Sigma-2 Receptors Play a Role in Cellular Metabolism: Stimulation of Glycolytic Hallmarks by CM764 in Human SK-N-SH Neuroblastoma

Abstract

Sigma-2 receptors are attractive antineoplastic targets due to their ability to induce apoptosis and their upregulation in rapidly proliferating cancer cells compared with healthy tissue. However, this role is inconsistent with overexpression in cancer, which is typically associated with upregulation of prosurvival factors. Here, we report a novel metabolic regulatory function for sigma-2 receptors. CM764 [6-acetyl-3-(4-(4-(2-amino-4-fluorophenyl)piperazin-1-yl)butyl)benzo[d ]oxazol-2 (3H)-one] binds with K i values of 86.6 6 2.8 and 3.5 6 0.9 nM at the sigma-1 and sigma-2 receptors, respectively. CM764 increased reduction of MTT [3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide] in human SK-N-SH neuroblastoma compared with untreated cells, an effect not due to proliferation. This effect was attenuated by five different sigma antagonists, including CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one], which has no significant affinity for sigma-1 receptors. This effect was also observed in MG-63 osteosarcoma and HEK293T cells, indicating that this function is not exclusive to neuroblastoma or to cancer cells. CM764 produced an immediate, robust, and transient increase in cytosolic calcium, consistent with sigma-2 receptor activation. Additionally, we observed an increase in the total NAD 1 /NADH level and the ATP level in CM764-treated SK-N-SH cells compared with untreated cells. After only 4 hours of treatment, basal levels of reactive oxygen species were reduced by 90% in cells treated with CM764 over untreated cells, and HIF1a and VEGF levels were increased after 3-24 hours of treatment. These data indicate that sigma-2 receptors may play a role in induction of glycolysis, representing a possible prosurvival function for the sigma-2 receptor that is consistent with its upregulation in cancer cells compared with healthy tissue.

Divergent Cytotoxic and Metabolically Stimulative Functions of Sigma-2 Receptors: Structure-Activity Relationships of 6-Acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79) Derivatives

“…Interestingly, for WA435 (X 5 S, R 5 NCS) the efficacy of the ligand increased when treated acutely compared with the 24 hours of continuous exposure (P 5 0.003 for comparison of the two conditions, Student's t test). It should be noted here that in our previous study using siramesine, removing siramesine (which cannot bind covalently to sigma receptors) after a 60-minute pretreatment of SK-N-SH neuroblastoma cells completely prevented the induction of cell death (Nicholson et al, 2016). This rules out the possibility that a 60-minute acute exposure to sigma-2 agonist is enough time to commit to a cell death program, such that cell death would occur after subsequent removal of even a reversibly bound ligand.…”

“…Effect of Ligand Treatment on Metabolic Reduction of MTT. We have recently described a novel, nontoxic, nonzero function for sigma-2 receptors using the SN79 derivative CM764 (Nicholson et al, 2016). Five of the ligands investigated in this series replicated this effect, inducing an increase in the reduction of MTT upon 24 hours of treatment compared with untreated cells.…”

“…This effect was blocked by various sigma-2 receptor antagonists. In addition to the increase in MTT reduction, the effect comprised an increased level of NAD 1 /NADH and ATP, a marked decrease in basal reactive oxygen species level, and an increase in hypoxia-inducible factor 1a, which may be connected to an observed increase in vascular endothelial growth factor expression (Nicholson et al, 2016). Thus, sigma-2 receptors may have a dual role with respect to cellular survival and may activate different pathways depending on the ligand.…”

“…Cleavage of Bid, caspase-3 activation, induction of an immediate rise in cytosolic calcium, and positive terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining have been proposed as markers of sigma-2 receptor agonist activity (Vilner and Bowen, 2000;Crawford and Bowen, 2002;Hazelwood and Bowen, 2006;Wang and Bowen, 2006;Zeng et al, 2014;Nicholson et al, 2015). Recently, we described a novel function of sigma-2 receptors, where a sigma-2 receptor ligand, CM764, induced an increase in various markers of glycolytic cellular metabolism as initially assessed by an increase in MTT reduction, without inducing changes in cell viability or cell proliferation (Nicholson et al, 2016). This effect was blocked by various sigma-2 receptor antagonists.…”

“…Furthermore, both cytotoxic effects and metabolic stimulative effects were induced by compounds within the same SN79-related structural class in various cell lines. CM572 induced programmed cell death (Nicholson et al, 2015), whereas CM764 induced metabolic stimulation (Nicholson et al, 2016). Thus, we further explored structural determinants of these divergent effects within this class.…”