Elsevier

Steroids

Volume 64, Issues 1–2, January–February 1999, Pages 22-27
Steroids

Rapid actions of estrogens
Estradiol modulates breast cancer cell apoptosis: a novel nongenomic steroid action relevant to carcinogenesis

https://doi.org/10.1016/S0039-128X(98)00106-8Get rights and content

Abstract

It is known that steroids can induce cell surface receptor aggregation followed by activation of receptor and nonreceptor tyrosine kinases. It has been shown recently that 17β-estradiol (E2) can stimulate the Src/p21ras/mitogen-activated protein kinase pathway in breast cancer cells, and this effect is supposed to mediate the E2-induced stimulation of breast cancer cell proliferation, possibly via activation of the c-fos and c-jun early genes or of genes involved in cell cycle control. Here we demonstrate the existence of an alternative mechanism of the cancer-promoting effect of E2. Human breast cancer cells (MCF-7) were exposed to the known proapoptotic agent vitamin E succinate (VES), added alone or together with different concentrations of E2. E2 conjugated with bovine serum albumin (E2-BSA), which cannot cross the plasma membrane of living cells, was also used in some experiments to assess whether E2 acted on the cell surface or at intracellular receptors. Apoptosis was analyzed by fluorescence-activated cell sorting after cell staining with propidium iodide and FITC-labeled annexin V. E2 showed a concentration-dependent stimulatory effect on spontaneous apoptosis but inhibited the VES-induced apoptosis. However, effects produced by the same molar concentrations of E2 were different when the hormone was free and when it was used in the form of the E2-BSA conjugate. The effects of E2 and E2-BSA were sensitive to genistein, a tyrosine kinase inhibitor. These data show that E2 modulates apoptosis of breast cancer cells, probably acting both at the cell surface and inside the cells. Tyrosine phosphorylation is involved in the signaling pathways mediating this E2 effect.

Introduction

Several lines of evidence suggest that 17β-estradiol (E2) has a role in proliferation of different tumor cell types [1], [2], [3], [4]. This E2 effect appears to be mediated, at least partly, by a nongenomic mechanism of action, involving receptors localized at the cell membrane. Signal transduction from these receptors to intracellular effectors implies protein tyrosine kinase (PTK) activation [5]. PTKs are efficiently inhibited by the isoflavonoid genistein [6], which is one of the common dietary phytoestrogens [7]. The inhibition of the PTK-mediated pathway may thus contribute to the protective action of dietary phytoestrogens against breast cancer [8], which was demonstrated by a recent case-control study [9].

E2 may promote cancer cell growth and division by stimulating different effector systems. For instance, E2 has been shown to regulate the production of fibroblast growth factor 1 in normal and cancer breast cells [10], to activate proteins involved in the cell cycle control, such as G1 cyclins [11], [12], [13] and to stimulate expression of genes required for cell division such as the proto-oncogene c-myc [14]. However, little attention has been paid to the possibility that E2-activated signal transduction pathways may interact with those pathways controlling apoptosis (programmed cell death), although several studies did reveal positive and negative effects of steroids on apoptosis of noncancer cells [15], [16], [17].

Here we review the E2-activatable cell signaling events that are susceptible to modulating programmed cell death. We also present preliminary experimental data showing that E2 modulates spontaneous and VES-induced apoptosis in the human breast cancer-derived MCF-7 cell line.

Section snippets

Steroid-induced PTK activation

The possibility that steroids may act at the cell surface by stimulating protein tyrosine phosphorylation, similar to peptidic hormones and growth factors, has been suggested by the finding that antibody- [18] or ligand-induced [19] aggregation of progesterone-receptor complexes on the cell surface of human spermatozoa elicits a biological response (acrosome reaction). Ligand-induced receptor aggregation is a common characteristic of PTK-coupled receptors. Subsequently, a direct demonstration

From PTK to activator protein-1 family

The PTK primarily involved in the transduction of E2-generated signals in MCF-7 cells is c-src [5]. This is an interesting finding because c-src activation occurs in many human breast and colon cancers and is believed to be directly involved in tumorigenesis [28], [29]. In MCF-7 cells, E2-activated c-src phosphorylates two substrates, Shc and p190 [26], the former then associating to the GRB2/mSos complex and stimulating p21ras activation [30]. This leads to stimulation of a serine/threonine

From AP-1 family to apoptosis

c-Fos and c-jun are signal-transducing transcription factors of the AP-1 family, normally involved in the regulation of the cell cycle, cell differentiation and transformation. The implication of c-jun in the programmed cell death was suggested by different kinds of observation, including overexpression of c-jun in lymphoblastoid cell lines [36] and pheochromocytoma cells [37] undergoing apoptosis as a result of withdrawal of growth factors, sustained activation of c-jun accompanying apoptosis

Effects of E2 and E2-BSA on spontaneous and VES-induced apoptosis of MCF-7 cells

To test the hypothesis that E2 can modulate human breast cancer cell apoptosis, we performed experiments in which cells of the MCF-7 human breast cancer-derived cell line were exposed to E2 or its membrane-impermeant conjugate E2-BSA, added either alone or together with the known proapoptotic agent VES. MCF-7 cells, routinely grown in modified Eagle’s medium with Earle’s balanced salts enriched with 10% fetal bovine serum and equilibrated with 5% CO2 in air, were maintained for 1 week before

Conclusion

Steroids exert nongenomic effects, involving PTK stimulation, in many cell types. In many tumor-derived cell lines, E2 activates the src nonreceptor PTK, resulting in tyrosine phosphorylation of Shc and subsequent activation of the ras/MAP-kinase cascade. MAP kinase has recently been shown to activate expression of the members of AP-1 family c-fos and c-jun proto-oncogenes that subsequently can regulate the expression of caspases and other effectors of apoptosis. These findings make up a mosaic

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