Liudi Zhang

Naringenin exerts anti-angiogenic effects in human endothelial cells: Involvement of ERRα/VEGF/KDR signaling pathway.

Naringenin (Nar), most abundant in oranges and tomatoes, are known for the hypocholesterolemic, anti-estrogenic, hypolipidemic, anti-hypertensive, and anti-inflammatory activities. Here, the present study was designed to investigate the in vitro and in vivo anti-angiogenesis of Nar. Inhibition of angiogenesis was determined in vitro by using proliferation, apoptosis, migration, and tube-formation assays in Nar-treated human endothelial cell. Finally, CAM assays were used to assess inhibitory effect of Nar on physiological angiogenesis in vivo. The data suggest that Nar should be a direct ERRα inhibitor capable of inhibiting angiogenesis in vitro and in vivo, including endothelial cell proliferation, survival, migration and capillary-like structures formation of HUVECs, as well as reduced neovascularization of the CAM. Furthermore, the effects exerted by Nar are cell cycle related and mediated by VEGF/KDR signaling pathway along with downregulation of certain proangiogenic inflammatory cytokines. Our data thus provide potential molecular mechanisms through which Nar manifests it as a promising anti-angiogenic and anti-cancer agent.

Downregulation of ERRα inhibits angiogenesis in human umbilical vein endothelial cells through regulating VEGF production and PI3K/Akt/STAT3 signaling pathway.

The human estrogen related receptor α (ERRα) is a pivotal regulator involved in energy homeostasis and mitochondrial biogenesis. It has been demonstrated that activation of ERRα in various breast cancer cells results in a significant increase of vascular endothelial growth factor (VEGF) mRNA and protein secretion. However, little is known about the relationship between ERRα and angiogenesis. Thus, the present study is aimed to investigate the effects and mechanism of ERRα suppression on the angiogenesis in human umbilical vein endothelial cells (HUVECs). Here we show that ERRα suppression powerfully inhibits proliferation, migration and capillary-like structures formation of HUVECs. Importantly, we demonstrate that these inhibitory effects are associated with the significantly reduced expression and production of VEGF. Results from further experiments using western blot and luciferase reporter assay exhibit that ERRα suppression inhibits hypoxia-inducible factor 1α (HIF-1α) expression, and phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription (STAT3) which up-regulated VEGF expression. In summary, we show that ERRα suppression inhibits angiogenesis in HUVECs and deserves further studies for application of rationale therapeutic target for patient with diseases related with aberrant angiogenesis.

Inhibition of estrogen related receptor α attenuates vascular smooth muscle cell proliferation and migration by regulating RhoA/p27 Kip1 and β-Catenin/Wnt4 signaling pathway

Abstract RhoA/p27Kip1 and &bgr;‐Catenin/Wnt4 signaling processes play central roles in proliferation and migration in vascular smooth muscle cells (VSMCs). ERR&agr;, a member of orphan nuclear receptors, is a potent prognostic factor in breast, ovarian, colon and other types of tumors. However, biological significance of ERR&agr; in VSMCs as well as the molecular mechanisms remains largely unknown. Therefore, the present study was designed to investigate whether ERR&agr; is involved in the proliferation and migration of VSMCs in vitro and neointimal formation in vivo. The specific ERR&agr; inverse agonist XCT790 (or ERR&agr; shRNA) resulted in a significant inhibition of proliferation and phenotypic switch in cultured rat aortic SMCs (RASMCs). Furthermore, cycle progression, cell cycle protein transcription as well as hyperphosphorylation of the retinoblastoma protein (Rb) in RASMCs were prevented by downregulation of ERR&agr;. Transwell assay demonstrated that migratory capacity of RASMCs was also inhibited the treatment of XCT790 (or ERR&agr; shRNA). At the molecular levels, RhoA/p27Kip1 and &bgr;‐Catenin/Wnt4 signaling pathways are involved in ERR&agr;‐mediated RASMCs growth and migration. Finally, inhibition of ERR&agr; significantly attenuated neointimal formation in rat artery after balloon injury. These results help to further understand vascular remodeling and suggest that ERR&agr; might be a potential target for the treatment of vascular proliferative diseases.

Characterization of a selective inverse agonist for estrogen related receptor α as a potential agent for breast cancer

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor that plays a primary role in the regulation of cellular energy homeostasis and osteogenesis. It is reported that ERRα is widely expressed in a range of tissues and accumulating evidence has supported that the high expression of ERRα correlates with poor prognosis of various human malignancies, including breast, endometrium, colon, prostate and ovary cancers. Herein is described the discovery of a novel selective inverse agonist (HSP1604) of ERRα, but not of ERRβ and ERRγ, as determined using transient transfection luciferase reporter assay and a time-resolved fluorescence resonance energy transfer (TR-FRET) co-activator assay. HSP1604 potently inhibits ERRα transcriptional activity with IC50=1.47±0.17μM in cell-based luciferase reporter assay and also decreases the protein level of ERRα and the mRNA levels of its downstream target genes such as pyruvate dehydrogenase kinase 4 (PDK4), pS2 and osteopontin. HSP1604 has also suppressed the proliferation of different human cancer cell lines and the migration of breast cancer cells with high expression of ERRα. Representative in vivo results show that HSP1604 suppresses the growth of human breast cancer xenograft in nude mice as doses at 30mg/kg or 100mg/kg administered every other day during 28-day period. HSP1604 thus has the potential both as a new agent to inhibit the growth of tumors and as a chemical probe of ERRα biology.

Inhibition of PDGF-BB-induced proliferation and migration in VSMCs by proanthocyanidin A2: Involvement of KDR and Jak-2/STAT-3/cPLA2 signaling pathways

Proanthocyanidin A2 (PA2), one of A-type proanthocyanidins, has been shown to harbor a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anti-HIV, anti-CDV and anti-?-glucosidase activities. However, little is known about the role for PA2 in regulating PDGF-induced VSMC proliferation and migration. In the present study, we investigated the possible effects of PA2 on PDGF-BB-induced proliferation, migration and inflammation in VSMCs in vitro to mimic a postangioplasty PDGF shedding condition. Herein, the data clearly show that PA2 markedly inhibited proliferation, migration and inflammatory responses at 0-30??g/ml concentration in VSMCs in vitro. 10-30??g/ml PA2 inhibited PDGF-mediated NAD(P)H oxidase activation and intracellular ROS formation in VSMCs. Furthermore, the effects exerted by PA2 involve the participation of KDR and Jak-2/STAT-3/cPLA2 signaling pathways. These data also highlight the possible therapeutic use of PA2 in vascular proliferative diseases, where abnormal proliferation and migration play important pathological roles.

A novel compound LingH2-10 inhibits the growth of triple negative breast cancer cells in vitro and in vivo as a selective inverse agonist of estrogen-related receptor α

Unlike other breast cancer subtypes, targeted therapies for triple negative breast cancer (TNBC) have yet to progress past clinical trial stage to approval. Accumulating evidences demonstrated that expression of estrogen-related receptor alpha (ERRα) indicated worse prognosis and correlated with poor outcome in breast cancers including TNBC. Therefore, ERRα modulators/regulators may be potential in the therapeutic treatment of breast cancers. In the current study, we presented a novel compound LingH2-10 that bound to ERRα, as identified using a time-resolved fluorescence resonance energy transfer assay (TR-FRET) with the IC50 value of 0.64±0.12μM. Further, functional activity was determined by transient transfection luciferase reporter assay in order to validate the utility of the binding affinity in a cellular context. LingH2-10 showed selective inhibition on ERRα transcriptional activity with the IC50 value of 0.58±0.09μM in cell-based luciferase reporter assay. Moreover, representative in vitro results showed that LingH2-10 suppressed the proliferation of various human cancer cells, and inhibited the migration of triple negative breast cancer cell MDA-MB-231. In addition, our results demonstrated that well known ERRα target genes such as PDK4, Osteopontin and pS2, were all significantly down modulated by LingH2-10. In vivo experiments showed that LingH2-10 (30mg/kg, every other day) observably suppressed the growth of MDA-MB-231 xenograft tumors by 42.02% compared to untreated xenograft tumors. Taken together, all these data suggested that LingH2-10, as a selective inverse agonist of ERRα, was a lead compound of anti-cancer agents for treating TNBC patients.

Downregulation of estrogen-related receptor alpha inhibits human cutaneous squamous cell carcinoma cell proliferation and migration by regulating EMT via fibronectin and STAT3 signaling pathways

Abstract Estrogen‐related receptor alpha (ERR&agr;), one of orphan nuclear receptors, has been recently revealed as an oncogenic regulator in a variety of cancers. However, the linking gain of ERR&agr; expression and cancer progression in cutaneous squamous cell carcinoma (cSCC) is largely unknown. Here, we showed that the mRNA and protein expression levels of ERR&agr; were markedly higher in A431 cells compared with human keratinocyte cell line HaCaT, and targeted inhibition of ERR&agr; by shRNA or its inverse agonist XCT790 significantly suppressed A431 cells proliferation and migration, while overexpression of ERR&agr; promoted cell proliferation and migration. In addition, the data revealed that ERR&agr; downregulation markedly inhibited the epithelial mesenchymal transition (EMT) of A431 cells with increasing the expression of E‐cadherin and decreasing fibronectin (FN) and vimentin. Results from further experiments using Western blot suggested that ERR&agr; suppression inhibited signal transducer and activator of transcription (STAT3) protein expression. In contrast, overexpression of ERR&agr; promoted EMT and the activation of STAT3 pathway. Moreover, treatment with specific STAT3 inhibitor reversed EMT markers in ERR&agr;‐overexpressing A431 cells. In tumor xenografts of A431 cells, we further showed that ERR&agr; depletion inhibited cSCC tumor growth in vivo. Taken together, these results demonstrate, for the first time, that ERR&agr; may function as an oncoprotein in cSCC to accelerate tumor aggressiveness by promoting EMT via FN and STAT3 pathway, and it could be a novel target for cSCC therapy.

Effects of FGFR gene polymorphisms on response and toxicity of cyclophosphamide-epirubicin-docetaxel-based chemotherapy in breast cancer patients

BackgroundThe chemotherapy resistance and toxicity of chemotherapy are major problems in breast cancer treatment. However, candidate biomarkers for predicting clinical outcomes and better prognosis remain lacking.MethodsIn this study, we analyzed possible impact of 8 genetic variants of fibroblast growth factor receptor1–4 (FGFR1–4) on the treatment response and toxicities in 211 breast cancer patients. DNA was extracted from peripheral blood cells, and the genotypes were examined using the TaqMan Pre-Designed SNP Genotyping Assays.ResultsThe FGFR4 rs1966265 and FGFR2 rs2981578 contributed to clinical outcome of breast cancer treated with docetaxel–epirubicin-cyclophosphamide (CET)-based chemotherapy. For rs1966265, AA genotype had significant correlation with the clinical response to neoadjuvant chemotherapy (NCT) when compared with GG and AG/GG genotype (P = 0.019 and P = 0.004, respectively). Moreover, A allele of FGFR2 rs2981578 had significant rates of response (P = 0.025). In addition, rs2420946 CC genotype was associated with higher frequency of toxicities compared with TT and CT/TT genotypes (P = 0.038 and P = 0.019, respectively). Also, rs2981578 AG genotype showed higher frequency of toxicities compared with GG genotype (P < 0.0001).ConclusionsThe results suggest these polymorphisms, especially rs1966265 and rs2981578, might be candidate pharmacogenomics factors to the response and prognosis prediction for individualized CET-based chemotherapy in breast cancer patients.