Elsevier

Cell Calcium

Volume 45, Issue 4, April 2009, Pages 340-345
Cell Calcium

F281, synthetic agonist of the sigma-2 receptor, induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

https://doi.org/10.1016/j.ceca.2008.12.005Get rights and content

Abstract

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca2+] ([Ca2+]i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca2+]i. In particular, CB-64D induces a Ca2+ response while PB28 supresses Ca2+ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC50 values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca2+ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca2+-entry through the channels activated by store depletion was also observed. After the increase in [Ca2+]i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.

Introduction

Sigma receptors are classified into the subtypes sigma-1 and sigma-2 [1], with different molecular weight (25 and 18–21.5 kDa, respectively), different patterns of tissue distribution, and subcellular localizations [2].

The sigma-1 receptor has been recently cloned from the tissues of guinea pig, rat, mouse and man [2]. It is characterized by two transmembrane regions and is anchored at rest to the membrane of the endoplasmic reticulum [3], [4].

The sigma-2 receptor, which this paper focuses on, is much less characterized and colocalizes with fluorescent markers of mitochondria, lysomes, endoplasmic reticulum, and the plasma membrane [5]. It has been linked to cancer biology by two important observations. Synthetic agonists of the sigma-2 receptor trigger a cell response leading to cell death [6] and inhibit the activity of the P-glycoprotein, responsible for the active extrusion of anticancer drugs [7]. Its precise physiological role remains unknown and has been implicated in the regulation of cell proliferation [8]. For example, the sigma-2 receptor agonists CB-64D and PB28 induced cytotoxicity in MCF-7 and MCF-7/Adr cell lines, from human breast adenocarcinoma [6], [9].

Recently, a clinical study has reported that high-grade transitional human cell carcinomas overexpress the sigma-2 receptor protein while normal tissue from bladder shows simultaneous normal expression [10]. The downstream cellular processes regulated by sigma-2 receptors have not yet been elucidated. Vilner and Bowen demonstrated that a number of sigma-2 receptor agonists, the most potent being CB-64D [11], produced an immediate, dose-dependent, and transient rise in [Ca2+]i of SK-N-SH cells (from a human neuroblastoma) [12].

More recently, we obtained quite different results by using PB28, synthesized in our laboratory. This molecule also induces cytotoxic effects in C6 (from a rat glioma), and SK-N-SH cell lines [13] and is the most specific agonist of the sigma-2 receptor known at present. PB28 per se has no effect on [Ca2+]i but inhibits the calcium release from the endoplasmic reticulum, elicited by a physiological agonist [14]. To summarize, CB-64D behaves as a physiological agonist while PB28 behaves as a regulator of the Ca2+ response.

Recently, we have synthesized the new analog F281 by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus (Fig. 1(A)) [15]. Although this bioisosteric substitution should not affect the ligand affinity at the sigma-2 receptor, F281 proved to be more cytotoxic than PB28 in a set of preliminary experiments. This very intriguing observation prompted us to investigate more thoroughly the interaction between F281 and SK-N-SH cells. In this paper special emphasis is given to the involvement of the agencies regulating [Ca2+]i. As a study model, SK-N-SH cells are again used because they express the sigma-1 receptor only in a low-affinity state [13].

Section snippets

Chemicals and cell culture

Cell culture reagents were purchased from Celbio S.r.l. (Milano, Italy). Thapsigargin and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (edelfosine or ET-18-OCH3) were obtained from Calbiochem (Milano, Italy); 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) -acetoxymethylester (AM), fura-2-AM, JC-1, and rhod-2-AM were purchased from Molecular Probes (Leiden, The Netherlands). All other reagents were from Sigma–Aldrich (Milano, Italy). Antimycin A, BAPTA-AM, carbonyl

Cytotoxicity of F281

We measured the effect of F281 (24 h incubation) on neuroblastoma SK-N-SH and adenocarcinoma LoVo cells; only the first cell line expresses sigma-2 receptors (data not shown). Cytotoxicity was estimated by measuring the release of d-lactic dehydrogenase into the medium. The dose response curves are shown in Fig. 1(B). The F281 concentration causing half-maximal cytotoxicity (EC50) was 65.4 ± 5.2 nM in SK-N-SH cells and 3.47 ± 0.35 μM in LoVo cells. These data suggest that sigma-2 receptor is involved

Discussion

In this study we have shown that 0.1 mM F281 induces a Ca2+ efflux either from mitochondria or from the endoplasmic reticulum, by opening the InsP3 receptor; this phenomenon triggers a Ca2+-entry through the channels activated by store depletion. After this Ca2+ response and within 10 min, we observed a sudden drop in metabolic activity and of intracellular [ATP] leading to cell death.

Particularly intriguing is the finding that F281 induces a Ca2+ efflux from mitochondria. So far, this phenomenon

Acknowledgement

This work was supported by a grant from the Università degli Studi di Bari (Italy).

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