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)

The cephalosporins are a family of semisynthetic antibiotics, some of which have structural features associated with substrates for the multidrug transporter, P-glycoprotein. The activity of a series of six cephalosporins in reversing multidrug resistance (MDR) was examined in MDR variants (Dx5 cells) of the human sarcoma line MES-SA. Dx5 cells express high levels of the mdr1 gene product P-glycoprotein and are 25- to 30-fold resistant to doxorubicin (DOX), etoposide (VP-16), and vinblastine (VBL). Cytotoxicity was measured by the MTT assay. Cefoperazone (1.0 mM) was the most effective modulator of MDR, lowering the IC50 for VP-16 by 29-fold (29x), for VBL by 16x, and for DOX by 14x. Ceftriaxone at 1.0 mM produced 10x modulation of VP-16 cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of resistance was concentration dependent, decreasing to 4x and 5x, respectively, for DOX with 0.25 mM cefoperazone and ceftriaxone. No modulation of cytotoxicity was observed in the parental MES-SA cells, which do not express mdr1. Cefazolin, cefotetan, cephradine, and ceftazidime were ineffective, producing less than 5x modulation of DOX at 1.0 mM. Among these cephalosporins, cefoperazone and ceftriaxone were the most highly protein bound in the media (30 and 52%), and the most lipid soluble, with octanol/water partitioning coefficients of -0.49 and -0.60. Varying the serum concentration in medium from 5 to 50% had less than a two-fold effect on the modulation of MDR by ceftriaxone. The ability to reverse MDR among these agents is associated with lipid solubility, high protein binding, a polycyclic planar geometry, and the presence of the piperazine group in cefoperazone. These data and the potential for achieving high tissue concentrations indicate that cefoperazone merits further study as a modulator of MDR.
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PMID:Reversal by cefoperazone of resistance to etoposide, doxorubicin, and vinblastine in multidrug resistant human sarcoma cells. 258 32

The cyanomorpholino derivative of doxorubicin (MRA-CN) is an anthracycline that is extremely potent and non-cross-resistant with doxorubicin (DOX) in multidrug-resistant cells. MRA-CN binds to and cross-links DNA and thus has been proposed to act as a targeted alkylating agent. In our study, the number of DNA interstrand and DNA-protein cross-links produced by MRA-CN was identical in multidrug-resistant Dx5 and parental MES-SA cells, as shown by alkaline elution analysis. The amount of cross-linking was directly proportional to drug concentration at concentrations from 10(-11) to 10(-7) M MRA-CN. Extensive DNA cross-linking was evident within 30 minutes of drug exposure. After 1 hour of drug exposure, the number of DNA cross-links increased for 90 minutes, reached a plateau, and then began to decrease after 120 minutes. Loss of cell viability was also observed as early as 3 hours after exposure to MRA-CN. The finding of the same number of DNA cross-links in MES-SA and Dx5 cells indicates that similar amounts of MRA-CN are likely to enter the nuclei of multidrug-resistant and sensitive cells. Other anthracyclines have major differences in nuclear distribution in sensitive and resistant cells. Several factors may contribute to the non-cross-resistance of MRA-CN in multidrug-resistant cells. (a) The lipophilicity of MRA-CN facilitates cell entry. (b) The substitution and loss of basicity at the amino nitrogen may reduce the affinity of the drug for the P-glycoprotein efflux pump, compared with that of DOX. (c) The detoxification function of P-glycoprotein may be less effective for drugs that produce rapid and irreversible cell damage, such as the DNA-targeted alkylation caused by MRA-CN.
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PMID:DNA cross-linking and cytotoxicity of the alkylating cyanomorpholino derivative of doxorubicin in multidrug-resistant cells. 317 56

An increasing body of evidence appears to implicate the lipid bilayer of multidrug resistant (MDR) cells with P-glycoprotein activity. Several cationic amphiphilic drugs (CADs) have been extensively described as modulators of MDR. These same agents are also known to (1) inhibit lysosomal acid sphingomyelinase (ASmase), a phospholipid degrading enzyme, and/or (2) induce phospholipidosis in animal tissues or cultured cell lines. In this report, we randomly selected 17 CADs and evaluated their potency in modulating MDR in the murine MDR P388/ADR leukemia cell line. We compared these results with their ability to inhibit ASmase and observed a significant dose-dependent linear relationship (95% central confidence interval), between ASmase inhibition and MDR reversal. This approach permitted us to identify three new modestly potent chemosensitizers: trimipramine, desipramine, and mianserine. Modulation of MDR was not cell line specific, since CADs at 10 microM increased doxorubicin (DOX) and vinblastine (VBL) (but not methotrexate, MTX) cytotoxicity in both P388/ADR and the human MDR cell lines MES-SA/Dx5 and K562/R7, but not in the parental drug-sensitive cells. Although all chemosensitizing CADs at 10 microM significantly increased Rhodamine-123 (Rho-123) accumulation in the human leukemia MDR cell line K562/R7 and most presented significant displacement of the photoaffinity labelling probe iodoarylazidoprazosin, no correlation between these observations and the ability of CADs to sensitize MDR cells to DOX and VBL was found. In conclusion, our study strongly suggests that the chemosensitizing potency of agents such as CADs may be due to a dual mechanism of action: direct antagonism of P-gp activity and indirect modulation of P-gp activity through the disruption of cellular lipid metabolism.
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PMID:Inhibition of lysosomal acid sphingomyelinase by agents which reverse multidrug resistance. 771 13

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.
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PMID:Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection. 791 96

The multidrug resistance (MDR) gene family has been shown to be highly expressed in several normal tissues including the canalicular membrane of the hepatocyte. We report that a cholestatic estrogen metabolite, 17 beta-estradiol glucuronide (E217G), is a substrate for the MDR transporter, P-glycoprotein. In cytotoxicity studies, the MDR sarcoma cell line Dx5 was 4.7-fold resistant to E217G, and the K562/R7 leukemia MDR cell line was 5.0-fold resistant to E217G relative to their parental cell lines. There was also a 2- to 3-fold accumulation defect of [3H]E217G in the MDR cells relative to their parental cell lines. E217G (100 microM) modulated resistance ot doxorubicin, taxol, vinblastine, and etoposide in the Dx5 cells, completely reversing the 30- to 60-fold resistance observed with these agents. E217G had no effect on the toxicity of these compounds in the parental cell line (MES-SA). In contrast, MDR cells were not resistant to the noncholestatic estrogen metabolite, estriol 3-glucuronide, and this metabolite did not modulate resistance to MDR substrates. ATP-dependent transport of [3H]E217G in rat canalicular membranes was inhibited by several MDR substrates including vinblastine, etoposide, verapamil, cyclosporine, and PSC-833.
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PMID:17 beta-estradiol glucuronide: an inducer of cholestasis and a physiological substrate for the multidrug resistance transporter. 810 46

We have previously described the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells. Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross-resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells. There were also differences in spermine and putrescine uptake rates between parental and MDR-positive with the MDR-positive cells having a lower Vmax and a higher Km. The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA. The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25-to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX). Cytotoxicity was measured by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Poly-SPM (50 microM) lowered the drug concentration IC50 values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC50 values without Poly-SPM. This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25 microM Poly-SPM was used. No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene. Poly-SPM had no influence on the IC50 of non-MDR chemotherapeutic agents such as cisplatin. The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of [3H]-VBL and [3H]-TAX in the Dx5 but not MES-SA cells. Pretreatment of the Dx5 cell with alpha-difluoromethylornithine (DFMO at 2 and 5 microM) for 24 h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells. DFMO pretreatment is known to upregulate the polyamine transporter(s). These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump. This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance.
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PMID:Reversal of doxorubicin, etoposide, vinblastine, and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine. 861 15

To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.
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PMID:Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells. 892 30

A variant of the multidrug-resistant human sarcoma cell line Dx5 was derived by co-selection with doxorubicin and the cyclosporin D analogue PSC 833, a potent inhibitor of the multidrug transporter P-glycoprotein. The variant DxP cells manifest an altered phenotype compared with Dx5, with decreased cross-resistance to Vinca alkaloids and no resistance to dactinomycin. Resistance to doxorubicin and paclitaxel is retained. The multidrug resistance phenotype of DxP cells is not modulated by 2 microM PSC 833 or cyclosporine. DxP cells manifest a decreased ability to transport [3H]cyclosporine. DNA heteroduplex analysis and sequencing reveal a mutant mdr1 gene (deletion of a phenylalanine at amino acid residue 335) in the DxP cell line. The mutant P-glycoprotein has a decreased affinity for PSC 833 and vinblastine and a decreased ability to transport rhodamine 123. Transfection of the mutant mdr1 gene into drug-sensitive MES-SA sarcoma cells confers resistance to both doxorubicin and PSC 833. Our study demonstrates that survival of cells exposed to doxorubicin and PSC 833 in a multistep selection occurred as a result of a P-glycoprotein mutation in transmembrane region 6. These data suggest that Phe335 is an important binding site on P-glycoprotein for substrates such as dactinomycin and vinblastine and for inhibitors such as cyclosporine and PSC 833.
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PMID:Multidrug-resistant human sarcoma cells with a mutant P-glycoprotein, altered phenotype, and resistance to cyclosporins. 903 18

The doxorubicin-selected, P-glycoprotein (P-gp)-expressing human sarcoma cell line MES-Dx5 showed the following levels of resistance relative to the non-P-gp-expressing parental MES-SA cells in a 72 h exposure to cytotoxic drugs: etoposide twofold, doxorubicin ninefold, vinblastine tenfold, taxotere 19-fold and taxol 94-fold. GF120918 potently reversed resistance completely for all drugs. The EC50s of GF120918 to reverse resistance of MES-Dx5 cells were: etoposide 7+/-2 nM, vinblastine 19+/-3 nM, doxorubicin 21+/-6 nM, taxotere 57+/-14 nM and taxol 91+/-23 nM. MES-Dx5 cells exhibited an accumulation deficit relative to the parental MES-SA cells of 35% for [3H]-vinblastine, 20% for [3H]-taxol and [14C]-doxorubicin. The EC50 of GF120918, to reverse the accumulation deficit in MES-Dx5 cells, ranged from 37 to 64 nM for all three radiolabelled cytotoxics. [3H]-vinblastine bound saturably to membranes from MES-Dx5 cells with a KD of 7.8+/-1.4 nM and a Bmax of 5.2+/-1.6 pmol mg(-1) protein. Binding of [3H]-vinblastine to P-gp in MES-Dx5 membranes was inhibited by GF120918 (K = 5+/-1 nM), verapamil (Ki = 660+/-350 nM) and doxorubicin (Ki = 6940+/-2100 nM). Taxol, an allosteric inhibitor of [3H]-vinblastine binding to P-gp, could only displace 40% of [3H]-vinblastine (Ki = 400+/-140 nM). The novel acridonecarboxamide derivative GF120918 potently overcomes P-gp-mediated multidrug resistance in the human sarcoma cell line MES-Dx5. Detailed analysis revealed that five times higher GF120918 concentrations were needed to reverse drug resistance to taxol in the cytotoxicity assay compared to doxorubicin, vinblastine and etoposide. An explanation for this phenomenon had not been found.
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PMID:The acridonecarboxamide GF120918 potently reverses P-glycoprotein-mediated resistance in human sarcoma MES-Dx5 cells. 1057 49

In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [(3)H]cyclosporine and [(3)H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [(3)H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [(3)H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.
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PMID:Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant P-glycoprotein with deleted phe(335). 1072 24


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