Vol. 59, Issue 2, 177-182, February 2001

Regulation of Human -Glucuronidase by A23187 and Thapsigargin in the Hepatoma Cell Line HepG2

Bernhard Sperker, Céline Tomkiewicz, Oliver Burk, Robert Barouki, and Heyo K. Kroemer

Institut für Pharmakologie, Ernst Moritz Arndt Universität Greifswald, Greifswald, Germany (B.S., H.K.K.); Institut National de la Santé et de la Recherche Médicale U490, Université René Descartes, Paris, France (C.T., R.B.); and Dr. Margarete Fischer-Bosch Institut für Klinische Pharmakolgie, Stuttgart, Germany (O.B.)

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 -glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in -glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human -glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of -glucuronidase activity. Therefore, we investigated regulation of -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--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 -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 -glucuronidase transcriptional rate. Our data demonstrate regulation of human -glucuronidase by xenobiotics. Down-regulation of -glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that -glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.