G2/M cell-cycle arrest and apoptosis by n-3 fatty acids in a pancreatic cancer model.
BACKGROUND
n-3 fatty acids (n-3FA) have anti-inflammatory and anti-proliferative effects including modulation of pro-inflammatory cascade mediators and cytokine elaboration (i.e., TNF-alpha, IL-10 and PGE(2)) in many cell lines. However, mechanisms of anti-proliferative effects have not been clearly defined.


METHODS
MIA PaCa-2 pancreatic cancer cells were treated either with n-3FA (treatment), media (control), or n-6FA (control) for all experiments. Cellular proliferation was evaluated with WST-1 reagent. Cells were stained with propidium iodide and analyzed by flow cytometry for cell-cycle arrest, which was further analyzed by cdc2 expression. Membrane and media lipid concentrations were analyzed by high-performance liquid chromatography. Apoptosis was evaluated by AnnexinV-FITC flow cytometry and reconfirmed by poly (ADP-ribose) polymerase (PARP) cleavage and B(cl)-2 expression.


RESULTS
Propidium iodide flow cytometry of MIA PaCa-2 dosed with n-3FA showed a decrease in cells in G1 phase (11-17%) and an increase cells in G2 phase (7-13%) from controls. cdc2 expression was also decreased at 24 h compared to controls. Annexin-V staining of n-3FA-treated cells demonstrated time-dependent increased apoptosis and PARP cleavage was present only in the n-3FA treatment group. Phospho-B(cl)-2 was also decreased in the n-3FA-treated cells compared to controls.


CONCLUSIONS
Co-incubation of MIA PaCa-2 cells with n-3FA results in both dose- and time-dependent cell-cycle arrest. Cells also progress to cell death via apoptosis. These data support the potential applicability for n-3FA as an antiproliferative and pro-apoptotic strategy.
Dekoj
Thomas
T
Department of Surgery, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Lee
Sang
S
Desai
Sagun
S
Trevino
Jose
J
Babcock
Tricia A
TA
Helton
W Scott
WS
Espat
N Joseph
NJ
D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000032:analysis; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D015525:Fatty Acids, Omega-3 / Q000494:pharmacology*; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D008563:Membrane Lipids / Q000032:analysis; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis
D017304:Annexin A5; D015525:Fatty Acids, Omega-3; D008563:Membrane Lipids; D019253:Proto-Oncogene Proteins c-bcl-2; D011065:Poly(ADP-ribose) Polymerases; D016203:CDC2 Protein Kinase