p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells
Introduction
Ellagic acid has been demonstrated [1], [2], [3], [4], [5] in animal models to inhibit tumor growth induced by several chemical carcinogens, including polycyclic aromatic hydrocarbons, N-nitrosamines, aflatoxins and aromatic amines. Other studies [6], [7] have demonstrated that ellagic acid, applied topically to mouse skin, could effectively inhibit TPA-induced ornithine decarboxylase activity, hydroperoxide production and DNA synthesis. In addition to earlier reports, recent studies have shown that oral administration of ellagic acid significantly reduced the level of lipid peroxidase and liver dihydroxy proline in animal models [8], [9]. The latter study [9] also indicated that oral administration of ellagic acid can circumvent carbon tetrachloride toxicity and subsequent lung fibrosis. Other than the few mentioned pharmacokinetic studies, the mechanisms of action of ellagic acid at the cellular and molecular level are still unclear. The focus of the present study was to understand the effect of ellagic acid on the cell cycle, DNA synthesis, apoptosis and overall cell proliferation inhibition. The cervical carcinoma cell line (CaSki) which carries the HPV-16 virus, whose E6 gene product complexes with wild type p53 causing loss of p53 function, was chosen to study the mechanisms of action of ellagic acid. To investigate whether the action of ellagic acid is exerted through modulation of the cell cycle and to test the functional state of p53, we analyzed the expression of p53 and p21 mRNA in ellagic acid treated and untreated CaSki cells in a time- and dose-dependent manner.
Section snippets
Ellagic acid purification
Ellagic acid purchased from Aldrich Chemicals was purified further by crystallization from ethanol until a purity of 99% was achieved using high-performance liquid chromatography (HPLC) (Fig. 1, Fig. 2). The highest concentration (10−3 M) of ellagic acid was made in DMSO American Type Culture Collection (ATCC) and lower concentrations (10−5–10−9 M) were made with DMSO. After confirming the purity of different dilutions of ellagic acid through HPLC, they were aliquoted, protected from light and
G1 arrest in ellagic acid treated cells
Cell cycle distribution analysis of ellagic acid treated (10−5 M) CaSki cells showed a minor G1 arrest at 24 h as determined by flow cytometry. Cell cycle analysis after 48 h of exposure to ellagic acid, however, showed an enhanced G1 peak. Such a cell cycle arrest was also observed at 48 h as a greatly reduced number of cells in the S phase. A pre-G1 apoptotic peak (31.8%) was very clear with an overlap of G1 phase cells after 72 h (Figs. 3A–D). The percentage of cells in S phase was observed
Discussion
Plant-derived ellagic acid, a phenolic compound found in raspberries and other plant food, has previously been identified as a potent anticarcinogenic agent [1], [2], [3], [4], [5], [6], [7], [8], [9]. In this article we have shown an effect of ellagic acid on cell proliferation inhibition, cell cycle arrest and induction of apoptosis in a time- and dose-dependent manner in cervical carcinoma cells (CaSki). CaSki cells were found to be sensitive to the effect of ellagic acid in inducing G1
Acknowledgements
We would like to express our sincere appreciation to Dr. Clifford Schweinfest (CMSB, HCC) for critical review of the data. We would also like to thank Dr. Sally Self and Cary Wiggins for assistance with the flow cytometry analysis.
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Present address: Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.