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)

H69AR is a multidrug-resistant small cell lung cancer cell line derived from a drug-sensitive cell line, H69, by selection in doxorubicin. It is cross-resistant to a wide variety of natural product-type antineoplastic agents but does not overexpress P-glycoprotein. In the present study, the levels of GSH and GSH-related enzymes in the H69AR cell line were determined and compared with those found in H69 cells. Unlike other drug-resistant cell lines, GSH levels were diminished 6-fold in H69AR cells (0.67 +/- 0.28 microgram/mg of protein), compared with H69 cells (4.23 +/- 1.17 micrograms/mg of protein) (p less than 0.01). This unusually low level of GSH may explain the pronounced collateral sensitivity of H69AR cells to buthionine sulfoximine (BSO), an inhibitor of the rate-limiting enzyme in GSH biosynthesis (ID50 of 4.4 microM BSO for H69AR cells versus ID50 of 300 microM BSO for H69 cells). BSO did not enhance doxorubicin cytotoxicity in the H69AR cell line, despite further depletion of GSH. GSH-reductase (EC 1.6.4.2) activity was elevated 2-fold in H69AR cells, compared with sensitive H69 cells (75.34 +/- 14.94 versus 38.62 +/- 5.06 nmol of NADPH/min/mg of protein) (p less than 0.05). Both selenium-dependent and -independent GSH-peroxidase (EC 1.11.1.9) activities were unchanged in the resistant H69AR cell line, compared with its parent cell line. gamma-Glutamyl transpeptidase (EC 2.3.2.2) activity was 5-fold elevated in H69AR cells, compared with H69 cells (2.50 +/- 0.44 versus 0.46 +/- 0.21 nmol of p-nitroaniline/min/mg of protein) (p less than 0.01), whereas GSH-S-transferase (EC 2.5.1.18) activity was 10-fold higher (201.98 +/- 43.62 versus 19.77 +/- 1.72 nmol of 1-chloro-2,4-dinitrobenzene/min/mg of protein in H69AR and H69 cells, respectively) (p less than 0.01). The GSH-S-transferases from both cell lines were purified by affinity chromatography and immunoblot analysis identified the GSH-S-transferases as belonging to the anionic pi class. GSH-S-transferases from the mu or alpha classes were not detectable in either cell line. In conclusion, marked differences in GSH levels and the activities of three of four GSH-related enzymes were observed between the multidrug-resistant H69AR cell line and its parent cell line. Further study is required to determine whether these changes are causally related to the development of drug resistance in this model system.
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PMID:Alterations in glutathione and glutathione-related enzymes in a multidrug-resistant small cell lung cancer cell line. 196 21

Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable synthetic tripeptide with an aldehyde at its C terminus specifically inhibits the activity of cysteine proteases. Since the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells is blocked by ALLN and ALLN has a cytotoxic effect on cells, we attempted to isolate ALLN-resistant cells that overproduce an ALLN-sensitive protease(s). However, we obtained an ALLN-resistant cell line that overproduced P-glycoprotein (Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1992) J. Biol. Chem. 267, 5731-5734). To circumvent the multidrug resistance (MDR) phenotype during selection, we have stepwise selected an ALLN-resistant cell line of CHO cells in the presence of verapamil, a competitive inhibitor of P-glycoprotein. These non-MDR ALLN-resistant cells overexpress a 35-kDa protein and have increased aldo-keto reductase activity. Partial amino acid sequences of the 35-kDa protein are highly homologous to members of the aldo-keto reductase superfamily. The aldo-keto reductases are NADPH-dependent oxidoreductases and catalyze reduction of a wide range of carbonyl compounds such as aldehydes, sugars, and ketones. Our findings support the concept that a physiological function for aldo-keto reductases may be detoxification.
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PMID:Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase. 844 54

The human colon carcinoma cell line, Caco-2, is widely used as a model for oral absorption of xenobiotics. The usefulness of Caco-2 cells has been limited, however, because they do not express appreciable quantities of CYP3A4, the principle cytochrome P450 present in human small bowel epithelial cells. We report that treatment of Caco-2 cells with 1 alpha,25-dihydroxyvitamin D3, beginning at confluence, results in a dose- and duration-dependent increase in CYP3A4 mRNA and protein, with little apparent effect on the expression of CYP3A5 or CYP3A7. This treatment also results in increases in NADPH cytochrome P450 reductase and P-glycoprotein (the MDR1 gene product) but has no detectable effect on expression of CYP1A1, CYP2D6, cytochrome b5, liver or intestinal fatty acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of serum. In the treated cells, the intrinsic formation clearance of 1'-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A enzymes) was estimated to be somewhat lower than that of human jejunal mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The 1'-OH-midazolam/4-OH-midazolam product ratio produced by the cells (approximately 5.3) is comparable to, but somewhat lower than, that observed in human jejunal microsomes (7.4-15.4), which may reflect the presence of CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D3 is less efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These observations, together with the time course of response, suggest that the vitamin D receptor may be involved in CYP3A4 regulation. The culture model we describe should prove useful in defining the role of CYP3A4 in limiting the oral bioavailability of many xenobiotics.
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PMID:Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha,25-dihydroxyvitamin D3. 914 12

In 10 human cancer cell lines, the activity of mitomycin C (MMC) was found to be determined by an interplay between activation by DT-diaphorase (DTD) and inactivation by glutathione S-transferase (GST). NADPH/cytochrome P-450 reductase was not responsible for MMC activation and expression of MDRI (Mr 170,000 P-glycoprotein), and MRP (multidrug resistance-associated protein) genes did not relate to MMC resistance. Gene expression analysis for NQO1 (DTD gene) and GSTpi predicted which enzyme activity predominated in a cell line, except K562 and K562/DOX. For tumors with DTD activity only, MMC given by itself was most active. In cell lines in which DTD action was predominant, tumor selectivity was achieved by enhancing DTD-mediated activation with m-iodobenzylguanidine and hyperglycemia, which reduced the intra-tumoral pH. KW2149, a novel MMC analogue activated by glutathione, was most active against tumors in which GSTpi predominated. These various enzyme-specific effects could be observed even in cell lines derived from tumors with multidrug resistance. Such MMC treatment based on cell enzymology may enhance significantly MMC efficacy, helping to overcome multidrug resistance.
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PMID:Molecular targeting of mitomycin C chemotherapy. 925 6

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.
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PMID:A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi. 947 99

Using Caco-2 cell monolayers expressing CYP3A4, we investigated the interplay between metabolism and transport on the first-pass intestinal extraction of the immunosuppressant sirolimus, a CYP3A4/P-glycoprotein (P-gp) substrate. Modified Caco-2 cells metabolized [(14)C]sirolimus to the predicted amounts of CYP3A4-mediated products based on CYP3A4 content, which was approximately 20% of that measured in human small intestinal mucosal homogenate. [(14)C]Sirolimus also degraded to the known ring-opened product, seco-rapamycin. Unexpectedly, a ring-opened dihydro metabolite (M2) was the major product detected in cells at all sirolimus concentrations examined (2-100 microM). Greater M2 formation after apical versus basolateral dosing (1.6-fold) was explained by higher intracellular content of sirolimus after apical dosing. M2 was not detected in incubations with human liver and intestinal microsomes but was readily detected with corresponding homogenates. M2 formation was NADPH-dependent but unaffected by the CYP3A4 inhibitors ketoconazole and troleandomycin. Although M2 was formed from purified seco-rapamycin (20 microM) in the homogenates, it was not detected in cells when seco-rapamycin was added to the apical compartment, because seco-rapamycin was essentially impermeable to the apical membrane. Sirolimus, seco-rapamycin (basolaterally dosed), and M2 were all actively secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 [(2R)-anti-5-[3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy]quinoline trihydrochloride]. Along with CYP3A4-mediated metabolism and P-gp-mediated secretion, we conclude that the following novel pathway, which occurs at least in the intestine, may contribute significantly to the first-pass extraction of sirolimus in humans: intracellular degradation of sirolimus to seco-rapamycin, metabolism of seco-rapamycin to M2 by an unidentified non-microsomal enzyme, and P-gp-mediated secretion of M2 and seco-rapamycin.
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PMID:Identification of a novel route of extraction of sirolimus in human small intestine: roles of metabolism and secretion. 1190 72

(R)-N-[4-[2-[[2-Hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]- 4-[4-(4-trifluoro-methylphenyl)thiazol-2-yl]benzenesulfonamide (1) is a potent and selective agonist of the human beta3-adrenergic receptor. We report herein the data from studies of the metabolism and excretion of 1 in rats. Five metabolites were identified in the bile of male Sprague-Dawley rats administered 3H-labeled 1 by either oral gavage (10 mg/kg) or intravenous injection (3 mg/kg). These included a pyridine N-oxide derivative (M2), a primary amine resulting from N-dealkylation and loss of the pyridinyl-2-hydroxyethyl group (M4), a carboxylic acid derived from N-dealkylation and loss of the pyridyl-2-hydroxyethyl amine (M5), and the corresponding taurine and isethionic acid conjugates (M1 and M3). Metabolites M1 and M3 also were identified in rats treated with M5 and were generated in incubations of M5 with rat liver subcellular fractions in the presence of ATP and coenzyme A with supplementary taurine or isethionic acid. These results suggest that M5 is the precursor of M1 and M3 and that the formation of these conjugated metabolites follows similar mechanisms of amino acid conjugation. On the other hand, M2, M4, and M5 were produced from 1 in an NADPH-dependent manner in incubations with liver microsomes from rats, dogs, monkeys, and humans. In human liver preparations, these routes of biotransformation were shown to be catalyzed by cytochrome P450 3A4. In a bidirectional transport assay, transport of 1 across a monolayer of cells expressing P-glycoprotein (Pgp) was observed to be similar to that of vinblastine, which is an established substrate of the transporter protein. This finding, together with the observation that the parent compound was excreted in the feces of bile duct-cannulated animals following intravenous dosing, suggests that 1 is subject to Pgp-mediated excretion from intestine of rats.
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PMID:Metabolism of a thiazole benzenesulfonamide derivative, a potent and elective agonist of the human beta3-adrenergic receptor, in rats: identification of a novel isethionic acid conjugate. 1206 36

Juvenile and adult female flounder (Platichthys flesus (L.)) were caught either in the estuary of the most polluted European river, the Elbe, or as controls in a reference site to study pollution-induced xenobiotic resistance in their livers in relation to pathological alterations. In juvenile fish, livers displayed reversible and irreversible degenerative toxipathic lesion types but never showed (pre)neoplastic changes. Tumour frequencies up to 70% were found macroscopically in livers of adult female flounder which had progressed to adenomas and carcinomas in the most polluted site. Because male adult flounder show only up to 50% of livers containing early preneoplastic foci but never malignancies, we focussed our study on female individuals. (Pre)neoplastic changes ranged from early eosinophilic foci to basophilic foci, adenomas and hepatocellular carcinomas. Adenomas were generally eosinophilic whereas carcinomas were mainly basophilic. These phenotypical sequential changes strongly resemble those found in chemically-induced liver carcinogenesis in mammals. Characteristic mutations known from mammalian cancers have not been found so far in these flounder livers. Therefore, we investigated whether epigenetic events had induced a metabolic "resistant phenotype" of (pre)malignant cancer cells during hepatocellular carcinogenesis. With a quantitative immunohistochemical approach, we studied expression of P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR), cytochrome P4501A1, glutathione-S-transferase-A which are key proteins in xenobiotic metabolism and elimination. Glucose-6-phosphate dehydrogenase (G6PDH) activity, the major source of the reducing power NADPH which is needed for biotransformation, oxyradical scavenging and biosynthesis, was detected as well. We observed upregulation of G6PDH activity already in early preneoplastic eosinophilic foci and subsequent further upregulation in basophilic foci and carcinomas. P-gp started to become overexpressed in basophilic foci and was overexpressed even more strongly in carcinomas and their invasively-growing protrusions (satellites). In carcinomas, P-gp protein was predominantly present in membranes of lysosomes which are the intracellular sites of deposition of xenobiotics. CYP450 was reduced whereas GST-A was increased in these carcinomas. Progression towards malignancy was positively correlated with levels of mitogenic organochlorines in these livers which are "fingerprint contaminants" of the river Elbe. We conclude that (pre)neoplastic hepatocytes in female flounder acquire growth advantages over normal hepatocytes by epigenetic metabolic adaptations during liver carcinogenesis as a result of chronic exposure to (pro)carcinogens in the polluted habitat.
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PMID:Clonal xenobiotic resistance during pollution-induced toxic injury and hepatocellular carcinogenesis in liver of female flounder (Platichthys flesus (L.)). 1514 37

In contrast with the Parkinson's-like effects associated with the mitochondrial neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroleptic agent haloperidol, there exist no reports on adverse central nervous system (CNS) effects with the structurally related N-substituted-4-arylpiperidin-4-ol derivative and antidiarrheal agent loperamide. Although this difference can be attributed to loperamide's P-glycoprotein substrate properties that prevent it from accessing the brain, an alternative possibility is that loperamide metabolism in humans is different from that of MPTP and haloperidol and does not involve bioactivation to a neurotoxic pyridinium species. In the current study, loperamide bioactivation was examined with particular focus on identification of pyridinium metabolites. A NADPH-dependent disappearance of loperamide was observed in both rat and human liver microsomes (human t(1/2) = 13 min; rat t(1/2) = 22 min). Loperamide metabolism was similar in human and rat and involved N-dealkylation to N-desmethylloperamide (M3) as the principal metabolic fate. Other routes of loperamide biotransformation included N- and C-hydroxylation to the loperamide-N-oxide (M4) and carbinolamide (M2) metabolites, respectively. Furthermore, the formation of an additional metabolite (M5) was also discernible in human and rat liver microsomes. The structure of M5 was assigned to the pyridinium species (LPP(+)) based on comparison of the liquid chromatography/tandem mass spectrometry characteristics to the pyridinium obtained from loperamide via a chemical reaction. Loperamide metabolism in human microsomes was sensitive to ketoconazole and bupropion treatment, suggesting P4503A4 and -2B6 involvement. Recombinant P4503A4 catalyzed all of the loperamide biotransformation pathways in human liver microsomes, whereas P4502B6 was only responsible for N-dealkylation and N-oxidation routes. The wide safety margin of loperamide (compared with MPTP and haloperidol) despite metabolism to a potentially neurotoxic pyridinium species likely stems from a combination of factors that include a therapeutic regimen normally restricted to a few days and the fact that loperamide and perhaps LPP(+) are P-glycoprotein substrates and are denied entry into the CNS. The differences in safety profile of haloperidol and loperamide despite a common bioactivation event supports the notion that not all compounds undergoing bioactivation in vitro will necessarily elicit a toxicological response in vivo.
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PMID:Identification of an N-methyl-4-phenylpyridinium-like metabolite of the antidiarrheal agent loperamide in human liver microsomes: underlying reason(s) for the lack of neurotoxicity despite the bioactivation event. 1531 35

Sirolimus is a recently marketed immunosuppressant that, in common with cyclosporine and tacrolimus, exhibits a low average oral bioavailability (approximately 20%). Likewise, sirolimus is a substrate for the major drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and the efflux transporter P-glycoprotein (P-gp), both of which are expressed in close proximity in epithelial cells lining the small intestine. Using CYP3A4-expressing Caco-2 cell monolayers, we examined the interplay between metabolism and transport on the intestinal first-pass extraction of sirolimus. Modified Caco-2 cells metabolized [14C]sirolimus to the same CYP3A4-mediated metabolites as human small intestinal and liver microsomes. [14C]Sirolimus also degraded to the known ring-opened product, seco-sirolimus. A ring-opened dihydro species (M2) was, surprisingly, the major product detected in cells at all sirolimus concentrations examined (2-100 micromol/L) and in incubations with human liver and intestinal homogenates but not in corresponding microsomes. M2 formation was NADPH-dependent but unaffected by prototypical CYP3A4 inhibitors. Although M2 was formed from purified seco-sirolimus (20 micromol/L) in the homogenates, it was not detected in cells when seco-sirolimus was added to the apical compartment because seco-sirolimus was essentially impermeable to the apical membrane. Sirolimus, seco-sirolimus (basolaterally dosed), and M2 were all secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 (zosuquidar trihydrochloride). Along with CYP3A4-mediated metabolism and P-gp-mediated efflux, a novel elimination pathway was identified that may also contribute to the first-pass extraction, and hence low oral bioavailability, of sirolimus. This new insight into the intestinal elimination of sirolimus, which was not identified using traditional drug metabolism/transport screening methods, may represent another source for the limited absorption of sirolimus.
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PMID:New insights into drug absorption: studies with sirolimus. 1538 26


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