Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the possible involvement of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and/or other glutathione S-conjugate export pump (GS-X pump) family members on the active efflux of irinotecan [(7-ethyl-10-[4-(1-piperidino)-1-pipertidino)-1-piperidino]carb onylox y camptothecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C2, C-A500, and KCP-4, which overexpress P-gp, MRP, and the unidentified GS-X pump, respectively. The carboxylate form of SN-38 exhibited significant ATP-dependent transport, with a Michaelis constant of 17 microM, into membrane vesicles from C-A500 but not from other cell lines. Among these KB-derived cells, significant ATP-dependent uptake of the carboxylate form of CPT-11 was only observed in KB-C2 vesicles. In addition, the uptake of the lactone and carboxylate forms of SN38-Glu into membrane vesicles from C-A500 and KB-C2, but not KCP-4, was ATP dependent, although the transport activity in C-A500 was much higher than that in KB-C2. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay revealed that the resistance of KB-C2 to CPT-11 and SN-38, compared with that of KB-3-1, was 6.3- and 6.8-fold, respectively; the corresponding figures for C-A500 were 12- and 27-fold, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectively. These results suggest that MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addition, a difference in substrate specificity among GS-X pump members was demonstrated.
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PMID:Active efflux of CPT-11 and its metabolites in human KB-derived cell lines. 991 83

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.
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PMID:Identification of the cyclosporin-binding site in P-glycoprotein. 992 80

Expression of P-glycoprotein (Pgp), the drug efflux pump which mediates multidrug resistance (MDR), has been widely reported in chronic lymphocytic leukaemia (CLL) and improved accumulation of daunorubicin has been reported using the MDR reversing agent cyclosporin A (CSA). We have investigated the effects on cell kill of the addition of CSA and its analogue PSC 833 to daunorubicin, doxorubicin, idarubicin, mitozantrone and fludarabine in samples from 51 patients with CLL using an MTT [3(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Pgp expression was assessed by immunocytochemistry using the JSB-1 monoclonal antibody. Of the 51 samples, 10 (20%) were Pgp positive and all of these samples were from treated patients. With the exception of mitozantrone, the addition of CSA and PSC 833 to cytotoxic agents failed to significantly improve cytotoxicity, even in the Pgp positive group. With mitozantrone significant responses were seen in both Pgp positive and negative groups suggesting that the responses were due to direct cytotoxicity of the cytotoxic-modifier combination rather than reversal of MDR. Both CSA and PSC 833 showed significant direct cytotoxicity (P = 0.004 and 0.04 for PSC 833 at 1000 ng/ml and 500 ng/ml respectively; P < 0.001 for both concentrations of CSA). The responses were disappointing compared to the highly significant improvements in cytotoxicity seen using cells from the Pgp positive CEM VLB 100 acute myeloid leukaemia cell line, and it was not possible to demonstrate the superiority of PSC 833 over CSA which is also seen in cell lines. Our data do not support a role for Pgp modifiers in CLL. Further studies using larger numbers of Pgp positive CLL cells and higher doses of PSC 833 would be useful.
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PMID:Effect on cell kill of addition of multidrug resistance modifiers cyclosporin A and PSC 833 to cytotoxic agents in chronic lymphocytic leukaemia. 993 32

Recently, it has been reported that continuous treatment with cyclosporin A or tacrolimus induces encephalopathy in transplant patients. The mechanism of immunosuppressant-induced encephalopathy is unclear. We investigated the cytotoxicity to brain capillary endothelial cells and the effect of these two drugs on P-glycoprotein function using mouse brain capillary endothelial (MBEC4) cells. The transcellular transport of [3H]sucrose was significantly increased and the cellular viability, based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion test, was decreased by cyclosporin A (approximately 50% at 5 microM; P<0.005), while tacrolimus showed a much smaller effect. These findings indicate that the toxicity of cyclosporin A was greater than that of tacrolimus. The uptake of [3H]vincristine, a substrate of P-glycoprotein, was increased by these two drugs. The expression of P-glycoprotein in MBEC4 cells was reduced, but there was no effect on mdr1b mRNA levels. The decrease in the expression of P-glycoprotein may be due to the inhibition of the turnover of P-glycoprotein, which involves translation. In conclusion, the direct cytotoxic effect on the brain capillary endothelial cells and the inhibition of P-glycoprotein may be partly involved in the occurrence of immunosuppressant-induced encephalopathy.
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PMID:Effect of cyclosporin A or tacrolimus on the function of blood-brain barrier cells. 1039 24

L-Canavanine (L-CAV) is a naturally occurring L-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 microM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 microM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.
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PMID:L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines. 1039 78

The resistance to doxorubicin (DOX) by some tumor cells is mainly due to the effect of P-glycoprotein encoded by the multidrug resistance-1 (mdr1) gene. We tried to prove the correlations between P-glycoprotein expression and the sensitivity for anticancer drugs including DOX and other cytotoxic drugs that are currently used for gastrointestinal cancer patients. We quantified the P-glycoprotein expression by flow cytometry techniques, and the sensitivity for anticancer drugs using a tetrazolium salt, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), assay in highly purified fresh human tumor cells obtained from 25 cancer patients. The inhibition rates were the lowest in DOX and mitomycin C (MMC), compared with other drugs. The most significant correlation between DOX and MMC was seen in the inhibition rates. A significant correlation was also seen between the inhibition rates for DOX and P-glycoprotein expression, whereas only a slight correlation between the sensitivity for MMC and P-glycoprotein expression was observed. We should therefore pay close attention to the effect of P-glycoprotein when treating cancer patients, especially if both the inhibition rates of DOX and MMC are low based on the findings of an MTT assay.
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PMID:P-glycoprotein-expressing tumor cells are resistant to anticancer drugs in human gastrointestinal cancer. 1045 34

A resistant descendant of the human stomach carcinoma cell line EPG85-257 was selected in the presence of increasing concentrations of daunorubicin (DRC). To avoid the expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), cells were cultured in the presence of verapamil. The resulting cells were used to evaluate an induced carbonyl reduction as a new determinant in DRC resistance. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) toxicity assay was performed to estimate sensitivity to DRC in both cell lines. IC50 values of DRC increased almost 8-fold in the resistant descendants compared to the parental cell line. P-gp transcripts were detectable in both cell lines at only very low levels, and no significant alterations between sensitive and resistant cells were observed. MRP mRNA expression was markedly higher compared to P-gp mRNA, but, as was the case with P-gp, MRP mRNA levels in sensitive and resistant cells showed no alteration. This was probably due to the effect of the presence of verapamil during cell selection. Another known drug resistance factor, the lung resistance-related protein (LRP), was not at all detectable. Interestingly, resistant cells possessed 6-fold higher levels of DRC carbonyl-reducing activity, leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL). The 6-fold higher DRCOL formation roughly parallels the 8-fold increase in DRC IC50 values during cell selection, and therefore may account for DRC resistance in these cells. The determination of specific carbonyl reducing enzymes, known to be involved in DRC detoxification, revealed that mRNA expression of carbonyl reductase (EC 1.1.1.184), aldose reductase (EC 1.1.1.21), and dihydrodiol dehydrogenase 2 (EC 1.3.1.20) increased in the resistant descendant. In contrast, the phase II-conjugating enzyme activities of glutathione S-transferases were significantly lower in resistant than in sensitive cells, whereas those of glucuronosyl transferase were not detectable in either cell line. Apparently, conjugating enzymes are not involved in DRC resistance in human stomach carcinoma cells. These studies indicate that DRC resistance in human stomach carcinoma cells may appear as a result of an induction of metabolic DRC inactivation via carbonyl reduction to the less active 13-hydroxy metabolite DRCOL.
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PMID:Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction. 1060 58

A new series of potential flavonoidic modulators of P-glycoprotein activity has been prepared. The flavanolignan silybin was first oxidised to dehydrosilybin and then C-alkylated with either prenyl or geranyl bromide. The resulting isoprenoid dehydrosilybins were shown to display high in vitro affinities for direct binding to P-glycoprotein, which ranged them among the best flavonoids ever tested.
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PMID:The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of P-glycoprotein. 1067 1

The title compound (6), its structure being imaginatively created, has been prepared through coupling of alizarine blue (2), a classical dye, and 3,4-di-O-acetyl-2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl bromide (3). Compound 6 has considerably higher and different antitumor activity from that of doxorubicin or its analogue (10), and, further, has properties to reverse multidrug resistance (by P-glycoprotein), to inhibit topoisomerase II, and to induce apoptosis.
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PMID:Preparation of 5-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyloxy)-6-hydroxynap htho[2,3- f]quinoline-7,12-dione (FT-Alz), a new-type, potentially antitumor substance with various biological activities. 1069 36

The interaction of the novel anticancer drug KRN5500, a spicamycin derivative, with human P-glycoprotein (P-gp) was analyzed from the viewpoint of cellular pharmacokinetics, i.e. by means of [3H]azidopine photoaffinity labeling, cellular accumulation and transcellular transport experiments. In this study, P-gp-overexpressing LLC-GA5-COL150 cells, porcine kidney epithelial LLC-PK1 cells transformed with human MDR1 cDNA, were used, since this cell line constructs monolayers with tight junctions, and would provide sufficient information for analyzing the cellular pharmacokinetics. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the growth-inhibitory effect of KRN5500 in LLC-GA5-COL150 cells was comparable to that in LLC-PK1 cells (IC50 = 79.4 and 72.7 nM, respectively), but the inhibition of [3H]azidopine binding by KRN5500 was concentration-dependent in the membrane fraction of LLC-GA5-COL150 cells. The cellular accumulation of [14C]KRN5500 after its basal application in LLC-GA5-COL150 cells was slightly lower than that in LLC-PK1 cells, and was restored by the multidrug resistance (MDR) modulator SDZ PSC 833. The basal-to-apical transport of [14C]KRN5500 in LLC-GA5-COL150 cells was also slightly higher than that in LLC-PK1 cells, and was inhibited by SDZ PSC 833. However, the basal-to-apical transport of [14C]KRN5500 in LLC-GA5-COL150 cells was only a little higher than the apical-to-basal transport. Consequently, these results demonstrated that KRN5500 interacted with, but was hardly transported via, P-gp. These observations suggested that KRN5500 may be useful even for the treatment of tumors exhibiting P-gp-mediated MDR.
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PMID:The novel anticancer drug KRN5500 interacts with, but is hardly transported by, human P-glycoprotein. 1076 13


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