| Eicosapentaenoic acid (EPA) has been demonstrated to induce apoptosis and cell cycle arrest in various cancer lines in vitro. In this study, we investigated the anti-tumor effects of EPA on hepatoma cell lines and the potential mechanisms of induction of apoptosis.; The three hepatoma cell lines tested have different p53 status: HepG2 has wild-type (wt) p53, Hep3B has its endogenous p53 deleted, and Huh7 has a mutated p53. Tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay showed that the viability of HepG2 cells was significantly decreased after exposure to EPA, and the cytotoxicity of EPA was time- and dose-dependent. Both Fas and Fas ligand (Fas-L) mRNA expression in HepG2 cells were up-regulated after exposure to EPA. On the other hand, an increase in the Fas-L mRNA expression in Hep3B and Huh7 cells was not observed after exposure to EPA. Most notably, EPA-induced apoptosis in HepG2 cells could be inhibited almost completely by Fas-L antisense oligonucleotide.; To have a better understanding of the function of wt p53 in human hepatocellular carcinoma (HCC), we have made an attempt to establish a cell line which has inducible and double-stable expression of wt p53 with a retrovirus-mediated Tet-Off system in Hep3B cell line (of which the p53 gene is deleted).; We then utilized the established high p53 producer, Hep3B/tet/p53-13 cell line, to further explore the function of wt p53 by controlling the “on or off” status or different expression levels.; Interestingly, Hep3B/tet/p53-15 cell line was extremely different from Hep3B/tet/p53-13 cell line in its responses to the induction of wt p53. We observed that the induced levels of wt p53 could dictate the response of the cells in such a way that a higher level of wt p53 expression would result in apoptosis whereas a lower level of wt p53 would only result in cell cycle arrest without apoptosis. Additionally, induction of p53 could up-regulate the expression of p21WAF1 in Hep3B/tet/p53-13 cells and Hep3B/tet/p53-15 cells.; In the last part of this study, we attempted to gain more insight into the molecular mechanism of EPA-induced cell killing by analyzing the apoptotic pathway with the established Hep3B/tet/p53-15 cell line. Our results showed that EPA resulted in significant loss of cell viability and induction of apoptotic death in the expressing-p53 cells, as evidenced by MTT assay, acridine orange (AO) staining and DNA fragmentation analysis.; Hep3B/tet/p53 cell lines are very useful tool for research into the function of p53 and the application of p53 as a possible target for gene therapy. (Abstract shortened by UMI.)... |
