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.

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.

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.

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.

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.

Optimized 2-methoxyestradiol invasomes fortified with apamin: a promising approach for suppression of A549 lung cancer cells.

Certain anticancer agents selectively target the nucleus of cancer cells. One such drug is 2-methoxyestradiol (2ME), which is used for treating lung cancer. To improve the therapeutic effectiveness of these agents, many new methods have been devised. 2ME was entrapped into the core of hydrophobic invasomes (INVA) covered with Phospholipon 90G and apamin (APA). The Box-Behnken statistical design was implemented to enhance the composition. Using Design-Expert software (Stat-Ease Inc., Minneapolis, MN), the INVA component quantities were optimized to obtain spherical particles with the smallest size, that is, a diameter of 167.8 nm. 2ME-INVA-APA significantly inhibited A549 cells and exhibited IC50 of 1.15 ± 0.04 µg/mL, which is lower than raw 2ME (IC50 5.6 ± 0.2 µg/mL). Post 2ME-INVA-APA administration, a significant rise in cell death and necrosis was seen among the A549 cells compared to those treated with plain formula or 2ME alone. This effect was indicated by increased Bax expression and reduced Bcl-2 expression, as well as mitochondrial membrane potential loss. Moreover, the cell cycle analysis showed that 2ME-INVA-APA arrests the G2-M phase of the A549 cells. Additionally, it was observed that the micellar formulation of the drug increased the cell count in pre-G1, thereby exhibiting phenomenal apoptotic potential. Furthermore, it up-regulates caspase-9 and p53 and downregulates TNF-α and NF-κß. Collectively, these findings showed that our optimized 2ME-INVA-APA could easily seep through the cell membrane and induce apoptosis in relatively low doses.

Combined Effects of 2-Methoxyestradiol (Hypoxia-Inducible Factor 1α Inhibitor) and Dasatinib (A Second-Generation Tyrosine Kinase Inhibitor) on Chronic Myelocytic Leukemia Cells.

Chronic myelocytic leukemia (CML) is a frequently encountered type of leukemia in China. Hypoxia-inducible factor 1 (HIF-1) serves as one of the most important factors of oxygen balance transcription. The activation of this gene mostly marks a poor outlook for cancer patients. To clarify the therapeutic effect of inhibiting this gene on CML, the present study is aimed at exploring the treatment effects of 2-methoxyestradiol (2-ME2), dasatinib alone, and combined both on K-562 cells and the possible mechanism of 2-ME2 in treating the disorder. The levels of HIF-1α, vascular endothelial growth factor (VEGF), and glutamate synthase 1 (GLU1) genes in K-562 cells were affected dose-dependently after 2-ME2 administration. 2-ME2 induced cell apoptosis by downregulating antiapoptotic protein expressions of Bcl-xl and Bcl-2. The therapeutic effect of single 2-ME2 was superior to single dasatinib, and the effect of combined therapy of both drugs produced better effectiveness than either of the single drug. Once the concentration of 2-ME2 exceeded 0.5 µM, downregulated C-myc gene expression could exert roles in anti-CML cell proliferation and inducing apoptosis. Dasatinib might participate in the inhibition of the C-myc pathway during this process whereas its effect remained not clear. Taken together, abnormal high expression of HIF-1α exerted an essential role in CML occurrence and development. Inhibition of this gene could markedly increase cell apoptosis in a dose-dependent fashion. Moreover, 2-ME2 could induce cell apoptosis by downregulating the C-myc gene and exert an apoptotic effect by downregulating Bcl-xl and Bcl-2 which act as antiapoptotic proteins.

Decreased Expression of HN1 Sensitizes Prostate Cancer Cells to Apoptosis Induced by Docetaxel and 2-Methoxyestradiol.

OBJECTIVE: Prostate cancer is one of the most frequently diagnosed cancer in men and ranks as the second most common cause of cancer-related deaths in developed countries. HN1 is a highly expressed gene in prostate cancer and controls the levels of several cell cycle regulatory proteins including Cyclin B1. Cyclin B1 is a cell cycle control protein but is also involved in Docetaxel and 2-Methoxyestradiol induced apoptosis. Since Cyclin B1 level may affect chemotherapy success and HN1-Cyclin B1 negative correlation has already been shown, so in this study, we investigated the possible role of HN1 in chemotherapeutic resistance in prostate cancer cells. METHODS: Androgen-dependent and independent prostate cancer cells were used in the study. A full-length human HN1 cDNA fragment was cloned to a mammalian expression vector and this construct was used for overexpression experiments. A siRNA that specifically targets HN1 was used for HN1 depletion experiments. Evaluation of apoptosis was performed by the level of PARP cleavage and an apoptosis kit that measure Caspase 3 activity. RESULTS: The HN1 protein level is decreased in the Docetaxel or 2-Methoxyestradiol treated LNCaP and PC-3 PCa cells whereas the Cyclin B1 level is increased. HN1 overexpression inhibited Docetaxel and 2-Methoxyestradiol induced apoptosis. In concordance, its depletion further stimulated apoptosis in drug-treated cells. However, silencing of Cyclin B1 along with HN1 abolished the increased apoptotic response induced by silencing of HN1 in Docetaxel or 2-Methoxyestradiol treated cells. CONCLUSION: HN1 is an anti-apoptotic molecule and inhibits Docetaxel and 2-Methoxyestradiol induced apoptosis by targeting Cyclin B1.

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.

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.

The potential protective effects of estradiol and 2-methoxyestradiol in ischemia reperfusion-induced kidney injury in ovariectomized female rats.

AIMS: Investigating the impact of 17ß estradiol (E2) and its endogenous non-hormonal metabolite 2-methoxyestradiol (2ME) on renal ischemia-reperfusion (RIR) induced kidney injury in ovariectomized (OVX) rats and the role of catechol-O-methyltransferase (COMT) in their effects. MAIN METHODS: Eighty female rats were allocated into eight groups. Control group, Sham group, OVX group, OVX and RIR group, OVX + RIR + E2 group, OVX + RIR + 2ME group, OVX + RIR + E2 + Entacapone group and OVX + RIR + 2ME + Entacapone group, respectively. Twenty-four hours post RIR, creatinine (Cr) and blood urea nitrogen (BUN) were determined in serum, while malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), Glutathione (GSH), myeloperoxidase (MPO), as well as the expressions of COMT, hypoxia inducible factor-1α (HIF-1α) and tyrosine hydroxylase (TH) were assessed in the kidney tissues. KEY FINDINGS: Serum Cr, BUN, MPO, as well as HIF-1α and TH expressions were significantly higher with concomitant decrease in COMT expression, SOD and CAT activities and GSH content observed in OVX and RIR group compared to sham group. E2 and 2ME treatment significantly ameliorated all parameters measured in OVX and RIR rats. On the other hand, Entacapone significantly decreased the effect of E2, with no effect on 2ME treatment. SIGNIFICANCE: E2 ameliorates RIR-induced kidney injury and this effect is mediated, at least in part, via its COMT-mediated conversion to 2ME. Thus, 2ME by the virtue of its pleiotropic pharmacological effects can be used as a safe and effective treatment of RIR injury.

The photodynamic reaction with IR-775 cyanine combined with 2-methoxyestradiol in ovarian (SKOV-3) and human breast adenocarcinoma (MDA MB-231) cell lines.

BACKGROUND: Photodynamic therapy (PDT) is known anticancer approach used mainly for topical skin cancer. However, it could serve as an excellent alternative to traditional anticancer therapies, such as chemotherapy or radiotherapy. AIMS: The study aimed to assess the effect of PDT, where the combination of cyanine with 2-methoxyestradiol (2-Me) was used on mammary and ovary adenocarcinoma human cell lines. MATERIALS AND METHODS: The cyanine IR-775 was used as the photosensitizer. Two human malignant adenocarcinoma cell lines - ovary and mammary adenocarcinoma (MDA MB-231 and SKOV-3) were investigated in photodynamic reaction (PDR), with the enhancement of 2-Me. PDR efficiency was evaluated by the MTT test. Photosensitizer intracellular distribution was assessed by fluorescent microscopy. Additionally, apoptotic and oxidative stress markers were investigated by immunocytochemistry staining. RESULTS AND CONCLUSIONS: It was observed that PDR enhanced by 2-Me is effective against two common but different types of cancer. The treatment decreased cells' viability by around 70%. Immunocytochemical staining of SOD2 and caspase-12 indicated apoptosis and oxidative stress induction in tested cell lines. The results suggest that the therapy could be involved in further oncological applications.