ORIGINAL ARTICLE Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells Samy A. F. Morad • Maria C. Messner • Jonathan C. Levin • Noha Abdelmageed • Hyejung Park • Alfred H. Merrill Jr. • Myles C. Cabot Received: 13 July 2012 / Accepted: 4 December 2012 / Published online: 21 December 2012 Ó Springer-Verlag Berlin Heidelberg 2012 Abstract Purpose 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 fragmen- tation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphor- ylation. 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)sphin- gomyelins, 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 demon- strated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotox- icity 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. Conclusion Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid cer- amidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies. Keywords Acid ceramidase Á Pancreatic cancer Á PSC 833 Á Ceramide Á Sphingolipid Abbreviations DHCer Dihydroceramide DHGlcCer Dihydroglucosylceramide DHSM Dihydrosphingomyelin dThd [ 3 H]thymidine FBS Fetal bovine serum GlcCer Glucosylceramide LC MS/MS Liquid chromatography/electrospray tandem mass spectrometry LSC Liquid scintillation counting MDR-1 Multidrug resistant-1 MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt P-gp P-glycoprotein Samy A. F. Morad and Maria C. Messner contributed equally to this work. S. A. F. Morad Á M. C. Messner Á J. C. Levin Á N. Abdelmageed Á M. C. Cabot (&) Department of Experimental Therapeutics, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404, USA e-mail: cabot@jwci.org S. A. F. Morad Department of Pharmacology, South Valley University, Qena, Egypt H. Park Á A. H. Merrill Jr. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA 123 Cancer Chemother Pharmacol (2013) 71:635–645 DOI 10.1007/s00280-012-2050-4