title: Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands.
abstract: Over-expression of σ 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 σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ 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 bcl₂ 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 α-tocopherol attenuated LPO. Interestingly, α-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.
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald, F.-L.-Jahn-Straße 17, 17487 Greifswald, Germany. Electronic address: korpis@uni-greifswald.de.
authors: Katharina
authors: K
authors: Korpis
authors: Institute of Pharmaceutical and Medicinal Chemistry, University of Münster Corrensstraße 48, 48149 Münster, Germany. Electronic address: fwebe_01@uni-muenster.de.
authors: Frauke
authors: F
authors: Weber
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald, F.-L.-Jahn-Straße 17, 17487 Greifswald, Germany. Electronic address: sbrun_02@uni-muenster.de.
authors: Stefanie
authors: S
authors: Brune
authors: Institute of Pharmaceutical and Medicinal Chemistry, University of Münster Corrensstraße 48, 48149 Münster, Germany. Electronic address: wuensch@uni-muenster.de.
authors: Bernhard
authors: B
authors: Wünsch
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald, F.-L.-Jahn-Straße 17, 17487 Greifswald, Germany. Electronic address: bednarsk@uni-greifswald.de.
authors: Patrick J
authors: PJ
authors: Bednarski
mesh_terms: D017209:Apoptosis; D001343:Autophagy / Q000187:drug effects*; D049109:Cell Proliferation; D006801:Humans; D008024:Ligands; D015227:Lipid Peroxidation; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D008024:Ligands
keywords: 2′,7′-dichlorofluorescein diacetate; 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; 3-MA; 3-methyladenine; 4,6-diamino-2-phenylindole; 4-difluoro-3,5-bis(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene; AI; Apoptosis; Autophagy; B(max); BODIPY 665/676 C(11); Cancer; DAPI; DCFDA; DMSO; DTG; Dulbeccos phosphate buffered saline; IC(50); K(d); K(i); LC3B; LPO; Lipid peroxidation; MMP; MTT; PARP; PBS; PI; PI3K; ROS; RPMI 8226 cells; SD; SEM; Sigma-receptor; TBH; apoptosis inhibitor; dimethylsulfoxide; ditolylguanidine; half maximal inhibitory drug concentration; inhibitory dissociation constant; ligand dissociation constant; maximal ligand binding sites; microtubule-associated protein light chain 3; mitochondrial membrane potential; phosphatidylinositol 3-kinase; poly(ADP-ribose) polymerase; propidium iodide; reactive oxygen species; standard deviation; standard error of mean; tertiary-butyl-hydroperoxide; z-VAD-FMK; z-Val-Ala-Asp(Ome)-CH(2)F