Involvement of AP-2 binding sites in regulation of human beta-glucuronidase.

The lysosomal hydrolase beta-glucuronidase (beta-gluc) can be used for the bioactivation of non-toxic glucuronide prodrugs of anticancer agents. The enzyme is present at high levels in many tumours and hence may lead to an enhanced drug targeting by tumour-selective release of the active anticancer drug. Individual expression and regulation of this enzyme is one factor modulating the bioactivation of glucuronide prodrugs. Nevertheless, in contrast to murine beta-gluc, which is inducible by androgens, the human enzyme has been regarded as an unregulated housekeeping gene due to a lacking TATA box and high G+C contents within the putative promotor sequence. Despite these facts, we were able to demonstrate downregulation of human beta-gluc expression by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. However, cis-acting elements responsible for this regulation have not yet been identified. We therefore characterised the 5'-untranslated region of the human beta-gluc gene using transient transfection assays with promotor-luciferase constructs in HepG2 cells and cloned fragments between 3,770 bp and 107 bp. A23187 reduced the beta-gluc promotor activity. This effect disappeared using fragments smaller than 356 bp. Using site-directed in vitro mutagenesis and gel-electrophoretic-mobility shift assays, we found evidence of an involvement of transcription factor activating protein-2 (AP-2) binding sites on the regulation of human beta-glucuronidase by A23187. Our studies provide a basis for the understanding of the transcriptional regulation of the human beta-glucuronidase gene and could be useful for the optimisation of glucuronide prodrug therapy.

Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells.

Anthracyclines such as doxorubicin remain among the most effective agents for the treatment of solid tumors and hematological malignancies. To overcome dose-limiting side effects like cardiotoxicity, an intensive effort has been undertaken to develop promising doxorubicin prodrugs that are specifically activated at the tumor site. One approach is the application of peptide prodrugs of doxorubicin. The enzyme cathepsin B catalyzes the activation of these prodrugs, and hence, the regulation of cathepsin B by antitumor agents could influence the efficacy of peptide prodrugs using this protease. In the present investigation, the effects of doxorubicin on cathepsin B expression in the human cervix carcinoma cell line HeLa were examined. Exposure to doxorubicin induced a time- and dose-dependent up-regulation of cathepsin B expression on mRNA, protein, and activity levels. In the cathepsin B gene promoter region, a potential nuclear factor kappaB (NF-kappaB) binding site could be identified. Pretreatment of HeLa cells with specific NF-kappaB inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein IkappaB could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicin-induced cathepsin B expression. Finally, binding of NF-kappaB subunits p50 and p65 to the NF-kappaB binding site in the cathepsin B gene promoter region could be demonstrated by electrophoretic mobility shift assay. In summary, our data clearly indicate that doxorubicin induces cathepsin B expression and activity via NF-kappaB. These findings contribute to a better understanding of tumor targeting with peptide prodrugs and help to define a possible mechanism of doxorubicin toxicity in tumor cells.

Expression of multidrug resistance proteins in rat and human chronic pancreatitis.

BACKGROUND AND AIMS: The expression of the ABC-transporters MDR-1, MRP1, and MRP-2 was investigated in healthy pancreas and in chronic pancreatitis tissue samples in rats and humans to evaluate their possible involvement in a multidrug resistance of the pancreas with consequences for the pharmacologic treatment of pancreatic diseases. METHODS: Human pancreatic tissue samples of healthy tissue and chronic pancreatitis were collected during pancreas surgery. In rats, the time-course of the expression of transporter proteins was studied 14, 28, and 56 days after experimental induction of chronic pancreatitis. The expression of MDR-1, MRP-1, MRP-2, and furthermore, LRP and PAP was investigated by RT-PCR, Real Time TaqManPCR, and immunohistochemistry. RESULTS: In rat pancreas, MDR-1 (P-gp) and MRP-1 but in human pancreas MDR-1 (P-gp), MRP-1 and MRP-2 were found to be expressed. Chronic pancreatitis lead to an increased transcription of mRNA of MDR-1 (rat and human) and much lower, MRP-2 (human). CONCLUSIONS: The expression of P-gp and related transporters could have impact on the metabolism, distribution, and availability of various compounds, including drugs, in the pancreas. The results indicate that this could be more pronounced in chronic pancreatitis.

Busulfan induces activin A expression in vitro and in vivo: a possible link to venous occlusive disease.

PURPOSE: Hepatic venous occlusive disease is a severe side effect after administration of busulfan before hematopoietic stem cell transplantation. The syndrome is characterized by liver enlargement, fluid retention, jaundice, and weight gain. Endothelial injury has been described as the precipitating factor. The link between busulfan administration and endothelial damage has not been established thus far. METHODS: Complementary deoxyribonucleic acid expression arrays were used to screen for busulfan responsive genes in ECV304 cells. Specific messenger ribonucleic acid and protein levels were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Serum samples of 15 pediatric patients with leukemia were analyzed for busulfan and cytokine levels. RESULTS: We identified a member of the transforming growth factor beta superfamily, activin A, to be induced in the human cell line ECV304 after exposure to busulfan in a time- and concentration-dependent manner. Maximum effects were observed at 120 and 168 hours for activin A messenger ribonucleic acid and protein, respectively. Preincubation with the protein kinase C inhibitor bisindolylmaleimide I (10 nmol/L) abolished activin A induction by busulfan (P <.05). Activin receptors were detected in ECV304. Both tissue factor and cyclooxygenase 2 were significantly induced by busulfan (P <.05). In a parallel in vivo study a significant increase in serum activin A concentration was found 4.5 hours after the second dose of busulfan. CONCLUSION: The data demonstrate that busulfan induces activin A both in vitro and in vivo. In view of the multiple targets of activin A (inflammation, proliferation, apoptosis, and coagulation), these findings may be of relevance to our understanding of venous occlusive disease.

Functional significance of a hereditary adenine insertion variant in the 5'-UTR of the endothelin-1 gene.

Endothelin-1 (ET-1) is known as a potent vasoconstrictor peptide and stimulator of cell proliferation. The human preproendothelin-1 mRNA contains a frequent adenine insertion polymorphism (allele frequency = 0.28) within the 5'-untranslated region (5'-UTR), 138 bp downstream of the transcription start site, which was assumed to be related to hypertension. This 5'-UTR variant could putatively influence the mRNA secondary structure and stability, efficacy of translation initiation, or binding of sequence-specific mRNA-binding proteins. By cloning the entire ET-1 gene 5'-UTR in a pGL3 vector and transfection of two cell lines, we studied the effects on luciferase expression in vitro. Luciferase activity was significantly increased in the insertion variant (I) compared to the wild-type (D) variant for both COS1 (2.97 +/- 0.12 versus 2.17 +/- 0.10; P = 0.002) and HepG2 cells (5.42 +/- 0.90 versus 3.68 +/- 0.37; P = 0.002). Investigations performed ex vivo using human umbilical vein endothelial cells were performed to examine the influence of genotypes on the formation of mRNA and protein. Preproendothelin-1-mRNA was quantified in relation to GAPDH by a realtime polymerase chain reaction. Homozygous I-carriers showed significant elevated mRNA levels compared to I/D and I/I-carriers (I/I 9.03 +/- 1.86, I/D 2.07 +/- 1.15, D/D 2.33 +/- 0.99; P = 0.001). ET-1 protein expression, determined by enzyme-linked immunosorbent assay, was increased among I-carriers (I/I 814 +/- 144, I/D 528 +/- 103, D/D 556 +/- 75 pg/ml; P = 0.001). The observed effects may be due to an enhanced mRNA stability because the half-life of mRNA consisting of the I-variant was prolonged (35.4 +/- 7.9 versus 19.9 +/- 4.5 min). We were able to show that the +138 I/D polymorphism is of functional importance for ET-1 expression, and this may have consequences for vessel tonus regulation.

The effects of the human MDR1 genotype on the expression of duodenal P-glycoprotein and disposition of the probe drug talinolol.

BACKGROUND AND OBJECTIVES: A single-nucleotide polymorphism (SNP) of the human multidrug-resistance gene in wobble position of exon 26 reportedly predicts expression and function of P-glycoprotein in human enterocytes and lymphocytes. Several other allelic variants of MDR1 have been identified, some of which lead to amino acid exchange with as yet unknown functional relevance. METHODS: In healthy white volunteers, we investigated the influence of the hereditary variants C3435T in exon 26 and G2677T/A (Ala893Ser/Thr) in exon 21 and the influence of 7 frequent or putative functional SNPs on duodenal MDR1 messenger ribonucleic acid (n = 32) and immunoreactive P-glycoprotein (n = 37) expression. Moreover, the disposition of the probe drug talinolol was evaluated in 55 subjects after oral administration (100 mg) and in 23 subjects after intravenous administration(30 mg). RESULTS: Duodenal MDR1 messenger ribonucleic acid and P-glycoprotein, as assessed by real-time polymerase chain reaction (TaqMan) and immunostaining, were not influenced by any MDR1 polymorphism studied. Talinolol disposition was not affected by the exon 26 mutation C3435T. In carriers of the TT/TA variants of G2677T/A, the area under the serum concentration-time curve values of oral talinolol were slightly but significantly elevated compared with those in carriers of at least 1 wild-type allele (P <.05, Kruskal-Wallis test; P =.014, Mann-Whitney U test). However, multiple comparisons with combinations of putative functional SNPs did not confirm a significant influence of the MDR1 genotype on talinolol disposition. CONCLUSIONS: We did not identify any influence of MDR1 genotypes on duodenal expression of P-glycoprotein and disposition of talinolol in humans.

Overexpression of glutathione S-transferase A1-1 in ECV 304 cells protects against busulfan mediated G2-arrest and induces tissue factor expression.

1. The antineoplastic drug busulfan is frequently used in preconditioning regimens for bone marrow transplantation. Pharmacokinetics vary tremendously between patients due to extensive metabolism in the liver via conjugation to glutathione catalysed by glutathione S-transferase (GST) A1-1. Since elevated busulfan plasma levels have been reported to be a risk factor for developing veno-occlusive disease (VOD), metabolism of busulfan may play a pivotal role in the induction of VOD. 2. Therefore, we developed a cell model to investigate the influence of busulfan metabolism on its biological effects. GSTA1-1 cDNA was transfected into the cell line ECV 304 and protein expression was demonstrated by Western blotting. Enzymatic activity could be detected by formation of tetrahydrothiophene. Additionally, effects of busulfan treatment on cell cycle and expression of tissue factor have been investigated. 3. A busulfan-induced G2-arrest was reduced in GSTA1-1-transfected cells, which consequently displayed a significantly higher activity of cdc2 kinase (24.1+/-1.5 AU mg(-1) protein) after busulfan treatment compared to controls (14.7+/-2.3 AU mg(-1) protein; P<0.01). Elevated basal expression of tissue factor in GSTA1-1-transfected ECV 304 cells could be 4 fold increased by busulfan treatment. 4. These data demonstrate that ECV 304 cells transfected with GSTA1-1 provide a valuable tool to assess busulfan metabolism in vitro. Furthermore, overexpression of GSTA1-1 leads to a partial protection against cell cycle effects of busulfan and affects tissue factor expression.

Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy.

ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g., in the gut or the blood-brain barrier) or contribute to drug excretion. Expression of transport proteins in the heart could be an important factor modifying cardiac concentrations of drugs known to be transported by P-gp (e.g., beta-blockers, cardiac glycosides, doxorubicin). We therefore investigated the expression and localization of P-gp in human heart. Samples from 15 human hearts (left ventricle; five non-failing, five dilated cardiomyopathy, and five ischemic cardiomyopathy) were analyzed for expression of P-gp using real-time RT-PCR, immunohistochemistry, and in situ hybridization. Immunohistochemistry revealed expression of P-gp in endothelium of both arterioles and capillaries of all heart samples. Although P-gp mRNA was detected in all samples, its expression level was significantly reduced in patients with dilated cardiomyopathy. We describe variable expression of P-gp in human heart and its localization in the endothelial wall. Thus, intracardiac concentrations of various compounds may be modified, depending on the individual P-gp level.

Effect of levothyroxine administration on intestinal P-glycoprotein expression: consequences for drug disposition.

OBJECTIVE: Thyroid function alters the pharmacokinetics of many drugs; one example is the cardiac glycoside digoxin. Because digoxin disposition is affected by intestinal expression of P-glycoprotein, we hypothesized that thyroid hormones may regulate P-glycoprotein and influence disposition of P-glycoprotein substrates. METHODS: Duodenal expression of P-glycoprotein measured by reverse transcriptase-polymerase chain reaction of MDR1 messenger ribonucleic acid (mRNA) and by immunohistochemical examination was studied in 8 healthy volunteers (4 men and 4 women; age range, 22-29 years; body weight, 59-89 kg) before and after coadministration with levothyroxine (200 microg orally for 17 days), which resulted in suppression of thyroid-stimulating hormone. The pharmacokinetics of the P-glycoprotein substrate talinolol was assessed after intravenous (30 mg) and oral (100 mg) administration. RESULTS: Duodenal MDR1 mRNA expression and immunoreactive P-glycoprotein were increased 1.4-fold (not significant; P =.078) and 3.8-fold (P <.01), respectively, after administration of levothyroxine. The changes in P-glycoprotein expression were associated with minor alterations in talinolol half-life after both oral and intravenous administration. CONCLUSIONS: Expression of intestinal P-glycoprotein in humans appears to be influenced by thyroid hormones. The functional consequences need to be addressed in patients with hyperthyroidism.

Expression of active human beta-glucuronidase in Sf9 cells infected with recombinant baculovirus.

Antibody directed enzyme prodrug therapy (ADEPT) using glucuronide prodrugs is an experimental approach to reduce systemic toxicity of anti-cancer agents. Bioactivation of such prodrugs is achieved by fusion proteins consisting of targeting moieties (e.g. ligands of tumor specific antigens) and human beta-glucuronidase. In order to test a large panel of possible beta-glucuronidase fusion proteins for their applicability in ADEPT, an easy, rapid and high-yield expression system like the baculovirus/insect cell expression system would be needed. A prerequisite for using such fusion proteins is functional and biochemical characterization of human beta-glucuronidase expressed in baculovirus-infected insect cells. Therefore, recombinant human beta-glucuronidase was expressed in Sf9 insect cells and characterized at the protein and functional level. As shown by Western blot analysis the recombinant enzyme consists of dimers with their monomers being linked via disulfide bonds. Posttranslational modifications of the monomers seem to be different as compared with mammalian cells or tissues. The enzyme is functionally active in cleaving the substrates 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, 4-methylumbelliferyl-beta-D-glucuronide and the glucuronide prodrug HMR 1826, respectively, with similar enzyme kinetic parameters as those found in human tissues. Our data demonstrate that beta-glucuronidase expressed in Sf9 cells displays the same enzymatic features as the protein expressed in mammalian cells. Therefore, we suggest that beta-glucuronidase fusion proteins produced in this cell line will be valuable tools for testing a large panel of various targeting moieties in human tumor xenograft models or may be used for ADEPT in man.

Verapamil decreases glucuronidase activity in the gut.

The present investigation addressed the role of verapamil for oral pharmacokinetics of morphine-6-beta-glucuronide (M6G). Male Sprague-Dawley rats received 62.5 mg kg(-1) M6G-dihydrate orally w/wo pre-treatment with 70 mg kg(-1) verapamil. Intravenous M6G (3.9 mg kg(-1) ) and oral morphine (52.7 mg kg(-1) morphine-hydrochloride) were also employed. Oral bioavailability of M6G and the fraction of M6G deglucuronidated to morphine were estimated from areas under the plasma-concentration vs. time curves (AUC) of morphine and its glucuronides. As initial results pointed towards inhibition of glucuronidases by verapamil, its capability to specifically inhibit E. coli and/or rat intestinal beta-glucuronidase was assessed using altered cleavage of the model substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG). Oral bioavailability of M6G was 2.1%; 13% of oral M6G was deglucuronidated to morphine. Co-administration of verapamil did not increase the AUC of M6G. AUCs of morphine and morphine-3-glucuronide were smaller in the verapamil group than in controls. Verapamil co-administration decreased the fraction of M6G deglucuronidated to morphine to 4.6%. In vitro experiments provided evidence that verapamil inhibits beta-glucuronidase from E. coli with an IC(50) of 30 microM, whereas no inhibition of the rat beta-glucuronidase from small intestine was seen. In conclusion, verapamil decreased intestinal deglucuronidation of M6G by inhibiting E. coli beta-glucuronidase. This indicates that verapamil is not suited as P-gp inhibitor in experiments involving glucuronides. An increase in the intestinal absorption of M6G due to P-gp-inhibition was not observed at the verapamil dose studied.

Dose optimization of a doxorubicin prodrug (HMR 1826) in isolated perfused human lungs: low tumor pH promotes prodrug activation by beta-glucuronidase.

HMR 1826 (N-[4-beta-Glucuronyl-3-nitrobenzyl-oxycarbonyl]doxorubicin) is a nontoxic glucuronide prodrug from which active doxorubicin is released by beta-glucuronidase. Preclinical studies aimed at dose optimization of HMR 1826, based on intratumoral pharmacokinetics, are important to design clinical studies. Using an isolated perfused human lung model, the uptake of doxorubicin into normal tissue and tumors after perfusion with 133 microg/ml (n = 6), 400 microg/ml (n = 10), and 1200 microg/ml (n = 6) HMR 1826 was compared. Extracellular tissue pH was measured, and enzyme kinetic studies were performed in vitro to investigate the effect of pH on the formation of doxorubicin. Extracellular pH was lower in tumors than in healthy tissue (6.46 +/- 0.35, n = 8 versus 7.30 +/- 0.33, n = 10; p < 0.001). In vitro, beta-glucuronidase activity was 10 times higher at pH 6.0 than at neutral pH. After perfusion with HMR 1826, there was a linear relationship between HMR 1826 concentrations in perfusate and normal lung tissue. After perfusion with 133, 400, and 1200 microg/ml HMR 1826, the final doxorubicin concentrations in normal and tumor tissue were 2.7 +/- 0.9, 11.1 +/- 5.4, and 21.8 +/- 8.4 microg/g (p < 0.05 for all comparisons), and 0.7 +/- 0.3, 8.6 +/- 2.0 microg/g (p < 0.01 versus 133 microg/g), and 8.7 +/- 4.9 microg/g, respectively. This agrees with the enzyme kinetic observations of saturation of beta-glucuronidase at 400 microg/ml HMR 1826 in the acidic environment of the tumor. Therefore, the escalation of the HMR 1826 dose most likely results in higher circulating concentrations than 400 microg/ml but does not increase the uptake of doxorubicin into tumors and, subsequently, antitumor efficacy. The isolated perfused human lung is an excellent model for preclinical investigations aimed at optimization of tissue pharmacokinetics of tumor-selective prodrugs.