Six of the ET-derived compounds, MUB, UBS, UB, AUB, UA and MUA exhibited significant anti-aromatase activity as measured in the microsome assay. However, this activity did not reach that of known aromatase inhibitor drugs previously tested in our laboratory [22
]. The test compounds AMUA, GA and EA showed no significant anti-aromatase activity in this assay. It is possible that a combination of these products may produce a more potent effect, as in vivo
they would not be found circulating in isolation. For example, a study by Kim et al showed that polyphenol-rich fractions of pomegranate inhibited aromatase activity in a microsomal assay up to 51% [23
], suggesting that combinations of polyphenols may be more effective than individual compounds. Future studies to examine the synergistic effects of these compounds would answer this question.
Of the urolithin compounds active in the microsomal assay, UB showed the highest anti-aromatase activity compared to the other compounds tested in the in-cell assay. This result may be due to a greater absorption of UB into the cells. A recent study by Larrosa et al demonstrated that both UA and UB were taken up intact in MCF-7 cells and metabolized to their sulfate and glucuronide forms, however, UB uptake was shown to be greater than that of UA [15
]. This result could explain the lower activity of UA in the live cell system. In addition to the differences in UB and UA absorption into the cells, urolithin B-sulfate and other isomers have been detected in the media but not the cell lysate in significant amounts, suggesting that the other forms of UA and UB were not as active in the in-cell assay perhaps because they must first undergo conversion to UB [15
]. The proliferation assays were done in a longer time frame (48 hours), and more activity was seen than the shorter term (3 hours) in-cell aromatase assay, suggesting longer term exposure could increase conversion and absorption.
In addition, the flavones chrysin and apigenin are identical in structure with the exception of an additional hydroxyl group on apigenin. In an aromatase assay, this small difference changed the EC50 of apigenin to 20 μM where chrysin was 7 μM, illustrating this point [24
]. Because MCF-7 is the parental line of the MCF-7aro cells utilized in our study, we suggest that urolithin B absorption is similar in both cells lines and the decreased uptake of UA may account for its lack of aromatase inhibition in the in-cell assay. We further tested UB to determine the nature of its inhibition of aromatase by kinetic analysis utilizing the microsome assay. Results indicated that UB is a competitive inhibitor of aromatase, with an approximate Kι of 5 μM.
Inhibition of testosterone-induced cell proliferation was observed with UB treatment as would be expected from the aromatase assay results. This suggests that the inhibition of aromatase, and therefore estrogen production in the cell, is one mechanism through which UB may inhibit breast cancer cell proliferation. However, because the MCF-7aro cell line also contains a functional estrogen receptor (ER), the antiproliferative effect may also be due in part to direct antagonism of the ER. To determine the roles of both aromatase and ER signaling in the antiproliferative mechanisms of urolithins in this cell line, proliferation assays using estrogen as an inducer were done. In this assay, UB showed very low activity, further suggesting its role as an inhibitor of aromatase.
Interestingly, the inhibition of testosterone-induced proliferation was also observed in cells treated with UA, GA, MUB and UBS, suggesting that these ET-derived compounds may inhibit breast cancer cell growth through aromatase-independent mechanisms. In the estrogen-induced proliferation assay, UBS and GA showed the highest anti-proliferative activity, suggesting that UBS may antagonize ER-signaling. GA on the other hand, had activity in both testosterone and estrogen-induced proliferation assays, suggesting multiple roles for this compound. Although these compounds may not be as readily taken up by the cells as UB, it is possible that their antiproliferative activity could be due conversion to other compounds in the medium. Future studies into the fate of these compounds and their effects on the cell cycle and cell signaling will be carried out to elucidate their mechanisms of action.
The ingestion of pomegranate juice can lead to concentrations of circulating urolithins reaching up to 18 μM in blood. Taken together with the results of current studies and reports of the presence of UA and UB in the blood and urine of human subjects following pomegranate ingestion, the results of these analyses suggest that pomegranate intake may be a viable strategy for the chemoprevention of breast cancer.