F281, synthetic agonist of the sigma-2 receptor, induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells
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).
References (34)
- et al.
A proposal for the classification of sigma binding sites
Trends Pharmacol. Sci.
(1992) - et al.
Correlation between sigma2 receptor protein expression and histopathologic grade in human bladder cancer
Cancer Lett.
(2006) - et al.
CB-64D and CB-184: ligands with high sigma2 receptor affinity and subtype selectivity
Eur. J. Pharmacol.
(1995) - et al.
The sigma-2 receptor agonist PB28 inhibits calcium release from the endoplasmic reticulum of SK-N-SH neuroblastoma cells
Cell Calcium
(2006) - et al.
Calcium response after stimulation by substance P of U373 MG cells: inhibition of store-operated calcium entry by protein kinase C
Cell Calcium
(2004) - et al.
JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis
FEBS Lett.
(1997) - et al.
Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum
Cell Calcium
(1988) - et al.
Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis
Gastroenterology
(2006) - et al.
2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels
Cell Calcium
(2003) - et al.
Calcium and mitochondria
FEBS Lett.
(2004)