Elsevier

Biochemical Pharmacology

Volume 67, Issue 6, 15 March 2004, Pages 1057-1064
Biochemical Pharmacology

Tamoxifen cytotoxicity in hepatoblastoma cells stably transfected with human CYP3A4

https://doi.org/10.1016/j.bcp.2003.10.027Get rights and content

Abstract

Tamoxifen can exert its effects through the competitive inhibition of estrogen receptors or other mechanisms. HepG2 cells lacking estrogen receptors and engineered to overexpress CYP3A4, the most important CYP to metabolize the drug, appear to be a good model to study the effects of tamoxifen metabolites. Tamoxifen altered cell cycle of transduced HepG2 cells, decreased G0/G1 cell numbers, diminished proliferation index and induced cell death mostly in cells overexpressing CYP3A4 but was without significant effect on cytotoxicity or proliferation of cells engineered to overexpress CYP2E1 or on empty vector transfected cells. Tamoxifen did not change MDR1 levels irrespectively on CYP450s expression, but inhibited by approximately 50% p-gp functions in all cell types. Drug treatment significantly increased dehydroepiandrosterone sulfotransferase activity and sulfotransferase inhibition significantly decreased tamoxifen cytotoxicity. Our results support the view that metabolic activation of tamoxifen in liver cells may proceed via CYP450-mediated metabolism and subsequent sulfotransferase-mediated activation and point to the role of CYP3A4 and dehydroepiandrosterone sulfotransferase in adverse tamoxifen effects.

Introduction

Anti-estrogens used mostly as anti-breast cancer drugs, can be classified into two major groups: triphenylethylene-like analogs of tamoxifen (Tam) with mixed estrogenic/anti-estrogenic properties or pure anti-estrogens [1]. Tamoxifen was approved in 1985 as an adjuvant in chemotherapy of postmenopausal women with node-positive breast cancer and is currently most widely prescribed for breast cancer therapy. Tam can exert its effects through competitive inhibition of estrogen receptors (ER) or non-receptor mechanisms. Detection of human uterine carcinomas or rat liver carcinogenesis related to Tam use stimulated research to elucidate mechanisms of drug action [2], [3]. It is known, that the drug undergoes metabolic conversion to Tam-N-oxide by the hepatic flavin-containing monooxygenase and into N-desmethyl- and 4-OH-Tam by cytochrome P450 [2], [4]. Although Tam metabolism is complex and yet not understood, hydroxylted derivatives were mostly considered as detoxication products while other compounds like arene oxides, their precursors or intermediates were supposed to be involved in drug-induced liver carcinogenesis due to their ability to covalently bind DNA and protein [4], [5]. Recent studies indicate however, that hydroxylated Tam metabolites, particularly the α-OH-Tam, may be transformed by sulfotransferases to sulfuric acid ester metabolites and form DNA adducts [6], [7], [8]. Thus, the α-OH-Tam, a major allylic alcohol metabolite of Tam, may be responsible for the adverse carcinogenic drug effects.

HepG2 cells lack functional estrogen receptors and may serve as a model to study Tam effects which are not related to ER-inhibition. HepG2 cells, however also lack CYP450 enzymes that can metabolically activate Tam, thus cells overexpressing CYP450s are more natural model to study non-anti-estrogen effects of Tam metabolites.

Section snippets

Cells and reagents

Human hepatoblastoma cells, subline-HepG2, transfected with an empty vector (C34 cells); HepG2 cells stably transfected with human CYP3A4 (3A4 cells) or cells overexpressing human CYP2E1 (2E1 cells) were used in this study. All cells originate from Department of Biochemistry, Mt. Sinai School of Medicine in New York and were kindly provided by Drs. D. Feierman and A. Cederbaum. Cells were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, glutamine (2 mM),

Results

Figure 1 shows Tam cytotoxicity measured with MTT test. C34 cells and cells overexpressing CYP2E1 or CYP3A4 were treated with different Tam concentrations for 48 hr. Important dose-dependent drug cytotoxicity was found only in cells overexpressing CYP3A4 but not in cells transfected with CYP2E1 or empty vector. Treatment with 20 μM Tam resulted in 60% toxicity in 3A4 cells. Linear regression coefficient was 4-fold lower (P<0.01) in 3A4 cells than in C34 cells.

Table 1, Fig. 2, Fig. 3 show Tam

Discussion

Tam has relatively low toxicity and is less harmful than most chemotherapeutics. On the other hand, increased occurrence of endometrial cancer has been reported in breast cancer patients and even in healthy women taking Tam [2]. Tam undergoes metabolic activation and drug metabolism supplies not only effective antitumor compounds but also numerous species that bind DNA. Animal studies show that Tam metabolism produce potent hepatocarcinogens [3], [14], [15]. In humans, the major pathway in Tam

Acknowledgements

This work was supported by a Grant 366511 from the Medical Academy of Bialystok.

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