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

Background: The sigma-2 receptor has been identified as a biomarker of proliferating cells in solid tumours. In the present study, we studied the mechanisms of sigma-2 ligand-induced cell death in the mouse breast cancer cell line EMT-6 and the human melanoma cell line MDA-MB-435. Methods: EMT-6 and MDA-MB-435 cells were treated with sigma-2 ligands. The modulation of multiple signaling pathways of cell death was evaluated. Results: Three sigma-2 ligands (WC-26, SV119 and RHM-138) induced DNA fragmentation, caspase-3 activation and PARP-1 cleavage. The caspase inhibitor Z-VAD-FMK partially blocked DNA fragmentation and cytotoxicity caused by these compounds. These data suggest that sigma-2 ligand-induced apoptosis and caspase activation are partially responsible for the cell death. WC-26 and siramesine induced formation of vacuoles in the cells. WC-26, SV119, RHM-138 and siramesine increased the synthesis and processing of microtubule-associated protein light chain 3, an autophagosome marker, and decreased the expression levels of the downstream effectors of mammalian target of rapamycin (mTOR), p70S6K and 4EBP1, suggesting that sigma-2 ligands induce autophagy, probably by inhibition of the mTOR pathway. All four sigma-2 ligands decreased the expression of cyclin D1 in a time-dependent manner. In addition, WC-26 and SV119 mainly decreased cyclin B1, E2 and phosphorylation of retinoblastoma protein (pRb); RHM-138 mainly decreased cyclin E2; and 10 μM siramesine mainly decreased cyclin B1 and pRb. These data suggest that sigma-2 ligands also impair cell-cycle progression in multiple phases of the cell cycle. Conclusion: Sigma-2 ligands induce 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 (B25 kDa) and sigma-2 (B21.5 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 . 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 proli-ferating mouse mammary adenocarcinoma cells was found to be B10-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 et al, , 2003Barbieri et al, 2003;Ostenfeld et al, 2005;Cassano et al, 2009). Although two of these studies 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 . 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 (G 1 ), a phase of DNA synthesis and replication (S), and a phase of cell growth and active synthesis of factors (G 2 ) 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 G 1 phase, leading to phosphorylation of the retinoblastoma protein (Rb). Phosphorylation of Rb releases the E2F transcription factors, which can then activate genes essential for G 1 -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 G 1 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 G 2 , 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 G 2 -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(Mach et al, , 2003(Mach et al, , 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 EC 50 s 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.

LDH assay
Lactate dehydrogenase (LDH) release assay was performed using the Cytotox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer's protocol.

Detection of intracellular caspase-3 activity
Activation of endogenous caspase-3 by sigma ligands was measured using the CellProbe HT caspase-3 whole-cell assay (Beckman Coulter, Fullerton, CA, USA) (see Supplementary Information for description of caspase-3 assay).

Flow cytometry
Flow cytometric analysis was performed using a FACScan (Becton Dickinson, Fullerton, CA, USA) equipped with an air-cooled argon laser. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling)-positive cells were detected with FITC-dUTP and quantified by flow cytometry using a TUNEL assay according to the manufacturer's protocol (Apo-direct Kit; BD Biosciences Pharmingen, San Diego, CA, USA). Briefly, the cells were detached from the culture dishes with trypsin, washed with phosphate-buffered saline (PBS) and then fixed in 1% paraformaldehyde. After washing with PBS twice, the cells were incubated with a DNA labelling solution containing terminal deoxynucleotidyl transferase and FITC -dUTP at 37 1C for 1 h. After washing with PBS, 7-amino-actinomycin D (7-AAD; BD Biosciences Pharmingen) was added to the cells to stain the nucleic acid at a concentration of 5 ml (0.25 mg) 7-AAD/ test (1 Â 10 6 cells). The cells were then analysed by flow cytometry.
Fluorescein isothiocyanate was excited at 488 nm and emission was collected with a 570-nm filter. 7-Amino-actinomycin D was excited at 488 nm and emission was collected using a 650-nm long-pass filter.

Transmission electron microscopy
MDA-MB-435 cells were plated in a 35-mm cell culture dish at a cell density of 3.2 Â 10 5 cells per dish for 24 h. The cells were then treated with 100 mM WC-26 or 10 mM siramesine for 0, 4, 8 and 16 h. The cells were quickly rinsed with PBS twice at room temperature and then fixed with 1 ml of 2.5% glutaraldehyde in 0.01 M Na cacodylate buffer at 4 1C until use. After rinsing with PBS, fixed cells were sequentially stained with osmium tetroxide and uranyl acetate, and then dehydrated and embedded in overturned gelatin capsules containing Polybed 812 resin (Polysciences, Warrington, PA, USA). The resin blocks were thin sectioned at 90 -100 nm on a Reichert-Jung Ultracut microtome, post-stained in uranyl acetate and lead citrate, viewed on a Zeiss 902 Electron Microscope, and recorded with Kodak EM film.

Statistical analysis
The results are expressed as the mean±s.d. based on three independent experiments performed in triplicate. Differences among groups were statistically analysed by two-tailed Student's t-test. A P-value of o0.05 was considered significant.

Sigma-2 ligands induced cytotoxicity
The chemical structures of the four sigma-2 ligands are shown in Figure 1A. Using the MTS assay, dose -response curves were generated after exposing EMT-6 and MDA-MB-435 cells to increasing concentrations of WC-26, SV119, RHM-138 and siramesine for 24 or 48 h. Cell kill increased with increasing dose of and exposure time to the four sigma-2 ligands ( Figure 1B). All EC 50 s were in the micromolar range (Table 1).

Sigma-2 ligands induced caspase-3 activation
To assess if caspase-mediated apoptosis was involved in the cytotoxic pathway, caspase-3 activity was measured by a whole-cell assay described under Materials and Methods. The EMT-6 cells were treated with 40 mM WC-26, 40 mM SV119 or 40 mM RHM-138 for 24 h. The data showed that caspase-3 activation in treated cells increased by 7-, 2.5-and 2.5-fold, respectively, over activation measured in untreated control cells (Figure 2A). MDA-MB-435 cells were also treated for 24 h with 80 mM WC-26, 80 mM SV119 or 50 mM RHM-138, and caspase-3 activities were shown to increase by 4.5-, 3.5-and 3-fold, respectively ( Figure 2A).
To determine whether caspases are responsible for the sigma-2 ligand-induced apoptosis and cell viability, the broad-spectrum caspase inhibitor, Z-VAD-FMK, was added to the culture medium 1 h before sigma-2 ligands. The effect of this inhibitor on sigma-2 ligand-induced DNA fragmentation was determined by TUNEL staining. The effect of the inhibitor on cell viability following treatment with sigma-2 ligands was determined by MTS assay and LDH assay. The results showed that DNA fragmentation induced by each of the three sigma-2 ligands was partially blocked by Z-VAD-FMK (100 mM) in EMT-6 cells ( Figure 3A). The decreased cell viability by the sigma-2 ligands was partially rescued by Z-VAD-FMK ( Figure 3B and C). These data suggest that caspase activation, to a certain extent, has a role in sigma-2 ligand-induced cell death. It is worth noting that Z-VAD-FMK partially blocked cell death induced by 20 mM siramesine but not by 10 mM siramesine ( Figure 3B), suggesting that the role of caspase in sigma-2 ligand-induced cell death is concentration-dependent.

Sigma-2 ligands induced autophagy
Excess levels of autophagy may be responsible for cell death (Platini et al, 2010). We therefore examined whether autophagy was involved in sigma-2 ligand-induced cell death. We first used transmission electron microscopy to examine the ultrastructure of MDA-MB-435 cells after treatment with 100 mM WC-26 or 10 mM siramesine. Compared with control cells (Figure 4A), 4-h treatment with 100 mM WC-26, the earliest time point examined, induced mitochondrial swelling ( Figure 4B, structure indicated by 1) and multilayer membrane structures ( Figure 4B, structures indicated by 2 and 3). Typical autophagy compartments, which have double membranes and partially degraded cytoplasmic material, were occasionally observed ( Figure 4C, structure 1). Siramesine at 10 mM also induced autophagy-like vacuoles ( Figure 4D). These ultrastructures suggest that autophagy may be induced by sigma-2 ligands. We further used western blot analysis to examine the processing of microtubule-associated protein light chain 3 (LC3), an autophagosome marker. Light chain 3 is expressed in most cell types as a full-length cytosolic protein, which exists in three isoforms (LC3A, LC3B and LC3C) and is proteolytically cleaved upon induction of autophagy, generating LC3-I. The carboxyterminal glycine of LC3-I then conjugates to phosphatidylethanolamine to generate processed LC3-II. Light chain 3-II is found on  Sigma-2 ligands induce tumour cell death C Zeng et al autophagosomes, where it has a role in both membrane fusion and selection of cargo for degradation . Western blot analysis demonstrated that LC3B-II was induced by all four sigma-2 ligands (100 mM WC-26, 100 mM SV119, 40 mM RHM-138 and 10 mM siramesine) over a 24-h period ( Figure 5). We also tested whether the mTOR signalling pathway is involved in sigma-2 ligand-induced autophagy. Western blot analysis showed that phosphorylation of p70S60 kinase and 4EBP-1, both of which are downstream effectors of mTORC1, were decreased by all four sigma-2 ligands, suggesting that the mTOR pathway is inhibited by sigma-2 ligands, which may in turn trigger autophagy ( Figure 5).

Sigma-2 ligands impaired the cell cycle in MDA-MB-435 cells
Deregulation of cell-cycle progression may cause cell death (Allan and Clarke, 2008;Clarke and Allan, 2009). Therefore, we examined the effects of sigma-2 ligands on the expression levels of all four major classes of cyclins (cyclin D1, E2, A and B1) and on the phosphorylation levels of Rb by western blot analysis. MDA-MB-435 cells were treated with 100 mM WC-26, 100 mM SV119, 40 mM RHM-138 or 10 mM siramesine for 0, 1, 2, 4, 8, 16 and 24 h. The data showed that all four sigma-2 ligands decreased the expression levels of cyclin D1 in a time-dependent manner ( Figure 6). In addition, the data showed that 100 mM WC-26 or 100 mM SV119 mainly decreased cyclin B1, E2 and phospho-Rb (pRb); 40 mM RHM-138 mainly decreased cyclin E2; and 10 mM siramesine mainly decreased cyclin B1 and pRb. It is well known that D-type cyclins are responsible for progression through G 1 phase; E-type cyclins for completion of G 1 phase and entry into S-phase; A-type cyclins for driving the transition from S-phase to M-phase (G 2 phase); and B-type cyclins for cell progression through M-phase (Malumbres and Barbacid, 2009). Our data suggest that all four sigma-2 ligands may block G 1 -phase progression by decreasing cyclin D1 expression. In addition, WC-26 and SV119 may block cell entry from G 1 to S-phase by decreasing cyclin E2, and block mitosis by decreasing cyclin B1; RHM-138 may block cell entry from G 1 to S phase by decreasing cyclin E2; and siramesine may block mitosis by decreasing cyclin B1. Collectively, our data suggest that all four sigma-2 ligands may impair the cell cycle in multiple phases (G 1 , S, G 2 and M), which may lead to apoptosis either directly or indirectly, as discussed below.

DISCUSSION
We studied the cell death mechanisms of three sigma-2 receptor ligands developed in our group and compared the results with the known sigma-2 agonist, siramesine. All four sigma-2 ligands induced cell death in EMT-6 and MDA-MB-435 cells. Our results indicated that (1) the apoptotic pathway is, in part, responsible for cell death; (2) the sigma-2 ligands induced autophagy; and (3) the sigma-2 ligands impaired cell-cycle progression. We first examined whether the sigma-2 ligands induced cell death through activating apoptosis. All three ligands induced Annexin-V-positive cells (Supplementary Figure 1), DNA fragmentation, caspase-3 activation and PARP-1 cleavage. In addition, at least one sigma-2 ligand, RHM-138, activated caspase-8 and 9 (Supplementary Figure 2). These results suggest that sigma-2 ligands induced apoptosis possibly through both intrinsic and extrinsic pathways. This conclusion is consistent with that reported from other laboratories Ostenfeld et al, 2005). Our data also showed that the broadspectrum caspase inhibitor, Z-VAD-FMK, partially inhibited DNA fragmentation and cytotoxicity of the sigma-2 ligands, suggesting that caspases are, in part, responsible for sigma-2 ligand (WC-26, SV119 and RHM-138)-induced cell death in EMT-6 and MDA-MB-435 cells. In previous studies, Crawford et al showed that the caspase inhibitors had no effect on sigma-2 ligand (CB-64D and CB-184) cytotoxicity or Annexin-V binding in human breast MCF-7 cells . Ostenfeld et al (2005) reported that caspase inhibitors failed to protect cells against siramesine-induced death in murine fibrosarcoma cells (WEHI-S) and MCF-7 cells. It appears that whether caspases are involved in sigma-2-induced cell death depends on the structure of the ligand, ligand concentration and tumour cell type. We examined whether sigma-2 ligands induced autophagy. Electron microscopic data showed that the sigma-2 ligands WC-26 and siramesine induced mitochondrial swelling and formation of multilayer membrane vacuoles ( Figure 4A -D). The sigma-2 ligands also increased the expression of the autophagosome marker LC3B, and decreased the downstream effectors of mTOR, p70S6K and 4EBP1 ( Figure 5). These results suggest that sigma-2 ligands induce autophagy most likely by inhibiting the mTOR pathway. These data are consistent with the previous report that siramesine induced autophagosome formation in MCF-7 cells (Ostenfeld et al, 2008). Autophagy has a dual role in cell survival and cell death. On one hand, autophagy is generally thought of as a survival mechanism through removal misfolded proteins and damaged organelles. On the other hand, excess levels of autophagy may lead to cell death (Platini et al, 2010). Ostenfeld et al (2008) showed that 3-methyladenine, an autophagy inhibitor, increased cytotoxicity induced by siramesine. They also showed that inhibition of autophagy by RNA interference-based depletion of the autophagy protein beclin 1 increased cell toxicity of siramesine. These data suggest that siramesine-induced autophagy is cytoprotective in MCF-7 cells and NIH3T3 cells. Studies of a variety of experimental systems indicate that whether autophagy is cytoprotective or cytotoxic is likely to be context-and cell typedependent (Platini et al, 2010). The role of our sigma-2 ligands, WC-26, SV119 and RHM-138, in autophagy in MDA-MB-435 cells needs to be further studied.
In the current work, we studied the effects of sigma-2 ligands on cell-cycle progression by examining the protein levels of the four major classes of cyclins. Our data showed that sigma-2 ligands altered cyclin protein expression levels and the phosphorylation  Figure 6). These data suggest that sigma-2 ligands may induce cell death by impairing cell-cycle progression. Regulation of cyclin levels has been extensively studied. Cyclin D1 protein is subject to transcriptional regulation and ubiquitinmediated proteolysis (Witzel et al, 2010). Unlike other cyclins, cyclin D1 is strongly dependent on extracellular mitogenic signals. For example, cyclin D1 is induced by the Ras-signalling pathway (Malumbres and Barbacid, 2001). It is possible that sigma-2 ligands decrease cyclin D1 by inhibiting the mitogenic signalling pathways or/and by increasing ubiquitin-dependent degradation processes. The Rb represses transcription by binding to transcription factors such as E2F-family members. D-type cyclins in complexes with CDK4 and/or CDK6 result in phosphorylation of Rb (Malumbres and Barbacid, 2005). This liberates E2F transcription factors and promotes the subsequent transcription of many important proteins such as cyclin E. In the present study, we show that the phosphorylation levels of Rb are decreased by WC-26, SV119 and siramesine. This could result from cyclin D1 reduction upon treatment with these ligands. The E-type cyclins are regulated transcriptionally by the phosphorylation status of Rb as well as by ubiquitin-dependent degradation mechanisms (Musgrove, 2006;Caldon and Musgrove, 2010). Our data show that WC-26 and SV119 decrease cyclin E2. This is probably due to reduction of phospho-Rb. By contrast, siramesine does not reduce cyclin E2. One possible reason is that siramesine decreases cyclin E2 by reducing phospho-Rb, but at the same time increases cyclin E2 by inhibiting its degradation. The net result is no change of cyclin E2 expression. A-and B-type cyclins are also regulated at the transcriptional level and by ubiquitin-mediated proteolysis (Miyazaki and Arai, 2007). The current study shows that WC-26, SV119 and siramesine caused a dramatic decrease in cyclin B1 expression. This could result from decreasing the transcription and/or increasing the ubiquitin-dependent degradation processes.
Taking these data together, the sigma-2 ligands decrease multiple cyclin protein levels. Decreasing cyclin levels has been proposed as a therapeutic strategy for cancer treatment (Freemantle et al, 2007). Our findings suggest that the sigma-2 ligands may serve as promising antitumour drugs by disrupting cell-cycle progression. Sigma-2 ligands induce apoptosis, autophagy and cell-cycle impairment. These pathways are inter-related. Autophagy and cellcycle arrest are known adaptive responses of cells to toxic insults (Clarke and Allan, 2009;Wyllie, 2010). Cells can remove the misfolded proteins and damaged organelles by autophagy. Cells can also activate cell-cycle checkpoints, which provide an opportunity to repair the damage. It is possible that upon sigma-2 ligand treatment, cells initiate autophagy and cell-cycle arrest to protect themselves from the toxic effects of the ligands. After these mechanisms fail to rescue the cells, they switch to facilitate cell death. Understanding the turning points where adaptive responses become contributors to toxicity is important for designing effective cancer therapeutic interventions. The PI3K/Akt/mTOR pathway is well known as one of the key molecular switches between apoptosis and autophagy (Moretti et al, 2007;Wyllie, 2010;Li et al, 2011). Our data suggest that sigma-2 ligands increase autophagy and apoptosis by suppressing the mTOR pathway. In addition, recent studies have revealed that the cell-cycle components can be directly involved in apoptosis. It is reported that CDK1 -cyclin B1 is a crucial regulator of not only mitosis but also apoptosis (Clarke and Allan, 2009). Cyclin-dependent kinase-1 -cyclin B1 phosphorylates the inhibitory site on caspase-9 (Thr125) during mitosis (Allan and Clarke, 2007). Phosphorylation of this site restrains caspase-9 activation and apoptosis. It is possible that the decrease in cyclin B1 upon treatment of the sigma-2 ligands reduces the phosphorylation level of caspase-9 at the inhibitory site and thus activates caspase-9 and apoptosis.  Previously we have shown that the sigma-2 fluorescent ligands rapidly internalise into cells and localise in the mitochondria, lysosomes, endoplasmic reticulum and plasma membranes (Zeng et al, 2007. It is possible that sigma-2 ligands trigger multiple signalling pathways by binding to sigma-2 receptors located in multiple cytoplasmic organelles and plasma membranes. For example, sigma-2 ligands may bind to mitochondria and damage their structure and function, as shown by mitochondrial swelling (Figure 4B and C), and trigger caspase-3-dependent orindependent apoptosis Ostenfeld et al, 2005). Sigma-2 ligands may bind to lysosomes, and cause lysosomal leakage and cell death as demonstrated previously by Ostenfeld et al (2005). The damage to cytoplasmic organelles by sigma-2 ligands may trigger autophagy, which may have either a survival or a detrimental role depending on the structure of the drugs and the tumour cell type.
Positron emission tomography imaging in rodent (Mach et al, 2001a;Kawamura et al, 2003) and human studies (unpublished data) has demonstrated that sigma-2 ligands selectively target tumours in vivo. Sigma-2 receptor ligands have shown efficacy in various tumour cells using cell culture and animal models (Ostenfeld et al, 2005;Kashiwagi et al, 2007Kashiwagi et al, , 2009) by multiple signalling pathways. These characteristics make sigma-2 ligands promising chemotherapeutic drugs for treating a variety of tumours.