Targeting an Old Foe for Cancer: A Molecular Dynamics Perspective to Unravel the Specific Binding Nature of 2-Methoxy Estradiol to Human ß-Tubulin Isotypes.

Microtubules, composed of α- and ß-tubulin subunits are crucial for cell division with their dynamic tissue-specificity which is dictated by expression of isotypes. These isotypes differ in carboxy-terminal tails (CTTs), rich in negatively charged acidic residues in addition to the differences in the composition of active site residues. 2-Methoxy estradiol (2-ME) is the first antimicrotubule agent that showed less affinity toward hemopoietic-specific ß1 isotype consequently preventing myelosuppression toxicity. The present study focuses on the MD-directed conformational analysis of 2-ME and estimation of its binding affinity in the colchicine binding pocket of various ß-tubulin isotypes combined with the α-tubulin isotype, α1B. AlphaFold 2.0 was used to predict the 3D structure of phylogenetically divergent human ß-tubulin isotypes in dimer form with α1B. The dimeric complexes were subjected to induced-fit docking with 2-ME. The statistical analysis of docking showed differences in the binding characteristics of 2-ME with different isotypes. The replicas of atom-based molecular dynamic simulations of the best conformation of 2-ME provided insights into the molecular-level details of its binding pattern across the isotypes. Furthermore, the MM/GBSA analyses revealed the specific binding energy profile of 2-ME in ß-tubulin isotypes. It also highlighed, 2-ME exhibits the lowest binding affinity toward the ß1 isotype as supported by experimental study. The present study may offer useful information for designing next-generation antimicrotubule agents that are more specific and less toxic.

Effect of 2-methoxyestradiol on mammary tumor initiation and progression.

BACKGROUND: The anti-cancer agent 2-methoxyestradiol (2-ME) has been shown to have anti-proliferative and anti-angiogenic properties. Previously, the effect of 2-ME on early- and late-stage breast cancer (BC) was investigated in vivo using a transgenic mouse model (FVB/N-Tg(MMTV-PyVT)) of spontaneous mammary carcinoma. Anti-tumor effects were observed in late-stage BC with no effect on early-stage BC. Given the contrasting results obtained from the different BC stages, we have now investigated the effect of 2-ME when administered before the appearance of palpable tumors. METHODS: Each mouse received 100 mg/kg 2-ME on day 30 after birth, twice per week for 28 days, while control mice received vehicle only. Animals were terminated on day 59. Lung and mammary tissue were obtained for immunohistochemical analysis of CD163 and CD3 expression, and histological examination was performed to analyze tumor necrosis. Additionally, blood samples were collected to measure plasma cytokine levels. RESULTS: 2-ME increased tumor mass when compared to the untreated animals (p = .0139). The pro-tumorigenic activity of 2-ME was accompanied by lower CD3+ T-cell numbers in the tumor microenvironment (TME) and high levels of the pro-inflammatory cytokine interleukin (IL)-1ß. Conversely, 2-ME-treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL-10 plasma levels, and low IL-6 and IL-27 plasma levels. CONCLUSION: Taken together, these findings suggest that 2-ME promotes early-stage BC development.

Establishment of cluster of differentiation 20 immobilized cell membrane chromatography for the screening of active antitumor components in traditional Chinese medicine.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignant tumors occurring in B or T lymphocytes, and no small molecule-positive drugs to treat NHL have been marketed. Cluster of differentiation 20 (CD20) is an important molecule regulating signaling for the life and differentiation of B lymphocytes and possesses the characteristics of a drug target for treating NHL. 2-Methoxyestradiol induces apoptosis in lymphoma Raji cells and CD20 protein is highly expressed by Raji lymphoma cells. Therefore, in this study, a CD20-SNAP-tag/CMC model was developed to validate the interaction of 2-methoxyestradiol with CD20. 2-Methoxyestradiol was used as a small molecule control compound, and the system was validated for good applicability. The cell membrane chromatography model was combined with high-performance liquid chromatography ion trap time-of-flight mass spectroscopy (HPLC-IT-TOF-MS) in a two-dimensional system to successfully identify, analyze, and characterize the potential active compounds of Schisandra chinensis (Turcz.) Baill. extract and Lysionotus pauciflorus Maxim. extract, including Schisandrin A, Schizandrol A, Schizandrol B, Schisantherin B, and Nevadensin, which can act on CD20 receptors. The five potential active compounds were analyzed by non-linear chromatography. The thermodynamic and kinetic parameters of their interaction with CD20 were also analyzed, and the mode of interaction was simulated by molecular docking. Their inhibitory effects on lymphoma cell growth were assessed using a Cell Counting Kit-8 (CCK-8). Nevadensin and Schizandrin A were able to induce apoptosis in Raji cells within a certain concentration range. In conclusion, the present experiments provide some bases for improving NHL treatment and developing small molecule lead compounds targeting CD20 with low toxicity and high specificity.

2-Methoxyestradiol and Hydrogen Peroxide as Promising Biomarkers in Parkinson's Disease.

Estrogens function in numerous physiological processes including controlling brain cell growth and differentiation. 2-Methoxestradiol (2-ME2), a 17ß-estradiol (E2) metabolite, is known for its anticancer effects as observed both in vivo and in vitro. 2-ME2 affects all actively dividing cells, including neurons. The study aimed to determine whether 2-ME2 is a potentially cancer-protective or rather neurodegenerative agent in a specific tissue culture model as well as a clinical setup. In this study, 2-ME2 activity was determined in a Parkinson's disease (PD) in vitro model based on the neuroblastoma SH-SY5Y cell line. The obtained results suggest that 2-ME2 generates nitro-oxidative stress and controls heat shock proteins (HSP), resulting in DNA strand breakage and apoptosis. On the one hand, it may affect intensely dividing cells preventing cancer development; however, on the other hand, this kind of activity within the central nervous system may promote neurodegenerative diseases like PD. Thus, the translational value of 2-ME2's neurotoxic activity in a PD in vitro model was also investigated. LC-MS/MS technique was used to evaluate estrogens and their derivatives, namely, hydroxy and methoxyestrogens, in PD patients' blood, whereas the stopped-flow method was used to assess hydrogen peroxide (H2O2) levels. Methoxyestrogens and H2O2 levels were increased in patients' blood as compared to control subjects, but hydoxyestrogens were simultaneously decreased. From the above, we suggest that the determination of plasma levels of methoxyestrogens and H2O2 may be a novel PD biomarker. The presented research is the subject of the pending patent application "The use of hydrogen peroxide and 17ß-estradiol and its metabolites as biomarkers in the diagnosis of neurodegenerative diseases," no. P.441360.

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.

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-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.

2-methoxyestradiol restrains non-small cell lung cancer tumorigenesis through regulating circ_0010235/miR-34a-5p/NFAT5 axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most prevalent and severe malignant tumors in the world and its molecular mechanism is still unclear. In recent years, increasing evidence indicates the significant roles of circRNAs in NSCLC. It has been determined that 2-methoxyestradiol (2-MeOE2) exerts antitumor roles in many cancers. However, the molecular mechanism of 2-MeOE2 in regulating the development of lung cancer needs further elucidation. METHODS: The expression levels of circ_0010235, microRNA-34a-5p (miR-34a-5p), and nuclear factor of activated T cells 5 (NFAT5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis and invasion were detected by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry and transwell assays, respectively. The interaction between miR-34a-5p and circ_0010235 or NFAT5 was predicted by bioinformatic software and confirmed by dual-luciferase reporter assay. RESULTS: Our data showed 2-MeOE2 hindered cell proliferation, invasion and induced apoptosis in NSCLC, which could be reversed by upregulation of circ_0010235 and NFAT5 or miR-34a-5p knockdown. Circ_0010235 and NFAT5 expression levels were increased, and miR-34a-5p expression level was decreased in NSCLC tissues and cells. In addition, 2-MeOE2 treatment suppressed the expression of circ_0010235 and NFAT5 while promoted the expression of miR-34a-5p. Furthermore, circ_0010235 functioned as a molecular sponge of miR-34a-5p to regulate NFAT5 expression. Knockdown of circ_0010235 or 2-MeOE2 treatment constrained tumor growth in vivo, and circ_0010235 depletion enhanced the inhibitory effect of 2-MeOE2 on tumor growth in vivo. CONCLUSION: These findings demonstrated that 2-MeOE2 retarded NSCLC progression by modulating the circ_0010235/miR-34a-5p/NFAT5 axis, thus providing a new perspective for 2-MeOE2 treatment in NSCLC.

CXCR4, regulated by HIF1A, promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation.

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45+ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.

PEGylated Polymeric Nanoparticles Loaded with 2-Methoxyestradiol for the Treatment of Uterine Leiomyoma in a Patient-Derived Xenograft Mouse Model.

Leiomyomas, the most common benign neoplasms of the female reproductive tract, currently have limited medical treatment options. Drugs targeting estrogen/progesterone signaling are used, but side effects and limited efficacy in many cases are major limitation of their clinical use. Previous studies from our laboratory and others demonstrated that 2-methoxyestradiol (2-ME) is promising treatment for uterine fibroids. However, its poor bioavailability and rapid degradation hinder its development for clinical use. The objective of this study is to evaluate the in vivo effect of biodegradable and biocompatible 2-ME-loaded polymeric nanoparticles in a patient-derived leiomyoma xenograft mouse model. PEGylated poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles loaded with 2-ME were prepared by nanoprecipitation. Female 6-week age immunodeficient NOG (NOD/Shi-scid/IL-2Rγnull) mice were used. Estrogen-progesterone pellets were implanted subcutaneously. Five days later, patient-derived human fibroid tumors were xenografted bilaterally subcutaneously. Engrafted mice were treated with 2-ME-loaded or blank (control) PEGylated nanoparticles. Nanoparticles were injected intraperitoneally and after 28 days of treatment, tumor volume was measured by caliper following hair removal, and tumors were removed and weighed. Up to 99.1% encapsulation efficiency was achieved, and the in vitro release profile showed minimal burst release, thus confirming the high encapsulation efficiency. In vivo administration of the 2-ME-loaded nanoparticles led to 51% growth inhibition of xenografted tumors compared to controls (P < 0.01). Thus, 2-ME-loaded nanoparticles may represent a novel approach for the treatment of uterine fibroids.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate inhibits store-operated Ca2+ entry in T lymphocytes and prevents experimental autoimmune encephalomyelitis.

Ca2+ signaling is one of the essential signaling systems for T lymphocyte activation, the latter being an essential step in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Store-operated Ca2+ entry (SOCE) ensures long lasting Ca2+ signaling and is of utmost importance for major downstream T lymphocyte activation steps, e.g. nuclear localization of the transcription factor 'nuclear factor of activated T cells' (NFAT). 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol (E2), blocks nuclear translocation of NFAT. The likely underlying mechanism is inhibition of SOCE, as shown for its synthetic sulfamate ester analogue 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564). Here, we demonstrate that another synthetic bis-sulfamoylated 2ME2 derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE, STX140), an orally bioavailable, multi-targeting anticancer agent and potent steroid sulfatase (STS) inhibitor, antagonized SOCE in T lymphocytes. Downstream events, e.g. secretion of the pro-inflammatory cytokines interferon-γ and interleukin-17, were decreased by STX140 in in vitro experiments. Remarkably, STX140 dosed in vivo completely blocked the clinical disease in both active and transfer experimental autoimmune encephalomyelitis (EAE) in Lewis rats, a T cell-mediated animal model for MS, at a dose of 10 mg/kg/day i.p., whereas neither 2ME2 nor Irosustat, a pure STS inhibitor, showed any effect. The STS inhibitory activity of STX140 is therefore not responsible for its activity in this model. Taken together, inhibition of SOCE by STX140 resulting in full antagonism of clinical symptoms in EAE in the Lewis rat, paired with the known excellent bioavailability and pharmaceutical profile of this drug, open potentially new therapeutic avenues for the treatment of MS.

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.

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 and mechanism of pirfenidone combined with 2-methoxy-estradiol perfusion through portal vein on hepatic artery hypoxia-induced hepatic fibrosis.

PURPOSE: The aim of this study was to explore the effect and mechanism of pirfenidone (PFD) combined with 2-methoxyestradiol (2-ME2) perfusion through portal vein on hepatic artery hypoxia-induced hepatic fibrosis. MATERIALS AND METHODS: Sprague-Dawley rats were divided into 5 groups (n â€‹= â€‹3/group): control group, hepatic artery ligation (HAL) group, HAL â€‹+ â€‹PFD (portal vein perfusion of PFD) group, HAL+2-ME2 (portal vein perfusion of 2-ME2) group and HAL â€‹+ â€‹PFD+2-ME2 group depending on whether they received HAL and/or portal vein perfusion (PFD and/or 2-ME2). Livers were harvested for pathology, western blotting (WB), and quantitative real-time PCR (qRT-PCR). RESULTS: Sirius red staining showed that portal vein perfusion of drugs resulted in degradation of liver fibrosis. Immunohistochemistry showed decreased hypoxia-inducible factor-1 α (HIF-1α) and α-smooth muscle actin (α-SMA) after portal intravenous drugs infusion compared with HAL group (P â€‹< â€‹0.05). WB analysis showed increased Smad7 in HAL â€‹+ â€‹PFD group compared with HAL group (P â€‹< â€‹0.05). qRT-PCR analysis showed decreased matrix metallo-proteinase 2 (MMP2), transforming growth factor ß1 (TGF-ß1), monocyte chemoattractant protein-1 (MCP-1), and Collagen I mRNA in HAL â€‹+ â€‹PFD group except for tissue inhibitor of metalloproteinase-1 (TIMP-1) compared with HAL group (P â€‹< â€‹0.05). Compared with HAL â€‹+ â€‹PFD group, the addition of 2-ME2 did not lead to better results in qRT-PCR analysis. CONCLUSIONS: The portal vein perfusion of PFD significantly reduced the hepatic artery hypoxia-induced fibrosis degree in treated rats by down-regulating the expression of HIF-1α, α-SMA, MMP2, TGF-ß1, MCP-1, and Collagen I, as well as up-regulating the TIMP-1 expression and Smad7 protein level. Combined 2-ME2 infusion was not better than PFD alone.

POLYDATIN AMELIORATES TRAUMATIC BRAIN INJURY-INDUCED SECONDARY BRAIN INJURY BY INHIBITING NLRP3-INDUCED NEUROINFLAMMATION ASSOCIATED WITH SOD2 ACETYLATION.

ABSTRACT: Traumatic brain injury (TBI) is a kind of disease with high morbidity, mortality, and disability, and its pathogenesis is still unclear. Research shows that nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) activation in neurons and astrocytes is involved in neuroinflammatory cascades after TBI. What is more, polydatin (PD) has been shown to have a protective effect on TBI-induced neuroinflammation, but the mechanisms remain unclear. Here, we speculated that PD could alleviate TBI-induced neuroinflammatory damage through the superoxide dismutase (SOD2)-NLRP3 signal pathway, and SOD2 might regulate NLRP3 inflammasome activation. The model of lateral fluid percussion for in vivo and cell stretching injury for in vitro were established to mimic TBI. NLRP3 chemical inhibitor MCC950, SOD2 inhibitor 2-methoxyestradiol, and PD were administered immediately after TBI. As a result, the expression of SOD2 acetylation (SOD2 Ac-K122), NLRP3, and cleaved caspase-1 were increased after TBI both in vivo and in vitro , and using SOD2 inhibitor 2-methoxyestradiol significantly promoted SOD2 Ac-K122, NLRP3, and cleaved caspase-1 expression, as well as exacerbated mitochondrial ROS (mtROS) accumulation and mitochondrial membrane potential (MMP) collapse in PC12 cells. However, using NLRP3 inhibitor MCC950 significantly inhibited cleaved caspase-1 activation after TBI both in vivo and in vitro ; meanwhile, MCC950 inhibited mtROS accumulation and MMP collapse after TBI. More importantly, PD could inhibit the level of SOD2 Ac-K122, NLRP3, and cleaved caspase-1 and promote the expression of SOD2 after TBI both in vivo and in vitro. Polydatin also inhibited mtROS accumulation and MMP collapse after stretching injury. These results indicated that PD inhibited SOD2 acetylation to alleviate NLRP3 inflammasome activation, thus acting a protective role against TBI neuroinflammation.

Adverse effects of 2-Methoxyestradiol on mouse oocytes during reproductive aging.

2-Methoxyestradiol (2-ME2) is a metabolite of 17ß-estradiol and is currently in clinical trials as an antitumor agent. Here we found 2-ME2 level remains stable in the local environment of ovaries but declines in serum in aging mice, and exogenous 2-ME2 impacts the meiotic maturation of mouse oocytes in dose-dependent manner. In vitro 2-ME2 application arrested oocytes at metaphase I (MI), with abnormal spindle structure and chromosome alignment. 2-ME2 exposure induced excessive production of reactive oxygen species (ROS) and malondialdehyde, as well as accelerated apoptosis progression. 2-ME2 unbalanced mitochondrial dynamics by increasing DRP1 and MFN1 while decreasing Opa1. Similar phenotypes were also observed in oocytes from mice injected intraperitoneally with 2-ME2. Taken together, this study indicates 2-ME2 exposure impairs oocyte meiotic maturation through inducing mitochondrial imbalance, oxidative stress and apoptosis. The gradual decline in oocyte quality and quantity may be associated with the stable 2-ME2 in ovaries during female reproductive aging.

Effects of 2-methoxyestradiol on hydrogen peroxide induced neuronal cell death and tau hyperphosphorylation.

AIMS: Alzheimer's Disease (AD) is characterized by progressive cognitive impairment, and memory loss. It has been shown that depletion of estrogens renders women vulnerable to AD with menopause women presenting higher risk for AD development than men. However, women under hormone replacement therapy (HRT) with 17ß-estradiol (E2) show lower risk for AD, implying that E2 may be protective. It has been shown that E2 exerts its effects through the estrogen receptor (ER) but also via its biologically active metabolites, 2-hydroxyestradiol (2OH), and 2-methoxyestradiol (2ME). We hypothesized that the neuroprotective effects of E2 are partly attributed to its metabolites. MATERIALS AND METHODS: SH-SY5Y neuronal cells were subjected oxidative stress (OS) cell death by hydrogen peroxide (H2O2), in the presence or absence of E2, 2ME and 2OH. Viability was assessed by trypan blue and thiazolyl blue tetrazolium bromide assays, intracellular OS with the Dichlorodihydrofluorescein Diacetate (DCFDA) assay, and Bax, p53 and PUMA quantified by RT-PCR. Tau hyperphosphorylation was studied by western blot. KEY FINDINGS: E2 and its metabolites 2OH and 2ME protect from cell death as assessed by the viability assays. Their effect was partly attributed to their antioxidant properties evidenced by the reduction of intracellular OS. Treatment with 2ME resulted in a reduction of Bax, but not p53 or PUMA in cells challenged with OS. Finally, 2ME was able to inhibit tau hyperphosphorylation as well. SIGNIFICANCE: E2 protects neuron cells partly through its metabolites. Further studies are needed to fully delineate the mechanism for this protection.

Construction of a high-throughput aorta smooth muscle-on-a-chip for thoracic aortic aneurysm drug screening.

Thoracic aortic aneurysm (TAA), in which arteries enlarge asymptomatically over time until dissection or rupture occurs, is a serious health risk. The mainstay of TAA treatment remains surgical repair due to the lack of effective drugs. The complex etiology and pathogenesis of TAA, including hemodynamic alterations and genetic factors, lead to inaccuracies in preclinical models for drug screening. Previously, our group designed an aorta smooth muscle-on-a-chip to emulate human aorta physiology and pathophysiology and screened three promising therapeutic drugs targeting mitochondrial dynamics in TAA. On this foundation, we updated the one-channel chip to an eighteen-well chip platform with four polydimethylsiloxane layers. Benefiting from this high-throughput chip, we rapidly screened multiple drugs simultaneously using distinct cell lines in vitro. In addition, we observed the abnormal activation of hypoxia-inducible factor 1-alpha (HIF-1alpha) in aortas from TAA patients by Western blot and bioinformatics analyses. Intriguingly, this phenomenon was replicated only when smooth muscle cells (SMCs) were strained on the chip. We then screened seven specific HIF-1alpha inhibitors and selected the two most effective drugs (2-methoxyestradiol and digoxin) by quantitative PCR and colorimetric methods. The results demonstrated that these two drugs can improve respiratory chain function and rescue the SMC contractile phenotype, showing applicability for the clinical treatment of TAA. This high-throughput aorta smooth muscle-on-a-chip will become a potential preclinical model for TAA drug screening.

Inhibition of JAK1/STAT3 pathway by 2-methoxyestradiol ameliorates psoriatic features in vitro and in an imiquimod-induced psoriasis-like mouse model.

Psoriasis is characterized by hyperproliferative keratinocytes, dilated capillaries and leukocyte infiltration. 2-Methoxyestradiol (2-ME) has shown significant inhibition on proliferation, angiogenesis and inflammation. To evaluate the anti-psoriatic potential of 2-ME, psoriasis-like dermatitis was induced by topical application of imiquimod (IMQ) on the dorsal skin of C57BL/6 mice for seven consecutive days, followed by treatment of vehicle or 2-ME ointment from Day 4 on. The psoriasis area and severity index (PASI) was assessed daily. On Day 8, skin histology and spleen index were assessed. The effects of 2-ME on the proliferation, apoptosis, cell cycle, vascular endothelial growth factor A (VEGFA), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways of HaCaT cells stimulated by interleukin-17 (IL-17A) were detected, together with its effect on the proliferation, tube formation and VEGF receptor expression of human umbilical vein endothelial cells (HUVECs). We found that topical 2-ME treatment significantly improved IMQ-induced psoriasis-like dermatitis and decreased the PASI scores, the activation of STAT3 in the skin (P < 0.05), and the spleen index in mice (P < 0.01). In vitro, 2-ME inhibited the proliferation of HaCaT cells by inducing apoptosis and G2/M phase arrest (P < 0.01). Moreover, 2-ME suppressed IL-17A-induced VEGFA (2.5 µM: P < 0.05; 5 µM: P < 0.01) and phosphorylation of STAT3 by blocking p-JAK1 in HaCaT cells and prevented tube formation (P < 0.01) and proliferation by targeting VEGF receptors 1 (VEGFR1) and 2 (VEGFR2) in HUVECs. We conclude that 2-ME alleviated psoriasis in vivo and in vitro by inhibiting JAK1/STAT3 pathway and was a promising therapeutic agent for psoriasis.

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.