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

Improved syntheses of aromatase inhibitors and neuroactive steroids efficient oxidations and reductions at key positions for bioactivity

An Henbest reduction, followed by the preparation of a silyl enol ether and oxidation in situ with m-CPBA has led to the neurosteroids 3[alpha]-hydroxy- and 3[alpha],21-dihydroxy-5[alpha]-pregnanolones. Using testosterone as starting material, a new short synthesis of an aromatase inhibitor, 4-OHA, has been achieved through hydroboration/oxidation followed by a Swern type oxidation and epimerization. Another aromatase inhibitor, androst-4-ene-3,6-17-trione, has been efficiently prepared using PCC on montmorillonite K10, under ultrasonic irradiation.http://www.sciencedirect.com/science/article/B6THR-3WC46V7-8/1/5f915790e78df65f4c988ab78bf4f17

Synthesis and biochemical studies of 17-substituted androst-3-enes and 3,4-epoxyandrostanes as aromatase inhibitors

http://www.sciencedirect.com/science/article/B6TC9-4T0WJXG-2/2/58305e64e3c068f352293a693f168db

Design e Síntese de Esteróides Inibidores da Aromatase

As estatísticas actuais efectuadas em vários países do Mundo Ocidental indicam que aproximadamente uma em cada dez mulheres contrai cancro da mama durante a sua vida e que cerca de 60% destes cancros estão directamente relaccionados com os níveis de estrogénios circulantes na corrente sanguínea, apesar da relação causa-efeito continuar desconhecida. A biossíntese dos estrogénios no organismo humano faz-se por intermédio da aromatase, uma enzima do grupo dos citocromos P450, e, por esse motivo, a sua inibição tem revelado ser uma abordagem terapêutica de elevado sucesso no tratamento dos cancros da mama estrogénio-dependentes, especialmente após a menopausa. No capítulo I da dissertação "DESIGN E SÍNTESE DE ESTERÓIDES INIBIDORES DA AROMATASE", além de algumas considerações gerais relativamente ao cancro da mama, são estudados os principais compostos desenvolvidos como inibidores da aromatase, apresentando-se o estado da arte relativamente à sua concepção e síntese. No capítulo II, no sentido de contornar algumas das limitações dos processos descritos na literatura, são planeadas e desenvolvidas duas novas e eficientes estratégias alternativas de síntese do Formestano. Este composto é um dos mais potentes e inócuos esteróides inibidores da aromatase da actualidade tendo sido introduzido recentemente em alguns países na prática clínica para o tratamento do cancro da mama. No capítulo III é estudada a reactividade química dos precursores do Formestano preparados nas estratégias de síntese desenvolvidas, sendo propostos mecanismos químicos para clivagens anormais detectadas em epóxidos. No capítulo IV são concebidos e preparados novos potenciais inibidores da aromatase contendo na sua estrutura funções químicas responsáveis pela interacção fármaco-receptor. A elucidação estrutural dos compostos preparados foi efectuada por espectroscopia de RMN 1H e 13C, de IV, de massa, por análise elementar e por difractometria de raios-X. Dez novas moléculas esteróides foram preparadas e nove estão actualmente em fase de screening de actividade anti-tumoral e anti-viral (HIV)

Apoptosis and autophagy in breast cancer cells following exemestane treatment.

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G(0)/G1 or in G(2)/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis

The Structural Diversity and Biological Activity of Steroid Oximes

Steroids and their derivatives have been the subject of extensive research among investigators due to their wide range of pharmacological properties, in which steroidal oximes are included. Oximes are a chemical group with the general formula R1R2C=N&minus;OH and they exist as colorless crystals and are poorly soluble in water. Oximes can be easily obtained through the condensation of aldehydes or ketones with various amine derivatives, making them a very interesting chemical group in medicinal chemistry for the design of drugs as potential treatments for several diseases. In this review, we will focus on the different biological activities displayed by steroidal oximes such as anticancer, anti-inflammatory, antibacterial, antifungal and antiviral, among others, as well as their respective mechanisms of action. An overview of the chemistry of oximes will also be reported, and several steroidal oximes that are in clinical trials or already used as drugs are described. An extensive literature search was performed on three main databases&mdash;PubMed, Web of Science, and Google Scholar

The Structural Diversity and Biological Activity of Steroid Oximes

Steroids and their derivatives have been the subject of extensive research among investigators due to their wide range of pharmacological properties, in which steroidal oximes are included. Oximes are a chemical group with the general formula R1R2C=N−OH and they exist as colorless crystals and are poorly soluble in water. Oximes can be easily obtained through the condensation of aldehydes or ketones with various amine derivatives, making them a very interesting chemical group in medicinal chemistry for the design of drugs as potential treatments for several diseases. In this review, we will focus on the different biological activities displayed by steroidal oximes such as anticancer, anti-inflammatory, antibacterial, antifungal and antiviral, among others, as well as their respective mechanisms of action. An overview of the chemistry of oximes will also be reported, and several steroidal oximes that are in clinical trials or already used as drugs are described. An extensive literature search was performed on three main databases—PubMed, Web of Science, and Google Scholar

Annexin V-PE labeling and mitochondrial transmembrane potential following exemestane treatment.

<p>(A) Annexin V-PE labelling of MCF-7aro cells treated with exemestane (15 µM) for 6 and 9 days of treatment. Cells cultured with T were considered as control and for 6 days present 3.54% and for 9 days 5.58% of Annexin V binding. Data presented in histograms were analysed with FlowJo Software (Tree Star, Inc), correspond to cells gated for negative 7-AAD staining and are representative of one independent assay. The grey filled line corresponds to control and the black line to exemestane at 15 µM. (B) Mitochondrial transmembrane potential (ΔΨm) of MCF-7aro cells treated with exemestane (15 µM) with or without 3-MA (1 mM), for 3 and 6 days. Viable cells and cells with ΔΨm loss were identified. Data presented in histograms were analysed with FlowJo Software (Tree Star, Inc) and are representative of one independent assay.</p

Effects of exemestane on mitochondrial transmembrane potential (ΔΨm) in MCF-7aro cells.

<p>Cells were treated with different concentrations of exemestane with or without 1 mM of 3-MA after 3 and 6 days. Treated cells were harvested and labeled with DiOC₆(3) and PI followed by flow cytometry analysis. Data are presented as viable cells and cells with ΔΨm loss. Cells cultured with T at 1 nM were considered as control and cells treated with T plus CCCP (10 µM) were considered as positive control for ΔΨm loss.The data represents means ± SEM of three independent experiments done in triplicate. The ratio treatment/control is presented in bold within brackets. Significant differences between the control versus treated cells, or between the control plus 3-MA versus cells with exemestane plus 3-MA are indicated by</p>***<p>(p<0.001).</p