title: Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells.
abstract: OBJECTIVE
  Acid ceramidase (AC) occupies an important place in the control of cancer cell proliferation. We tested the influence of AC inhibition on the effects of PSC 833, a P-glycoprotein antagonist with potent ceramide-generating capacity, to determine whether AC could be a therapeutic target in pancreatic cancer.
  
  
  METHODS
  Ceramide metabolism was followed using (3)H-palmitate, and molecular species were determined by mass spectroscopy. Apoptosis was measured by DNA fragmentation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphorylation. AC was measured in intact cells using fluorescent substrate.
  
  
  RESULTS
  Exposure of human PANC-1 or MIA-PaCa-2 cells to PSC 833 promoted increases in de novo (dihydro)ceramides, (dihydro)glucosylceramides, and (dihydro)sphingomyelins, demarking ceramide generation and robust metabolism. Despite the multifold increases in (dihydro)ceramide levels, cells were refractory to PSC 833. However, PSC 833 produced a dose-dependent decrease in DNA synthesis and dose- and time-dependent decreases in RB phosphorylation, consistent with cell cycle arrest as demonstrated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotoxicity was accompanied by formation of acridine orange-stained acidic vesicles and an increase in LC3 expression, indicative of autophagic response. Cell death was not reversed by preexposure to myriocin, which blocks PSC 833-induced ceramide generation.
  
  
  CONCLUSIONS
  Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid ceramidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies.
authors.affiliation: Department of Experimental Therapeutics, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404, USA.
authors.forename: Samy A F
authors.initials: SA
authors.lastname: Morad
authors.forename: Maria C
authors.initials: MC
authors.lastname: Messner
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Levin
authors.forename: Noha
authors.initials: N
authors.lastname: Abdelmageed
authors.forename: Hyejung
authors.initials: H
authors.lastname: Park
authors.forename: Alfred H
authors.initials: AH
authors.lastname: Merrill
authors.forename: Myles C
authors.initials: MC
authors.lastname: Cabot
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors; D055573:Acid Ceramidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000165:Acridine Orange; D000577:Amides / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D002855:Chromatography, Thin Layer; D003524:Cyclosporins / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D023381:Endpoint Determination; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D013058:Mass Spectrometry; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D013107:Sphingolipids / Q000378:metabolism
chemical_list: C559741:(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000577:Amides; D000970:Antineoplastic Agents; D003524:Cyclosporins; D004273:DNA, Neoplasm; D005231:Fatty Acids, Unsaturated; D013107:Sphingolipids; D055573:Acid Ceramidase; D000165:Acridine Orange; C070272:valspodar