Background: Conjugated eicosapentaenoic acid (cEPA) containing conjugated double bonds, which is prepared by alkaline treatment of eicosapentaenoic acid (EPA), selectively inhibited the activities of both mammalian DNA polymerases (pols) and human DNA topoisomerases (topos).
Methods: Human colon carcinoma cell line, HCT116, was cultured and performed drug and small interfering RNA (siRNA) treatment, flow cytometry analysis, BrdU incorporation analysis, and western blot analysis.
Results: The levels of bromodeoxyuridine (BrdU) incorporation labeling during DNA synthesis were decreased in time- and dose-dependent manners in HCT116 cells, treated with cEPA. The level of chromatin association of RPA70, a subunit of the single-stranded DNA (ssDNA)-binding protein, was increased following cEPA exposure, suggesting that the replication forks were stalled in response to inhibition of replicative pol activity by cEPA in the cells. cEPA also induced the activation of ataxia-telangiectasia and Rad3-related (ATR) protein in HCT116 cells, and activated the G1 checkpoint pathway in the cells, which was down-regulated by a small interfering RNA (siRNA) against ATR protein. Moreover, caffeine, a known ATR kinase inhibitor, abrogated the cEPA-induced G1 checkpoint in HCT116 cells.
General significance: cEPA could inhibit the activity of replicative pols, such as pols alpha, delta and epsilon, affect the DNA replication fork including ssDNA, and then activate the G1 checkpoint pathway by the induction of RPA and ATR expression levels in cancer cells.