Aromatase In Breast Cancer Cells Research Articles

Cellulose nanocrystals derived from chicory plant: an un-competitive inhibitor of aromatase in breast cancer cells via PI3K/AKT/mTOP signalling pathway

A significant contributing factor in the development of breast cancer is the estrogens. The synthesis of estrogens is primarily facilitated by aromatase (CYP19), a cytochrome P450 enzyme. Notably, aromatase is expressed at a higher level in human breast cancer tissue compared with the normal breast tissue. Therefore, inhibiting aromatase activity is a potential strategy in hormone receptor-positive breast cancer treatment. In this study, Cellulose Nanocrystals (CNCs) were obtained from Chicory plant waste through a sulfuric acid hydrolysis method with the objective of investigating that whether the obtained CNCs could act as an inhibitor of aromatase enzyme, and prevent the conversion of androgens to estrogens. Structural analysis of CNCs was carried out using Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), while morphological results were obtained using AFM, TEM, and FE-SEM. Furthermore, the nano-particles were found to be spherical in shape with a diameter range of 35-37 nm and displayed a reasonable negative surface charge. Stable transfection of MCF-7 cells with CYP19 has demonstrated the ability of CNCs to inhibit aromatase activities and prevent cell growth by interfering with the enzyme activities. Spectroscopic results revealed the binding constant of CYP19-CNCs and (CYP19-Androstenedione)-CNCs complexes to be 2.07 × 103 L/gr and 2.06 × 104 L/gr, respectively. Conductometry and CD data reported different interaction behaviors among CYP19 and CYP19-Androstenedione complexes at the presence of CNCs in the system. Moreover, the addition of CNCs to the solution in a successive manner resulted in the enhancement of the secondary structure of the CYP19-androstenedione complex. Additionally, CNCs showed a marked reduction in the viability of cancer cells compared to normal cells by enhancing the expression of Bax and p53 at protein and mRNA levels, and by decreasing mRNA levels of PI3K, AKT, and mTOP, as well as protein levels of PI3Kg-P110 and P-mTOP, in MCF-7 cells after incubation with CNCs at IC50 concentration. These findings confirm the decrease in proliferation of breast cancer cells associated with induction of apoptosis through down-regulation of the PI3K/AKT/mTOP signaling pathway. According to the provided data, the obtained CNCs are capable of inhibiting aromatase enzyme activity, which has significant implications for the treatment of cancer. Communicated by Ramaswamy H. Sarma

Aromatase (CYP19) inhibition by biflavonoids obtained from the branches of Garcinia gardneriana (Clusiaceae).

Overexpression of aromatase in breast cancer cells may substantially influence its progression and maintenance. In this sense, the inhibition of aromatase is a key target for the treatment of breast cancer in postmenopausal women. Although several flavonoids had already demonstrated the capacity of inhibiting aromatase activity, the role of biflavonoids as aromatase inhibitors is poorly studied. In this work, the biflavonoids isolated from Garcinia gardneriana, morelloflavone (1), Gb-2a (2) and Gb-2a-7-O-glucose (3) were submitted to in vitro assay to evaluate the aromatase modulatory effect. As results, it was demonstrated that all biflavonoids were able to inhibit the enzyme, with IC50 values ranging from 1.35 to 7.67 μM. This demonstrates that biflavonoids are an important source of scaffolds for the development of new aromatase inhibitors, focusing on the development of new anticancer agents.

Anti-tumor efficacy of new 7α-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cells

The majority of breast cancer cases are estrogen receptor positive (ER+). Although, third-generation aromatase inhibitors (AIs) are used as first-line treatment in post-menopausal women, they cause endocrine resistance and bone loss, which limits their success. Therefore, there is a demand to discover new potent molecules, with less toxicity that can circumvent these drawbacks. Our group has previously demonstrated that new 7α-substituted steroidal molecules, 7α-(2ξ,3ξ-epoxypropyl)androsta-1,4-diene-3,17-dione (3), 7α-allylandrost-4-ene-3,17-dione (6), 7α-allylandrost-4-en-17-one (9), 7α-allyl-3-oxoandrosta-1,4-dien-17β-ol (10) and 7α-allylandrosta-1,4-diene-3,17-dione (12) are potent AIs in placental microsomes. In this work, it was investigated their anti-aromatase activity and in vitro effects in sensitive and resistant breast cancer cells. All the steroids efficiently inhibit aromatase in breast cancer cells, allowing to establish new structure-activity relationships for this class of compounds. Moreover, the new AIs can inhibit breast cancer cell growth, by causing cell cycle arrest and apoptosis. The effects of AIs 3 and 12 on sensitive cells were dependent on aromatase inhibition and androgen receptor (AR), while for AI 9 and AI 10 were AR- and ER-dependent, respectively. In addition, it was shown that all the AIs can sensitize resistant cancer cells being their behavior similar to the sensitive cells. In summary, this study contributes to the understanding of the structural modifications in steroidal scaffold that are translated into better aromatase inhibition and anti-tumor properties, providing important information for the rational design/synthesis of more effective AIs. In addition, allowed the discovery of new potent 7α-substituted androstane molecules to inhibit tumor growth and prevent endocrine resistance.

The effects of Nobiletin, Hesperetin, and Letrozole in a combination on the activity and expression of aromatase in breast cancer cells.

Nobiletin (NOB) and hesperetin (HES) are the citrus polymethoxyflavone and flavonone. Aromatase or cytochrome P450 (CYP19) enzyme is a key enzyme in estrogen biosynthesis. The objective of this study was to investigate the combinational effects of HES, NOB and letrozole (LET) as aromatase inhibitors on the activity and expression of aromatase in MCF-7 cells. In this study, aromatase enzyme activity based on the conversion of androgen substrate testosterone to 17β-Estradiol was determined. Estradiol concentrations were measured using an electrochemiluminescence immunoassay. CYP19 gene expression was determined by quantitative real-time PCR. Our findings demonstrated that none of combinations including LET+NOB, LET+HES, LET+NOB+HES, and NOB+HES had no significant effects on aromatase activity and expression. The present study showed for the first time that the combination of HES, NOB, and LET had no effects on activity and expression of aromatase in MCF-7 breast cancer cells.

Relationship of BTG2 expression and anastrozole resistance in breast cancer.

220 Background: The B-cell translocation gene-2 (BTG2) belongs to a class of proteins known as the Tob and BTG antiproliferative protein family. It was shown that estrogen and progesterone suppress BTG2 expression for the development of mammary gland. We demonstrated that proliferation rate of low level BTG2 expression in MCF7 was strongly inhibited by the administration of tamoxifen. In postmenopausal breast cancer patients, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Based on these results, we hypothesized that BTG2 expression affects the sensitivity against aromatase inhibitior. Methods: We used tetracycline-inducible BTG2 expression model in MCF7 stably transfected with the human aromatase gene (MCF7/tet/aro) as in vitro models of aromatase-driven breast cancer. The effects of BTG2 expression and administration of anastrozole in breast cancer cells were assessed by proliferation assays. Results: Administration of androstendion increased 79.1% of cellular proliferation, suggested that introduced aromatase gene worked well. Elevated level of BTG2 mRNA expression by tetracycline treatment was confirmed by Quantitative-RTPCR. Anastrozole treatment (100nM) reduced 37.8% of cellular proliferation ability, whereas the concomitant administration of tetracycline and anastorozole reduced 59.0% of cellular proliferation. These results suggested that the inhibitory effect of anastrozol for cellular proliferation was enhanced under the condition of BTG2 expression. Conclusions: Our results suggested loss of BTG2 expression may be affects the sensitivity against aromatase inhibitor.

Abstract 2430: Aromatase expression inhibits anoikis and promotes bone metastasis of ERα positive breast cancer cells

Abstract Aromatase is the rate-limiting enzyme that catalyzes the final step in estrogen biosynthesis. In postmenopausal women the ovary ceases to make estrogen. Although it has been shown that estrogen is produced locally in breast cancer (BCa) tissue of postmenopausal women for the proliferation and survival of ERα (+) BCa cells, there is little information on the role and the regulation of aromatase in breast cancer cells for their survival when they intravasate into the circulation where estrogen level is very low. Human breast cancer CAMA-1 cell along with aromatase-transfected MCF-7 (MCF-7/Aro) and ZR-75-1 (ZR-75-1/Aro/CL10) cells were cultured in suspension to mimic circulating tumor cells. Interestingly, all the three ERα(+) BCa cell lines expressed more aromatase in suspension culture compare to the adhesion culture. Moreover both endogenous and transfected aromatase were upregulated in suspension culture. Additionally, a higher expression of ERα was also observed in suspension culture, which could increase the sensitivity of the breast caner cells to intracrine estrogen. We found that anoikis was shown to be inhibited by the supplementation of testosterone, which is converted to estradiol by aromatase. Conversely, addition of the aromatase inhibitor letrozole blocked the testosterone-induced anoikis inhibition. The increase of pro-apoptotic protein BAX and BAD were observed in suspension culture, while the treatment of estrogen inhibited the expression of BAX and BAD in both adhesion culture and suspension culture. More interestingly, mice with intracardiacally inoculated ZR-75-1/Aro cells presented distant bone metastasis in the mandible and tibiae/femora after 2-weeks of inoculation without estrogen supplement, while the control ZR-75-1 cells were incapable of forming metastasis without estrogen. Our findings provide an important foundation for future investigation on how hormone-dependent BCa cells survive in circulation during the process of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2430. doi:10.1158/1538-7445.AM2011-2430

Functional Activation of the Estrogen Receptor-α and Aromatase by the HDAC Inhibitor Entinostat Sensitizes ER-Negative Tumors to Letrozole

Approximately 25% of breast cancers do not express the estrogen receptor-α (ERα) and consequently do not respond to endocrine therapy. In these tumors, ERα repression is often due to epigenetic modifications such as methylation and histone deacetylation. For this reason, we investigated the ability of the histone deacetylase inhibitor entinostat (ENT) to trigger reexpression of ERα and aromatase in breast cancer cells, with the notion that this treatment would restore sensitivity to the aromatase inhibitor (AI) letrozole. ENT treatment of tumor cells increased expression of ERα and aromatase, along with the enzymatic activity of aromatase, in a dose-dependent manner both in vitro and in vivo. Notably, ERα and aromatase upregulation resulted in sensitization of breast cancer cells to estrogen and letrozole. Tumor growth rate was significantly lower in tumor xenografts following treatment with ENT alone and in combination with letrozole than in control tumors (P > 0.001). ENT plus letrozole also prevented lung colonization and growth of tumor cells, with a significant reduction (P > 0.03) in both visible and microscopic foci. Our results show that ENT treatment can be used to restore the letrozole responsiveness of ER-negative tumors. More generally, they provide a strong rationale for immediate clinical evaluation of combinations of histone deacetylase and aromatase inhibitors to treat ER-negative and endocrine-resistant breast cancers.

COX-2 inhibitor nimesulide analogs are aromatase suppressors in breast cancer cells

Cyclooxygenase-2 (COX-2) inhibitor nimesulide derivatives compounds A and B decreased aromatase activity in breast cancer cells via a novel mechanism different to aromatase inhibitors (AIs), and were defined as “aromatase suppressors”. Breast carcinoma cells (MCF-7aro and T47Daro) transfected with aromatase full gene were used to explore the mechanisms of the two compounds. They dose and time-dependently suppressed aromatase activity in MCF-7aro and T47Daro cells in the nanomole range. However, they neither directly inhibited aromatase, nor improved aromatase degradation even at much higher concentrations. They could also suppress androgen stimulated cell growth, but did not affect estrogen enhanced cell proliferation. These results suggest that compounds A and B selectively interfere with aromatase in breast cancer cells, but not estrogen receptor (ER) downstream to disrupt androgen mediated cell growth. Interestingly, compound B effectively inhibited LTED (long-term estrogen deprived MCF-7aro cell) cell growth, which is a model for AIs resistance, with an IC 50 of 4.68 ± 0.54 μM. The results indicate that compound B could potentially overcome AI resistance in breast cancer cell and could be used as a lead to design more potent derivatives.

Abstract LB-203: Aromatase expression promotes breast cancer tumorigenesis and bone metastasis

Abstract Aromatase P450 (CYP19) is the rate-limiting enzyme catalyzing the final step in estrogen biosynthesis, and an important target in breast cancer therapy. In post-menopausal women, ovaries cease to make estrogen, yet the incidence of breast cancer increases with age as aromatase present in breast adipose stroma and epithelium catalyzes local estrogen formation. The circulating estrogen level in postmenopausal women is very low and it reflects the sum of local estrogen biosynthesis in various sites. Although breast cancer cells have been shown to express aromatase prompting the hypothesis that intracrine action of estrogen in breast cancer cells may drive tumor progression, there has been little evidence in addressing the hypothesis. We stably transfected the ER positive (ER+) breast cancer ZR-75-1 cells with an human aromatase expression vector and obtained a few clones expressing a moderately higher level of aromatase in comparison with the control vector-transfected cells. One of the clones called ZR-75-1/Aro/Clone10 (Cl10) was selected for further study due to its increased intracellular estradiol (E2) concentration compared to other clones. The Cl10 cells formed tumors after being xenografted into the inguinal mammary fat pad of female athymic mice while the control cells were incapable of forming tumors without estrogen supplementation. Implantation of testosterone pellet further stimulated the growth of the tumor and a cell line, ZR-75-1/Aro/Clone10/TT1 (TT1) was established from the tumor. Both Cl10 and TT1 cells expressed similar levels of aromatase. Interestingly, the aromatase expression increased when the cells were cultured in suspension suggesting circulating ER+ breast cancer cells may up-regulate intracrine estrogen activity for survival after leaving the estrogen-rich adipose stroma at primary site. To investigate the importance of intracrine estrogen in promoting tumorigenesis and metastasis, we implanted the Cl10 and TT1 cells orthotopically and intracardiacally (I.C.) into female athymic mice. Notably, both cell lines generated orthotopic tumors with no estrogen supplementation after three weeks of inoculation. More interestingly, the mice with I.C. inoculated aromatase-expressing cells presented distant metastasis to bones in mandible and legs after two weeks of inoculation detected by whole mouse fluorescence and bioluminescence imaging as both cells were stably transfected with a luciferase and GFP expression vector. We will determine whether growth of the orthotopic tumors and bone metastases can be inhibited with systemic administration of an aromatase inhibitor and whether bone metastasis is osteoclastic or osteoblastic. Our studies show that a moderate expression of aromatase in breast cancer cells is sufficient to generate intracrine estrogenic action and promote tumorigenesis and skeletal metastasis in ER+ breast cancer cells. These aromatase-expressing models should be useful for future investigation on how ER+ breast cancer cells up-regulate aromatase expression in suspension and induces bone remodeling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-203.

Rapid Estradiol/ERα Signaling Enhances Aromatase Enzymatic Activity in Breast Cancer Cells

In situ estrogen production by aromatase conversion from androgens plays an important role in breast tumor promotion. Here, we show that 17beta-estradiol (E2) can rapidly enhance aromatase enzymatic activity through an increase of aromatase protein phosphorylation in breast cancer cell lines. In vivo labeling experiments and site-directed mutagenesis studies demonstrated that phosphorylation of the 361-tyrosine residue is crucial in the up-regulation of aromatase activity under E2 exposure. Our results demonstrated a direct involvement of nonreceptor tyrosine-kinase c-Src in E2-stimulated aromatase activity because inhibition of its signaling abrogated the up-regulatory effects induced by E2 on aromatase activity as well as phosphorylation of aromatase protein. In addition, from our data it emerges that aromatase is a target of cross talk between growth factor receptors and estrogen receptor alpha signaling. These findings show, for the first time, that tyrosine phosphorylation processes play a key role in the rapid changes induced by E2 in aromatase enzymatic activity, revealing the existence of a short nongenomic autocrine loop between E2 and aromatase in breast cancer cells.

Expression of transcription factor CREB1 in human breast cancer and its correlation with prognosis

It has been suggested that the cAMP responsive element-binding protein (CREB) may act as a transcription regulator of aromatase in breast cancer cells. However, there is little knowledge on the expression of CREB1 in human breast cancer and its clinical significance. The current study investigated the expression pattern of CREB1 in human breast cancer at the mRNA and protein level and correlated it with the clinical outcome. CREB1 staining was primarily seen in the nucleus of both normal and tumour cells. At the mRNA level, we found a significantly higher level of CREB1 in breast tumour tissues (n=120) as compared to non-neoplastic mammary tissues (n=33, p=0.0092). When compared between different histological types CREB1 expression was significantly higher in ductal carcinoma as compared to lobular and other breast carcinoma. Patients with a poor prognosis and with metastasis had a markedly raised level of CREB1 compared to patients who were disease free. In addition, node-positive tumours had higher levels of CREB1 than node-negative tumours (p=0.0018). Finally, patients with high levels of CREB1 had a significantly shorter disease-free survival [95.3 (68.4-122.3, 95% CI) months] compared with those with lower levels [133.9 (123.5-144.2) months, p=0.0193]. This study demonstrates that the level of CREB1 in breast cancer patients is elevated and is significantly raised in patients with a poor prognosis, metastatic disease and nodal involvement. We conclude that the level of CREB1 expression is aberrant in human breast cancer and is associated with disease progression in breast cancer patients.

Role of Androgens on MCF-7 Breast Cancer Cell Growth and on the Inhibitory Effect of Letrozole

Previous work has shown that androgens inhibit breast cancer cells and tumor growth. On the other hand, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Here, we report that androgens, such as the aromatizable androstenedione and the non-aromatizable 5alpha-dihydrotestosterone, inhibit MCF-7 cell proliferation. This effect is observed only in the absence or at a low concentration of estrogens and is evident in cells with low aromatase activity. Growth of a new aromatase stably transfected MCF-7 cell line (Ac1) was stimulated by conversion of androstenedione into estrogens and was sensitive to aromatase inhibitors. We show that blockade of the androgen receptor (AR) in these cells by the antiandrogen casodex or by the anti-AR small interfering RNA inhibited the antiproliferative effect of dihydrotestosterone and letrozole (aromatase inhibitor). We also show that suppression of the estrogen-induced antiapoptotic protein Bcl-2 may be involved in the antiproliferative effects of androgens and letrozole. These effects can be reversed by casodex. In conclusion, the results suggest that aromatase inhibitors may exert their antiproliferative effect not only by reducing the intracellular production of estrogens but also by unmasking the inhibitory effect of androgens acting via the AR.

The Red Wine Polyphenol Resveratrol Displays Bilevel Inhibition on Aromatase in Breast Cancer Cells

Estrogen plays a crucial role in the development of breast cancer, and the inhibition of estrogen synthesis has been an important target for the prevention and treatment of this disease. The rate-limiting reaction of the hormone biosynthesis is catalyzed by cytochrome P450 (CYP) 19 enzyme or aromatase. It has been of genuine interest to uncover an aromatase-inhibitory compound from a dietary source. Resveratrol is a polyphenolic compound that can be isolated from grape peel. Because of its structural resemblance to estrogen, resveratrol's agonistic and antagonistic properties on estrogen receptor have been examined and demonstrated. In the present study, the effect of resveratrol on the expression and enzyme activity of aromatase was investigated. By assaying on MCF-7 cells stably transfected with CYP19 (MCF-7aro cells), resveratrol inhibited the aromatase activity with an IC(50) value of 25 microM. Kinetic analysis indicated that both competitive and noncompetitive inhibition might be involved. The administration of 10 nmol/l testosterone-a substrate of aromatase-produced a 50% increase in the MCF-7aro cell number. This cell proliferation specifically induced by testosterone was significantly reduced by 10 microM resveratrol. In addition, 50 microM resveratrol significantly reduced the CYP19-encoding mRNA abundance in SK-BR-3 cells. The transcriptional control of CYP19 gene is tissue specific, and promoter regions I.3 and II have previously been shown to be responsible for CYP19 expression in breast cancer cells. Luciferase reporter gene assays revealed that resveratrol could repress the transcriptional control dictated by the promoter regulation. The present study illustrated that pharmacological dosage of resveratrol inhibited aromatase at both the enzyme and mRNA levels.

Transcriptional regulation of aromatase expression in human breast tissue

Aromatase (CYP19) is the estrogen synthetase that converts androgen to estrogen. The expression of aromatase in breast cancer cells and surrounding stromal cells is up regulated compared to non-cancerous cells. In situ estrogen synthesis is thought to stimulate breast cancer growth in both an autocrine and a paracrine manner. A complex mechanism is involved in the control of human aromatase expression, in that seven promoters have been identified and found to be utilized in a tissue-selective manner. Increased aromatase expression in breast tumors is, in part, attributed to changes in the transcriptional control of aromatase expression. While promoter I.4 is the main promoter that controls aromatase expression in non-cancer breast tissue, promoters II and I.3 are the dominant promoters that drive aromatase expression in breast cancer tissue. During the last several years, our laboratory performed a series of studies to examine the transcription regulatory mechanism of aromatase expression in breast cancer cells. We functionally characterized promoters II and I.3, and carried out DNase 1 footprinting analysis that identified two regulatory elements, S1 and CREaro. Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that four orphan/nuclear receptors, ERRα-1, EAR-2, COUP-TFI and RARγ, bind to the S1 element, and that CREB1, Snail (SnaH) and Slug proteins bind to the CREaro element. Studies from this and other laboratories have revealed that in cancer tissue versus normal tissue, several positive regulatory proteins (e.g. ERRα-1 and CREB1) are present at higher levels and several negative regulatory proteins (e.g. EAR-2, COUP-TFI, RARγ, Snail and Slug proteins) are present at lower levels. This may explain why the activity of promoters II and I.3 is up regulated in cancer tissue. An understanding of the molecular mechanisms of aromatase expression between non-cancerous and cancerous breast tissue, at the transcriptional level, may help in the design of a therapy based on the suppression of aromatase expression in breast cancer tissue.

Control of aromatase in breast cancer cells and its importance for tumor growth

Three approaches have been taken to elucidate further the biological importance of intratumoural aromatase activity. (i) MCF 7 and T47D hormone-dependent breast cancer cell lines both showed detectable aromatase activity in vitro. The up-regulation of this by TGFα indicates the possible existence of an autocrine growth stimulatory loop involving aromatase. (ii) Both tamoxifen and cytotoxic chemotherapy caused the suppression of aromatase activity in breast carcinomas in vivo. Aromatase activity prior to treatment did not predict for clinical response to tamoxifen. (iii) Transfection of aromatase into MCF 7 cells led to their growth being stimulated by low doses of androgens and this was inhibited by the aromatase inhibitor CGS 16949A.