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.

2-methoxyestradiol inhibits the malignant behavior of triple negative breast cancer cells by altering their miRNome.

Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with no effective targeted treatment currently available. Estrogen and its metabolites influence the growth of mammary cancer. Previously, we demonstrated the anti-cancer effects of 2-methoxyestradiol (2ME2) on mammary carcinogenesis. Materials and methods: In the present study, we investigated the effects of 2ME2 on TNBC cells. TNBC (MDA-MB-231 and MDA-MB-468) and non-tumorigenic breast (MCF10A) cell lines were used to determine the effects of 2ME2 on cell proliferation (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS assay), cell cycle (flow cytometric assay), migration (transwell migration assay), invasion (matrigel invasion assay), apoptosis (annexin V/propidium iodide assay), colony formation (soft agar assay), and miRNome (human miRNA profiling array). The miRNome data were analyzed using the c-BioPortal and Xena platforms. Moreover, Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and reactome pathway analyses were performed. Results: We found that 2ME2 effectively inhibited cell proliferation and induced apoptosis. Furthermore, 2ME2 treatment arrested TNBC cells in the S-phase of the cell cycle. Treatment with 2ME2 also significantly decreased the aggressiveness of TNBC cells by inhibiting their migration and invasion. In addition, 2ME2 altered the miRNA expression in these cells. In silico analysis of the miRNome profile of 2ME2-treated MDA-MB-468 cells revealed that miRNAs altered the target genes involved in many different cancer hallmarks. Conclusion: 2ME2 inhibits triple negative breast cancer by impacting major cellular processes like proliferation, apoptosis, metastasis, etc. It further modifies gene expression by altering the miRNome of triple negative breast cancer cells. Overall, our findings suggest 2ME2 as a potent anti-cancer drug for the treatment of TNBC.

Sulfamoylated Estradiol Analogs Targeting the Actin and Microtubule Cytoskeletons Demonstrate Anti-Cancer Properties In Vitro and In Ovo.

The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico-an ESE-15-one and an ESE-16 one-with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class.

2-methoxyestradiol sensitizes tamoxifen-resistant MCF-7 breast cancer cells via downregulating HIF-1α.

The clinical studies for breast cancer (BC) are now assessing the efficacy of 2-Methoxyestradiol (2-ME), a naturally occurring derivative of estradiol. Our study aimed to explore the potential of combining the 2-ME and tamoxifen (TAM) on sensitization of TAM-resistant cells using LCC2 the TAM-resistant cells as a model and comparing the results to the sensitive cells MCF-7. Sulphorhodamine-B (SRB) assay is used to examine the 2-ME chemo-sensitizing impact on the cytotoxicity of TAM on LCC2 cells. Colorimetric assay kits were used to assess the level of the apoptosis-related markers caspases 3, Bcl2, and Bax in cell lysate. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was measured using western blotting. Total cholesterol and triglyceride (TG) levels were examined colorimetrically, using the BIOLABO kit. The use of 2-ME enhanced the cytotoxic effects of TAM and effectively reversed TAM resistance. This was achieved by inhibiting the expression of HIF-1α, while concurrently increasing the levels of apoptotic marker caspase-3, as well as the pro-apoptotic protein Bax. Additionally, there was a reduction in the levels of Bcl2, an anti-apoptotic protein. Furthermore, a reduction in TG and cholesterol levels was noted. Our findings show that HIF-1α plays an important role in TAM resistance and that suppression of HIF-1α by 2-ME-mediated sensitization of BC-resistant cells to TAM. Therefore, the concurrent administration of TAM/2-ME might potentially serve as a viable therapeutic approach to address TAM resistance and enhance the overall therapy efficacy for patients with BC.

2-Methoxyestradiol, an Endogenous 17ß-Estradiol Metabolite, Induces Antimitogenic and Apoptotic Actions in Oligodendroglial Precursor Cells and Triggers Endoreduplication via the p53 Pathway.

The abnormal growth of oligodendrocyte precursor cells (OPCs) significantly contributes to the progression of glioblastoma tumors. Hence, molecules that block OPC growth may be of therapeutic importance in treating gliomas. 2-Methoxyestradiol (2ME), an endogenous tubulin-interacting metabolite of estradiol, is effective against multiple proliferative disorders. Based on its anti-carcinogenic and anti-angiogenic actions, it is undergoing phase II clinical trials. We hypothesize that 2ME may prevent glioma growth by targeting OPC growth. Here, we tested this hypothesis by assessing the impact of 2ME on the growth of an OPC line, "Oli-neu", and dissected the underlying mechanism(s). Treatment with 2ME inhibited OPC growth in a concentration-dependent manner, accompanied by significant upregulation in the expression of p21 and p27, which are negative cell-cycle regulators. Moreover, treatment with 2ME altered OPC morphology from multi-arm processes to rounded cells. At concentrations of 1uM and greater, 2ME induced apoptosis, with increased expressions of caspase 3, PARP, and caspase-7 fragments, externalized phosphatidylserine staining/APOPercentage, and increased mitochondrial activity. Flow cytometry and microscopic analysis demonstrated that 2ME triggers endoreduplication in a concentration-dependent fashion. Importantly, 2ME induced cyclin E, JNK1/2, and p53 expression, as well as OPC fusion, which are key mechanisms driving endoreduplication and whole-genome duplication. Importantly, the inhibition of p53 with pifithrin-α rescued 2ME-induced endoreduplication. The pro-apoptotic and endoreduplication actions of 2ME were accompanied by the upregulation of survivin, cyclin A, Cyclin B, Cyclin D2, and ppRB. Similar growth inhibitory, apoptotic, and endoreduplication effects of 2ME were observed in CG4 cells. Taken together, our findings provide evidence that 2ME not only inhibits OPC growth and triggers apoptosis, but also activates OPCs into survival (fight or flight) mode, leading to endoreduplication. This inherent survival characteristic of OPCs may, in part, be responsible for drug resistance in gliomas, as observed for many tubulin-interacting drugs. Importantly, the fate of OPCs after 2ME treatment may depend on the cell-cycle status of individual cells. Combining tubulin-interfering molecules with drugs such as pifithrin-α that inhibit endoreduplication may help inhibit OPC/glioma growth and limit drug resistance.

[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.

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.

2-Methoxyestradiol ameliorates doxorubicin-induced cardiotoxicity by regulating the expression of GLUT4 and CPT-1B in female rats.

The clinical usage of doxorubicin (DOX) is hampered due to cardiomyopathy. Studies reveal that estrogen (E2) modulates DOX-induced cardiotoxicity. Yet, the exact mechanism is unclear. The objective of the current study is to evaluate the influence of E2 and more specifically its metabolite 2-methoxyestradiol (2ME) on cardiac remodeling and the reprogramming of cardiac metabolism in rats subjected to DOX cardiotoxicity. Seventy-two female rats were divided into groups. Cardiotoxicity was induced by administering DOX (2.5 mg/kg three times weekly for 2 weeks). In some groups, the effect of endogenous E2 was abolished by ovariectomy (OVX) or by using the estrogen receptor (ER) blocker Fulvestrant (FULV). The effect of administering exogenous E2 or 2ME in the OVX group was studied. Furthermore, the influence of entacapone (COMT inhibitor) on induced cardiotoxicity was investigated. The evaluated cardiac parameters included ECG, histopathology, cardiac-related enzymes (creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH)), and lipid profile markers (total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL)). The expression levels of key metabolic enzymes (glucose transporter-4 (GLUT4) and carnitine palmitoyltransferase-1B (CPT-1B)) were assessed. Our results displayed that co-treatment of E2 and/or 2ME with DOX significantly reduced DOX-induced cardiomyopathy and enhanced the metabolism of the heart through the maintenance of GLUT4 and CPT-1B enzymes. On the other hand, co-treatment of DOX with OVX, entacapone, or FULV increased the toxic effect of DOX by further reducing these important metabolic enzymes. E2 and 2ME abrogate DOX-induced cardiomyopathy partly through modulation of GLUT 4 and CPT-1B enzymes.

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.

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.

The role of estrogen metabolites in human ovarian function.

Estrogens produced by the ovary play diverse roles in controlling physiological changes in the function of the female reproductive system. Although estradiol acts through classical nuclear receptors, its metabolites (EMs) act by alternative pathways. It has been postulated that EMs act through paracrine-autocrine pathways to regulate key processes involved in normal follicular growth, corpus luteum (CL) development, function, and regression. The present review describes recent advances in understanding the role of EMs in human ovarian physiology during the menstrual cycle, including their role in anovulatory disorders and their action in other target tissues.

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.

[Regulación de la expresión de IL-33 e IL-17 por la modulación farmacológica de HIF-1 en un modelo murino de inflamación alérgica pulmonar].

Objective: To evaluate the effect of pharmacological modulation of HIF-1 on the expression of IL-33 and IL-17 in a murine model of allergic pulmonary inflam- mation (API) with different degrees of severity. Methods: 5 mice/group received ovalbumin (OVA) 1(mild), 2(moderate) or 3(severe) challenges via i.t. prior to allergen sensitization, in addition to the HIF-1 induction or inhibition groups, received EDHB (OVA+EDHB) i.p. or 2ME (OVA+2ME) i.t. respectively. Control groups received saline solution (SS) in the same way. HE (inflammatory infiltrate), PAS (mucus production) and immunohistochemical staining for HIF-1a, IL-33, IL-17 were performed, quantitatively analyzing by digital pathology. Results: We obtained different degrees of severity with a greater number of challenges, increasing the expression of HIF-1, correlating with the expression of IL-33/IL-17. Increasing or decreasing, respectively by pharmacological modulation. Conclusions: The above suggests that the high expression of HIF-1 favors the production of IL-33 and IL-17 contributing to the damage in lung tissue and the severity of the disease and these can be regulated through the modulation of HIF- 1.

Objetivo: Evaluar el efecto de la modulación farmacológica de HIF-1 en la expresión de IL-33 e IL-17 en un modelo murino de inflamación alérgica pulmonar (IAP) con diferentes grados de severidad. Métodos: 5 ratones/grupo recibieron ovoalbúmina (OVA) 1(leve), 2(moderada) o 3(severa) retos vía i.t. previa sensibilización como alergeno, además los grupos de inducción o inhibición de HIF-1a, recibieron EDHB (OVA+EDHB) i.p. o 2ME (OVA+2ME) i.t. respectivamente. Los grupos controles recibieron solución salina (SS) de igual forma. Se realizaron tinciones de HE (infiltrado inflamatorio), PAS (producción de moco) e inmunohistoquímicas de HIF-1a, IL-33, IL-17, analizando cuantitativamente por patología digital. Resultados: Obtuvimos diferentes grados de severidad a mayor número de retos, incrementando la expresión de HIF-1, correlacionando con la expresión de IL- 33/IL-17. Aumentando o disminuyendo, respectivamente por la modulación farmacológica. Conclusiones: Lo anterior sugiere que la alta expresión de HIF-1 favorece la producción de IL-33 e IL-17 contribuyendo al daño en el tejido pulmonar y la severi- dad de la enfermedad y estas pueden ser reguladas a través de la modulación de HIF-1.

Protective Effects of 2-Methoxyestradiol on Acute Isoproterenol-Induced Cardiac Injury in Rats.

Myocardial injury (MI) is an important pathological driver of mortality worldwide., and arises as a result of imbalances between myocardial oxygen demand and supply. In MI, oxidative stress often leads to inflammatory changes and apoptosis. Current therapies for MI are known to cause various adverse effects. Consequently, the development of new therapeutic agents with a reduced adverse event profile is necessary. In this regard, 2-methoxyestradiol (2ME), the metabolic end-product of oestradiol, possesses anti-inflammatory and antioxidant properties. The aim of this research is to assess the impact of 2ME on cardiac injury caused by isoproterenol (ISO) in rats. Animals were separated into six groups; controls, and those receiving 2ME (1 mg/kg), ISO (85 mg/kg), ISO + 2ME (0.25 mg/kg), ISO + 2ME (0.5 mg/kg), and ISO + 2ME (1 mg/kg). 2ME significantly attenuated ISO-induced changes in electrocardiographic changes and the cardiac histological pattern. This compound also decreased lactate dehydrogenase activity, creatine kinase myocardial band and troponin levels. The ability of 2ME to act as an antioxidant was shown by a decrease in malondialdehyde concentration, and the restoration of glutathione levels and superoxide dismutase activity. Additionally, 2ME antagonized inflammation and cardiac cell apoptosis, a process determined to be mediated, at least partially, by suppression of Gal-3/TLR4/MyD88/NF-κB signaling pathway. 2ME offers protection against acute ISO-induced MI in rats and offers a novel therapeutic management option.

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.

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.

2-Methoxyestradiol inhibits the proliferation level in keloid fibroblasts through p38 in the MAPK/Erk signaling pathway.

BACKGROUND: The MAPK/Erk signaling pathway is a classic pathway in cell proliferation. Our former study showed that keloid tissue revealed a higher proliferation level than physiological scars and normal skin. As a natural metabolite of estradiol, 2-methoxyestradiol (2ME2) showed an inhibition proliferation effect on tumor cells. AIM: In this study, the treatment effect of 2ME2 and its potential mechanisms are explored. METHODS: Six keloid patients and six non-keloid patients were randomly selected from the Department of Plastic Surgery at our hospital during June 2021 to December 2021. Six groups were established: normal skin fibroblasts (N); keloid fibroblasts (K); keloid fibroblasts treated with 2ME2 (K + 2ME2); keloid fibroblasts treated with dimethyl sulfoxide (DMSO) (K + DMSO); keloid fibroblasts treated with doramapimod (K + IN); keloid fibroblasts treated with doramapimod (p38 inhibitor) and 2ME2 (K + IN+2ME2). The fibroblast activity and key factor expression of the MAPK/Erk signaling pathway were measured. RESULTS: In the results, 2ME2 significantly inhibited keloid fibroblast activity and key factor expression (except STAT1). CONCLUSION: The proliferation levels were reduced by both the p38 inhibitor and 2ME2, indicating 2ME2 may achieve an antiproliferation effect by targeting p38 in keloid fibroblasts.

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.

HIF-1α participates in secondary brain injury through regulating neuroinflammation.

A deeper understanding of the underlying biological mechanisms of secondary brain injury induced by traumatic brain injury (TBI) will greatly advance the development of effective treatments for patients with TBI. Hypoxia-inducible factor-1 alpha (HIF-1α) is a central regulator of cellular response to hypoxia. In addition, growing evidence shows that HIF-1α plays the important role in TBI-induced changes in biological processes; however, detailed functional mechanisms are not completely known. The aim of the present work was to further explore HIF-1α-mediated events after TBI. To this end, next-generation sequencing, coupled with cellular and molecular analysis, was adopted to interrogate vulnerable events in a rat controlled cortical impact model of TBI. The results demonstrated that TBI induced accumulation of HIF-1α at the peri-injury site at 24 h post-injury, which was associated with neuronal loss. Moreover, gene set enrichment analysis unveiled that neuroinflammation, especially an innate inflammatory response, was significantly evoked by TBI, which could be attenuated by the inhibition of HIF-1α. Furthermore, the inhibition of HIF-1α could mitigate the activation of microglia and astrocytes. Taken together, all these data implied that HIF-1α might contribute to secondary brain injury through regulating neuroinflammation.