Letrozole-, Anastrozole-, and Tamoxifen-Responsive Genes in MCF-7aro Cells: A Microarray Approach

Abstract

Antiestrogens and aromatase inhibitors are important drugs in the treatment of estrogen-dependent breast cancer. To investigate the effects of these drugs on gene expression in breast cancer cells, we treated estrogen receptor -positive MCF-7 cells stably transfected with the aromatase gene (known as MCF-7aro cells) with testosterone, 17B-estradiol, two aromatase inhibitors (letrozole and anastrozole), and an antiestrogen (tamoxifen). We found that testosterone or 17B-estradiol induced the proliferation of MCF-7aro cells at a rate six times faster than the untreated cells. In addition, the testosterone-induced proliferation of MCF-7aro cells was effectively suppressed by letrozole, anastrozole, or tamoxifen. Microarray analyses on Affymetrix Human Genome U133A GeneChips (Affymetrix, Santa Clara, CA) were carried out using total RNA isolated from the control and treated cells. At the false discovery rate of 0.05 and a minimum fold-change criteria of 1.5, 104 genes were identified that were up-regulated and 109 genes were identified that were down-regulated by both androgen and estrogen. More than 50% of these hormone-regulated genes were counterregulated by all three inhibitors and >90% were counterregulated by at least one of the inhibitors. Comparing the effect of each inhibitor on gene expression, we observed that letrozole and anastrozole are more similar in terms of the genes they affect compared with treatment with tamoxifen. To validate the gene expression profiles identified from microarray analyses, the expression patterns of 13 representative genes were examined by Northern analysis. Finally, the genes identified as statistically significant were classified based on their expression patterns and biological function/pathways. The results of this study provide us with a better understanding of the actions of both aromatase inhibitors and antiestrogens at the molecular level. We believe that the results of this study serve as the first step in identifying unique expression patterns following drug treatment, and that this will ultimately be useful in customizing patient treatment strategies for hormone-dependent breast cancer.

C-6α- vs C-7α-Substituted Steroidal Aromatase Inhibitors: Which Is Better? Synthesis, Biochemical Evaluation, Docking Studies, and Structure–Activity Relationships

“…The AIs 9 and 13 demonstrate to be the most potent AIs, since they presented IC 50 values of 0.060 and 0.055 μM, respectively, being these IC 50 values similar to those of formestane (0.042 μM) and exemestane (0.050 μM) . The antiaromatase activity in MCF-7aro cells, an aromatase-overexpressing ER + breast cancer cell line that is considered the best in vitro cell model to study AIs, , was also studied for the most potent AIs. As presented in Table , except for compounds 5 and 12 that induced the formation of crystals in cell culture, the results demonstrate that the majority of these steroids present an antiaromatase activity higher than 80%.…”

“…It this study, for the compounds that presented an aromatase inhibition in placental microsomes higher than 80%, their antiaromatase activity in an ER-positive (ER + ) aromatase-overexpressing human breast cancer cell line (MCF-7aro) was also evaluated, according to the methods by Thompson and Siiteri and Zhou et al with modifications. , These cells were prepared by stable transfection of MCF-7 cells with the human placental aromatase gene and Geneticin selection, , reason why they correspond to a good model to study AIs in ER + breast cancer . They were maintained with Eagle’s minimum essential medium supplemented with 1 mmol/L sodium pyruvate, 1% penicillin–streptomycin–amphotericin B, 100 μg/mL G418, and 10% heat-inactivated fetal bovine serum (Gibco Invitrogen Co., Paisley, Scotland, U.K.).…”

“…21,22 These cells were prepared by stable transfection of MCF-7 cells with the human placental aromatase gene and Geneticin selection, 71,72 reason why they correspond to a good model to study AIs in ER + breast cancer. 56 They were maintained with Eagle's minimum essential medium supplemented with 1 mmol/L sodium pyruvate, 1% penicillin−streptomycin−amphotericin B, 100 μg/mL G418, and 10% heat-inactivated fetal bovine serum (Gibco Invitrogen Co., Paisley, Scotland, U.K.). Briefly, confluent MCF-7aro cells plated in a 24-well plate were cultured in serum-free medium containing the inhibitors at 10 μM, with 50 nM [1β-3 H] androstenedione as substrate and also 500 nM progesterone (that was used to suppress 5α-reductase activity, which also uses androgen as substrate) and incubated at 37 °C for 1 h. The aromatase reaction was finished by addition of 100 μL of 20% trichloroacetic acid (TCA).…”

Molecular effects of oestrogen deprivation in breast cancer

“…Thus, amongst the genes most significantly downregulated are KIAA0101, ZWINT, PDZK1, UBE2C, IRS1 and trefoil factors 1 and 3. These are classical examples of oestrogen-regulated genes whose expression has been shown either to be induced by oestrogen or reduced by antioestrogen treatments in experimental systems [22][23][24][25]31]. Significantly downregulated probes also included those for genes associated with promoting proliferation, for example CDC2, thymidylate synthetase, cyclin B1, PCNA and CKS2.…”

“…Oestrogen regulated genes: KIA0101, TFF1, TFF3, IRS1 and SERPINA3 are genes whose expression is stimulated in a variety of oestrogen-sensitive systems [22][23][24][25]. The average change in expression produced by letrozole in breast cancers is summarized in Figure 3c.…”