Sigma-2 ligands induce tumour cell death by multiple signalling pathways.

Sigma receptors are a distinct class of receptors that are found in many tumours and normal tissues. Sigma receptors have been associated with many cellular and organ processes, including motor function, endocrine function, proliferation, immunoregulation and ion channel modulation (Walker et al, 1990; Hellewell et al, 1994; Megalizzi et al, 2010). Radioligand binding studies and biochemical analyses have shown that there are at least two types of sigma receptors, sigma-1 (∼25 kDa) and sigma-2 (∼21.5 kDa). The sigma-1 receptor has been cloned from many species (Hanner et al, 1996; Seth et al, 1997). Recently the progesterone receptor membrane component 1 (PGRMC1) protein complex was identified as the putative sigma-2 receptor-binding site (Xu et al, 2011). Sigma-2 receptors are expressed in high density in nearly all human and rodent tumour cell lines (Vilner et al, 1995). Sigma-2 receptor densities have been correlated with the grade of some human and bovine tumours (Bem et al, 1991; Colabufo et al, 2006; Roperto et al, 2010). The density of sigma-2 receptors in proliferating mouse mammary adenocarcinoma cells was found to be ∼10-fold higher than that in the corresponding quiescent tumour cells both in vitro and in vivo (Mach et al, 1997; Wheeler et al, 2000). These observations have led to the development of the sigma-2 ligands as molecular probes for diagnostic imaging of solid tumours (Wheeler et al, 2000; Mach et al, 2001a; Kawamura et al, 2003), and the identification of sigma-2 receptors as a potential target for treatment of cancer (John et al, 1998; Caveliers et al, 2001). Apoptosis (programmed cell death) is a physiological process that is important for development, homeostasis and suppression of oncogenesis (Jacobson et al, 1997). Deregulated apoptosis has been implicated in many diseases, including ischemic stroke and cancer (Reed, 2002). It is well known that the caspase family has a central role in both the intrinsic and extrinsic pathways of apoptosis. Caspase-3, the ‘executioner' caspase, is a key enzyme, which degrades proteins such as gelsolin and lamin, leading to apoptotic cell death (Reed, 2002). Sigma-2 ligands with different chemical structures have been reported to induce apoptosis in several tumour cell lines (Brent et al, 1996; Crawford and Bowen, 2002; Crawford et al, 2002, 2003; Barbieri et al, 2003; Ostenfeld et al, 2005; Cassano et al, 2009). Although two of these studies (Crawford and Bowen, 2002; Ostenfeld et al, 2005) have shown that prolonged exposure to sigma-2 ligands kills cancer cells by a caspase-independent apoptotic pathway, a complete understanding of how sigma-2-selective ligands induce cell death has not yet been established. Autophagy is a lysosomal degradation pathway for cytoplasmic materials (Glick et al, 2010). At basal levels, autophagy maintains cellular homeostasis by removing misfolded or aggregated proteins, and by clearing damaged cellular organelles. Autophagy is strongly induced upon nutrient deprivation and enhances cell survival by degrading nonessential components of the cell to generate nutrients for vital biological processes. Autophagy begins with an isolation membrane, also known as a phagophore. The phagophore then expands to engulf cytoplasmic materials, forming a closed double-membrane autophagosome. Autophagosomes fuse with endosomal and/or lysosomal vesicles, promoting degradation of autophagosomal contents by lysosomal acid proteases. The degradation products can be re-used for cellular metabolism. By contrast, excessive autophagy can result in non-apoptotic cell death (Platini et al, 2010). Mammalian target of rapamycin (mTOR) promotes cell growth and inhibits autophagy (Janku et al, 2011). Mammalian target of rapamycin kinase forms two distinct multiprotein complexes called mTORC1 and mTORC2. Mammalian target of rapamycin-C1 activity results in phosphorylation of S6 ribosomal protein kinase (S6K), phosphorylation of eukaryotic initiation factor 4E-binding protein (4EBP1) and subsequent activation of the protein translational machinery in the cell. Mammalian target of rapamycin-C2 mediates Akt activation, which in turn stimulates and activates mTORC1. Because of its dual role in cell survival and cell death, both inhibition and activation of the autophagic lysosomal pathway are novel strategies for treating cancer (Platini et al, 2010). In fact, Ostenfeld et al (2008) have proposed combination therapy of siramesine, a sigma-2 ligand, with drugs that inhibit autophagy as a strategy for treating cancer. The cell cycle can be described by four successive cellular phases: a phase of cell growth to prepare for DNA replication (G1), a phase of DNA synthesis and replication (S), and a phase of cell growth and active synthesis of factors (G2) required for mitosis (M) (Malumbres and Barbacid, 2009). Progression through the cell cycle is regulated by sequential waves of different cyclin/cyclin-dependent kinase (CDK) activities. Cyclins are synthesised and destroyed at specific time points during the cell cycle, thus regulating CDK kinase activities in a timely manner. Cyclin-dependent kinase–cyclin complexes directly involved in cell-cycle control include three interphase CDKs (CDK2, CDK4 and CDK6), a mitotic CDK (CDK1) and four classes of cyclins (cyclins A, B, D and E). Mitogenic signals first induced the expression of D-type cyclins (D1, D2 and D3). The D-type cyclins bind to and activate CDK4 and CDK6 during G1 phase, leading to phosphorylation of the retinoblastoma protein (Rb). Phosphorylation of Rb releases the E2F transcription factors, which can then activate genes essential for G1–S transition and S-phase, including E-type cyclins (Witzel et al, 2010). The E-type cyclins are required to activate Cdk2 for proper completion of the G1 phase, as well as for initiating DNA replication. Once cells enter S-phase, Cdk2–cyclin E complexes need to be silenced to avoid re-replication of DNA. The A-type cyclins accumulate during S-phase. Cyclin-dependent kinase-2–cyclin A complexes have been reported to phosphorylate numerous proteins that are required for proper completion and exit from S-phase. During G2, the A-type cyclins are degraded by ubiquitin-mediated proteolysis whereas the B-type cyclins are actively synthesised. As a consequence, Cdk1 binds to the B-type cyclins – an association believed to regulate several events during both the G2–M transition and progression through mitosis. Finally, inactivation of Cdk1–cyclin B complexes is required for proper exit from mitosis. This inactivation is accomplished by degradation of the B-type cyclins through the proteolytic pathway mediated by ubiquitination. Cell-cycle malfunction may cause cell death (Allan and Clarke, 2008; Clarke and Allan, 2009). Recent results indicate that regulation of apoptosis is directly linked with cell-cycle checkpoints by common components (Clarke and Allan, 2009). Few studies have investigated the effects of sigma-2 ligands on the cell cycle. Our group has previously reported the synthesis and in vitro characterisation of a number of structurally diverse ligands with a high affinity for sigma-2 receptors (Mach et al, 2001b, 2003, 2004). By screening these sigma ligands for their cytotoxicity, we identified three potent sigma-2-selective ligands, WC-26, SV119 and RHM-138, that killed mouse breast cancer EMT-6 and human MDA-MB-435 melanoma tumour cells with EC50s in micromolar range after a 48-h exposure. The aim of the present study was to explore the mechanism by which these three sigma-2-selective ligands induce cell death. The data presented in this report indicate that treatment of EMT-6 and MDA-MB-435 cancer cells with these sigma-2 ligands induces multiple mechanisms of cell death, including apoptosis, autophagy and cell-cycle impairment.