Pomegranate ellagitannin-derived compounds exhibit antiproliferative and antiaromatase activity in breast cancer cells in vitro

Abstract

Estrogen stimulates the proliferation of breast cancer cells and the growth of estrogen-responsive tumors. The aromatase enzyme, which converts androgen to estrogen, plays a key role in breast carcinogenesis. The pomegranate fruit, a rich source of ellagitannins (ET), has attracted recent attention due to its anticancer and antiatherosclerotic properties. On consumption, pomegranate ETs hydrolyze, releasing ellagic acid, which is then converted to 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one ("urolithin") derivatives by gut microflora. The purpose of this study was to investigate the antiaromatase activity and inhibition of testosterone-induced breast cancer cell proliferation by ET-derived compounds isolated from pomegranates. A panel of 10 ET-derived compounds including ellagic acid, gallagic acid, and urolithins A and B (and their acetylated, methylated, and sulfated analogues prepared in our laboratory) were examined for their ability to inhibit aromatase activity and testosterone-induced breast cancer cell proliferation. Using a microsomal aromatase assay, we screened the panel of ET-derived compounds and identified six with antiaromatase activity. Among these, urolithin B (UB) was shown to most effectively inhibit aromatase activity in a live cell assay. Kinetic analysis of UB showed mixed inhibition, suggesting more than one inhibitory mechanism. Proliferation assays also determined that UB significantly inhibited testosterone-induced MCF-7aro cell proliferation. The remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than UB. These studies suggest that pomegranate ET-derived compounds have potential for the prevention of estrogen-responsive breast cancers.

Figure 1. Chemical structures of test compounds

Figure 2. Inhibition of aromatase by urolithins

In vitro human placental aromatase assay was performed in the presence of 0.1 μM ³H-androstenedione and urolithin compounds (47 μM). The aromatase activity of vehicle-treated microsomes was set at 100%. The measurements were performed in triplicate. Data is expressed as percentage of activity remaining, mean ± SEM, p ≤ 0.01.

Figure 3. “In Cell” inhibitory effect of urolithin compounds on aromatase in MCF-7aro cells

MCF-7aro cells were maintained in MEM medium and switched to serum-free medium upon assay. Tritiated androstenedione (0.1 μM) and urolithins (0, 2.35 and 4.7μM) were administered and incubated for 3 hours. Values are means ± SEM, n = 3, *p ≤ 0.05, **p ≤ 0.01.

Figure 4. Effect of urolithin compounds on MCF-7aro cell proliferation

MCF-7aro cells were seeded in 96-well plates and maintained in MEM supplemented with 10% charcoal dextran-treated serum. Proliferation was measured after 48 hours under the influence of testosterone (A) or estrogen (B) + urolithins at the indicated concentrations using the CellTiter-Glo® Luminescent Cell Viability Assay. “TFM” = testosterone free medium, “T” = testosterone, “EFM” = estrogen free medium, “E2” = estrogen. Values are expressed as mean ± SEM, n = 9, asterisk indicates significant difference from hormone alone (0) group (p ≤ 0.01).

Figure 5. Aromatase inhibition kinetic profile by urolithin B compound

The enzyme assay was performed in the presence of 0, 23.5 and 47μM of urolithin B with increasing concentrations of [³H]-androstenedione (0 – 200 nM). The assay was performed in triplicate. (A) Lineweaver-Burk plot (1/v versus 1/ [androstenedione] (B) secondary plot (slope versus [UB]) used to determine Ki value for UB.