Orphan nuclear receptor TLX contributes to androgen insensitivity in castration-resistant prostate cancer via its repression of androgen receptor transcription.

The metastatic castration-resistant prostate cancer (CRPC) is a lethal form of prostate cancer, in which the expression of androgen receptor (AR) is highly heterogeneous. Indeed, lower AR expression and attenuated AR signature activity is shown in CRPC tissues, especially in the subset of neuroendocrine prostate cancer (NEPC) and prostate cancer stem-like cells (PCSCs). However, the significance of AR downregulation in androgen insensitivity and de-differentiation of tumor cells in CRPC is poorly understood and much neglected. Our previous study shows that the orphan nuclear receptor TLX (NR2E1), which is upregulated in prostate cancer, plays an oncogenic role in prostate carcinogenesis by suppressing oncogene-induced senescence. In the present study, we further established that TLX exhibited an increased expression in metastatic CRPC. Further analyses showed that overexpression of TLX could confer resistance to androgen deprivation and anti-androgen in androgen-dependent prostate cancer cells in vitro and in vivo, whereas knockdown of endogenous TLX could potentiate the sensitivity to androgen deprivation and anti-androgen in prostate cancer cells. Our study revealed that the TLX-induced resistance to androgen deprivation and anti-androgen was mediated through its direct suppression of AR gene transcription and signaling in both androgen-stimulated and -unstimulated prostate cancer cells. We also characterized that TLX could bind directly to AR promoter and repress AR transcription by recruitment of histone modifiers, including HDAC1, HDAC3, and LSD1. Together, our present study shows, for the first time, that TLX can contribute to androgen insensitivity in CRPC via repression of AR gene transcription and signaling, and also implicates that targeting the druggable TLX may have a potential therapeutic significance in CRPC management, particularly in NEPC and PCSCs.

Nuclear receptor profiling in prostatospheroids and castration-resistant prostate cancer.

Nuclear receptors (NRs), which belong to a superfamily of transcription factors and consist of a total of 48 members in humans, govern the expression of genes involved in a board range of developmental, reproductive, metabolic and immunological programs. Given the significant importance of androgen receptor and a few known NRs in the progression of prostate cancer, we surveyed the expression profiles of the entire NR superfamily in three-dimensional cultured prostatospheroids derived from different prostate cancer cell lines and a tumor xenograft model of castration-resistant prostate cancer VCaP-CRPC by quantitative real-time RT-PCR. Our results revealed that prostatospheroids and castration-relapse VCaP-CRPC xenografts, both contained enriched populations of prostate cancer stem/progenitor-like cells (PCSCs), displayed distinct expression patterns of NRs. Intriguingly, most of these differentially expressed NRs were orphan NRs and showed upregulation. Pairwise analysis identified five orphan NRs (including RORß, TLX, COUP-TFII, NURR1 and LRH-1) that showed common upregulation in both mRNA and protein levels in the prostatospheroids and castration-relapse VCaP-CRPC xenografts, and overexpression of these orphan NRs could increase cancer stem cell marker expressions and enhance spheroid formation capacity in prostate cancer cells, suggesting that these orphan NRs might perform positive roles in the growth regulation of PCSCs and castration-resistant prostate cancer. Together, our NR expression dataset not only revealed the distinct physiologic status and regulatory roles governed by the networks of specific NRs but also some of these identified orphan NRs could be the potential therapeutic targets for PCSCs or castration-resistant prostate cancer.

The emerging roles of orphan nuclear receptors in prostate cancer.

Orphan nuclear receptors are members of the nuclear receptor (NR) superfamily and are so named because their endogenous physiological ligands are either unknown or may not exist. Because of their important regulatory roles in many key physiological processes, dysregulation of signalings controlled by these receptors is associated with many diseases including cancer. Over years, studies of orphan NRs have become an area of great interest because their specific physiological and pathological roles have not been well-defined, and some of them are promising drug targets for diseases. The recently identified synthetic small molecule ligands, acting as agonists or antagonists, to these orphan NRs not only help to understand better their functional roles but also highlight that the signalings mediated by these ligand-independent NRs in diseases could be therapeutically intervened. This review is a summary of the recent advances in elucidating the emerging functional roles of orphan NRs in cancers, especially prostate cancer. In particular, some orphan NRs, RORγ, TR2, TR4, COUP-IFII, ERRα, DAX1 and SHP, exhibit crosstalk or interference with androgen receptor (AR) signaling in either normal or malignant prostatic cells, highlighting their involvement in prostate cancer progression as androgen and AR signaling pathway play critical roles in this process. We also propose that a better understanding of the mechanism of actions of these orphan NRs in prostate gland or prostate cancer could help to evaluate their potential value as therapeutic targets for prostate cancer.

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21(WAF1) (/) (CIP1) ) and SIRT1 genes.

Oncogene-induced senescence is an important tumour-suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene-induced senescence. We determined that TLX exhibited an increased expression in high-grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over-expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin-induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene-induced senescence induced either by activated oncogene H-Ras(G12V) or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence-associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene-induced senescence, and also suggests that targeting the senescence-regulatory TLX is of potential therapeutic significance in prostate cancer.

Estrogen-related receptor α decreases RHOA stability to induce orientated cell migration.

Several physiopathological processes require orientated cellular migration. This phenomenon highly depends on members of the RHO family of GTPases. Both excessive and deficient RHO activity impair directional migration. A tight control is thus exerted on these proteins through the regulation of their activation and of their stability. Here we show that the estrogen-related receptor α (ERRα) directly activates the expression of TNFAIP1, the product of which [BTB/POZ domain-containing adapter for Cullin3-mediated RhoA degradation 2 (BACURD2)] regulates RHOA protein turnover. Inactivation of the receptor leads to enhanced RHOA stability and activation. This results in cell disorientation, increased actin network, and inability to form a lamellipodium at the migration edge. As a consequence, directional migration, but not cell motility per se, is impaired in the absence of the receptor, under pathological as well as physiological conditions. Altogether, our results show that the control exerted by ERRα on RHOA stability is required for directional migration.

Coadministrating luteolin minimizes the side effects of the aromatase inhibitor letrozole.

Aromatase inhibitors (AIs) have been used as adjuvant therapeutic agents for breast cancer. Their adverse side effect on blood lipid is well documented. Some natural compounds have been shown to be potential AIs. In the present study, we compared the efficacy of the flavonoid luteolin to the clinically approved AI letrozole (Femara; Novartis Pharmaceuticals, East Hanover, NJ) in a cell and a mouse model. In the in vitro experimental results for aromatase inhibition, the Ki values of luteolin and letrozole were estimated to be 2.44 µM and 0.41 nM, respectively. Both letrozole and luteolin appeared to be competitive inhibitors. Subsequently, an animal model was used for the comparison. Aromatase-expressing MCF-7 cells were transplanted into ovariectomized athymic mice. Luteolin was given by mouth at 5, 20, and 50 mg/kg, whereas letrozole was administered by intravenous injection. Similar to letrozole, luteolin administration reduced plasma estrogen concentrations and suppressed the xenograft proliferation. The regulation of cell cycle and apoptotic proteins-such as a decrease in the expression of Bcl-xL, cyclin-A/D1/E, CDK2/4, and increase in that of Bax-was about the same in both treatments. The most significant disparity was on blood lipids. In contrast to letrozole, luteolin increased fasting plasma high-density lipoprotein concentrations and produced a desirable blood lipid profile. These results suggested that the flavonoid could be a coadjuvant therapeutic agent without impairing the action of AIs.

Ion channel TRPM8 promotes hypoxic growth of prostate cancer cells via an O2 -independent and RACK1-mediated mechanism of HIF-1α stabilization.

The growth adaptation of cancer cells to a hypoxic tumour microenvironment is mostly regulated by hypoxia-induced transcription factor HIF-1. HIF-1 transcriptional activity is strictly controlled by protein levels of the HIF-1α subunit, which is tightly regulated by a well-characterized O2 -dependent ubiquitin ligase-proteasomal degradation pathway. The cold-sensitive Ca(2+) channel protein TRPM8 exhibits increased expression in advanced prostate cancer. However, its exact functional roles in prostate cancer growth regulation are unclear and controversial. In this work, we show that TRPM8 promotes in vitro hypoxic growth capacities, drug resistance, and in vivo tumourigenicity, accompanied with enhanced HIF-1α protein levels. These effects are further potentiated by TRPM8 agonists but suppressed by TRPM8 gene knockdown and blocking with antagonists or TRPM8 antibody. TRPM8-induced suppression of HIF-1α ubiquitination and enhanced HIF-1 transactivation were attenuated by forced RACK1 expression and TRPM8 overexpression reduced phospho-RACK1 levels, thus affecting its dimerization status, and promoted RACK1 binding to HIF-1α and calcineurin. These data indicate that TRPM8-induced increase of HIF-1α protein in hypoxia- or normoxia-exposed prostate cancer cells was mediated through a newly characterized Ca(2+) -dependent but O2 -independent mechanism involving binding of RACK1 to HIF-1α and RACK1-mediated ubiquitination of HIF-1α. Collectively, our study not only provides a mechanistic insight into how TRPM8 promotes the hypoxic growth adaptation of cancer cells via its promotion of RACK1-mediated stabilization of HIF-1α but also suggests a potential therapeutic strategy for prostate cancer by targeting TRPM8.

Celecoxib increases miR-222 while deterring aromatase-expressing breast tumor growth in mice.

BACKGROUND: Breast cancer is one of the most deadly diseases in women. Inhibiting the synthesis of estrogen is effective in treating patients with estrogen-responsive breast cancer. Previous studies have demonstrated that use of cyclooxygenase (COX) inhibitors is associated with reduced breast cancer risk. METHODS: In the present study, we employed an established mouse model for postmenopausal breast cancer to evaluate the potential mechanisms of the COX-2 inhibitor celecoxib. Aromatase-expressing MCF-7 cells were transplanted into ovariectomized athymic mice. The animals were given celecoxib at 1500 ppm or aspirin at 200 ppm by oral administration with androstenedione injection. RESULTS: Our results showed that both COX inhibitors could suppress the cancer xenograft growth without changing the plasma estrogen level. Protein expression of ERα, COX-2, Cyclin A, and Bcl-xL were reduced in celecoxib-treated tumor samples, whereas only Bcl-xL expression was suppressed in those treated with aspirin. Among the breast cancer-related miRNAs, miR-222 expression was elevated in samples treated with celecoxib. Further studies in culture cells verified that the increase in miR-222 expression might contribute to ERα downregulation but not the growth deterrence of cells. CONCLUSION: Overall, this study suggested that both celecoxib and aspirin could prevent breast cancer growth by regulating proteins in the cell cycle and apoptosis without blocking estrogen synthesis. Besides, celecoxib might affect miR expression in an undesirable fashion.

Assessing the effect of food mycotoxins on aromatase by using a cell-based system.

Zeranol, aflatoxin B1, zearalenone are mycotoxins that are commonly found as food contaminants. The chemical structures of zeranol and zearalenone resemble estrogen, and may disrupt hormone metabolism. The biosynthesis of estrogen is catalyzed by aromatase or CYP19. In the present study, effect of these mycotoxins on aromatase was evaluated by using 4 cell lines, i.e. the CYP19-overexpressing cells MCF-7aro, the placental cells JEG-3, the breast cells MCF-7, and the brain cells T98G. Our data indicated that zearalenone was a competitive inhibitor of aromatase with a K(i) value of 1 µM. As aromatase expression is promoter-specific and regulated by alternate splicing, we employed three cell lines for investigation. Our results showed that zearalenone and zeranol could suppress aromatase expression through promoters II and I.3. For aromatase transcription dictated by promoters I.f and I.1, the expression was not affected. Taken together, zearalenone was a potential aromatase inhibitor among the three mycotoxins tested. Furthermore, this 4-cell line approach could be employed in principle to screen for compounds inhibiting or inducing estrogen synthesis.

ERRα augments HIF-1 signalling by directly interacting with HIF-1α in normoxic and hypoxic prostate cancer cells.

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up-regulation of hypoxia-inducible factor 1 (HIF-1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen-related receptor-α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα-over-expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF-1α protein expression and HIF-1 signalling. These effects could also be observed in ERRα-over-expressing cells grown under normoxia, suggesting that ERRα could function to pre-adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF-1α via its AF-2 domain. A ubiquitination assay showed that this ERRα-HIF-1α interaction could inhibit ubiquitination of HIF-1α and thus reduce its degradation. Such ERRα-HIF-1α interaction could be attenuated by XCT790, an ERRα-specific inverse agonist, resulting in reduced HIF-1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF-1α, suggesting ERRα as a potential therapeutic target for cancer treatment.

FK-16 derived from the anticancer peptide LL-37 induces caspase-independent apoptosis and autophagic cell death in colon cancer cells.

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF-/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.

Increased expression of activated endothelial nitric oxide synthase contributes to antiandrogen resistance in prostate cancer cells by suppressing androgen receptor transactivation.

Development of antiandrogen-resistance in advanced prostate cancer involves multiple androgen receptor (AR)-dependent and -independent pathways. Here, we demonstrated that endothelial nitric oxide synthase (eNOS) exhibited an overexpression pattern in hormone-refractory prostate cancer and several models of advanced hormone-resistant prostate cancer. We further established a novel in vitro model of antiandrogen-resistant prostate cancer (LNCaP-BC) by long-term bicalutamide treatment. Besides antiandrogen-resistant and other enhanced malignant growth phenotypes, LNCaP-BC cells exhibited an increased activated eNOS expression and NO production, and suppressed AR transactivation status. Treatment with a NOS inhibitor L-NAME could re-sensitize the growth response to bicalutamide and enhance the AR transactivation in LNCaP-BC cells. Together, our present findings indicate that increased NO production by acquired increased expression of activated eNOS could contribute to the antiandrogen-resistant growth of prostate cancer cells, via a mechanism of NO-mediated suppression of AR activity, and also targeting eNOS could be a potential therapeutic strategy for antiandrogen-resistant prostate cancer.

The citrus flavonone hesperetin prevents letrozole-induced bone loss in a mouse model of breast cancer.

Aromatase is a key enzyme in estrogen synthesis, and aromatase inhibitors (AIs) have been developed for treating estrogen-responsive breast cancer. Because of its nondiscriminatory inhibition of estrogen synthesis, patients treated with AIs also contract diseases typically associated with estrogen deficiency, such as bone deterioration. Our laboratory found that the citrus flavonone hesperetin could inhibit aromatase, and the selective estrogen receptor modulator nature of flavonoid might counteract the undesirable effect of AIs. In the present study, we employed an established postmenopausal model for breast carcinogenesis to examine the drug interaction between hesperetin and letrozole, one of the AIs. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells (MCF-7aro). Hesperetin was administered in the diet at 5000 ppm, and letrozole was injected sc at different doses. Results showed that either hesperetin or letrozole could reduce plasma estrogen level and inhibit tumor growth. Most importantly, the letrozole-induced bone loss measured as bone volume fraction was reversed by hesperetin without compromising on the deterrence of MCF-7aro tumor growth. Taken together, the present study suggested that hesperetin could be a potential cotherapeutic agent to AI.

Transient receptor potential channel TRPC5 is essential for P-glycoprotein induction in drug-resistant cancer cells.

An attractive strategy to overcome multidrug resistance in cancer chemotherapy is to suppress P-glycoprotein (P-gp), which is a pump overproduced in cancer cells to remove cytotoxic drugs from cells. In the present study, a Ca(2+)-permeable channel TRPC5 was found to be overproduced together with P-gp in adriamycin-resistant breast cancer cell line MCF-7/ADM. Suppressing TRPC5 activity/expression reduced the P-gp induction and caused a remarkable reversal of adriamycin resistance in MCF-7/ADM. In an athymic nude mouse model of adriamycin-resistant human breast tumor, suppressing TRPC5 decreased the growth of tumor xenografts. Nuclear factor of activated T cells isoform c3 (NFATc3) was the transcriptional factor that links the TRPC5 activity to P-gp production. Together, we demonstrated an essential role of TRPC5-NFATc3-P-gp signaling cascade in P-gp induction in drug-resistant cancer cells.

The citrus flavonone hesperetin inhibits growth of aromatase-expressing MCF-7 tumor in ovariectomized athymic mice.

Aromatase is responsible for the rate-determining reaction in estrogen synthesis and is a prime target for treating estrogen-receptor-positive breast cancer. Previous in vitro study has demonstrated that apigenin (APG), naringenin (NGN) and hesperetin (HSP) are three of the most potent natural aromatase inhibitors. Because the enzyme inhibition could potentially block breast cancer development, we employed an established postmenopausal breast cancer model to examine the chemopreventive effect of these flavonoids in vivo. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells. Dietary administration of HSP at 1000 ppm and 5000 ppm significantly deterred the xenograft growth, while a null effect was observed in mice treated with APG or NGN. Further study illustrated that plasma estrogen in HSP-treated mice was reduced. Messenger RNA expression of the estrogen-responsive gene pS2 was also decreased in the tumors of mice treated with 1000 and 5000 ppm HSP. On the other hand, western analysis indicated that cyclin D1, CDK4 and Bcl-x(L) were reduced in the tumors. This study suggested that HSP could be a potential chemopreventive agent against breast carcinogenesis through aromatase inhibition.

17-Beta-estradiol induces neoplastic transformation in prostatic epithelial cells.

Although estrogens have been long implicated in the prostate carcinogenesis, direct evidence showing their carcinogenicity on prostatic epithelial cells has not yet been clearly demonstrated. In this study, we treated an immortalized, non-transformed and androgen-responsive rat prostatic epithelial cell line NRP-152 with 17ß-estradiol (E2) at concentrations 1-3 microM for period 2-6 weeks. After in vitro treatment, we evaluated the anchorage-independent growth of E2-treated NRP-152 cells by soft agar assay and isolated the colonies formed by the transformed E2-NRP-152 cells in soft agar for further growth phenotype characterization. Our results showed that the isolated E2-NRP-152 clones displayed neoplastic transformation phenotype, as demonstrated by their capacity of forming colonies in soft agar and tumors in immunodeficient nude mice, while losing their spheroid formation capacity in Matrigel 3D-culture. Western blot and RT-PCR analyses showed that the transformed E2-NRP-152 cells expressed increased levels of ERα and several putative prostate cancer stem cell markers (integrins α2ß1, CD44, CD133, ABCG2 and CXCR4) but decreased levels of ERß and AR. Comet assay revealed that E2-treatment also induced formation of comet cells, indicating that E2 caused DNA damage to the NRP-152 cells. Our present findings demonstrated that in vitro E2 exposure could neoplastically transform the rat prostatic epithelial cells, indicating that E2 is carcinogenic to the prostatic epithelial cells.

Identification of differently expressed genes in chemical carcinogen-induced rat bladder cancers.

Possible altered gene expression patterns in bladder tumour carcinogenesis in rat bladder cancers induced by BBN [N-butyl-N-(4-hydroxybutyl)nitrosamine] was examined by cDNA microarray analysis of gene expression profiles. Thirty Sprague-Dawley rats were given drinking water containing 0.05% BBN ad libitum for 24 to 28 weeks. Equal numbers of control rats were given tap water without BBN. After treatment, the rat bladders were excised for RNA extraction and histopathological examinations. Total RNAs were extracted from rat transitional cell carcinoma (TCC) tissues and micro-dissected normal rat bladder epithelia. The atlas glass rat microarray was used, which included oligonucleotides of 1081 rat genes. Some of the up-regulated genes in rat bladder TCCs were further confirmed by Northern blotting. Our results showed that the transcriptions of 30 genes were significantly elevated in the rat bladder TCCs, and these included fly proto-oncogene, Lipocortin 2, COX IV, COX V a, and cathepsin D. Also, 15 genes were significantly down-regulated in the rat bladder TCCs and they included B7.1, TNFr1, APOA1 and VHL. The results of cDNA microarray analysis demonstrated that normal rat bladder epithelia and bladder TCC exhibited different and specific gene statement profiles. The increased expressions of the identified genes may play an important role in the chemically induced bladder carcinogenesis.

Dietary administration of the licorice flavonoid isoliquiritigenin deters the growth of MCF-7 cells overexpressing aromatase.

Licorice is the sweet-tasting rhizomes of a bean plant and is quite commonly used in Western countries for culinary purposes, while it is a medicinal herb in China. Many flavonoids have been isolated from licorice, and their pharmacological properties may be applicable in preventive medicine. Overexposure to estrogen has been implicated in the etiology of breast cancer, and cytochrome P450 (CYP) 19 enzyme, or aromatase, catalyzes the rate-limiting reaction. Phytocompounds that are able to inhibit this enzyme may potentially suppress breast cancer development. In the present study the licorice flavonoid isoliquiritigenin (ILN) was shown to be an aromatase inhibitor in recombinant protein and MCF-7 cells stably transfected with CYP19 (MCF-7aro). ILN displayed a K(i) value of around 3 muM, and it also blocked the MCF-7aro cell growth pertaining to the enzyme activity in vitro. Subsequently, the compound administered in diet was given to ovariectomized athymic mice transplanted with MCF-7aro cells. This mouse model is widely accepted for studying postmenopausal breast cancer. The phytochemical significantly deterred the xenograft growth without affecting the body weight. Subsequently, the flavonoid's effect on CYP19 transcriptional control in vitro was also investigated. At the mRNA level, ILN could also suppress the expression in wild-type MCF-7 cells. Reporter gene assay and real-time PCR verified that the transactivity of CYP19 driven by promoters I.3 and II was suppressed in these cells. Deactivation of C/EBP could be the underlying molecular mechanism. Our study demonstrated that ILN was an inhibitor of aromatase and a potential chemopreventive agent against breast cancer.