Ionizing radiation downregulates estradiol synthesis via endoplasmic reticulum stress and inhibits the proliferation of estrogen receptor-positive breast cancer cells

Breast cancer is a major threat to women’s health and estrogen receptor-positive (ER+) breast cancer exhibits the highest incidence among these cancers. As the primary estrogen, estradiol strongly promotes cellular proliferation and radiotherapy, as a standard treatment, exerts an excellent therapeutic effect on ER+ breast cancer. Therefore, we herein wished to explore the mechanism(s) underlying the inhibitory effects of radiation on the proliferation of ER+ breast cancer cells. We used the ER+ breast cancer cell lines MCF7 and T47D, and their complementary tamoxifen-resistant cell lines in our study. The aforementioned cells were irradiated at different doses of X-rays with or without exogenous estradiol. CCK8 and clone-formation assays were used to detect cellular proliferation, enzyme-linked immunosorbent assay (ELISA) to determine estradiol secretion, western immunoblotting analysis and quantitative real-time PCR to evaluate the expression of proteins, and immunofluorescence to track endoplasmic reticulum stress-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays to explore the protein expression in tumors using immunohistochemistry. We found that ionizing radiation significantly reduced the phosphorylation of estrogen receptors and the secretion of estradiol by ER+ breast cancer cells. CYP19A (aromatase) is an enzyme located in the endoplasmic reticulum, which plays a critical role in estradiol synthesis (aromatization), and we further demonstrated that ionizing radiation could induce endoplasmic reticulum stress with or without exogenous estradiol supplementation, and that it downregulated the expression of CYP19A through ER-phagy. In addition, ionizing radiation also promoted lysosomal degradation of CYP19A, reduced estradiol synthesis, and inhibited the proliferation of tamoxifen-resistant ER+ breast cancer cells. We concluded that ionizing radiation downregulated the expression of CYP19A and reduced estradiol synthesis by inducing endoplasmic reticulum stress in ER+ breast cancer cells, thereby ultimately inhibiting cellular proliferation.

Regulation of estradiol synthesis by aromatase interacting partner (AIPB)

Estradiol is essential for the development of female sex characteristics and fertility. Postmenopausal women and breast cancer patients have high estradiol. Aromatase catalyzes estradiol synthesis; however, the factors regulating aromatase activity are unknown. We identified a new 22-kDa protein, aromatase interacting partner in breast (AIPB), from the endoplasmic reticulum of human breast tissue. AIPB expression is reduced in tumorigenic breast and further reduced in triple negative tumors. Like aromatase, AIPB expression is induced by nonsteroidal estrogen. We found that AIPB and aromatase interact in nontumorigenic and tumorigenic breast tissues and cells. Conditional AIPB over expression decreased estradiol and blocking AIPB availability with an AIPB binding antibody increased estradiol in tumorigenic cells. Estradiol synthesis is highly increased in AIPB knockdown cells, suggesting that the newly identified AIPB protein is important for aromatase activity and a key modulator of estradiol synthesis. Thus, a change in AIPB protein expression may represent an early event in tumorigenesis and be predictive of an increased risk of developing breast cancer.

FGF21 Promotes Proliferation and Estradiol Synthesis in Porcine Granulosa Cells

Background: The proliferation and estradiol synthesis in granulosa cells (GCs) directly promotes follicular development. Previous studies had found that FGF21 regulated the hypothalamic-pituitary-gonad axis in response to the control of fertility. However, the functions and mechanisms of FGF21 in GCs are unclear.Results: Here, we found that the mRNA and protein levels of FGF21 in the ovarian tissue of high-yielding sows (Sus scrofa) was higher than that in low-yielding sows. Moreover, FGF21 was predominantly expressed in porcine GCs. Additionally, ELISA assay showed estradiol was significantly increased when overexpression of FGF21 in porcine GCs. Meanwhile, overexpressed FGF21 up-regulated both the mRNA and protein levels of key estradiol synthesis-related genes in porcine GCs, including StAR, CYP11A1 and CYP19A1. Corresponsingly, knockdown FGF21 inhibited estradiol levels and its synthesis-related genes expression. Besides, overexpression of FGF21 promoted the proliferation of porcine GCs, displayed as increasing the percentage of S-phase cells in cell cycle and EdU positive cells, including cell viability, and upregulated cell cycle genes, including cell cycle protein B (Cyclin B) and protein E (Cyclin E). Corresponsingly, knockdown FGF21 in porcine GCs suppressed the cell cycle and cell viability, as well as EdU positive cell number.Conclusions: These findings highlight that FGF21 is associated with the development of GCs and may be a novel underlying regulator of porcine follicular development.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = −6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

lncRNA MALAT1 Regulates Mouse Granulosa Cell Apoptosis and 17β-Estradiol Synthesis via Regulating miR-205/CREB1 Axis

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a known long noncoding RNA, was reported to play a crucial role in follicular growth and ovarian disease. However, the physiological function of MALAT1 in mouse granulosa cells (mGCs) remains largely unclear. The aims of this study were to determine the biological function and molecular mechanism of MALAT1 in mGCs. We knocked down MALAT1 in mGCs by using siRNA against MALAT1. We found that knockdown of MALAT1 promoted apoptosis and caspase-3/9 activities in mGCs. Enzyme-linked immunosorbent assay demonstrated that knockdown of MALAT1 significantly decreased the production of estradiol (E2) and progesterone (P4) in mGCs. Mechanistically, MALAT1 serves as a competing endogenous RNA (ceRNA) to sponge microRNA-205 (miR-205), thereby facilitating its downstream target of cyclic AMP response element- (CRE-) binding protein 1 (CREB1). Furthermore, CREB1 overexpression or miR-205 downregulation partially recovered the effect of MALAT1 depletion in mGCs. In summary, these findings suggested that MALAT1 regulated apoptosis and estradiol synthesis of mGCs through the miR-205/CREB1 axis.

Comparison of In Vitro Endocrine Activity of Phthalates and Alternative Plasticizers

Because of the deleterious effects of phthalates, regulations have been taken to decrease their use, and the needs for alternatives are increasing. Due to the concerns about the endocrine-disrupting properties of phthalates, it was deemed necessary to particularly investigate these effects for potential substitutes. In this study, we compared the in vitro endocrine activity of several already used potential alternative plasticizers (DEHT, DINCH, and TOTM) or new substitutes (POLYSORB® isosorbide and POLYSORB® ID 46) to one of 2 phthalates, DEHP and DINP. Effects of these chemicals on 3 common mechanisms of endocrine disruption, i.e., interaction with estrogen receptors (ER), androgen receptors (AR), or steroidogenesis, were studied using extensively used in vitro methods. In the E-Screen assay, only DEHP moderately induced MCF-7 cell proliferation; none of the other tested substances were estrogenic or antiestrogenic. No androgenic or antiandrogenic activity in MDA-kb2 cells was shown for any of the tested phthalates or alternatives. On the other hand, both DEHP and DINP, as well as DEHT, DINCH, and TOTM, disrupted steroidogenesis in the H295R assay, mainly by inducing an increase in estradiol synthesis; no such effect was observed for POLYSORB® isosorbide and POLYSORB® ID 46.