Coffee consumption during pregnancy increases the risk of preeclampsia in rats by inhibiting 2-methoxyestradiol production.

Preeclampsia (PE) is a pregnancy-specific disease that causes maternal symptoms such as high blood pressure and adverse pregnancy outcomes. 2-methoxyestradiol (2-MeO-E2), an endogenous metabolite of 17ß-estradiol (E2) formed by Catechol-O-Methyltransferase (COMT), plays an important role in pregnancy. Our earlier studies have shown that polyphenols present in coffee can inhibit COMT activity, which may inhibit the formation of 2-MeO-E2 and contribute to PE. Therefore, the current study aims to investigate the possible effect and mechanism of coffee intake during pregnancy on PE in SD rats. Coffee is administered with or without cotreatment of 2-MeO-E2 to pregnant rats from the10th to the18th day of pregnancy. The results show that pregnant rats with coffee intake had prominent fetal growth restriction, hypertension and proteinuria, which can be ameliorated by co-treatment of 2-MeO-E2. In addition, coffee treatment leads to significantly decreased serum 2-MeO-E2. Therefore, the PE symptoms induced by coffee treatment is probably mediated by decreased 2-MeO-E2. Our findings provide new mechanistic insight into how coffee intake could lead to increased risk of PE, and demonstrate the effectiveness of 2-MeO-E2 supplementation as a potential therapeutic agent for PE.

Inhibition of STAT3 by 2-Methoxyestradiol suppresses M2 polarization and protumoral functions of macrophages in breast cancer.

BACKGROUND: Breast cancer metastasis remains the leading cause of cancer-related deaths in women worldwide. Infiltration of tumor-associated macrophages (TAMs) in the tumor stroma is known to be correlated with reduced overall survival. The inhibitors of TAMs are sought after for reprogramming the tumor microenvironment. Signal transducer and activator of transcription 3 (STAT3) is well known to contribute in pro-tumoral properties of TAMs. 2-Methoxyestradiol (2ME2), a potent anticancer and antiangiogenic agent, has been in clinical trials for treatment of breast cancer. Here, we investigated the potential of 2ME2 in modulating the pro-tumoral effects of TAMs in breast cancer. METHODS: THP-1-derived macrophages were polarized to macrophages with or without 2ME2. The effect of 2ME2 on macrophage surface markers and anti-inflammatory genes was determined by Western blotting, flow cytometry, immunofluorescence, qRT‒PCR. The concentration of cytokines secreted by cells was monitored by ELISA. The effect of M2 macrophages on malignant properties of breast cancer cells was determined using colony formation, wound healing, transwell, and gelatin zymography assays. An orthotopic model of breast cancer was used to determine the effect of 2ME2 on macrophage polarization and metastasis in vivo. RESULTS: First, our study found that polarization of monocytes to alternatively activated M2 macrophages is associated with the reorganization of the microtubule cytoskeleton. At lower concentrations, 2ME2 treatment depolymerized microtubules and reduced the expression of CD206 and CD163, suggesting that it inhibits the polarization of macrophages to M2 phenotype. However, the M1 polarization was not significantly affected at these concentrations. Importantly, 2ME2 inhibited the expression of several anti-inflammatory cytokines and growth factors, including CCL18, TGF-ß, IL-10, FNT, arginase, CXCL12, MMP9, and VEGF-A, and hindered the metastasis-promoting effects of M2 macrophages. Concurrently, 2ME2 treatment reduced the expression of CD163 in tumors and inhibited lung metastasis in the orthotopic breast cancer model. Mechanistically, 2ME2 treatment reduced the phosphorylation and nuclear translocation of STAT3, an effect which was abrogated by colivelin. CONCLUSIONS: Our study presents novel findings on mechanism of 2ME2 from the perspective of its effects on the polarization of the TAMs via the STAT3 signaling in breast cancer. Altogether, the data supports further clinical investigation of 2ME2 and its derivatives as therapeutic agents to modulate the tumor microenvironment and immune response in breast carcinoma.

Endogenous estrogen metabolites as oxidative stress mediators and endometrial cancer biomarkers.

BACKGROUND: Endometrial cancer is the most common gynecologic malignancy found in developed countries. Because therapy can be curative at first, early detection and diagnosis are crucial for successful treatment. Early diagnosis allows patients to avoid radical therapies and offers conservative management options. There are currently no proven biomarkers that predict the risk of disease occurrence, enable early identification or support prognostic evaluation. Consequently, there is increasing interest in discovering sensitive and specific biomarkers for the detection of endometrial cancer using noninvasive approaches. CONTENT: Hormonal imbalance caused by unopposed estrogen affects the expression of genes involved in cell proliferation and apoptosis, which can lead to uncontrolled cell growth and carcinogenesis. In addition, due to their ability to cause oxidative stress, estradiol metabolites have both carcinogenic and anticarcinogenic properties. Catechol estrogens are converted to reactive quinones, resulting in oxidative DNA damage that can initiate the carcinogenic process. The molecular anticancer mechanisms are still not fully understood, but it has been established that some estradiol metabolites generate reactive oxygen species and reactive nitrogen species, resulting in nitro-oxidative stress that causes cancer cell cycle arrest or cell death. Therefore, identifying biomarkers that reflect this hormonal imbalance and the presence of endometrial cancer in minimally invasive or noninvasive samples such as blood or urine could significantly improve early detection and treatment outcomes.

[Protective effects of 2-methoxyestradiol against hypoxic pulmonary hypertension in neonatal rats]. / 2-ç”²æ°§åŸºé›ŒäºŒé†‡å¯¹æ–°ç”Ÿå¤§é¼ ç¼ºæ°§æ€§è‚ºåŠ¨è„‰é«˜åŽ‹çš„ä¿æŠ¤ä½œç”¨.

OBJECTIVES: To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. METHODS: Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 µg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. RESULTS: Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). CONCLUSIONS: 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.

2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 µM (vs. 6.79 µM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of ß-tubulin proteins in LTED cells than in MCF-7 cells. Among the ß-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

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.

Zeolite Nanoparticles Loaded with 2-Methoxystradiol as a Novel Drug Delivery System for the Prostate Cancer Therapy.

The estrogen metabolite 2-methoxyestradiol (2ME) is a promissory anticancer drug mainly because of its pro-apoptotic properties in cancer cells. However, the therapeutic use of 2ME has been hampered due to its low solubility and bioavailability. Thus, it is necessary to find new ways of administration for 2ME. Zeolites are inorganic aluminosilicates with a porous structure and are considered good adsorbents and sieves in the pharmaceutical field. Here, mordenite-type zeolite nanoparticles were loaded with 2ME to assess its efficiency as a delivery system for prostate cancer treatment. The 2ME-loaded zeolite nanoparticles showed an irregular morphology with a mean hydrodynamic diameter of 250.9 ± 11.4 nm, polydispersity index of 0.36 ± 0.04, and a net negative surface charge of -34 ± 1.73 meV. Spectroscopy with UV-vis and Attenuated Total Reflectance Infrared Fourier-Transform was used to elucidate the interaction between the 2ME molecules and the zeolite framework showing the formation of a 2ME-zeolite conjugate in the nanocomposite. The studies of adsorption and liberation determined that zeolite nanoparticles incorporated 40% of 2ME while the liberation of 2ME reached 90% at pH 7.4 after 7 days. The 2ME-loaded zeolite nanoparticles also decreased the viability and increased the mRNA of the 2ME-target gene F-spondin, encoded by SPON1, in the human prostate cancer cell line LNCaP. Finally, the 2ME-loaded nanoparticles also decreased the viability of primary cultures from mouse prostate cancer. These results show the development of 2ME-loaded zeolite nanoparticles with physicochemical and biological properties compatible with anticancer activity on the human prostate and highlight that zeolite nanoparticles can be a good carrier system for 2ME.

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.

Effect of Interaction between Chromium(VI) with 17ß-Estradiol and Its Metabolites on Breast Cancer Cell Lines MCF-7/WT and MDA-MB-175-VII: Preliminary Study.

The number of factors initiating and stimulating the progression of breast cancer are constantly increasing. Estrogens are a risk factor for breast adenocarcinoma, the toxicity of which increases as a result of metabolism and interaction with other factors. Due to the presence of environmental exposure to estrogens and metalloestrogens, we investigated how interactions between estrogens and toxic chromium(VI)[Cr(VI)] affect breast cancer lines and investigated whether estrogens play a protective role. The aim of the study was to investigate the effect of 17ß-estradiol and its metabolites: 2-methoxyestradiol (2-MeOE2), 4-hydroxyestradiol (4-OHE2), and 16α-hydroxyestrone (16α-OHE1) in exposure to Cr(VI) on cell viability and DNA cell damage. Two estrogen-dependent breast cancer cell lines, MCF 7/WT and MDA-MB-175-VII, were examined. In addition, the expression of Cu-Zn superoxide dismutase (SOD1) was determined immunocytochemically to elucidate the mechanism of oxidative stress. The effects of single substances and their mixtures were tested in the model of simultaneous and 7-day estrogen pre-incubation. As a result, the viability of MCF-7 and MDA-MB-175-VII cells is lowered most by Cr(VI) and least by 17ß-E2. In the combined action of estrogens and metalloestrogens, we observed a protective effect mainly of 17ß-E2 against Cr(VI)-induced cytotoxicity. The highest expression of SOD1 was found in MCF-7/WT cells exposed to 17ß-E2. Moreover, high apoptosis was caused by both Cr(VI) itself and its interaction with 4-OHE2 and 2-MeOE2. The direction and dynamics of changes in viability are consistent for both lines.