Cyclin G2 promotes cell cycle arrest in breast cancer cells responding to fulvestrant and metformin and correlates with patient survival

Definition of cell cycle control proteins that modify tumor cell resistance to estrogen (E2) signaling antagonists could inform clinical choice for estrogen receptor positive (ER+) breast cancer (BC) therapy. Cyclin G2 (CycG2) is upregulated during cell cycle arrest responses to cellular stresses and growth inhibitory signals and its gene, CCNG2, is directly repressed by E2-bound ER complexes. Our previous studies showed that blockade of HER2, PI3K and mTOR signaling upregulates CycG2 expression in HER2+ BC cells, and that CycG2 overexpression induces cell cycle arrest. Moreover, insulin and insulin-like growth factor-1 (IGF-1) receptor signaling strongly represses CycG2. Here we show that blockade of ER-signaling in MCF7 and T47D BC cell lines enhances the expression and nuclear localization of CycG2. Knockdown of CycG2 attenuated the cell cycle arrest response of E2-depleted and fulvestrant treated MCF7 cells. These muted responses were accompanied by sustained inhibitory phosphorylation of retinoblastoma (RB) protein, expression of cyclin D1, phospho-activation of ERK1/2 and MEK1/2 and expression of cRaf. Our work indicates that CycG2 can form complexes with CDK10, a CDK linked to modulation of RAF/MEK/MAPK signaling and tamoxifen resistance. We determined that metformin upregulates CycG2 and potentiates fulvestrant-induced CycG2 expression and cell cycle arrest. CycG2 knockdown blunts the enhanced anti-proliferative effect of metformin on fulvestrant treated cells. Meta-analysis of BC tumor microarrays indicates that CCNG2 expression is low in aggressive, poor-prognosis BC and that high CCNG2 expression correlates with longer periods of patient survival. Together these findings indicate that CycG2 contributes to signaling networks that limit BC.     

Effects of estradiol on the endocytic transport of vitamin D carrier protein in hepatocytes

Background

The possible modulation of receptor-mediated endocytosis (RME) by sex steroids is not well understood, especially in terms of the different receptor–ligand systems and cell types that may exhibit such regulation. The main objective of the current study was to examine the short-term effects of 17β-estradiol (E2) on RME of an extracellular carrier protein for calciferols, vitamin D-binding protein (DBP).

Methods

Murine male and female primary hepatocytes were treated for 30 min in the absence (controls) or presence of Ε2 (1 μM). Labeled DBP was then added, and its endocytosis was measured after an incubation of 10 min at 37 °C using standard ELISA techniques. To obtain further insight into potential molecular mechanisms, fulvestrant and 17α-ethinyl estradiol (EE) were also analyzed. And as part of comparative analyses, a second nutrient carrier protein, vitamin A-binding protein (RBP), was also analyzed.

Results

The results provide the first evidence for an estradiol-dependent stimulation of DBP endocytosis (p < 0.05 relative to controls without Ε2). This stimulation, however, was only observed in female hepatocytes. Uptake of RBP was enhanced to a similar extent as DBP by estradiol. In normal (non-estradiol treated) male and female hepatocytes such changes in DBP or RBP endocytosis were not observed. Both fulvestrant and EE exhibited a significant (p < 0.05), but incomplete, inhibition of Ε2-dependent stimulation of endocytosis.

Conclusions

The results provide novel evidence for Ε2 effects on endocytic transport; and for gender-related differences in E2-enhanced transport. These Ε2 effects may be partly dependent on estrogen receptors; but possible, additional or alternative mechanisms are also proposed.

General significance

Endocytic transport is a fundamental function whose regulation has implications for cell signaling, growth, survival, differentiation, and death. This study helps delineate a possible endocrine regulatory pathway involving modulation of endocytosis by a steroid hormone. It also provides a potential, new relation between different hormonal regulators, e.g., estradiol effects on cellular assimilation of calciferols.     

Inhibition of soluble epoxide hydrolase by fulvestrant and sulfoxides

The soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of epoxy-fatty acids, signaling molecules involved in numerous biologies. Toward finding novel inhibitors of sEH, a library of known drugs was tested for inhibition of sEH. We found that fulvestrant, an anticancer agent, is a potent (K_I = 26 nM) competitive inhibitor of sEH. From this observation, we found that alkyl-sulfoxides represent a new kind of pharmacophore for the inhibition of sEH.     

The effect of dehydroepiandrosterone administration on intestinal calcium absorption in ovariectomized female rats

[Purpose] Dehydroepiandrosterone (DHEA) administration reportedly recovers osteoporosis, a bone disorder associated with bone deficiency in postmenopausal women. However, the physiological mechanism of DHEA in osteoporosis remains elusive, especially in terms of intestinal calcium absorption. Therefore, we investigated the effect of DHEA administration on calcium absorption in ovariectomized (OVX) female rats using an estrogen receptor antagonist.[Methods] Female Sprague-Dawley rats (n=23, 6 weeks old) were randomized into three groups: OVX control group (OC, n=7), OVX with DHEA treatment group (OD, n=8), and OVX with DHEA inhibitor group (ODI, n=8) for 8 weeks.[Results] Intestinal calcium accumulation, as well as the rate of absorption, demonstrated no significant differences during the experimental period among investigated groups. The bone mineral density (BMD) of the tibia at the proximal metaphysis was higher in the OD group than that in the OC group (p<0.05); however, BMD of the ODI group showed no significant difference from investigated groups. Furthermore, the BMD of the tibia at the diaphysis did not significantly differ among these groups.[Conclusion] We revealed that DHEA administration does not involve intestinal Ca absorption, although this treatment improves BMD levels in OVX rats. These observations indicate that the effect of DHEA on the bone in postmenopausal women is solely due to its influence on bone metabolism and not intestinal calcium absorption.     

Emerging principles for the development of resistance to antihormonal therapy: implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E₂ and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E₂), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E₂. Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E₂ in Phase II antihormone-resistant tumors reversed the E₂-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E₂-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E₂ and fulvestrant.     

Estrogen induces death of tamoxifen-resistant MCF-7 cells: contrasting effect of the estrogen receptor downregulator fulvestrant

A common problem in breast cancer therapy is resistance to the antiestrogen tamoxifen. However, tamoxifen-resistant breast tumors can still respond to other hormonal therapies. In animal models of tamoxifen-resistant breast cancer cells, physiological levels of estrogen can induce tumor regression. Recently, the estrogen receptor downregulator fulvestrant was shown to promote tumor growth of tamoxifen-resistant cells when added in combination with physiological levels of estrogen. Here, we show, using a cell culture model, that continuous exposure of tamoxifen-resistant cells to physiological levels of estrogen leads to cell death. Addition of the estrogen receptor downregulator fulvestrant prevents estrogen-induced death in a dose-dependent manner. Our data indicate that endogenous levels of estrogen affect the response of tamoxifen-resistant cells to fulvestrant. These results suggest that failure of fulvestrant to inhibit tumor growth in some tamoxifen-resistant patients may be due to endogenous estrogen levels. Moreover, these studies support short-term treatment with estrogen as a second-line hormonal therapy for tamoxifen-resistant breast cancer.     

氟维司群纳米聚合物胶束的制备与表征

目的:本文目的是制备氟维司群纳米聚合物胶束,并对其体外特性进行表征。方法:采用生物可降解材料甲氧基聚乙二醇-b-聚D,L-丙交酯(m PEG-b-PDLLA),并用固体分散法制备氟维司群聚合物胶束。利用透射电子显微镜与马尔文激光粒度测定仪分析胶束的形态与粒径。用X射线单晶体衍射仪定性测试胶束包封性。建立并验证氟维司群高效液相色谱(HPLC)分析方法,定量测定胶束载药量与包封率。采用透析袋法分析胶束体外释放情况。结果:氟维司群聚合物胶束形态圆整、分散均匀无粘连,粒径为89.97±4.33 nm,多分散指数为0.162±0.023,载药量与包封率达8.95%±0.86%与97.25%±0.86%。胶束释药具有明显的缓释特点。结论:成功制备氟维司群胶束并显著提高其水溶性,表现良好的缓释行为,能够开发为氟维司群的新型纳米制剂。