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Verapamil regulates activity and mRNA-expression of human β-glucuronidase in HepG2 cells

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Abstract

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as β-glucuronidase (β-gluc). Regulation of β-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of β-gluc activity by the calcium channel blocker verapamil.

To further explore this phenomenon, we investigated the effect of verapamil on β-gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2.

Treatment of HepG2 cells with verapamil revealed down-regulation of β-gluc activity, protein, and mRNA level down to 50% of the control with EC50 values of 25 μM. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human β-glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by β-glucuronidase.

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Abbreviations

β-gluc:

β-Glucuronidase

MEM:

Minimal essential medium

DMEM:

Dublecco’s modified Eagle’s medium

SD:

Standard deviation

RT-PCR:

Real-time PCR

AP2:

Activating protein 2

nt:

Nucleotide

NFκB:

Nuclear factor κB

SP1:

Specificity protein 1

HMR 1826:

N-[4-β-glucuronyl-3-nitrobenzyloxycarbonyl]doxorubicin

bp:

Base pair

M6G:

Morphine-6-glucuronide

MUG:

4-Methylumbelliferyl-β-D-glucuronide

MU:

4-Methylumbelliferone

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

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Acknowledgement

This study was supported by the grant Kr 945 7-1 from the Deutsche Forschungsgemeinschaft.

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Authors and Affiliations

  1. Department of Pharmacology, Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Friedrich Loefflerstrasse 23d, 17487, Greifswald, Germany

    M. Grube, C. Kunert-Keil, B. Sperker & H. K. Kroemer

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  1. M. Grube
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  2. C. Kunert-Keil
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  3. B. Sperker
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  4. H. K. Kroemer
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Correspondence to H. K. Kroemer.

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Grube, M., Kunert-Keil, C., Sperker, B. et al. Verapamil regulates activity and mRNA-expression of human β-glucuronidase in HepG2 cells. Naunyn-Schmiedeberg's Arch Pharmacol 368, 463–469 (2003). https://doi.org/10.1007/s00210-003-0837-x

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  • DOI: https://doi.org/10.1007/s00210-003-0837-x

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