Sigma-2 receptor agonists activate a novel apoptotic pathway and potentiate antineoplastic drugs in breast tumor cell lines

We have reported previously that sigma-2 receptors are expressed in high densities in a variety of tumor cell types (B. J. Vilner et al., Cancer Res., 55: 408-413, 1995) and that various sigma ligands have cytotoxic effects (B. J. Vilner et al., J. Neurosci., 15: 117-134, 1995). Other investigators have demonstrated increased expression of sigma-2 receptors in rapidly proliferating tumors (R. H. Mach et al., Cancer Res., 57: 156-161, 1997) and the ability of some sigma ligands to inhibit proliferation (P. J. Brent and G. T. Pang, Eur. J. Pharmacol., 278: 151-160, 1995). We demonstrate here the ability of sigma-2 receptor agonists to induce cell death by a mechanism consistent with apoptosis. In breast tumor cell lines that are sensitive (MCF-7) and resistant (MCF-7/Adr-, T47D, and SKBr3) to antineoplastic agents, incubation with the sigma-2 subtype-selective agonists CB-64D and CB-184 produced dose-dependent cytotoxicity (measured by lactate dehydrogenase release into medium). The EC₅₀ for this response was similar across cell lines, irrespective of p53 genotype and drug-resistance phenotype. CB-64D and the subtype nonselective sigma-2 agonists haloperidol and reduced haloperidol induced terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining in MCF-7 and T47D cells, indicating that cell death occurs via apoptosis. Apoptosis was also indicated by increases in Annexin V binding caused by CB-64D. In MCF-7 cells, cytotoxicity and Annexin V binding induced by the antineoplastics doxorubicin and actinomycin D was partially or completely abrogated by certain specific and general inhibitors of caspases. In contrast, caspase inhibitors had no effect on sigma-2 receptor-mediated (CB-64D and CB-184) cytotoxicity or Annexin V binding. Marked potentiation of cytotoxicity was observed when a subtoxic dose of CB-184 was combined with doxorubicin or actinomycin D, both in drug-sensitive (MCF-7) and drug-resistant (MCF-7/Adr-) cell lines. Haloperidol potentiated doxorubicin only in drug-resistant cells. These findings suggest the involvement of a novel p53- and caspase-independent apoptotic pathway used by sigma-2 receptors, which is distinct from mechanisms used by some DNA-damaging, antineoplastic agents and other apoptotic stimuli. These observations further suggest that sigma-2 receptors may be targets that can be therapeutically exploited in the treatment of both drug-sensitive and drug-resistant metastatic tumors.