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 Damages DNA in Glioblastoma Cells by Regulating nNOS and Heat Shock Proteins.

Gliomas are the most prevalent primary tumors of the central nervous system (CNS), accounting for over fifty percent of all primary intracranial neoplasms. Glioblastoma (GBM) is the most prevalent form of malignant glioma and is often incurable. The main distinguishing trait of GBM is the presence of hypoxic regions accompanied by enhanced angiogenesis. 2-Methoxyestradiol (2-ME) is a well-established antiangiogenic and antiproliferative drug. In current clinical studies, 2-ME, known as Panzem, was examined for breast, ovarian, prostate, and multiple myeloma. The SW1088 grade III glioma cell line was treated with pharmacological and physiological doses of 2-ME. The induction of apoptosis and necrosis, oxidative stress, cell cycle arrest, and mitochondrial membrane potential were established by flow cytometry. Confocal microscopy was used to detect DNA damage. The Western blot technique determined the level of nitric oxide synthase and heat shock proteins. Here, for the first time, 2-ME is shown to induce nitro-oxidative stress with the concomitant modulation of heat shock proteins (HSPs) in the SW1088 grade III glioma cell line. Crucial therapeutic strategies for GMB should address both cell proliferation and angiogenesis, and due to the above, 2-ME seems to be a perfect candidate for GBM therapy.

Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer.

Lung cancer is one of the most common cancers worldwide, causing nearly one million deaths each year. Herein, we present the effect of 2-methoxyestradiol (2-ME), the endogenous metabolite of 17ß-estradiol (E2), on non-small cell lung cancer (NSCLC) cells. We observed that 2-ME reduced the viability of lung adenocarcinoma in two-dimensional (2D) and three-dimensional (3D) spheroidal A549 cell culture models. Molecular modeling was carried out aiming to visualize amino acid residues within binding pockets of the acyl-protein thioesterases, namely 1 (APT1) and 2 (APT2), and thus to identify which ones were more likely involved in the interaction with 2-ME. Our findings suggest that 2-ME acts as an APT1 inhibitor enhancing protein palmitoylation and oxidative stress phenomena in the lung cancer cell. In order to support our data, metabolomics of blood serum from NSCLC patients was also performed. Moreover, computational analysis suggests that 2-ME as compared to other estrogen metabolism intermediates is relatively safe in terms of its possible non-receptor bioactivity within healthy human cells due to a very low electrophilic potential and hence no substantial risk of spontaneous covalent modification of biologically protective nucleophiles. We propose that 2-ME can be used as a selective tumor biomarker in the course of certain types of lung cancers and possibly as a therapeutic adjuvant or neoadjuvant.

DNA methylation profile in patients with indolent systemic mastocytosis.

BACKGROUND: Mastocytosis is a clinically heterogeneous, usually acquired disease of the mast cells with a survival time that depends on the onset of the disease and ranges from skin-limited to systemic disease, including indolent and more aggressive variants. The crucial element in pathogenesis is the presence of oncogenic KIT somatic mutation D816V. Further epigenetic alterations are responsible for regulating the expression of genes. It is essential to identify indicators of disease progression, and the specific clinical picture to establish an appropriate therapeutic strategy. OBJECTIVE: The aim of this study was to analyze the relation of mastocytosis symptoms and epigenetic changes, and to identify epigenetic predictors of the disease. METHODS: Global DNA methylation profile analysis was performed in peripheral blood collected from 73 patients with indolent systemic mastocytosis (ISM) and 43 healthy adult volunteers. Levels of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were determined using an ELISA-based method, while the methylation of the Alu and LINE-1 repeats were assayed with the quantitative methylation-specific PCR technique. A questionnaire interview was conducted among the study participants to collect data on possible epigenetic modifiers. Additionally, the methylation profile was compared between three human mast cell lines: ROSA KIT D816V, ROSA KIT WT, and HMC-1.1 KIT V560G, in order to assess the association between KIT mutations and methylation profile. RESULTS: A significantly lower level of DNA hydroxymethylation (5-hmC) in the blood was found in patients with ISM as compared to the controls (0.022% vs. 0.042%, p = 0.0001). Differences in the markers of global DNA methylation (5-mC, Alu, LINE-1) were not statistically significant, although they did indicate generally higher DNA methylation in patients with mastocytosis. The 5-hmC level was significantly associated with allergy (p = 0.011) in patients with ISM, showing a higher level of 5-hmC in patients with allergy as compared to patients without allergy. The in vitro study revealed significant differences between the studied cell lines at the level of 5-mC, Alu, and LINE-1. CONCLUSIONS: This study confirms that epigenetic changes are involved in mastocytosis, and suggests that allergy may be an important epigenetic modifier of the disease. A possible association between KIT mutations and methylation status observed in human mast cell lines requires further investigation in human studies. CLINICAL IMPLICATIONS: Epigenetic alterations are involved in mastocytosis pathology. The possible role of allergy as an important epigenetic modifier suggests the more impaired function of mast cells in ISM patients without allergy. CAPSULE SUMMARY: Decreased DNA demethylation in the blood DNA of patients with ISM confirms that epigenetic alterations are involved in mastocytosis pathology. We observed a possible role of allergy as an important epigenetic modifier. There is a possible association between KIT mutations and the methylation status observed in human mast cell lines.

Regulation of Mitochondrial Dynamics in Parkinson's Disease-Is 2-Methoxyestradiol a Missing Piece?

Mitochondria, as "power house of the cell", are crucial players in cell pathophysiology. Beyond adenosine triphosphate (ATP) production, they take part in a generation of reactive oxygen species (ROS), regulation of cell signaling and cell death. Dysregulation of mitochondrial dynamics may lead to cancers and neurodegeneration; however, the fusion/fission cycle allows mitochondria to adapt to metabolic needs of the cell. There are multiple data suggesting that disturbed mitochondrial homeostasis can lead to Parkinson's disease (PD) development. 2-methoxyestradiol (2-ME), metabolite of 17ß-estradiol (E2) and potential anticancer agent, was demonstrated to inhibit cell growth of hippocampal HT22 cells by means of nitric oxide synthase (NOS) production and oxidative stress at both pharmacologically and also physiologically relevant concentrations. Moreover, 2-ME was suggested to inhibit mitochondrial biogenesis and to be a dynamic regulator. This review is a comprehensive discussion, from both scientific and clinical point of view, about the influence of 2-ME on mitochondria and its plausible role as a modulator of neuron survival.

Regulation of mitochondrial dynamics in 2-methoxyestradiol-mediated osteosarcoma cell death.

Osteosarcoma (OS) is one of the most malignant tumors of childhood and adolescence. Research on mitochondrial dynamics (fusion/fission) and biogenesis has received much attention in last few years, as they are crucial for death of cancer cells. Specifically, it was shown that increased expression of the cytoplasmic dynamin-related protein 1 (Drp1) triggers mitochondrial fission (division), which activates BAX and downstream intrinsic apoptosis, effectively inhibiting OS growth. In the presented study, human OS cells (metastatic 143B OS cell line) were incubated with 2-methoxyestradiol (2-ME) at both physiologically and pharmacologically relevant concentrations. Cell viability was determined by the MTT assay. Confocal microscopy and western blot methods were applied to examine changes in Drp1 and BAX protein levels. Mitochondrial Division Inhibitor 1, MDIVI-1, was used in the study to further examine the role of Drp1 in 2-ME-mediated mechanism of action. To determine quantitative and qualitative changes in mitochondria, electron microscopy was used. 2-ME at all used concentrations increased mitochondrial fission and induced autophagy in OS cells. At the concentration of 1 µM 2-ME increased the area density of mitochondria in OS cells. Subsequent, upregulated expression of Drp1 and BAX proteins by 2-ME strongly suggests the activation of the intrinsic apoptosis pathway. We further observed 2-ME-mediated regulation of glycolytic state of OS cells. Therefore, we suggest that changes of mitochondrial dynamics may represent a novel mechanism of anticancer action of 2-ME. This finding may open new approaches to improve the efficacy of chemotherapy in the treatment of OS, however, it has to be confirmed by in vivo studies.