Estrogen-Induced Apoptosis of Breast Epithelial Cells Is Blocked by NO/cGMP and Mediated by Extranuclear Estrogen Receptors

Gihani T Wijewickrama,Gregory R J Thatcher,Irida Kastrati,Praneeth D Edirisinghe

doi:10.1210/en.2010-0378

Gihani T Wijewickrama, Gregory R J Thatcher + Show 2 more

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https://doi.org/10.1210/en.2010-0378

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Journal: EndocrinologyJN	Publication Date: Oct 13, 2010
Citations: 23	License type: cc-by

Affiliation: University of Illinois at Chicago

Abstract

Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17β-estradiol (E(2)) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E(2). Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E(2) + NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E(2)-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal.

Highlights

Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cyclic GMP (cGMP)
NO concentrations generated by exogenous sources or forced overexpression of inducible NO synthase (NOS) (iNOS) may be used to kill tumor cells; paradoxically, iNOS is overexpressed in many tumors, and overexpression of iNOS often correlates with poor prognosis, leading to iNOS inhibition as a proposed target for cancer therapy [8, 9]. cGMP-independent posttranslational nitrosation of caspase and pol(ADP-ribose) polymerase are reported as antiapoptotic cell defenses [10, 11]
In serum-free medium (SFM), the increase in apoptotic nuclei at 48 h was reported to be inhibited by addition of epidermal growth factor (EGF), insulin, or extracellular matrix components, changes accompanied by modulation of proapoptotic (Bax) and antiapoptotic factors (Bcl-2)

Summary

Introduction

Via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. Nitric oxide (NO), produced by the NO synthase (NOS) family of proteins, modulates a variety of important physiological responses, including cell proliferation, invasion, migration, expression of angiogenic factors, and apoptosis Regulation of these responses is mediated by activation of the primary NO effector soluble guanylyl cyclase (sGC) to produce the secondary messenger cyclic GMP (cGMP), and by NO-based chemical modifications of proteins and lipids, or reactions with metals and free radicals. NO steady-state concentration has emerged as a key determinant of biological function [5], whereby lower concentrations promote cell survival and proliferation via cGMP-dependent pathways, and higher levels favor cell cycle arrest, apoptosis, and senescence [6] The latter cytotoxic effects have been associated with activation of cells involved in inflammatory responses and induction of inducible NOS (iNOS) [7]. In normal cells, inhibition by E2 of apoptosis induced by oxidative or other stress is documented: for example, in cardiomyocytes, E2 prevented apoptosis via activation of phosphatidylinositol 3 kinase (PI3K) and serine-threonine kinase (Akt) phosphorylation [17, 18]

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