Abstract
A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human β-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in β-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human β-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of β-glucuronidase activity. Therefore, we investigated regulation of β-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-β-d-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of β-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased β-glucuronidase transcriptional rate. Our data demonstrate regulation of human β-glucuronidase by xenobiotics. Down-regulation of β-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that β-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.
Footnotes
- Received May 15, 2000.
- Accepted September 28, 2000.
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Send reprint requests to: Dr. Bernhard Sperker, Institut für Pharmakologie, Ernst Moritz Arndt Universität Greifswald, Friedrich Loeffler Str. 23d, D-17487 Greifswald, Germany. E-mail: sperker{at}uni-greifswald.de
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This work was supported by Grant No. Kr 945/4–3 from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by the Robert Bosch-Stiftung (Stuttgart, Germany). Parts of the work have been presented at the XIIIth International Congress of Pharmacology 1998 (Munich, Germany) [Naunyn-Schmiedeberg's Arch Pharmacol 358 (2 Suppl.):R534 (1998)] and at the 8th Annual Meeting of the German Society for Clinical Pharmacology and Therapeutics 1998, Greifswald, Germany [Eur J Clin Pharmacol 54:A24 (1998)].
- The American Society for Pharmacology and Experimental Therapeutics
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