Effect of 4-hydroxytamoxifen isomers on growth and ultrastructural aspects of normal human breast epithelial (HBE) cells in culture

Abstract

In the search for a breast cancer prevention strategy which would avoid undesirable effects of orally administered tamoxifen, the percutaneous administration of the highly active metabolite 4OHTamoxifen (4OHTam) has been proposed. Percutaneous 4OHTam penetrates the skin to reach breast tissues. It, thus, avoids the hepatic first pass effect, and offers an optimal local/systemic effect. However, trans-4OHTamoxifen can spontaneously isomerize into the cis-isomer, which may have estrogen agonist action. The aim of this study was to examine the effect of cis-4OHTam on normal human breast epithelial (HBE) cells in culture.

Spontaneous isomerization of trans- into cis-4OHTam occurred within 24–48 h, but stabilized rapidly at a trans/cis ratio of 70/30, whether in stock solution, culture medium or cultured cells. The cis-4OHTam did not stimulate HBE cell growth according to histometric cell counts and scanning electron microscopy analysis, but inhibited E₂-induced cell growth, albeit two to three times less than trans-4OHTam.

In conclusion, spontaneous isomerization of trans- to cis-4-OHTam is limited and 4OHTam retains a marked antiestrogenic effect. It may prove to be a useful alternative to tamoxifen in breast cancer prevention, especially if administered percutaneously.

Introduction

The antiproliferative effect of antiestrogens of the triphenylethylene series has been well documented in breast cancer cell lines [1], [2], [3] and such antiestrogens are currently used as hormonal adjuvant therapy in breast cancer [4], [5], [6]. Considering their cytostatic effect first observed on cancer cells, and then on normal breast epithelial cells [7], [8], the antiestrogen tamoxifen (Tam) and its active metabolite 4-hydroxytamoxifen (4OHTam) [9] have been proposed as hormonal therapy for primary prevention in patients at high risk for breast cancer [10], [11].

Moreover, we have previously shown that 4OHTam, may be administered percutaneously directly on the breast. It penetrates the skin and reaches breast tissue [12], [13], thereby avoiding the hepatic first pass effect of oral administration.

It has therefore been suggested that 4OHTam, percutaneously administered to the breast, could be of value for primary prevention of breast cancer in patients at high risk since it offers an optimal local/systemic antiestrogenic effect [14], [15]. However, spontaneous isomerization from the trans- to the cis-isomers of 4OHTam may occur [16], [17], [18]. Since the cis-isomers of the triphenylethylene series may have estrogen agonist action [16], [17], [19], it was essential to examine the effect of cis-4OHTam on normal breast cells. Cultures of normal human breast epithelial (HBE) cells are routinely obtained in our laboratory [20], [21], [22], [23]. These cells remain hormone-dependent in culture [20], [21], [22]. Presence of ER has been demonstrated qualitatively by immunocytochemistry [23] and quantitatively as well by binding assay and by EIA, both of which indicate similar ER amounts (ranging from 140 to 220 fmol/mg of DNA; unpublished data). Moreover, estradiol stimulates [7], [22] whereas antiestrogens inhibit [7] cell multiplication. In addition, progestins inhibit cell growth [22] and induce progesterone-dependent 17β-hydroxysteroid dehydrogenase activity [20], [22]. These HBE cells were therefore used to compare the action of the cis- and trans-isomers of 4OHTam on both the growth and the ultrastructural aspects of normal human breast cells.