title: Lysosomal membrane permeabilization is an early event in Sigma-2 receptor ligand mediated cell death in pancreatic cancer.
abstract: BACKGROUND
  Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer.
  
  
  RESULTS
  Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43.
  
  
  CONCLUSIONS
  Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.
authors.affiliation: Department of Surgery, Washington University School of Medicine, S, Euclid Avenue, St, Louis, MO, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Hornick
authors.forename: Suwanna
authors.initials: S
authors.lastname: Vangveravong
authors.forename: Dirk
authors.initials: D
authors.lastname: Spitzer
authors.forename: Carmen
authors.initials: C
authors.lastname: Abate
authors.forename: Francesco
authors.initials: F
authors.lastname: Berardi
authors.forename: Peter
authors.initials: P
authors.lastname: Goedegebuure
authors.forename: Robert H
authors.initials: RH
authors.lastname: Mach
authors.forename: William G
authors.initials: WG
authors.lastname: Hawkins
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D005260:Female; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D018613:Microscopy, Confocal; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism; D017480:Receptors, sigma / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D008024:Ligands; D017382:Reactive Oxygen Species; D017480:Receptors, sigma; C106107:sigma-2 receptor; D053148:Caspase 3