Deregulation of spindle checkpoint signaling is one potential source for chromosomal instability and aneuploidy found in many human primary tumors and tumor cell lines (2
). In an effort to find small molecules with anti-spindle checkpoint activity, we executed a newly designed HTS based on different cell-to-substrate attachment properties of round mitotic and flat interphase cells (7
). The discovery of the dietary flavonoid fisetin as a drug that overcomes chemically induced mitotic block provides a proof-of-principle for the screening strategy. Our results show that fisetin interferes with kinetochore accumulation of several key mitotic proteins and inhibits Aurora B kinase in cultured cells and in vitro
. As Aurora B activity has been shown to be required for the maintenance of spindle checkpoint signaling, we propose that fisetin may exerts its antimitotic activity, at least partially, through inhibition of Aurora B. It also remains possible that Aurora B function is inhibited indirectly in the cells, e.g. by fisetin inhibiting an enzyme required for the activation of the kinase.
Fisetin and other plant-derived flavonoids have been shown to exhibit antiproliferative effects in several normal and tumor cell lines primarily through activation of the programmed cell death pathways (12
). In case of fisetin, these potential chemopreventive activities can be partially explained by the previously reported inhibition of Cdk1/2/4/6, nuclear factor-kappa B and Topo II activities (14
). This also indicates that the drug most probably targets multiple proteins and pathways simultaneously. We demonstrate that fisetin treatment rapidly compromised a mitotic block imposed by the microtubule-depolymerizing drug nocodazole, the microtubule-stabilizing drug taxol or the Eg5 inhibitor monastrol, indicating that fisetin's mechanisms of action are independent of microtubule–kinetochore interactions and/or spindle morphology. Moreover, rapid bypass of a chemically induced mitotic block was observed with all cell lines tested this far, proposing perturbation of a conserved pathway. Finally, the fact that proteasome inhibition prevented the mitotic exit induced by fisetin suggests that the drug's molecular targets reside upstream of onset of anaphase where a set of spindle checkpoint kinases, including Aurora B kinase, BubR1, Plk1 and Cdk1, work to maintain spindle checkpoint activity.
The cellular effects of fisetin on untreated mitotic cells we report here phenocopy those caused by Aurora B RNAi (30
), dominant-negative Aurora B mutants (23
) and experimental Aurora drugs such as ZM-class of inhibitors (31
), Hesperadin (32
) and VX-680 (33
); cells bypassed the spindle checkpoint and exited M phase despite presence of multiple unaligned chromosomes and failed in cytokinesis leading to formation of progeny cells with 4N DNA content. These findings suggest that the inhibitory effect of fisetin in mitotic cells is primarily on Aurora B kinase rather than Aurora A, which when silenced or genetically perturbed does not compromise the spindle checkpoint or induce cytokinesis errors (34
). In support of this argument, in vitro
kinase assays showed that fisetin inhibits Aurora B more strongly than Aurora A (). Furthermore, phosphorylation of CenpA at serine 7, a specific Aurora B phosphorylation site, was significantly reduced by fisetin in cells (), whereas no major effects were observed on Aurora A autophosphorylation (). The observed resistance of anaphase cells to fisetin is most probably a consequence of drug kinetics; the full effect of fisetin may require >10 min, which is the average duration of anaphase in unperturbed cells. Alternatively, spatial modulation of Aurora B activity, or that of other possible fisetin targets, in anaphase cells may include protein modifications that prevent fisetin–kinase interaction in late mitotic cells (36
Comparison of the anti-Aurora B potencies of fisetin (IC50
~2.0 μM) and ZM447439 [IC50
~300 nM in the present study and IC50
130 nM in the original report (27
)] indicates that fisetin is less effective in suppression of the Aurora B kinase activity in vitro
. Also, the cellular effects of fisetin in the anti-microtubule drug pre-challenged cell populations are slightly different from those of ZM447439, a selective Aurora inhibitor ZM447439 overrides taxol-induced M phase arrest more readily compared with nocodazole block when used at 1–5 μM concentration (27
), whereas fisetin causes premature escape from both nocodazole and taxol blocks at 30 μM concentration. This discrepancy is most probably due to differences in the effective fisetin and ZM447439 drug concentrations as increasing ZM447439 concentration over 10 μM causes forced mitotic exit from nocodazole block (27
). It is also probably that fisetin-induced forced mitotic exit depends on perturbed function of other proteins besides Aurora B. For instance, inhibition of Cdk1 during mitosis could provide an explanation for the observed forced mitotic exit. However, the inhibition of Cdk1 by fisetin was reported to occur at a higher drug concentration of 60 μM (14
) compared with the concentration used here that resulted in complete override of the spindle checkpoint. Moreover, our in vitro
kinase assays demonstrate notably lower IC50
value for Aurora B inhibition compared with that of Cdk1 and Aurora A. Topo II is another confirmed fisetin target (17
) whose activity is essential for normal mitosis where it is required for decatenation of entangled DNA strands and for proper DNA condensation. However, treatment of cells with anti-Topo II drugs such as VM-26 (37
) or VP-16 (our unpublished data) does not override chemically activated spindle checkpoint but instead appears to cause a transient metaphase arrest (38
). In contrast, Topo II-depleted Drosophila
S2 and HeLa cells can successfully satisfy the spindle checkpoint without any significant mitotic delay (40
). Interestingly, in the same study, Topo II RNAi caused an indirect inhibition of Aurora B kinase activity through BubR1-mediated action (40
). We conclude that the observed inhibition of Aurora B activity by fisetin can provide a sufficient explanation for the forced mitotic exit but we cannot exclude the possibility that fisetin inappropriately turns off the spindle checkpoint by simultaneous inhibition of more than one mitotic target involved in this process such as Mps1, Bub1 and BubR1.
Previous reports with FACS analyses of fisetin-treated (10–60 μM; 24–48 h) human prostate (PC-3) or colon (HT-29) cancer cells have shown a significant accumulation of cells in G2
/M phase followed by apoptosis (14
). In light of our findings that fisetin rapidly overrides mitotic control and abrogates cytokinesis even in unperturbed cells, we propose that the earlier FACS analyses showing increase in the G2
/M peaks after fisetin treatment are, at least partially, due to formation of G1
cells with 4N DNA content after forced mitotic exit without cytokinesis. In agreement with previous studies, we show that fisetin treatment triggers cell suicide mechanisms in many cancer cell lines leading to apoptosis starting 24 h after exposure to the drug. It appears that certain cell types are more sensitive to the induction of cell death by fisetin as they undergo massive apoptosis rapidly after addition of the drug without showing significant increase in polyploidy (e.g. A549 and DU145 cells) whereas some cell types show accumulation of 4N and 8N populations followed by moderate levels of apoptosis (e.g. PC3). Interestingly, the non-carcinogenic MCF-10A cells were less sensitive to the fisetin-induced cell killing, which proposes that cell line-specific variations exists either in the mode of action of fisetin, in its pharmacokinetics, or in cellular processes of the drug response pathways. Future testing of broader cell line panels for the antiproliferative effects of fisetin and other flavonoids is, however, essential before any conclusions can be made on cancer cell-specific killing.
Due to its reported cardioprotective, memory-enhancing and anticarcinogenic properties fisetin is often added to nutritional supplements at very high concentrations under the assumption that it will possess enhanced beneficial health effects. The concentration of fisetin in these dietary supplements can far exceed the daily dose attained from a typical vegetarian diet. Importantly, previous studies suggested that maternal ingestion of flavonoids might induce breaks and translocations affecting the mixed lineage leukemia gene in the fetus, and eventually lead to infant leukemia, through possible effects on the catalytic activities of Topo II (42
). Also, Aurora kinases are known to have essential roles in embryonic cell divisions (43
) and their inhibition may disturb normal fetal development. Our finding that fisetin inhibits the activity of Aurora B kinase in cells provides a new mechanism of action whereby fisetin may elicit its previously reported antiproliferative effects and suggest that flavonoids in general may contribute to the preventive effect of a plant-based diet on serious diseases, including solid tumors. However, the biological effects of ingesting flavonoids at high concentrations are not known and precaution should be taken when using nutritional supplements rich with fisetin, especially during pregnancy.