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

<p>Abstract</p> <p>Background</p> <p>Aromatase, 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 <b>3a </b>and <b>4a</b>, 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.</p> <p>Results</p> <p>The new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G₀/G₁phase and inducing cell death with features of apoptosis and autophagic cell death.</p> <p>Conclusion</p> <p>Our <it>in vitro </it>studies showed that the two steroidal AIs, <b>3a </b>and <b>4a</b>, 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/G₁phase 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.</p

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death-1

for 72 hr. Wright staining shows that cells treated with 4a have condensed and marginalized chromatin (arrowheads) and cytoplasm vacuolization (arrows) (C) in comparison to the control cells (A). Nuclear morphological changes in MCF-7aro cells were demonstrated by Hoechst 33258 staining under the fluorescence microscope. Untreated cells exhibited normal nuclear morphology and the presence of abundant mitotic figures (open arrows) (B). Treatment with 4a induced chromatin condensation (arrows) (D).<p><b>Copyright information:</b></p><p>Taken from "New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death"</p><p>http://www.biomedcentral.com/1471-2121/9/41</p><p>BMC Cell Biology 2008;9():41-41.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515307.</p><p></p

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death-3

H the indicated concentrations of the compounds in medium containing 1 nM T. Cells cultured with testosterone represented maximum cell proliferation and were considered as control. 3a and 4a induced a decrease in cell proliferation, evaluated by the thymidine incorporation assay, in a time- and dose-dependent manner. Results are the mean ± SE of three independent experiments whereas cultures were performed in triplicate. Significant inhibition relative to the control level is denoted by * (< 0.001), ** (P < 0.01) and θ (P < 0.05).<p><b>Copyright information:</b></p><p>Taken from "New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death"</p><p>http://www.biomedcentral.com/1471-2121/9/41</p><p>BMC Cell Biology 2008;9():41-41.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515307.</p><p></p

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death-6

72 hr incubation with the compounds, cells were treated with MDC for 1 hr at 37°C, washed with PBS and analysed by fluorescence microscopy. The formation of autophagic vacuoles in MCF-7aro cells treated with compound 4a was indicated by punctuated MDC labelling in the cytoplasm.<p><b>Copyright information:</b></p><p>Taken from "New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death"</p><p>http://www.biomedcentral.com/1471-2121/9/41</p><p>BMC Cell Biology 2008;9():41-41.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515307.</p><p></p

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death-5

an increase in annexin V binding in MCF-7aro cells, in comparison to 9.2% of the control (medium containing 1 nM T, open histograms). Numbers in histograms are percent of annexin V-positive cells after treatment with the compounds. The histograms correspond to cells gated for negative 7-AAD staining. Data are representative of triplicate cultures and the figure is representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death"</p><p>http://www.biomedcentral.com/1471-2121/9/41</p><p>BMC Cell Biology 2008;9():41-41.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515307.</p><p></p

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death-8

Arrows indicate multiblebbing cells and arrowheads indicate perinuclear vesicles in the cytoplasm.<p><b>Copyright information:</b></p><p>Taken from "New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death"</p><p>http://www.biomedcentral.com/1471-2121/9/41</p><p>BMC Cell Biology 2008;9():41-41.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515307.</p><p></p