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Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells

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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 3H-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.

Conclusion

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.

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Abbreviations

DHCer:

Dihydroceramide

DHGlcCer:

Dihydroglucosylceramide

DHSM:

Dihydrosphingomyelin

dThd:

[3H]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

PI:

Propidium iodide

S1-P:

Sphingosine 1-phosphate

SM:

Sphingomyelin

SPT:

Serine palmitoyltransferase

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Acknowledgments

This work was supported by the National Cancer Institute (CA143755), the National Institute of General Medical Sciences (GM77391), and NIH GM069338 (Lipid Maps). We thank Matthew Bush for compiling the typescript and for creating the figures.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Experimental Therapeutics, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA, 90404, USA

    Samy A. F. Morad, Maria C. Messner, Jonathan C. Levin, Noha Abdelmageed & Myles C. Cabot

  2. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA

    Hyejung Park & Alfred H. Merrill Jr.

  3. Department of Pharmacology, South Valley University, Qena, Egypt

    Samy A. F. Morad

Authors
  1. Samy A. F. Morad
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  2. Maria C. Messner
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  3. Jonathan C. Levin
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  4. Noha Abdelmageed
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  5. Hyejung Park
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  6. Alfred H. Merrill Jr.
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  7. Myles C. Cabot
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Correspondence to Myles C. Cabot.

Additional information

Samy A. F. Morad and Maria C. Messner contributed equally to this work.

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Morad, S.A.F., Messner, M.C., Levin, J.C. et al. Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells. Cancer Chemother Pharmacol 71, 635–645 (2013). https://doi.org/10.1007/s00280-012-2050-4

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