Several mechanisms have been proposed for apigenin's effect on cell growth including modulation of the MAP kinase pathway [20, 21], inhibition of ornithine decarboxylase , improvement in cell-cell communication through increased gap junctions , and cell cycle arrest. Cell-cycle checkpoints at G2/M as well as G1/S are critical in maintaining DNA integrity and regulating the passage of cells through the cell cycle. It is well known that loss of these checkpoints is involved in the transformation into and progression of cancer cells. A protein kinase complex consisting of a catalytic subunit, cdc2, and the cyclin B protein performs the central and rate-limiting function in the transition from G2 to M phase . The cdc2/cyclin B complex responds to DNA damage and causes a delay in cell cycle progression to allow DNA repair before cells enter mitosis. The complex accomplishes this through phosphorylation of cytoskeleton proteins such as lamins and histone H1 . Cdc2 binding to cyclin B in and of itself is not enough, however, for checkpoint progression. Dephosphorylation of cdc2 at the Tyr-15 site through cdc25c phosphatase is required for activation of the complex [24, 26]. Cdc25A also contributes to the cellular phosphatase pool required to dephosphorylate cdc2 fully . Cyclin A, known mainly for its role in G1/S transition, is also required for the entry of cells into mitosis [28, 29].
In the current study, treatment of human pancreatic cancer cells with apigenin resulted in inhibition of DNA synthesis and cell proliferation through G2/M cell cycle arrest. A decrease in levels of cyclin A, cyclin B, cdc25 A and cdc25C all appear to be involved. It is not clear why levels of phosphorylated cdc2 were decreased given that reduced levels of cdc25c should have the opposite effect, however, the reduced levels of cyclin B appear to be the main mechanism involved in the arrest of cells at G2/M.
Our results are congruent with previous studies in which cell cycle arrest has been shown to result from apigenin treatment in other cell lines. G2/M arrest through inhibition of cyclin B associated cdc2 has been shown to occur in epidermal and fibroblast cells [8, 30], breast cancer cells , oral cancer cells , melanoma , mouse keratinocytes , endothelial cells , prostate cancer cells , and colon cancer cell lines . Interestingly, inhibition of cyclin B appears to be concentration-related as lower doses have less effect while higher doses (>70 μM) as we used in our study decrease cyclin B levels .
A considerable amount of attention has been given to the role of p53 in cell cycle checkpoint control. P53 blocks cells at G2/M through direct inhibition of cdc2 kinase . All four cell lines used in this study contain mutated p53, which implies that the G2/M arrest seen after apigenin treatment is through a p53-independent pathway. Other studies have found this to be the case as well. Apigenin was found to inhibit growth in both p53 wild-type and mutated breast cancer cells , p53 mutated oral squamous cell cancer cells , and mutated colon cancer cell lines .