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

Abstract

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.

Figure 1

Sigma-2 ligands decreased viability in EMT-6 and MDA-MB-435 cells. (A) Chemical structures of the sigma-2 ligands. (B) EMT-6 or MDA-MB-435 cells were treated with increasing concentrations of the sigma-2 ligands WC-26, SV119, RHM-138 and siramesine for 48 h. Cell viability was determined by MTS assay. The bars represent the mean±s.e.m. of at least three independent experiments.

Figure 2

Sigma-2 ligands induced caspase-3 activation. (A) EMT-6 and MDA-MB-435 cells were treated for 24 h with the sigma-2 ligands at concentrations that resulted in the highest level of caspase-3 activation (40 μM WC-26, 40 μM SV119 or 40 μM RHM-138 for EMT-6 cells; 80 μM WC-26, 80 μM SV119 or 50 μM RHM-138 for MDA-MB-435 cells). Caspase-3 activation was determined by the CellProbe HT caspase-3 whole-cell assay. ^*P<0.001 compared with untreated control. The bars indicate the mean±s.d. of the representative data of at least three independent experiments. (B and C) Sigma-2 ligands induced procaspase-3 and PARP-1 cleavage as shown by western blot analysis in EMT-6 cells (B) and MDA-MB-435 cells (C). EMT-6 cells were treated for 0–24 h prior to assay with WC-26 (40 μM), SV119 (100 μM) or RHM-138 (40 μM). MDA-MB-435 cells were treated for 0–24 h with WC-26 (80 μM), SV119 (100 μM) or RHM-138 (40 μM).

Figure 3

The broad-spectrum caspase inhibitor, Z-VAD-FMK, partially blocked DNA fragmentation, viability and cytotoxicity induced by sigma-2 ligands. (A) EMT-6 cells were pre-treated for 1 h with Z-VAD-FMK (100 μM), and then treated with WC-26 (40 μM) for 48 h, SV119 (100 μM) for 16 h or RHM-138 (40 μM) for 16 h. The percentage of TUNEL-positive cells was analysed by flow cytometry (^*P⩽0.001 compared with the no inhibitor control). (B and C) MDA-MB-435 cells were pre-treated for 1 h with Z-VAD-FMK (100 μM), and then treated with WC-26 (100 μM), SV119 (100 μM), RHM-138 (100 μM) or siramesine (10 or 20 μM) for 18 h. Viability of the cells was measured by MTS assay (B). Cytotoxicity to the cells was measured by LDH assay. (^*P<0.05 compared with the no inhibitor control). The bars represent the mean±s.d. of the representative data of at least three independent experiments.

Figure 4

WC-26 and siramesine induced the formation of autophagosomes in MDA-MB-435 cells studied by transmission electron microscopy. (A) Cells without any treatment showed normal mitochondria ultrastructure (arrows). (B) Treatment with 100 μM WC-26 for 4 h induced mitochondrial swelling (indicated by 1), multilayer membrane compartments containing mitochondria and other cytoplasm contents (indicated by 2), and various kinds of multilayer membrane structures (indicated by 3). (C) Treatment with 100 μM WC-26 for 8 h induced autophagic vacuoles (indicated by 1) and mitochondrial swelling (indicated by 2). (D) Treatment with 10 μM siramesine for 16 h induced the formation of various vacuoles.

Figure 5

Sigma-2 ligands induced autophagy in MDA-MB-435 cells. Cells were treated with WC-26 (100 μM), SV119 (100 μM), RHM-138 (40 μM) or siramesine (10 μM) for 0–24 h. Autophagosome markers, LC3B and the downstream effectors of mTOR, p70S6K and 4EBP1, were analysed by western blotting.

Figure 6

Sigma-2 ligands impaired cell-cycle progression in MDA-MB-435 cells. Cells were treated with WC-26 (100 μM), SV119 (100 μM), RHM-138 (40 μM) or siramesine (10 μM) for 0–24 h. The expression levels of cyclin D1, pRb, cyclin E2, cyclin A and cyclin B1were analysed by western blotting.