Chrysoeriol Prevents TNFα-Induced CYP19 Gene Expression via EGR-1 Downregulation in MCF7 Breast Cancer Cells

Estrogen overproduction is closely associated with the development of estrogen receptor-positive breast cancer. Aromatase, encoded by the cytochrome P450 19 (CYP19) gene, regulates estrogen biosynthesis. This study aimed to identify active flavones that inhibit CYP19 expression and to explore the underlying mechanisms. CYP19 expression was evaluated using reverse transcription PCR, quantitative real-time PCR, and immunoblot analysis. The role of transcription factor early growth response gene 1 (EGR-1) in CYP19 expression was assessed using the short-hairpin RNA (shRNA)-mediated knockdown of EGR-1 expression in estrogen receptor-positive MCF-7 breast cancer cells. We screened 39 flavonoids containing 26 flavones and 13 flavanones using the EGR1 promoter reporter activity assay and observed that chrysoeriol exerted the highest inhibitory activity on tumor necrosis factor alpha (TNFα)-induced EGR-1 expression. We further characterized and demonstrated that chrysoeriol inhibits TNFα-induced CYP19 expression through inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated EGR-1 expression. Chrysoeriol may be beneficial as a dietary supplement for the prevention of estrogen receptor-positive breast cancer, or as a chemotherapeutic adjuvant in the treatment of this condition.

FOXO1 Negates the Cooperative Action of FOXL2C134W and SMAD3 in CYP19 Expression in HGrC1 Cells by Sequestering SMAD3

Adult granulosa cell tumor (aGCT) is a rare type of ovarian cancer characterized by estrogen excess. Interestingly, only the single somatic mutation FOXL2C134W was found across virtually all aGCTs. We previously reported that FOXL2C134W stimulates CYP19 transcription synergistically with SMAD3, leading to elevated estradiol synthesis in a human granulosa cell line (HGrC1). This finding suggested a key role for FOXL2C134W in causing the typical estrogen overload in patients with aGCTs. We have now investigated the effect of FOXO1, a tumor suppressor, on CYP19 activation by FOXL2C134W in the presence of SMAD3. Intriguingly, FOXO1 antagonized the positive, synergistic effect of FOXL2C134W and SMAD3 on CYP19 transcription. Similar to FOXL2C134W, FOXO1 binds SMAD3 but not the proximal FOXL2C134W binding site (−199 bp) of the CYP19 promoter identified in our earlier studies. The results of a competitive binding assay suggested a possible underlying mechanism in which FOXO1 sequesters SMAD3 away from FOXL2C134W, thereby negating the cooperative action of FOXL2C134W and SMAD3 in inducing CYP19 expression. To our knowledge, this study is the first to demonstrate the ability of FOXO1 to restore an altered CYP19 expression by FOXL2C134W and SMAD3 and provides insight as to why FOXO1 deficiency promotes GCT development in mice.

Regulation by 3,5,3ʹ-tri-iodothyronine and FSH of cytochrome P450 family 19 (CYP19) expression in mouse granulosa cells

Cytochrome P450 family 19 (CYP19) plays an important role in follicular development, which is regulated by FSH. Although 3,5,3′-tri-iodothyronine (T3) combines with FSH to induce preantral follicle growth and granulosa cell development, the mechanism involved remains unclear. The aim of the present study was to determine the cellular and molecular mechanisms by which thyroid hormone (TH) and FSH regulate CYP19 expression and sterol biosynthesis during preantral follicle growth. Mice were injected subcutaneously (s.c.) with eCG (Equine chorionic gonadotropin). The results showed that eCG increased CYP19 expression in ovarian cells. CYP19 expression in granulosa cells was increased after FSH treatment, and this response was enhanced by T3. Knockdown of CYP19 significantly decreased granulosa cell viability and hormone-stimulated proliferation. In addition, CYP19 knockdown also blocked T3- and FSH-induced oestradiol (E2) synthesis in granulosa cells. Furthermore, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was required for T3 and FSH regulation of CYP19 expression. In conclusion, the results of the present study indicate that CYP19 is important for T3- and FSH-induced granulosa cell development in the early stages. CYP19 could be a downstream effector of the PI3K/Akt pathway in regulating TH and FSH during follicular development and sterol biosynthesis. The findings suggest that CYP19 is a novel mediator of T3- and FSH-induced follicular development.