As2O3. Phosphorus radioisotope assay was used to detect the activity of PKC on cell membrane or in cytoplasm. DNA ladders of ventriclar myocardium in Tyrode's solution after exposed to As2O3 were evaluated.RESULTS The cytosolic [Ca2+]i did not change when incubated guinea pig's ventriclar myocardium in Tyrode's solution with 0.5μmol•L−1As2O3, after incubated in2μmol•L−1As2O3 for 6min, [Ca2+]i began to increase and was continuous increasing for about 9min. After exposed to 10μmol•L−1As2O3 for less than 3 min, the [Ca2+]i kept on increasing till the end (about 27min) of the examination and could not recover. The increasing of [Ca2+]i could be partly blocked by calcium channel blocking agents—verapamil and nifedipin. The PKC of myocardial cells began to rise after incubated in an As2O3 changing concentration culture media for 3 hours, which initial As2O3 was 10μmol•L−1, and the DNA ladders appeared after incubated in it for10 hours. No DNA ladders appeared when incubated in an As2O3 culture media in which the initial As2O3 concentration was under 5μmol•L−1.CONCLUSIONS As2O3 can active PKC, increase cytosolic [Ca2+]i and promote apoptosis of myocardial cells. The promoting apoptosis action of As2O3 is closely related to the lasting activation of PKC. The initial action target of As2O3 is cell membrane.

Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.

The Influence of Anti‐irritants on Captopril Hydrophilic Gel

In this study, we compared the irritation inhibition of various types of anti‐irritants such as antihistamines (cyprohetadine, diphenhydramine, and promethazine), alpha‐hydroxy acids (gluconolactone and gluco‐δ‐lactone), corticosteroids (betamethasone and clobetasol), and ion channel modulating agents (amiloride, ethacrynic acid, nifedipine, and verapamil) on the adverse dermatological reaction caused by captopril gel using noninvasive bioengineering methods including measuring the transepidermal water loss (TEWL) and the color change of skin surface [such as change chroma (delta C) and difference in color (delta E) between the gel‐treated site and the untreated site]. In addition, the influence of these anti‐irritants on the penetration capacity of captopril through the rabbit skin was also investigated. The results showed that the TEWL, change chroma (delta C), and difference in color (delta E) of skin were significantly reduced via incorporating diphenhydramine and clobetasol, indicating that both substances had potent irritation inhibition activity. Moreover, these substances had no effect on the percutaneous absorption of captopril gel. However, flux of the captopril with anti‐irritants was about 480 µg/cm2/h and the required minimum administration area to obtain the minimum effective concentration was about 15 cm2, indicating that this formulation could possibly be developed for a transdermal drug delivery system.

Intravesical pH: a potentially important variable affecting efficacy and the further development of anthracycline chemotherapy for superficial bladder cancer.

To assess, using epirubicin-sensitive and multidrug resistant (MDR) derivatives of human bladder cancer cell lines in vitro, the probable effect of intravesical pH changes, with and without the MDR antagonist verapamil, on the uptake, intracellular distribution and cytotoxicity of epirubicin during intravesical chemotherapy. Incubations for cytotoxicity testing were carried out in buffered medium containing epirubicin, at pH values of 6.0-8.5, with verapamil where appropriate. The cytotoxicity of epirubicin, with and without verapamil, was determined using the tetrazolium cytotoxicity assay. Intracellular epirubicin fluorescence was assessed using flow cytometry and confocal microscopy. Flow cytometric total intracellular epirubicin fluorescence was measured at pH 6.0, 6.4, 6.8, 7.2, and 7.6, and confocal microscopy was carried out at pH 6.0 and 8.0. The MDR-reversing agent verapamil was added at 100 micro g/mL to some incubations. Epirubicin cytotoxicity in resistant cell lines appears considerably enhanced by adding verapamil and further improved, especially in MDR cells, by alkalinization of the drug solution to pH 8.0. Flow cytometry results showed striking and consistent differences in epirubicin handling with pH. Sensitive cells can be induced to absorb considerably more drug at alkaline pH, whilst resistant cells show no such behaviour. Nuclear drug fluorescence was greater in sensitive cells at alkaline pH, but cytoplasmic drug fluorescence in the resistant cells was little changed by pH. Adding verapamil to resistant cells restored the sensitive phenotype of drug handling. Buffering epirubicin to an alkaline pH before intravesical application should increase its intrinsic cytotoxicity. The potential for synergy at certain drug combinations will be enhanced by applying these findings. MDR reversal and fatty acid augmentation of drug uptake are discussed as examples.

Calcium channel antagonist verapamil inhibits neointimal formation and enhances apoptosis in a vascular graft model

Background: The potential of the calcium channel antagonist verapamil to cause apoptosis (programmed cell death) is of considerable importance in arterial injury where the loss of smooth muscle cells may contribute to a reduction in intimal hyperplasia development. The aim of this study was to determine whether verapamil induces vascular cell apoptosis after carotid artery synthetic grafting. Methods: Thirty-two adult-female Merino sheep received gelatin sealed fusiform shape-Dacron grafts into the left common carotid artery at day 0. After operation animals were randomly allocated to either a control group or one of three treatment groups (groups 2, 3, and 4). Group 1 animals (n = 9) received no treatment. For the treatment groups, intravenous verapamil was given at a rate of 0.5 mg/kg per day in two divided doses. Group 2, 3, and 4 sheep were treated for 1, 2, and 4 weeks, respectively. Animals were sacrificed at 4 weeks. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-fluorescent labelling. Proliferating cells and their phenotype were determined by doublestaining with antiproliferation cellular nuclei antigen and anti-α-actin or anti-HAM-56. Results: There were significantly more apoptotic cells in the perigraft adventitia in the 4-week treatment group than in the control group ( P <0.05). The average number of proliferating cells at 2 and 4 weeks in the intima were significantly less than in the control ( P <0.05). The average numbers of macrophages inside graft matrix in the 2 and 4 weeks treatment groups were significantly less than for the control ( P <0.05). The number of proliferating cells inside the graft was significantly lower at 4 weeks compared with control ( P <0.05). There was negative correlation between intimal PCNA expression and perigraft apoptotic expression level ( P <0.05). Conclusion: The antihypertensive agent verapamil inhibits intimal hyperplasia through enhancing adventitial cell apoptosis and inhibiting intimal cell proliferation after vascular grafting.

The effects of chloroquine and verapamil on digestive vacuolar pH of P.falciparum either sensitive or resistant to chloroquine

In the preceding paper, we present a novel method for measuring the digestive vacuolar pH (pH vac) of the malarial parasite Plasmodium falciparum, and show that, surprisingly, pH vac is lower for chloroquine resistant (CQR) Dd2 parasites relative to chloroquine sensitive (CQS) HB3. These data may have important consequences for elucidating mechanisms of antimalarial drug resistance and for developing new antimalarial therapy. Additional issues central to a better understanding of antimalarial pharmacology and antimalarial drug resistance require detailed comparative data on the effects of key drugs and other compounds on parasite biophysical parameters such as pH vac, measured under close-to-physiologic conditions. Since the methods we develop in the previous paper allow us to record fluorescence signals from spatially well-defined regions of the living parasite while they are under continuous perfusion, it is relatively straightforward for us to test how antimalarial drugs (e.g. chloroquine, CQ) and other compounds (e.g. the chemoreversal agent verapamil [VPL]) affect pH vac. In this paper, we measure both short term (i.e. initial perfusion conditions) and longer-term effects of CQ and VPL for living, intraerythrocytic CQS (HB3) and CQR (Dd2) malarial parasites under constant perfusion with physiologically relevant buffers. We find that VPL normalizes pH vac for Dd2 to a value near that measured for HB3, but has no effect on pH vac for HB3. Longer term CQ exposure is found to alter pH vac for HB3 but not Dd2, and short-term exposure to the drug has no significant effect in either strain. The results may help resolve longstanding debate regarding the effects of CQ and VPL on parasite physiology, and further support our evolving hypothesis for the mechanism of CQ resistance.

MDR3 P-glycoprotein, a Phosphatidylcholine Translocase, Transports Several Cytotoxic Drugs and Directly Interacts with Drugs as Judged by Interference with Nucleotide Trapping

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [alpha-(32)P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with one MDR3 null allele).

Total synthesis of ningalin B utilizing a heterocyclic azadiene Diels-Alder reaction and discovery of a new class of potent multidrug resistant (MDR) reversal agents.

A concise, efficient approach to the total synthesis of ningalin B (1) based on a heterocyclic azadiene Diels-Alder strategy (1,2,4,5-tetrazine-->1,2,-diazine-->pyrrole) ideally suited for construction of the densely functionalized pyrrole core found in the natural product is detailed. Examination of the natural product and a number of synthetic intermediates revealed that while lacking inherent cytotoxic activity, many reverse the multidrug-resistant (MDR) phenotype, resensitizing a human colon cancer cell line (HCT116/VM46) to vinblastine and doxorubicin at lower doses than the prototypical agent verapamil.

Effects of the multidrug-resistance-reversing agents verapamil and CL 347,099 on the efficacy of ivermectin or moxidectin against unselected and drug-selected strains of Haemonchus contortus in jirds (Meriones unguiculatus).

The development of anthelmintic resistance is making parasite control in small ruminants problematic. Following the discovery that the drug transporter P-glycoprotein may be involved in macrocyclic lactone resistance in Haemonchus contortus, we determined the effect of two multidrug-resistance modulators, verapamil and CL347,099, on the efficacy of ivermectin and moxidectin against unselected and drug-selected strains of H. contortus. CL347,099 is an analog of verapamil that has multidrug-resistance properties but weaker calcium-channel-blocking activity than the parent drug. The combinations of verapamil with either ivermectin or moxidectin significantly reduced worm counts of the selected strains as compared with the untreated controls, whereas ivermectin or moxidectin alone did not significantly reduce worm counts as compared with the untreated controls. The CL347,099 plus moxidectin combination was significantly more efficacious than moxidectin alone against the ivermectin-selected strain. The drug-combination regimes were without adverse effect on the jirds. However, higher levels of verapamil (> or =40 mg/kg) produced some toxicity.

Enhanced absorption of pour-on ivermectin formulation in rats by co-administration of the multidrug-resistant-reversing agent verapamil.

The effect of verapamil, a multidrug-resistance (Mdr)-reversing agent on the absorption of a pour-on formulation of ivermectin was evaluated in rats. Absorption of ivermectin was effectively enhanced (40%) by the presence of verapamil, suggesting that absorption of ivermectin involves Mdr-P-glycoprotein and that verapamil should act as a competitive inhibitor for the transport and extrusion of ivermectin by P-glycoprotein. This hypothesis is consistent with other studies describing verapamil as a blocking agent of P-glycoprotein involved in the efflux of ivermectin in a resistant strain of Haemonchus contortus.

A multidrug-resistant breast cancer cell line induced by weekly exposure to doxorubicin.

Attempts to study drug resistance in vitro have focused on continuous exposure of cell lines to cytotoxic agents that results in marked resistance. In an effort to examine drug resistance in an environment that is more typical of a clinical setting, monolayered human breast cancer cells (MCF-7) were exposed weekly for one hour to doxorubicin at a concentration that is achieved in vivo (0.1 microg/ml). After 20 weeks, the treated cell line (C20) was found to be resistant to doxorubicin by a factor of 2.0-2.5 at the 10 and 1% cell survival level compared to parent MCF-7 cells. Additionally, cross resistance to other chemotherapeutic agents including etoposide, vincristine, cisplatin, and mitomycin C was observed. Similar to other models of in vitro drug resistance, no modification of radiosensitivity was observed in C20 cells. The p170 glycoprotein was not overexpressed on C20 cells as assessed by the anti-p170 glycoprotein monoclonal antibody C219 staining nor was mRNA for the mdr-1 or MRP gene overexpressed. In addition, the mdr-blocking agent verapamil had no effect on the level of resistance encountered in tissue culture. C20 cells did not differ from the parent cell line with respect to cell cycle distribution, doubling time, GSH, GSH peroxidase, GSH reductase or GSH transferase levels. After a one-hour exposure to doxorubicin, lower intracellular doxorubicin levels were found in C20 cells compared to the parent line which provides at least a partial explanation for resistance, although the mechanism for this is unclear. Although the magnitude of resistance observed in the C20 cell line is low compared to other in vitro models of drug resistance, the modest level of drug resistance is probably sufficient to explain drug resistance seen clinically. The model of non-mdr mediated drug resistance presented may be a more relevant model for the evaluation of drug resistance in vitro.

Suppression of Lymphocyte Mitogenesis by Tamoxifen: Studies on Protein Kinase C, Calmodulin and Calcium

The effect of tamoxifen (TX; 1.0 μM) on the mitogenic response of rat lymphocytes was compared with the effect of drugs that are known to act on protein kinase C (PKC), calmodulin (CM), and calcium (Ca²⁺). The calcium ionophore A23187 (0.2 μM) was mitogenic on its own which was not influenced by TX. The agents modulating PKC or CM (phorbol-myristate-13-acetate; R24571, chlorpromazine) influenced mitogenesis differently than did TX. General inhibition of lymphocyte proliferation was seen with the Ca²⁺ antagonist agents (EGTA, TMB-8) as with TX. The antiproliferative effect of TX was partially reversed by the increase of Ca²⁺ in the culture medium when T cell mitogens were used, but not in the case of lipid A, a B lymphocyte mitogen. However, the concanavalin A-induced Ca²⁺ influx was further elevated by TX which differed from the effect of the Ca²⁺ channel-blocking agent verapamil. The results suggest that TX resets the threshold stimulus necessary for mitogenesis and is completely reversible.

Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics.

The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamilbodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

Biotransformation of Organic Sulfides. Part 11. Preparation of Functionalised Phenyl Propyl Sulfoxides Using Helminthosporium Species and Mortierella Isabellina

Para-substituted phenyl 3-chloropropyl and phenyl 3-hydroxypropyl sulfides have been oxidised to the corresponding (R)-sulfoxides by biotransformation with Mortierella isabellina ATCC 42613, and to the (S)-sulfoxides by biotransformation with Helminthosporium species NRRL 4671 or Acinetobacter calcoaceticus NCIMB 9871. The stereoselectivity of these oxidations was consistent with that predicted by examination of previously published models for sulfoxidation by these biocatalysts. In several cases ((R)- and (S)-p-cyanophenyl 3-chloropropyl sulfoxide, (S)-p-cyanophenyl 3-hydroxypropyl sulfoxide, and (R)-p-methoxyphenyl 3-chloropropyl sulfoxide) enantiopure materials were obtained by crystallisation of the biotransformation products. The latter are valuable chiral starting materials for the synthesis of analogues of the cardiovascular agent verapamil.

Total Syntheses of Ningalin A, Lamellarin O, Lukianol A, and Permethyl Storniamide A Utilizing Heterocyclic Azadiene Diels−Alder Reactions

Concise, efficient total syntheses of ningalin A (1), lamellarin O (2), lukianol A (3), and permethyl storniamide A (5) are detailed on the basis of a common heterocyclic azadiene Diels−Alder strategy (1,2,4,5-tetrazine → 1,2-diazine → pyrrole) ideally suited for construction of the densely functionalized pyrrole cores found in the three classes of marine natural products. Examination of the natural products and a number of synthetic intermediates revealed that some including lamellarin O (2) and lukianol A (3) exhibit modest cytotoxic activity against both wild-type and multidrug-resistant tumor cell lines. Fundamentally more important, a new class of agents including permethyl storniamide A (5) and its precursor 30, which lack inherent cytotoxic activity, are disclosed which reverse the multidrug-resistant (MDR) phenotype, resensitizing a human colon cancer cell line (HCT116/VM46) to vinblastine and doxorubicin at lower doses than the prototypical agent verapamil.

Secretion of sparfloxacin from the human intestinal Caco-2 cell line is altered by P-glycoprotein inhibitors.

The mechanism of intestinal secretion of the difluorinated quinolone sparfloxacin was investigated with the epithelial cell line Caco-2 and was compared to that of the P-glycoprotein (P-gp) substrate vinblastine. The P-gp inhibitors verapamil and progesterone significantly increased the epithelial cell accumulation of both vinblastine and sparfloxacin. This increase is likely to result from an inhibition of drug secretion since both vinblastine uptake and sparfloxacin uptake are known to proceed through a passive transmembrane diffusion. The unidirectional fluxes across cell monlayers grown on permeable filters indicated that a net secretion of sparfloxacin and vinblastine occurred across Caco-2 cells. These secretions were significantly inhibited by the MDR-reversing agent verapamil. We conclude that the P-gp is likely to be involved in the intestinal elimination of the difluorinated quinolone sparfloxacin.

Use of the comet test in the evaluation of multidrug resistance of human cell lines.

The comet test is a reported method for measuring DNA damage in individual mammalian cells. In the present report, the ability of this test to detect multidrug resistance (MDR) was evaluated. For this purpose, two human leukemia, well-characterized parental cell lines, HL60 and CEM, and their derived multidrug-resistant cells, HL60/DNR and CEM/VBL, were cultured with or without different anti-cancer agents. To evaluate the comet test, two DNA-damaging agents were used: daunorubicin (DNR), which is involved in MDR, and ambamustine (AMBA), which is independent from MDR. Moreover, in order to evaluate the specificity of the comet test, the activity of vinblastine (VBL), an MDR-related, DNA-independent anti-cancer drug, was also tested. Finally, the specificity of the comet test in detecting MDR was confirmed by culturing parental or resistant cells with DNR with or without the revertant agent verapamil (VER). Results confirm that the comet test is able to predict cellular chemoresistance when DNA damaging agents are tested. Finally, experiments on the role of the comet test in evaluating certain aspects of DNA repair are discussed.

Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids.

Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400+/-70 microm), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100+/-50 microm), which consist entirely of proliferating cells. After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity. At a depth 80 microm from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids. The P-glycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect. Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 microm distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence. This was corroborated by whole-cell patch-clamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids.

Development of an intrinsic P‐glycoprotein‐mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids

Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400 ± 70 μm), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100 ± 50 μm), which consist entirely of proliferating cells. After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity. At a depth 80 μm from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids. The P-glycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect. Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 μm distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence. This was corroborated by whole-cell patchclamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids. Int. J. Cancer 75:855–863, 1998. © 1998 Wiley-Liss, Inc.