Sperm utilize tumor necrosis factor alpha to shut down a 2-methoxyestradiol nongenomic pathway that accelerates oviductal egg transport in the rat.

In brief: Mating shuts down the 2-methoxyestradiol (2ME) nongenomic pathway that accelerates oviductal egg transport in the rat. This study shows that sperm cells, but not vaginocervical stimulation, utilize TNF-α to shut down this 2ME nongenomic pathway. Abstract: The transport of oocytes or embryos throughout the oviduct to the implantation site in the uterus is defined as egg transport. In the rat, 2-methoxyestradiol (2ME) accelerates egg transport through the oviduct via a nongenomic pathway. Mating is known to shut down this 2ME pathway and then trigger an estradiol genomic pathway that accelerates egg transport. Here, we tested whether intrauterine insemination (IUI) or vaginocervical stimulation (VCS) shuts down the 2ME nongenomic pathway that accelerates egg transport, and if these mating components require tumor necrosis factor alpha (TNF-α). Levels of TNF-α and the mRNA for TNF-α receptors were measured in the oviduct of IUI or VCS rats. The tissue distribution of TNF-α receptor proteins and the concentration of the mRNA for catechol-O-methyl transferase (Comt) and 2ME were also analyzed in the oviduct. Finally, we assessed whether 2ME accelerates egg transport in IUI or VCS rats previously treated with the TNF-α antagonist W9P9QY. Results show that IUI, but not VCS, increased TNF-α and their receptors in the oviduct. IUI and VCS did not change the tissue distribution of TNF-α receptors; however, both decreased the oviductal concentration of Comt and 2ME. IUI and VCS each blocked the 2ME-induced egg transport acceleration; however, only the IUI was antagonized by the TNF-α antagonist. We concluded that IUI and VCS inhibit the 2ME nongenomic pathway that accelerates egg transport; however, the vias of action are distinct, with a TNF-α increase on spermatozoa presence being required for the shutdown of the 2ME pathway.

Effect of 2-methoxyestradiol treatment on early- and late-stage breast cancer progression in a mouse model.

The prevalence of breast cancer (BC) continues to increase and is the leading cause of cancer deaths in many countries. Numerous in vitro and in vivo studies have demonstrated that 2-methoxyestradiol (2-ME) has antiproliferative and antiangiogenic effects in BC, thereby inhibiting tumour growth and metastasis. We compared the effect of 2-ME in early- and late-stage BC using a transgenic mouse model-FVB/N-Tg(MMTV-PyVT)-of spontaneously development of aggressive mammary carcinoma with lung metastasis. Mice received 100 mg/kg 2-ME treatment immediately when palpable mammary tumours were identified (early-stage BC; Experimental group 1) and 28 days after palpable mammary tumours were detected (late-stage BC; Experimental group 2). 2-ME was administered via oral gavage three times a week for 28 days after initiation of treatment, whereas control mice received the vehicle containing 10% dimethyl sulfoxide and 90% sunflower oil for the same duration as the treatment group. Mammary tumours were measured weekly over the 28 days and at termination, blood, mammary and lung tissue were collected for analysis. Mice with a tumour volume threshold of 4000 mm3 were killed before the treatment regime was completed. 2-ME treatment of early-stage BC led to lower levels of mammary tumour necrosis, whereas tumour mass and volume were increased. Additionally, necrotic lesions and anti-inflammatory CD163-expressing cells were more frequent in pulmonary metastatic tumours in this group. In contrast, 2-ME treatment of late-stage BC inhibited tumour growth over the 28-day period and resulted in increased CD3+ cell number and tumour necrosis. Furthermore, 2-ME treatment slowed down pulmonary metastasis but did not increase survival of late-stage BC mice. Besides late-stage tumour necrosis, none of the other results were statistically significant. This study demonstrates that 2-ME treatment has an antitumour effect on late-stage BC, however, with no increase in survival rate, whereas the treatment failed to demonstrate any benefit in early-stage BC.

2-Methoxyestradiol ameliorates paraquat-induced pulmonary fibrosis by inhibiting the TGF-ß1/Smad2/3 signaling pathway.

Paraquat (PQ) is a highly effective and highly toxic herbicide that is highly toxic to both humans and animals. Pulmonary fibrosis is the primary cause of fatality in patients with PQ poisoning, there is no effective drug treatment yet. 2-Methoxyestradiol (2ME) is a natural metabolite of estradiol with anti-tumor, anti-angiogenesis, and anti-proliferative effects. Whether 2ME has the potential to inhibit pulmonary fibrosis induced by PQ is unclear. This study aims to investigate the potential effects and mechanism of 2ME on PQ-induced pulmonary fibrosis. C57BL/6 mice and A549 cells were exposed to PQ to establish pulmonary fibrosis model. In vivo, Hematoxylin and eosin (H&E) staining was utilized to assess the pathological characteristics. Masson's trichrome staining was employed to evaluate the collagen deposition. Western blot and immunohistochemistry were conducted to determine the expressions of fibrosis markers. In vitro, the expressions of epithelial-mesenchymal transition (EMT) markers were detected using western blot and immunofluorescence to evaluated the potential inhibition of PQ-induced EMT by 2ME. And proteins associated with the TGF-ß1/Smad2/3 signaling pathway were measured by western blot in vivo and in vitro. The result found that 2ME can ameliorated PQ-induced pulmonary fibrosis and inhibit the activation of TGF-ß1/Smad2/3 signaling pathway. These findings suggest that 2ME may serve as a potential therapeutic agent for treating PQ-induced pulmonary fibrosis.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate (STX140) Inhibits Proliferation and Invasion via Senescence Pathway Induction in Human BRAFi-Resistant Melanoma Cells.

The endogenous estradiol derivative 2-Methoxyestradiol (2-ME) has shown good and wide anticancer activity but suffers from poor oral bioavailability and extensive metabolic conjugation. However, its sulfamoylated derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (STX140), has superior potential as a therapeutic agent, acts by disrupting microtubule polymerization, leading to cell cycle arrest and apoptosis in cancer cells and possesses much better pharmaceutical properties. This study investigated the antiproliferative and anti-invasive activities of STX140 in both SKMEL-28 naïve melanoma (SKMEL28-P) cells and resistant melanoma cells (SKMEL-28R). STX140 inhibited cell proliferation in the nanomolar range while having a less pronounced effect on human melanocytes. Additionally, STX140 induced cell cycle arrest in the G2/M phase and sub-G1, reduced migration, and clonogenic potential in monolayer models, and inhibited invasion in a 3D human skin model with melanoma cells. Furthermore, STX140 induced senescence features in melanoma and activated the senescence machinery by upregulating the expression of senescence genes and proteins related to senescence signaling. These findings suggest that STX140 may hold potential as a therapeutic agent for melanoma treatment.

Radiosensitization of Breast Cancer Cells with a 2-Methoxyestradiol Analogue Affects DNA Damage and Repair Signaling In Vitro.

Radiation resistance and radiation-related side effects warrant research into alternative strategies in the application of this modality to cancer treatment. Designed in silico to improve the pharmacokinetics and anti-cancer properties of 2-methoxyestradiol, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) disrupts microtubule dynamics and induces apoptosis. Here, we investigated whether pre-exposure of breast cancer cells to low-dose ESE-16 would affect radiation-induced deoxyribonucleic acid (DNA) damage and the consequent repair pathways. MCF-7, MDA-MB-231, and BT-20 cells were exposed to sub-lethal doses of ESE-16 for 24 h before 8 Gy radiation. Flow cytometric quantification of Annexin V, clonogenic studies, micronuclei quantification, assessment of histone H2AX phosphorylation and Ku70 expression were performed to assess cell viability, DNA damage, and repair pathways, in both directly irradiated cells and cells treated with conditioned medium. A small increase in apoptosis was observed as an early consequence, with significant repercussions on long-term cell survival. Overall, a greater degree of DNA damage was detected. Moreover, initiation of the DNA-damage repair response was delayed, with a subsequent sustained elevation. Radiation-induced bystander effects induced similar pathways and were initiated via intercellular signaling. These results justify further investigation of ESE-16 as a radiation-sensitizing agent since pre-exposure appears to augment the response of tumor cells to radiation.

Intramuscular injection of sotagliflozin promotes neovascularization in diabetic mice through enhancing skeletal muscle cells paracrine function.

Diabetes mellitus is associated with series of macrovascular and microvascular pathological changes that cause a wide range of complications. Diabetic patients are highly susceptible to hindlimb ischemia (HLI), which remains incurable. Evidence shows that skeletal muscle cells secrete a number of angiogenic factors to promote neovascularization and restore blood perfusion, this paracrine function is crucial for therapeutic angiogenesis in diabetic HLI. In this study we investigated whether sotagliflozin, an anti-hyperglycemia SGLT2 inhibitor, exerted therapeutic angiogenesis effects in diabetic HLI in vitro and in vivo. In C2C12 skeletal muscle cells, we showed that high glucose (HG, 25 mM) under hypoxia markedly inhibited cell viability, proliferation and migration potentials, which were dose-dependently reversed by pretreatment with sotagliflozin (5-20 µM). Sotagliflozin pretreatment enhanced expression levels of angiogenic factors HIF-1α, VEGF-A and PDGF-BB in HG-treated C2C12 cells under hypoxia as well as secreted amounts of VEGF-A and PDGF-BB in the medium; pretreatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME2, 10 µM) or HIF-1α knockdown abrogated sotagliflozin-induced increases in VEGF-A and PDGF-BB expression, as well as sotagliflozin-stimulated cell proliferation and migration potentials. Furthermore, the conditioned media from sotagliflozin-treated C2C12 cells in HG medium enhanced the migration and proliferation capabilities of vascular endothelial and smooth muscle cells, two types of cells necessary for forming functional blood vessels. In vivo study was conducted in diabetic mice subjected to excising the femoral artery of the left limb. After the surgery, sotagliflozin (10 mg/kg) was directly injected into gastrocnemius muscle of the left hindlimb once every 3 days for 3 weeks. We showed that intramuscular injection of sotagliflozin effectively promoted the formation of functional blood vessels, leading to significant recovery of blood perfusion in diabetic HLI mice. Together, our results highlight a new indication of SGLT2 inhibitor sotagliflozin as a potential therapeutic angiogenesis agent for diabetic HLI.

2-methoxyestradiol inhibits melanoma cell growth by activating adaptive immunity.

Background: The overall survival of melanoma patients remains poor despite advancements in surgical treatment and targeted therapies. Therefore, there is a need to develop new therapeutic strategies for melanoma. 2-methoxyestradiol (2-ME) is a major metabolite of estrogen that has been shown to have anti-tumor effects against many malignancies. However, the effects and mechanisms of action of 2-ME against melanoma remain unclear.Materials and methods: Melanoma cells (B16) were treated with 2-ME in vitro. Cell proliferation was detected by CCK8 and clone formation, transwell was carried out to measure the migration of B16 cells with or without 2-ME. Flow cytometry was performed to measure the apoptosis and cell cycle. C57BL/6 mice were used for tumor-bearing of B16 cells, tumor volumes were measured once a day, and sacrificed after it was over 2000 mm3, then immunofluorescence was implemented to examine the marker of CD3, CD8 and PD-L1.Results: In our study, we found that 2-ME significantly affected the proliferation, migration, apoptosis, and cell cycle of melanoma in vitro. Our results also showed that 2-ME had strong anti-tumor effects against melanoma in vivo and increased the infiltration of tumor-specific cytotoxic lymphocytes CD8+ T cells in the tumor microenvironment. Besides, PD-L1 expression in tumor cells was significantly higher in the 2-ME-treated group than in the control group, indicating that 2-ME could exhibit stronger anti-tumor effects against melanoma if combined with PD-1 blockade therapy.Conclusion: 2-ME suppresses melanoma in vivo and in vitro and is a promising synergistic enhancer of PD-1 blockade immunotherapy.

2-Methoxyestradiol Inhibits Radiation-Induced Skin Injuries.

Radiation-induced skin injury (RISI) is a main side effect of radiotherapy for cancer patients, with vascular damage being a common pathogenesis of acute and chronic RISI. Despite the severity of RISI, there are few treatments for it that are in clinical use. 2-Methoxyestradiol (2-ME) has been reported to regulate the radiation-induced vascular endothelial-to-mesenchymal transition. Thus, we investigated 2-ME as a potent anti-cancer and hypoxia-inducible factor 1 alpha (HIF-1α) inhibitor drug that prevents RISI by targeting HIF-1α. 2-ME treatment prior to and post irradiation inhibited RISI on the skin of C57/BL6 mice. 2-ME also reduced radiation-induced inflammation, skin thickness, and vascular fibrosis. In particular, post-treatment with 2-ME after irradiation repaired the damaged vessels on the irradiated dermal skin, inhibiting endothelial HIF-1α expression. In addition to the increase in vascular density, post-treatment with 2-ME showed fibrotic changes in residual vessels with SMA+CD31+ on the irradiated skin. Furthermore, 2-ME significantly inhibited fibrotic changes and accumulated DNA damage in irradiated human dermal microvascular endothelial cells. Therefore, we suggest that 2-ME may be a potent therapeutic agent for RISI.

Cell Fate following Irradiation of MDA-MB-231 and MCF-7 Breast Cancer Cells Pre-Exposed to the Tetrahydroisoquinoline Sulfamate Microtubule Disruptor STX3451.

A tetrahydroisoquinoline (THIQ) core is able to mimic the A and B rings of 2-methoxyestradiol (2ME2), an endogenous estrogen metabolite that demonstrates promising anticancer properties primarily by disrupting microtubule dynamic instability parameters, but has very poor pharmaceutical properties that can be improved by sulfamoylation. The non-steroidal THIQ-based microtubule disruptor 2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (STX3451), with enhanced pharmacokinetic and pharmacodynamic profiles, was explored for the first time in radiation biology. We investigated whether 24 h pre-treatment with STX3451 could pre-sensitize MCF-7 and MDA-MB-231 breast cancer cells to radiation. This regimen showed a clear increase in cytotoxicity compared to the individual modalities, results that were contiguous in spectrophotometric analysis, flow cytometric quantification of apoptosis induction, clonogenic studies and microscopy techniques. Drug pre-treatment increased radiation-induced DNA damage, with statistically more double-strand (ds) DNA breaks demonstrated. The latter could be due to the induction of a radiation-sensitive metaphase block or the increased levels of reactive oxygen species, both evident after compound exposure. STX3451 pre-exposure may also delay DNA repair mechanisms, as the DNA damage response element ataxia telangiectasia mutated (ATM) was depressed. These in vitro findings may translate into in vivo models, with the ultimate aim of reducing both radiation and drug doses for maximal clinical effect with minimal adverse effects.

2-Methoxyestradiol synergizes with Erlotinib to suppress hepatocellular carcinoma by disrupting the PLAGL2-EGFR-HIF-1/2α signaling loop.

Erlotinib, an EGFR tyrosine kinase inhibitor has been introduced into cancer chemotherapy. However, the therapeutic effects of erlotinib in hepatocellular carcinoma (HCC) remain vaguely understood. Our previous study found that a hypoxia-mediated PLAGL2-EGFR-HIF-1/2α signaling loop in HCC decreased response to erlotinib. The current study has demonstrated that the combination of erlotinib and 2ME2 exerted synergistic antitumor effects against HCC. Further investigation showed that erlotinib increased the expression level of EGFR, HIF-2α, and PLAGL2, which contributes to the insensitivity of hypoxic HCC cells to erlotinib. The simultaneous exposure to 2ME2 effectively inhibited the expression level of EGFR, HIF-2α, and PLAGL2 that was induced by erlotinib. This contributes to the synergistic effect of the two therapeutic agents. Furthermore, the combination of erlotinib and 2ME2 induced apoptosis and inhibited the stemness of hypoxic HCC cells. Our findings potentially explain the mechanism of HCC insensitivity to erlotinib and provide a new strategy of combining EGFR and HIF1/2α inhibitors for HCC treatment.

Synthesis and biological evaluation of new 2-methoxyestradiol derivatives: Potent inhibitors of angiogenesis and tubulin polymerization.

Here, we report the structural optimization of a hit natural compound, 2-ME2 (2-methoxyestradiol), which exhibited inhibitory activity but low potency on tubulin polymerization, anti- angiogenesis, MCF-7 proliferation and metastasis in vitro and in vivo. A novel series of 3,17-modified and 17-modified analogs of 2-ME2 were synthesized and investigated for their antiproliferative activity against MCF-7 and another five different human cancer cell lines leading to the discovery of 9i. 9i bind to tubulin colchicine site tightly, inhibited tubulin polymerization and disrupted cellular microtubule networks. Cellular mechanism studies revealed that 9i could induce G2/M phase arrest by down-regulated expression of p-Cdc2, P21 and cell apoptosis by regulating apoptosis-related proteins (Parp, Caspase families) in a dose-dependent manner. Importantly, 9i significantly inhibited HUVEC tube formation, proliferation, migration and invasion. The inhibitory effect against angiogenesis in vivo was confirmed by zebrafish xenograft. Furthermore, 9i could effectively inhibit the proliferation and metastasis of MCF-7 cells in vitro and in zebrafish xenograft. The satisfactory physicochemical property and metabolic stability of 9i further indicated that it can act as a promising and potent anti-angiogenesis, inhibiting proliferation and metastasis of breast cancer agent via targeting tubulin colchicine binding site.

Evidence for 2-Methoxyestradiol-Mediated Inhibition of Receptor Tyrosine Kinase RON in the Management of Prostate Cancer.

2-Methoxyestradiol (2-ME2) possesses anti-tumorigenic activities in multiple tumor models with acceptable tolerability profile in humans. Incomplete understanding of the mechanism has hindered its development as an anti-tumorigenic compound. We have identified for the first-time macrophage stimulatory protein 1 receptor (MST1R) as a potential target of 2-ME2 in prostate cancer cells. Human tissue validation studies show that MST1R (a.k.a RON) protein levels are significantly elevated in prostate cancer tissues compared to adjacent normal/benign glands. Serum levels of macrophage stimulatory protein (MSP), a ligand for RON, is not only associated with the risk of disease recurrence, but also significantly elevated in samples from African American patients. 2-ME2 treatment inhibited mechanical properties such as adhesion and elasticity that are associated with epithelial mesenchymal transition by downregulating mRNA expression and protein levels of MST1R in prostate cancer cell lines. Intervention with 2-ME2 significantly reduced tumor burden in mice. Notably, global metabolomic profiling studies identified significantly higher circulating levels of bile acids in castrated animals that were decreased with 2-ME2 intervention. In summary, findings presented in this manuscript identified MSP as a potential marker for predicting biochemical recurrence and suggest repurposing 2-ME2 to target RON signaling may be a potential therapeutic modality for prostate cancer.

DPP4 Inhibition, NPY1-36, PYY1-36, SDF-1α, and a Hypertensive Genetic Background Conspire to Augment Cell Proliferation and Collagen Production: Effects That Are Abolished by Low Concentrations of 2-Methoxyestradiol.

By reducing their metabolism, dipeptidyl peptidase 4 inhibition (DPP4I) enhances the effects of numerous peptides including neuropeptide Y1-36 (NPY1-36), peptide YY1-36 (PYY1-36), and SDF-1α Studies show that separately NPY1-36, PYY1-36 and SDF-1α stimulate proliferation of, and collagen production by, cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs), particularly in cells isolated from genetically hypertensive rats. Whether certain combinations of these factors, in the absence or presence of DPP4I, are more profibrotic than others is unknown. Here we contrasted 24 different combinations of conditions (DPP4I, hypertensive genotype and physiologic levels [3 nM] of NPY1-36, PYY1-36, or SDF-1α) on proliferation of, and [3H]-proline incorporation by, CFs, PGVSMCs, and GMCs. In all three cell types, the various treatment conditions differentially increased proliferation and [3H]-proline incorporation, with a hypertensive genotype + DPP4I + NPY1-36 + SDF-1α being the most efficacious combination. Although the effects of this four-way combination were similar in male versus female CFs, physiologic (1 nM) concentrations of 2-methoxyestradiol (2ME; nonestrogenic metabolite of 17ß-estradiol), abolished the effects of this combination in both male and female CFs. In conclusion, this study demonstrates that CFs, PGVSMCs, and GMCs are differentially activated by various combinations of NPY1-36, PYY1-36, SDF-1α, a hypertensive genetic background and DPP4I. We hypothesize that as these progrowth conditions accumulate, a tipping point would be reached that manifests in the long term as organ fibrosis and that 2ME would obviate any profibrotic effects of DPP4I, even under the most profibrotic conditions (i.e., hypertensive genotype with high NPY1-36 + SDF-1α levels and low 2ME levels). SIGNIFICANCE STATEMENT: This work elucidates combinations of factors that could contribute to long-term profibrotic effects of dipeptidyl peptidase 4 inhibitors and suggests a novel drug combination that could prevent any potential profibrotic effects of dipeptidyl peptidase 4 inhibitors while augmenting the protective effects of this class of antidiabetic agents.

Treatment with 2-methoxyestradiol increases endothelial nitric oxide synthase activity via scavenger receptor class BI in human umbilical vein endothelial cells.

Concentrations of 2-methoxyestradiol (2ME2), a principal metabolite of estradiol, are significantly lower in women with severe preeclampsia. Nitric oxide (NO) released by endothelial nitric oxide synthase (eNOS) plays an important role in regulating cardiovascular homeostasis. Importantly, high-density lipoprotein (HDL) stimulates eNOS activity via endothelial human scavenger receptor class B type I (hSR-BI/CLA-1). Here, we aimed to determine the effect of 2ME2 on hSR-BI/CLA-1 expression in human umbilical vein endothelial cells (HUVECs). hSR-BI/CLA-1 expression was measured by real-time PCR, western blotting and reporter gene assays; eNOS activity was assessed by the measurement of eNOS phosphorylation. Both the mRNA and protein concentrations of hSR-BI/CLA-1 were significantly increased by 2ME2 in HUVECs. 2ME2 also dose-dependently increased the transcriptional activity of the hSR-BI/CLA-1 promoter. The effect of 2ME2 treatment on the promoter activity of hSR-BI/CLA-1 was abrogated by treatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, as was the increase in HDL-induced eNOS activation. Notably, constitutively active Akt increased the activity of the hSR-BI/CLA-1 promoter, whereas dominant-negative Akt abolished the effect of 2ME2 treatment on hSR-BI/CLA-1 promoter activity. The nuclear Sp1 protein concentration was significantly increased by exposure to 2ME2 and Sp1 overexpression increased the promoter activity of the hSR-BI/CLA gene. Furthermore, knockdown of Sp1 inhibited the effect of 2ME2 treatment on hSR-BI/CLA-1 protein expression. These results indicate that 2ME2 treatment increases HDL-dependent eNOS phosphorylation by upregulating endothelial hSR-BI/CLA-1 expression, suggesting that 2ME2 has a potential therapeutic value in the treatment of preeclampsia.

The Major Heat Shock Proteins, Hsp70 and Hsp90, in 2-Methoxyestradiol-Mediated Osteosarcoma Cell Death Model.

2-Methoxyestradiol is one of the natural 17ß-estradiol derivatives and a potential novel anticancer agent currently being under evaluation in advanced phases of clinical trials. However, the mechanism of anticancer action of 2-methoxyestradiol has not been yet fully established. In our previous studies we have demonstrated that 2-methoxyestradiol selectively induces the expression and nuclear translocation of neuronal nitric oxide synthase in osteosarcoma 143B cells. Heat shock proteins (Hsps) are factors involved in the regulation of expression and activity of nitric oxide synthases. Herein, we chose osteosarcoma cell lines differed in metastatic potential, metastatic 143B and highly metastatic MG63.2 cells, in order to further investigate the anticancer mechanism of 2-methoxyestradiol. The current study aimed to determine the role of major heat shock proteins, Hsp90 and Hsp70 in 2-methoxyestradiol-induced osteosarcoma cell death. We focused on the implication of Hsp90 and Hsp70 in control under expression of neuronal nitric oxide synthase, localization of the enzyme, and further generation of nitro-oxidative stress. To give the insight into the role of Hsp90 in regulation of anticancer efficacy of 2-methoxyestradiol, we used geldanamycin as a potent Hsp90 inhibitor. Herein, we evidenced that inhibition of Hsp90 controls the protein expression of 2-methoxyestradiol-induced neuronal nitric oxide synthase and inhibits enzyme nuclear translocation. We propose that decreased level of neuronal nitric oxide synthase protein after a combined treatment with 2-methoxyestradiol and geldanamycin is directly associated with the accompanying upregulation of Hsp70 and downregulation of Hsp90. This interaction resulted in abrogation of anticancer efficacy of 2-methoxyestradiol by geldanamycin.

Effect of Interaction between 17ß-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone with Chromium (VI) on Ovary Cancer Line SKOV-3: Preliminary Study.

Ovarian cancer is the leading cause of death from gynecologic malignancies. Some estrogens, as well as xenoestrogens, such as chromium (VI) (Cr(VI)), are indicated as important pathogenic agents. The objective of this study was to evaluate the role of estradiol and some its metabolites upon exposure to the metalloestrogen Cr(VI) in an in vitro model. The changes in cell viability of malignant ovarian cancer cells (SKOV-3 resistant to cisplatin) exposed to 17ß-estradiol (E2) and its two metabolites, 2-methoxyestradiol (2-MeOE2) and 16α-hydroxyestrone (16α-OHE1), upon exposure to potassium chromate (VI) and its interactions were examined. The single and mixed models of action, during short and long times of incubation with estrogens, were applied. The different effects (synergism and antagonism) of estrogens on cell viability in the presence of Cr(VI) was observed. E2 and 16α-OHE1 caused a synergistic effect after exposure to Cr(VI). 2-MeOE2 showed an antagonistic effect on Cr(VI). The examined estrogens could be ranked according to the most protective effect or least toxicity in the order: 2-MeOE2 > E2 > 16α-OHE1. Early pre-incubation (24 h or 7 days) of cells with estrogens caused mostly an antagonistic effect-protective against the toxic action of Cr(VI). The beneficial action of estrogens on the toxic effect of Cr(VI), in the context of the risk of ovarian cancer, seems to be important and further studies are needed.

2-Methoxyestradiol attenuates chronic-intermittent-hypoxia-induced pulmonary hypertension through regulating microRNA-223.

Pulmonary hypertension (PH) is prevalent in patients with obstructive sleep apnea (OSA) syndrome, and coexistence of PH and OSA indicates a worse prognosis and higher mortality. Chronic intermittent hypoxia (CIH) is the key pathogenesis of OSA. Also, microRNA-223 (miR-223) plays a role in the regulation of CIH-induced PH process. However, the detailed mechanism of CIH inducing PH is still unclear. This study aimed to investigate the pathological process of CIH associated PH and explore the potential therapeutic methods. In this study, adult Sprague-Dawley rats were exposed to CIH or normoxic (N) conditions with 2-methoxyestradiol (2-Me) or vehicle treatment for 6 weeks. The results showed that 2-Me treatment reduced the progression of pulmonary angiogenesis in CIH rats, and alleviated proliferation, cellular migration, and reactive oxygen species formation was induced by CIH in pulmonary artery smooth muscle cells (PASMCs). CIH decreased the expression of miR-223, whereas 2-Me reversed the downregulation of miR-223 both in vivo and in vitro. Furthermore, the antiangiogenic effect of 2-Me observed in PASMCs was abrogated by miR-223 inhibitor, while enhanced by miR-223 mimic. These findings suggested that miR-223 played an important role in the process of CIH inducing PH, and 2-Me might reverse CIH-induced PH via upregulating miR-223.

The anticancer effects of 2-methoxyestradiol on human huh7 cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) is associated with a poor prognosis. 2-methoxyestradiol (2-ME) is currently under preclinical evaluation as a treatment for many malignancies, but the utility of the drug in terms of HCC treatment remains unclear. Here, we explored the effect of 2-ME on human huh7 cell proliferation and apoptosis and discuss the possible molecular mechanisms involved. The MTT assay showed that proliferation was markedly inhibited by 2-ME (at 5, 10, 15, and 20 µmol/L) in a time- and dose-dependent manner. Moreover, flow cytometry indicated that 2-ME induced cell cycle arrest at the G2/M phase, and early apoptosis. We used Western blotting and PCR to detect the expression of vascular endothelial growth factor (VEGF) and Bcl-2; 2-ME decreased the mRNA/protein expression levels of both effectors. Furthermore, 2-ME remarkably suppressed xenograft tumor growth in nude mice, and no visible toxicity was observed in either the liver or kidneys. Immunohistochemically, the Bcl-2 and VEGF expression levels were significantly lower than those of controls. Thus, 2-ME inhibited huh7 cell proliferation, promoted apoptosis, and suppressed xenograft tumor growth in nude mice, perhaps reflecting the effects of the drug on VEGF and Bcl-2 expression.

Autophagy mediates 2-methoxyestradiol-inhibited scleroderma collagen synthesis and endothelial-to-mesenchymal transition induced by hypoxia.

OBJECTIVES: To investigate whether autophagy mediates 2-methoxyestradiol (2-ME)-inhibited hypoxia-induced fibrosis and endothelial-to-mesenchymal transition (endoMT) in SSc. METHODS: Autophagy in the skin of SSc patients was assessed by transmission electron microscopy. SSc skin fibroblasts and human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic (1% O2) conditions with 2-ME or autophagy inhibitor. Collagen I and connective tissue growth factor (CTGF) in fibroblasts and vascular endothelial (VE)-cadherin, CD31, vimentin and α-smooth muscle actin (α-SMA) in HUVECs were examined by western blotting. Autophagic markers were evaluated by confocal microscopy and immunofluorescence. RESULTS: SSc skins presented increased autolysosomes, LC3-II, collagen I and CTGF. Hypoxia-challenged fibroblasts and HUVECs formed more autophagosomes and autolysosomes, with increased LC3 and decreased P62. Meanwhile, hypoxia increased collagen I and CTGF in fibroblasts and increased vimentin and α-SMA but decreased VE-cadherin and CD31 in HUVECs. Bafilomycin A1 increased LC3-II and P62 in fibroblasts and HUVECs and decreased collagen I and CTGF in fibroblasts and vimentin and α-SMA in HUVECs, while upregulating VE-cadherin and CD31. 3-methyladenine decreased autophagy and fibrosis in fibroblasts and endothelial-to-mesenchymal transition in HUVECs. 2-ME-treated HUVECs showed more autophagosomes and fewer autolysosomes while 2-ME-treated fibroblasts showed fewer of both. Moreover, 2-ME decreased LC3-II and increased P62 in fibroblasts and increased both in HUVECs. Inhibition of autophagy by 2-ME showed the same effect with bafilomycin A1 on fibroblast collagen synthesis as well as endothelial and mesenchymal markers in HUVECs. CONCLUSION: Autophagy mediated hypoxia-induced fibroblast collagen synthesis and endoMT in SSc, which could be reversed by 2-ME.

Detection of the effects and potential interactions of FSH, VEGFA, and 2-methoxyestradiol in follicular angiogenesis, growth, and atresia in mouse ovaries.

Ovarian follicular development is a complex process that requires codevelopment of the perifollicular vascular network, which is closely regulated by angiogenic factors, gonadotropins, sex steroids, and their metabolites. To detect the effects of vascular endothelial growth factor 120 (VEGF120), follicle-stimulating hormone (FSH), and 2-methoxyestradiol (2ME2) on follicular angiogenesis during development and atresia, we treated sexually immature and mature female mice with VEGF120, FSH, 2ME2, and FSH receptor (FSHR) antagonist singly or in combination via intraperitoneal injection. The number of follicles and their perifollicular angiogenesis and atresia rates at different developmental stages were examined in paraffin sections after hematoxylin and eosin staining. The results showed that the exogenous factors have specific and precise effects on developmental, angiogenesis, and atresia processes in follicles of different sizes in mature and immature mice. Perifollicular angiogenesis was regulated by VEGFA and closely related to follicular development and atresia. 2ME2 affected angiogenesis through VEGFA and might regulate atresia directly. FSH might control VEGFA function via both transcriptional and posttranscriptional mechanisms because FSHR was required for achieving VEGFA functions at all the follicular development stages. The present study presents insights into the mechanisms of FSH, 2ME2, and VEGFA in follicular development and disorders and provides a foundation for the development of new therapeutic strategies.