TY - JOUR
T1 - Downregulation of neutral ceramidase by gemcitabine
T2 - Implications for cell cycle regulation
AU - Wu, Bill X.
AU - Zeidan, Youssef H.
AU - Hannun, Yusuf A.
N1 - Funding Information:
This study was supported by NIH grant CA87584 and GM43825. We would like to thank the Lipidomics Core at the Medical University of South Carolina for expert assistance.
PY - 2009/8
Y1 - 2009/8
N2 - 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.
AB - 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.
KW - Cell cycle arrest
KW - Ceramide
KW - Gemcitabine
KW - Neutral ceramidase
KW - Sphingolipid
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U2 - 10.1016/j.bbalip.2009.03.012
DO - 10.1016/j.bbalip.2009.03.012
M3 - Article
C2 - 19345744
AN - SCOPUS:67650239637
VL - 1791
SP - 730
EP - 739
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
SN - 1388-1981
IS - 8
ER -