Aromatase Activity In Placental Microsomes Research Articles

New steroidal aromatase inhibitors: suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death.

BackgroundAromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.ResultsThe new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G0/G1 phase and inducing cell death with features of apoptosis and autophagic cell death.ConclusionOur in vitro studies showed that the two steroidal AIs, 3a and 4a, are potent inhibitors of breast cancer cell proliferation. Moreover, it was also shown that the antiproliferative effects of these two steroids on MCF-7aro cells are mediated by disrupting cell cycle progression, through cell cycle arrest in G0/G1 phase and induction of cell death, being the dominant mechanism autophagic cell death. Our results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer.

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.

Estrogen synthetase. demonstration that the high molecular weight form of nadph-cytochrome c reductase from human placental microsomes is required for androgen aromatization

NADPH-cytochrome c reductase from human placental microsomes was solubilized using deoxycholate and purified by 2',5'ADP-Sepharose 4B affinity chromatography in deoxycholate buffer followed by exclusion chromatography on Bio-Gel A-1.5 m in detergent-free buffer. Two principal peaks of reductase activity elute from the Bio-Gel column: the first peak (A form) comes at the void volume and has a principal monomer molecular weight of 73,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); the second peak (B form) elutes at 80,000 dallons with a principal monomer molecular weight of 68,000 by SDS-PAGE. The two fractions contain flavoproteins and are immunochemically identical by Ouchterlony double diffusion but apparently differ in net charge since they have different electrophoretic mobilities under non-denaturing conditions. The following results show that only the A form of the purified human placental microsomal NADPH-cytochrome c reductase is necessary for the expression of estrogen synthetase (aromatase) activity in placental microsomes, although aromatase activity was not detected in the purified protein. In a preparation of detergent solubilized placental microsomes which lacked both reductase and aromatase activities, but contained cytochrome P-450, aromatase activity could be regained by adding the A form of the reductase to these microsomes. Also, only the A form stimulated placental microsomal aromatase when mixed with a normal microsomal homogenate containing both reductase and aromatase activities. Rabbit anti-sera prepared against either the A form or a mixture of A and B forms inhibit equally both NADPH-cytochrome c reductase and aromatase activities in placental microsomes but do not inhibit NADH-cytochrome c reductase or estrogen 17β-hydroxysteroid dehydrogenase activities. The other aromatase component protein has not yet been positively identified, but its likely identity as cytochrome P-450 is consistent with these results.