Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation

Bill X. Wu, Youssef Zeidan, Yusuf A. Hannun

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Gemcitabine (GMZ) is a chemotherapeutic agent with well established effects on cell growth arrest and apoptosis. In this study, we investigated the potential roles of bioactive sphingolipids in mediating the growth suppressing effects of GMZ on a polyoma middle T transformed murine endothelial cell line. After 12-hour GMZ (0.6 μM) treatment, cell growth was arrested at the G0/G1 phase as detected by flow cytometric cell cycle analysis and MTT cell viability analysis, and this was accompanied by dephosphorylation of the retinoblastoma protein (Rb). Furthermore, GMZ treatment resulted in increased levels of specifically the very long chain ceramides as determined by mass spectrometry. Mechanistically, GMZ did not appear to affect the activities of many enzymes of ceramide metabolism; however, GMZ caused a selective reduction in the protein levels of neutral ceramidase (NCDase), as indicated by Western blot analysis, with a concomitant decrease in NCDase activity. The significance of NCDase loss on cell cycle regulation was investigated by specific knockdown of the enzyme using small interfering RNA (siRNA). Interestingly, NCDase siRNA transfection was sufficient to induce a cell cycle arrest at G0/G1 and an increase in total ceramide levels, with significant elevation in very long chain ceramides (C24:1 and C24:0). NCDase siRNA also induced Rb dephosphorylation. These data provide evidence for a novel mechanism of action for GMZ and highlight downregulation of NCDase as a critical step in GMZ-mediated ceramide elevation and cell cycle arrest.

Original languageEnglish (US)
Pages (from-to)730-739
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1791
Issue number8
DOIs
StatePublished - Aug 2009
Externally publishedYes

Fingerprint

gemcitabine
Neutral Ceramidase
Ceramides
Cell Cycle
Down-Regulation
Small Interfering RNA
Cell Cycle Checkpoints
Growth
Retinoblastoma Protein
Cell Cycle Resting Phase
Sphingolipids
G1 Phase
Enzymes
Transfection
Mass Spectrometry
Cell Survival
Endothelial Cells
Western Blotting
Apoptosis
Cell Line

Keywords

  • Cell cycle arrest
  • Ceramide
  • Gemcitabine
  • Neutral ceramidase
  • Sphingolipid

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Downregulation of neutral ceramidase by gemcitabine : Implications for cell cycle regulation. / Wu, Bill X.; Zeidan, Youssef; Hannun, Yusuf A.

In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Vol. 1791, No. 8, 08.2009, p. 730-739.

Research output: Contribution to journalArticle

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