Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands

Over-expression of sigma receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential sigma-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the sigma(1) and sigma(2) receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of sigma receptors, both compounds inhibited cell proliferation with IC50 values in the low mu M range. No chiral differentiation between either the sigma receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl(2) family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant alpha-tocopherol attenuated LPO. Interestingly, alpha-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells. (C) 2013 Elsevier Ltd. All rights reserved.