Distinct modulation of alkaline phosphatase isoenzymes by 17β-estradiol and xanthohumol in breast cancer MCF-7 cells
Introduction
Breast cancer is one of the most frequent neoplasms among women, with high morbidity and mortality rates in the western world. Estrogens are relevant risk factors for breast cancer development [1]. In previous studies, our group investigated the role of 17β-estradiol (E2) in up-regulating several signalling pathways in breast cancer cell lines [2]. According to the literature, one of the genes that can be modulated by E2 is alkaline phosphatase (ALP) [3], [4], [5], [6].
ALP is a phosphate hydrolase, which enables the transfer of inorganic phosphates to an acceptor substrate [7], [8]. There are several isoenzymes of ALP encoded in four distinct loci, which catalyze identical reactions. Many studies report that ALP dephosphorylates proteins involved in cell growth and differentiation, apoptosis and cell migration [9], [10]. Abnormal expression of ALP isoenzymes has been found in malignant tissues, being often established as a useful prognostic indicator [11], [12].
Estrogens mediate ALP gene expression in cervix and breast cancer cell lines [13]. Estrogens bind to specific estrogen receptors (ER) in cell nuclei, resulting in modulation of target gene transcription. ER are also present in cell membranes, suggesting a nongenomic estrogen pathway [1]. These nongenomic pathways are involved in regulating ionic channels and G-protein-associated membrane receptors [14]. The genomic or nongenomic character of ALP modulating effects by estrogens is presently unknown.
Lupulus prenylated flavonoids, such as xanthohumol (XN), have been reported to present anti-carcinogenic properties, preventing both initiation and progression of cancer [15], [16]. A few studies indicate a putative association between XN and estrogen signalling [15], [17]. The evident role of ALP in tumorigenesis, as well as the fact that this enzyme might be modulated by estrogens and by XN, two compounds associated with breast cancer development and progression, renders this enzyme an interesting target to investigate in breast cancer cells.
Section snippets
Chemicals
All cell culture reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). 17β-estradiol (E2) was obtained from Sigma Aldrich (Portugal). Antibodies against tissue non-specific alkaline phosphatase (TNS-ALP), intestinal ALP (IALP) and β-actin (Santa Cruz Biotechnology, USA) were used for immunoblotting. The pure estrogen antagonist ICI 182,780 (ICI) was kindly provided by AstraZeneca (Portugal). Concentrations for E2 and ICI used were based on previous studies [18], [19].
Expression of ERα and ERβ in MCF-7 cell cultures
The expression of estrogen receptor (ER)α and ERβ isoforms in MCF-7 cells was first determined by RT-PCR. MCF-7 cells expressed both isoforms after incubation with ethanol (EtOH), 17β-estradiol (E2), 17β-estradiol + ICI (E2 + ICI) and xanthohumol (XN) (Fig. 1A). A significant decrease in the two ER isoforms was found after incubation with ICI (*p < 0.05). Slightly reduced ERα and ERβ expressions were observed after incubation with XN, although not reaching statistical significance.
17β-estradiol and XN resulted in decreased expression of ALP enzymes in MCF-7 cells
Expression of
Discussion
The aberrant expression of ALP genes in cancer cells suggests that this enzyme may be involved in tumorigenesis. Using breast cancer MCF-7 cells, we showed that E2 resulted in a down-regulation of ALP expression and activity. Our findings are in contrast to the ones described in ovarian cancer Ishikawa cells [5], [6]. One reason for this paradox is the distinct response to estrogens of the two cell types used. In fact, despite that both Ishikawa and MCF-7 cells are estrogen-dependent cell
Acknowledgments
This study was partially supported by grants from iBeSa, Institute of Beverages and Health, Portugal. The authors are grateful to iBeSa for providing the xanthohumol and to AstraZeneca (Portugal) for providing ICI 182,780, the pure estrogen antagonist.
References (32)
- et al.
Ontogenic and phylogenic studies of intestinal, hepatic, and placental alkaline phosphatases. Evidence that intestinal alkaline phosphatase is a late evolutionary development
Gastroenterology
(1986) - et al.
Purification and some properties of the fast migrating alkaline phosphatase in FL-amnion cells (the Kasahara isoenzyme) and its cDNA cloning
Clin. Chim. Acta
(1990) - et al.
Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines
Food Chem. Toxicol.
(1999) - et al.
Com-1/P8 in oestrogen regulated growth of breast cancer cells, the ER-beta connection
Biochem. Biophys. Res. Commun.
(2005) - et al.
The multifaceted mechanisms of estradiol and estrogen receptor signaling
J. Biol. Chem.
(2001) - et al.
Estrogen receptor pathways to AP-1
J. Steroid Biochem. Mol. Biol.
(2000) - et al.
Oestrogen-mediated tyrosine phosphorylation of caveolin-1 and its effect on the oestrogen receptor localisation: an in vivo study
Mol. Cell. Endocrinol.
(2005) - et al.
Estrogen carcinogenesis in breast cancer
N. Engl. J. Med.
(2006) - et al.
17 beta-estradiol-mediated vessel assembly and stabilization in tumor angiogenesis requires TGF beta and EGFR crosstalk
Angiogenesis
(2003) - et al.
Studies on the effects of biomedicinal agents on serum concentration of Ca2+, P and ALP activity in osteoporosis-induced rats
J. Vet. Sci.
(2003)