Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Anthracyclines possessing either a 9-alkyl modification in the A-ring of the tetracyclic aglycone and/or specific changes to the amino sugar moiety retain effective cytotoxic activity against multidrug resistant (MDR) cell lines. To obtain a better understanding of the structural features responsible for this potentially valuable behaviour, we used the MTT tetrazolium dye reduction assay to calculate resistance factors (RF = the ratio of ID50 for the drug-resistant line to that for the parental line) for the EMT6/P mouse mammary tumour and its MDR variant EMT6/AR1.0, and the H69/P human small cell lung cancer line and its MDR counterpart H69/LX4. Both MDR lines exhibit marked resistance to doxorubicin, MDR 1 gene amplification, hyperexpression of the membrane P-glycoprotein and reduced drug accumulation. RF values for doxorubicin were 34 and 131 in the EMT6 and H69 cell line pairs, respectively. The 9-alkyl-substituted anthracyclines were confirmed as having RF values 9- to 15-fold lower than those for doxorubicin. The 9-ethyl analogues Ro 31-1966 (RF for EMT6 2.2, RF for H69 4.7) and Ro 31-1749 (RF for EMT6 3.9, RF for H69 9.5) were superior to the previously studied 9-methyl analogue Ro 31-1215 (RF for EMT6 8.1 RF for H69 12.4). A clear trend for RF values to decrease with increasing 9-alkyl chain length was also noted in the structurally more complex aclacinomycin series. For example, 13-methyl-aclacinomycin (RF for EMT6 1.0, RF for H69 2.2) featuring a 9-isopropyl moiety was superior to the 9-alkyl-containing aclacinomycin A (RF for EMT6 4.7, RF for H69 5.8), and this was in turn more effective than the 9-methyl analogue sulfurmycin A (RF for EMT6 6.4, RF for H69 14.2). The trisaccharide moiety was not an essential feature for activity against MDR lines in the aclacinomycins, as shown by the low RF value with aklavine (RF for EMT6 2.1, RF for H69 2.5). However, a small change in one of the sugar moieties of aclacinomycin A, as in marcellomycin, resulted in a considerable increase in RF values (RF for EMT6 18.5, RF for H69 25.3). The complex anthracyclines AD 32 (RF for EMT6 6.5, RF for H69 11.7) and particularly tetrahydropyranyl-doxorubicin (RF for EMT6 1.4, RF for H69 3.2) were effective against MDR lines.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Further examination of 9-alkyl- and sugar-modified anthracyclines in the circumvention of multidrug resistance. 133 31

We investigated the chemosensitisation of the parental EMT6 mouse mammary tumour cell line by low doses of cyclosporin A (CsA). This cell line has not previously been exposed to cytotoxic drugs but expresses low levels of P-glycoprotein. We produced greater than 2-fold sensitisation to doxorubicin, colchicine and vincristine using 0.084 microM (0.1 micrograms/ml) CsA. Cellular accumulation of doxorubicin and daunorubicin was also increased by this dose. In the MDR subline EMT6/AR1.0, much higher doses of CsA were required to effect optimal restoration of doxorubicin or daunorubicin accumulation. The effects of CsA on the parent line could not be increased by extended preincubation of cells with the sensitiser. These effects of CsA in the EMT6 parent cell line occur at a dose that is 1 order of magnitude lower than those previously reported to produce significant chemosensitisation.
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PMID:Chemosensitisation of a drug-sensitive parental cell line by low-dose cyclosporin A. 174 45

We have established a subline (EMT6/VRP) of the mouse tumour cell line EMT6/P with acquired resistance to the calcium transport blocker verapamil (VRP). The subline was 4-fold resistant to the cytoxicity of VRP alone compared with the parent line but of similar sensitivity to adriamycin, vincristine or colchicine. EMT6/VRP cells growing in 75 micrograms ml-1 VRP were morphologically different from and larger in diameter than EMT6/P cells, but these two parameters reverted almost to normal within 3 days of VRP removal, although resistance was retained. Expression of an mRNA coding for P-glycoprotein was similar in EMT6/VRP and the parent cell line, although considerable hyperexpression was seen in a multidrug resistant subline, EMT6/AR1.0. Cellular accumulation of both 3H-daunorubicin and 3H-VRP were greater in EMT6/VRP than in the parent line. Sensitisation to adriamycin by 3.3 micrograms ml-1 VRP was, however, somewhat reduced in EMT6/VRP (i.e. to 6.1-fold) compared with the 11-fold sensitisation seen in the parent line. It is clear that resistance to VRP seen in this cell line occurs via a different mechanism from the resistance to drugs such as adriamycin, vincristine and colchicine seen in multidrug resistant cell lines.
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PMID:Characterisation of a mouse tumour cell line with in vitro derived resistance to verapamil. 196 61

Deoxyspergualin, a synthetic analogue of the immunosuppressive anti-tumour antibiotic spergualin, has been shown to possess potent in vitro and in vivo anti-tumour activity and is currently in the National Cancer Institute (NCI) decision network. Deoxyspergualin shows similarities in properties and mechanisms of action to the natural-product immunosuppressive agents cyclosporin A and FK506, each of which can act as a modifier of multidrug resistance. We therefore decided to examine the comparative activity of deoxyspergualin in parent and multidrug-resistant cells. Deoxyspergualin contains the polyamine spermidine within its structure. Bovine serum copper amine oxidase catalyses the oxidative deamination of spermidine to produce an aminoaldehyde, ammonia and hydrogen peroxide. These aminoaldehydes are believed to be responsible for the toxicity of polyamines in vitro in the presence of bovine serum. For this reason, all experiments were carried out in medium containing bovine serum and in medium containing horse serum (which is low in copper amine oxidase content). We used the tetrazolium (MTT) colorimetric assay to determine drug sensitivity and tritiated daunorubicin accumulation together with inhibition of azidopine binding to study specific mechanisms of resistance modulation. The murine cell lines EMT6/P and EMT6/AR1.0 and the human cell lines H69/P and H69/LX4 were, respectively, 32-, 32-, 372- and 483-fold more sensitive to spermidine and 175-, 133-, 321- and 444-fold more sensitive to spermine in the presence of calf serum than in the presence of horse serum. However, these large differential effects were not seen for deoxyspergualin. It appears that in the presence of horse serum, deoxyspergualin may exert its effect by a mechanism other than polyamine oxidation. Deoxyspergualin did not enhance the accumulation of [3H]-daunorubicin in EMT6/AR1.0 cells. Furthermore, deoxyspergualin (1-20 microM) did not restore the sensitivity of EMT6/AR1.0 or H69/LX4 cells to that of the parent lines. P-glycoprotein (Pgp) in membranes prepared from H69/LX4 cells was photo-affinity-labeled with [3H]-azidopine. Deoxyspergualin did not inhibit this labeling. Although deoxyspergualin appears to exert its immunosuppressive effect via a mechanism similar to that of cyclosporin A and FK506, it does not share their ability to modify Pgp-mediated multidrug resistance. However, its lack of cross-resistance and potent in vivo anti-tumour activity make deoxyspergualin a promising candidate for development as an anti-cancer agent.
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PMID:The activity of deoxyspergualin in multidrug-resistant cells. 755 42

In order to address the association of enhanced drug efflux with the multidrug-resistant (MDR) phenotype, we have studied the cellular pharmacokinetics of anthracyclines in the P-glycoprotein (Pgp)-positive MDR cell lines H69/LX4 (human small cell lung cancer) and EMT6/AR1.0 (mouse mammary tumour). Both doxorubicin (DOX) and daunorubicin (DNR) were accumulated to a lesser extent and effluxed at a higher rate by MDR cells than by their drug-sensitive counterparts. In contrast, the 9-alkyl substituted compound, aclacinomycin A (ACL), was accumulated and effluxed from parent and MDR cells at an identical rate. In experiments designed to examine energy-dependent efflux, DOX and DNR were shown to be efficiently effluxed against the concentration gradient in the presence of glucose. However, in the same experiments the analogues ACL and Ro 31-3294 (9-alkyl and morpholinyl substituted), which have previously been shown to retain activity against MDR cell lines, were accumulated and effluxed at identical rates in parent and MDR EMT6 cells. Hence, 9-alkyl and morpholinyl substituted compounds appear to behave less favourably as substrates for energy-driven drug efflux by Pgp-positive MDR cells than do DOX or DNR. Resistance modifiers verapamil and cyclosporin A appeared to abolish energy-dependent efflux for DOX and DNR in both the EMT6 and H69 MDR lines whereas they had no effect on the cellular efflux of ACL. The altered cellular pharmacology in MDR cell lines may provide a rational basis for the use of modified anthracycline analogues (e.g. 9-alkyl and morpholinyl (substituted) and resistance of modifying agent in the treatment of tumours expressing a Pgp-mediated phenotype.
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PMID:The efflux of anthracyclines in multidrug-resistant cell lines. 790 89

Rhodamine 123 (Rh123) is a fluorescent dye which locates in the mitochondria of cells. It is a substrate for P-glycoprotein (Pgp) and can, therefore, be used as a molecular probe in studies of the multidrug resistance (MDR) phenotype. However, not all MDR cells overexpress Pgp. In some, the MDR phenotype is associated with expression of an alternative transporter molecule, the multidrug resistance-associated protein (MRP). We have studied the accumulation and efflux of Rh123 in MDR cells having both Pgp-mediated and MRP-associated phenotypes. In the mouse tumour parental cell line, EMT6/P, Rh123 accumulates rapidly to reach plateau levels by 90 min. Confocal microscopy confirms a localisation to the mitochondria. In the MDR subline, EMT6/AR1.0, which overexpresses Pgp and which is 10-fold resistant to Rh123 cytotoxicity, accumulation is dramatically reduced. Efflux of Rh123 from both resistant and parental lines is rapid but can be inhibited by reduced temperature or by the presence of cyclosporin A (5 micrograms/ml). Efflux from the parental line is probably due to the presence of very low, but detectable, levels of Pgp but the existence of other mechanisms cannot be ruled out. In contrast, the human lung cancer parental cell line COR-L23/P, and its MRP-associated (but Pgp-negative) MDR subline, COR-L23/R (which is 23-fold resistant to Rh123 cytotoxicity), accumulate Rh123 at similar rates for the first 30 min. The curves then diverge so that, at 180 min, the resistant cells contain only 70% of the Rh123 of parental cells. Confocal microscopy demonstrates a similar distribution of fluorescence in resistant and parental cells. Essentially no efflux of Rh123 occurs from parental cells, whereas 70% of the content is lost from resistant cells over a period of 150 min. Such efflux may again be inhibited by reduced temperature but cyclosporin A (5 micrograms/ml) has little effect. These observations should be borne in mind when interpreting Rh123 efflux data in terms of MDR mechanisms.
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PMID:A comparison of rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes. 799 26

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.
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PMID:Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines. 809 7

The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.
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PMID:In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576. 1121 78

Intratumoral injection of controlled-release microsphere formulations of anticancer compounds has the potential to selectively increase tumour exposure to drugs. This work aimed to evaluate the therapeutic effect and toxicity of microsphere formulations containing the anticancer drug, doxorubicin, in a murine tumour model. The effect of co-administration of verapamil, a P-glycoprotein modulator or chemosensitizer, was investigated. Initial in-vitro studies confirmed the ability of verapamil to enhance the accumulation of both doxorubicin and [(99mTc)]sestamibi, also a P-glycoprotein substrate, in EMT6 murine breast sarcoma cells and a doxorubicin-selected multidrug-resistant variant, EMT6/AR1.0. Ex-vivo studies using confocal microscopy demonstrated release of doxorubicin from microspheres and diffusion of the drug through tissue. For in-vivo studies, EMT6 and EMT6/AR1.0 cells were grown in BALB/c mice. Following intratumoral injection of doxorubicin-loaded microspheres, alone or in combination with verapamil-loaded microspheres, the tumour diameter was measured serially as an indication of therapeutic effect, while the weight, appearance, and behaviour of the mice were monitored as an indication of general toxicity. Intratumoral injections of doxorubicin-loaded microspheres were tolerated much better than systemic administration of equivalent drug concentrations. There was a modest (up to 34%) delay of tumour growth compared with groups receiving no treatment or blank microspheres. Co-injection of verapamil microspheres with doxorubicin microspheres produced a moderate increase in toxicity but no further delay in tumour growth. Controlled-release microsphere formulations of anticancer agents administered intratumorally were an efficient way to deliver high drug doses to the tumour with little systemic toxicity.
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PMID:Delivery of an anticancer drug and a chemosensitizer to murine breast sarcoma by intratumoral injection of sulfopropyl dextran microspheres. 1295 95

The objectives of this study were to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines and to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using an ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line, MDA435/LCC6/MDR1, and a mouse cell line, EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLNs resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared with free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane-associated Pgp when delivered as Dox-PLNs, and in this form, the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticle formulations.
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PMID:A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system. 1654 67


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