Antiepileptic drugs inhibit cell growth in the human breast cancer cell line MCF7

https://doi.org/10.1016/j.mce.2003.10.032Get rights and content

Abstract

Several antiepileptic drugs (AEDs) are associated with anti-cancer activity. At the same time, many AEDs alter endocrine function with phenytoin (PHT) and phenobarbital (PB) causing-reduced free fractions of sex-steroid hormones, while VPA induces hyperandrogenism. Changes in sex-steroid hormone levels are known to affect apoptosis in endocrine tissue. The aim of the study was to investigate the influence of the antiepileptic drugs PHT, PB, VPA and lamotrigine (LTG) on estrogen-stimulated cell growth of human breast cancer cells (MCF-7), and to evaluate whether this effect could be related to a direct estrogen receptor (ER) binding. VPA reduced cell growth at therapeutically relevant concentrations; half-maximum effect of VPA on cell growth was 230 μM. PHT (100 μM) and PB (10 μM) reduced cell growth by 47 and 21%, respectively. None of the drugs had affinity to isolated estrogen receptors, and excess of estrogen was not able to abolish the growth inhibition provoked by VPA. However, sub-therapeutic concentrations of VPA (100 μM) mimicked estrogen by inducing cell growth (11%) in an estrogen-depleted medium, an effect that was abolished by adding an estrogen receptor antagonist. In conclusion; the estrogen receptor appear to be indirectly activated by sub-therapeutic concentrations of VPA, but therapeutic concentrations of VPA inhibits cell growth by mechanisms that do not seem to involve the estrogen receptor or estrogen stimulation.

Introduction

It has recently become evident that several antiepileptic drugs (AEDs) are associated with anti-cancer activity (Cinatl Jr. et al., 1997; Göttlicher et al., 2001), and have influence on cell growth and apoptosis in several cancer cell types in vitro (for review, see Blaheta and Cinatl Jr., 2002; Kawagoe et al., 2002). At the same time, many AEDs affect endocrine function with phenytoin (PHT) and phenobarbital (PB) reducing free fractions of sex-steroid hormones (for review, see Lambert, 2001), while valproate (VPA) induces hyperandrogenism (Isojarvi et al., 1993, Isojarvi et al., 1996, Isojarvi et al., 2001, Murialdo et al., 1998). Changes in sex-steroid hormone levels are known to affect apoptosis in endocrine tissue. In the ovary and testis, estrogen in physiological concentrations has been shown to act as an anti-apoptotic agent while androgens are pro-apoptotic in the ovary (Billig et al., 1993, Pentikainen et al., 2000). Recently it became evident that VPA at therapeutic serum concentrations acts as an apoptotic agent in ovarian follicles (Taubøll et al., 2003). In dogs and rats, chronic VPA treatment leads to testicular atrophy (Walker et al., 1990; Røste et al., 2001).

So far, no one has investigated if the estrogen-signalling pathway is affected by VPA, and the initial events leading to hormonal changes after AED treatment are only partly known. We therefore wanted to compare four commonly used AEDs, which affect the sex-steroid hormone balance in different ways, for their effect on the estrogen-signalling pathway. PHT and PB, which are known to reduce the effect of oral contraceptives (Morrell, 1999) and reduce the free fraction of sex hormones, VPA, which are known to induce hyperandrogenism (Isojarvi et al., 1993, Isojarvi et al., 2001; O’Donovan et al., 2002), and lamotrigine (LTG) that does not seem to have endocrine effects (Stephen et al., 2001; Røste et al., in press). For this purpose, the human breast cancer cell line MCF-7 was used. These cells contain estrogen receptor-α (ERα); (Jørgensen et al., 2000; Register and Adams, 1998) and are highly responsive to estrogens (Lippman and Bolan, 1975).

The aim of this study was (1) to evaluate if the antiepileptic drugs PHT, PB, VPA or LTG affect 17β-estradiol stimulated cell growth in human breast cancer cells MCF-7, which are known to proliferate in response to estrogen-like stimuli (Soto et al., 1995), and (2) to evaluate if such an effect can be related to competitive binding to the isolated estrogen receptor-α.

Section snippets

Chemicals and media

Standard commercial available solutions of PB (Nycomed Pharma, Norway; 100 mg/ml) and PHT (Nycomed Pharma, Norway; 50 mg/ml), were used as stock solutions and further diluted in absolute ethanol to achieve the different concentrations used. Sodium salt of VPA was obtained from Sigma–Aldrich, Germany, dissolved in distilled water, and ethanol was added for the comparison with a solvent control. LTG was kindly supplied from GSK Norway (Oslo), dissolved in methyl cellulose 0.25% in distilled water,

Results

PHT, PB and VPA were all able to reduce cell growth (Fig. 1), while LTG had no effect (results not shown). PHT (100 μM) caused a significant reduction of the cell growth by 47%. PB (10 μM) reduced the cell growth significantly by 21%, but elevation of the PB concentration to 100 μM had no further inhibitory effect on the cell growth. Therapeutic concentrations of VPA (250 μM) reduced cell growth significantly by 40%, 500 μM VPA reduced the cell growth by 70%, and the growth was further reduced by

Discussion

Both VPA, PB and PHT reduced cell growth of MCF-7 cells. LTG had no effect on cell growth when tested at concentrations up to 6.6 μM, which is in the lower end of the therapeutic range (results not shown). The growth inhibition provoked by VPA, PB and PHT was not caused by cytotoxicity or induction of apoptosis as evaluated by microscopic examination of cells stained with propidium iodide and Hoechst 33342, nor was there any reduction in general redox capacity as evaluated with the Alamar Blue

Acknowledgements

This study was supported by grant from the Research Council of Norway. We are grateful to Dr. A.M. Soto for kindly providing us with MCF-7 cells.

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