Homocysteine-induced decrease in HUVEC cells' resistance to oxidative stress is mediated by Akt-dependent changes in iron metabolism.

PURPOSE: Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases and also promotes neuronal death in various neurodegenerative diseases. There is evidence that iron can mediate homocysteine (Hcy) toxicity. Thus, the aim of this study was to investigate the effect of Hcy on iron metabolism in HUVEC and SH-SY5Y cells. METHODS: HUVEC and SH-SY5Y cells were treated with 3 mM Hcy for a defined time. RESULTS: We demonstrate that Hcy induced the upregulation of ferritins type L and H in HUVEC cells in a time-dependent manner and had no effect on the ferritins in SH-SY5Y cells. The change in ferritin expression was preceded by a significant decrease in the cellular level of the active form of Akt kinase in HUVEC but not in SH-SY5Y cells. An increase in ferritin L and H protein levels was observed in the Akt1, Akt2, Akt3 siRNA transfected cells, while in the cells transfected with FOXO3a siRNA, a decrease in both ferritins levels was noticed. Moreover, in the HUVEC cells treated with Hcy for 6 days, the active form of kinase Akt returned to the control level and it was accompanied by a drop in ferritin L and H protein levels. Cytotoxicity of hydrogen peroxide significantly increased in HUVEC cells pre-treated with Hcy for 24 h. CONCLUSIONS: These data indicate that Hcy induces an increase in cellular ferritin level, and the process is mediated by alterations in Akt-FOXO3a signaling pathway.

Potential Health Benefits of Olive Oil and Plant Polyphenols.

Beneficial effects of natural plant polyphenols on the human body have been evaluated in a number of scientific research projects. Bioactive polyphenols are natural compounds of various chemical structures. Their sources are mostly fruits, vegetables, nuts and seeds, roots, bark, leaves of different plants, herbs, whole grain products, processed foods (dark chocolate), as well as tea, coffee, and red wine. Polyphenols are believed to reduce morbidity and/or slow down the development of cardiovascular and neurodegenerative diseases as well as cancer. Biological activity of polyphenols is strongly related to their antioxidant properties. They tend to reduce the pool of reactive oxygen species as well as to neutralize potentially carcinogenic metabolites. A broad spectrum of health-promoting properties of plant polyphenols comprises antioxidant, anti-inflammatory, anti-allergic, anti-atherogenic, anti-thrombotic, and anti-mutagenic effects. Scientific studies present the ability of polyphenols to modulate the human immune system by affecting the proliferation of white blood cells, and also the production of cytokines or other factors that participate in the immunological defense. The aim of the review is to focus on polyphenols of olive oil in context of their biological activities.

Capping Agent-Dependent Toxicity and Antimicrobial Activity of Silver Nanoparticles: An In Vitro Study. Concerns about Potential Application in Dental Practice.

Objectives: In dentistry, silver nanoparticles (AgNPs) have drawn particular attention because of their wide antimicrobial activity spectrum. However, controversial information on AgNPs toxicity limited their use in oral infections. Therefore, the aim of the present study was to evaluate the antibacterial activities against a panel of oral pathogenic bacteria and bacterial biofilms together with potential cytotoxic effects on human gingival fibroblasts of 10 nm AgNPs: non-functionalized - uncapped (AgNPs-UC) as well as surface-functionalized with capping agent: lipoic acid (AgNPs-LA), polyethylene glycol (AgNPs-PEG) or tannic acid (AgNPs-TA) using silver nitrate (AgNO3) as control. Methods: The interaction of AgNPs with human gingival fibroblast cells (HGF-1) was evaluated using the mitochondrial metabolic potential assay (MTT). Antimicrobial activity of AgNPs was tested against anaerobic Gram-positive and Gram-negative bacteria isolated from patients with oral cavity and respiratory tract infections, and selected aerobic Staphylococci strains. Minimal inhibitory concentration (MIC) values were determined by the agar dilution method for anaerobic bacteria or broth microdilution method for reference Staphylococci strains and Streptococcus mutans. These strains were also used for antibiofilm activity of AgNPs. Results: The highest antimicrobial activities at nontoxic concentrations were observed for the uncapped AgNPs and the AgNPs capped with LA. It was found that AgNPs-LA and AgNPs-PEG demonstrated lower cytotoxicity as compared with the AgNPs-TA or AgNPs-UC in the gingival fibroblast model. All of the tested nanoparticles proved less toxic and demonstrated wider spectrum of antimicrobial activities than AgNO3 solution. Additionally, AgNPs-LA eradicated Staphylococcus epidermidis and Streptococcus mutans 1-day biofilm at concentration nontoxic to oral cells. Conclusions: Our results proved that a capping agent had significant influence on the antibacterial, antibiofilm activity and cytotoxicity of AgNPs. Clinical significance: This study highlighted potential usefulness of AgNPs against oral anaerobic Gram-positive and Gram-negative bacterial infections and aerobic Staphylococci strains provided that pharmacological activity and risk assessment are carefully performed.

A Proposed Molecular Mechanism of High-Dose Vitamin D3 Supplementation in Prevention and Treatment of Preeclampsia.

A randomized prospective clinical study performed on a group of 74 pregnant women (43 presenting with severe preeclampsia) proved that urinary levels of 15-F(2t)-isoprostane were significantly higher in preeclamptic patients relative to the control (3.05 vs. 2.00 ng/mg creatinine). Surprisingly enough, plasma levels of 25-hydroxyvitamin D3 in both study groups were below the clinical reference range with no significant difference between the groups. In vitro study performed on isolated placental mitochondria and placental cell line showed that suicidal self-oxidation of cytochrome P450scc may lead to structural disintegration of heme, potentially contributing to enhancement of oxidative stress phenomena in the course of preeclampsia. As placental cytochrome P450scc pleiotropic activity is implicated in the metabolism of free radical mediated arachidonic acid derivatives as well as multiple Vitamin D3 hydroxylations and progesterone synthesis, we propose that Vitamin D3 might act as a competitive inhibitor of placental cytochrome P450scc preventing the production of lipid peroxides or excess progesterone synthesis, both of which may contribute to the etiopathogenesis of preeclampsia. The proposed molecular mechanism is in accord with the preliminary clinical observations on the surprisingly high efficacy of high-dose Vitamin D3 supplementation in prevention and treatment of preeclampsia.

Chasing Graphene-Based Anticancer Drugs: Where are We Now on the Biomedical Graphene Roadmap?

Graphene and graphene-based materials have attracted growing interest for potential applications in medicine because of their good biocompatibility, cargo capability and possible surface functionalizations. In parallel, prototypic graphene-based devices have been developed to diagnose, imaging and track tumor growth in cancer patients. There is a growing number of reports on the use of graphene and its functionalized derivatives in the design of innovative drugs delivery systems, photothermal and photodynamic cancer therapy, and as a platform to combine multiple therapies. The aim of this review is to introduce the latest scientific achievements in the field of innovative composite graphene materials as potentially applied in cancer therapy. The "Technology and Innovation Roadmap" published in the Graphene Flagship indicates, that the first anti-cancer drugs using graphene and graphene-derived materials will have appeared on the market by 2030. However, it is necessary to broaden understanding of graphene-based material interactions with cellular metabolism and signaling at the functional level, as well as toxicity. The main aspects of further research should elucidate how treatment methods (e.g., photothermal therapy, photodynamic therapy, combination therapy) and the physicochemical properties of graphene materials influence their ability to modulate autophagy and kill cancer cells. Interestingly, recent scientific reports also prove that graphene nanocomposites modulate cancer cell death by inducing precise autophagy dysfunctions caused by lysosome damage. It turns out as well that developing photothermal oncological treatments, it should be taken into account that near-infrared-II radiation (1000-1500 nm) is a better option than NIR-I (750-1000 nm) because it can penetrate deeper into tissues due to less scattering at longer wavelengths radiation.

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.

Diallyl trisulfide is more cytotoxic to prostate cancer cells PC-3 than to noncancerous epithelial cell line PNT1A: a possible role of p66Shc signaling axis.

Diallyl trisulfide (DATS) is an organosulfur compound isolated from garlic, and has been shown to have anticancer activity both in vitro and in vivo. The aim of this study was to compare cytotoxic effects of DATS on prostate cancer cells PC-3 and noncancerous human prostate epithelial cells PNT1A. PC-3 prostate cancer and noncancerous human prostate epithelial cells PNT1A were used in the study. We observed that PNT1A cells had higher resistance to DATS-induced cell death than PC-3 cells. Investigating signaling pathways involved in the cell death we observed that p66Shc phosphorylation at serine 36 and extracellular signal-regulated kinase 1/2 activation induced by DATS, were significantly attenuated in PNT1A cells as compared to PC-3 cells. Moreover, DATS-induced Akt inactivation was also significantly reduced in PNT1A cells. In addition to that, DATS-induced reactive oxygen species generation was nearly completely abolished in PNT1A cells. Interestingly, DATS induced only slight decrease in the level of ferritin H, whereas ferritin L was elevated. These data suggest that cytotoxicity of DATS toward PNT1A cells is strongly reduced as opposed to PC-3 cancer cells, which corresponds to the lower activation of prodeath signaling pathway mediated by the adaptor protein p66Shc in the noncancerous PNT1A cells.

[Isoprostanes - important marker of the oxidative stress estimation in patients with chronic kidney disease]. / Izoprostany - wazny wskaznik stresu oksydacyjnego u chorych z objawami przewleklej choroby nerek.

UNLABELLED: The discovery of isoprostanes, which are products of non-enzymatic lipid peroxidation, resulted in the research on the new role of free radicals in physiology and pathophysiology. Isoprostane quantitative analysis is a great achievement in the evaluation of free radical impact on many diseases in human. Isoprostanes were also found to be elevated in end-stage renal disease, another condition associated with increased oxidative stress. The aim of the study was to evaluate the influence of nephroprotective treatments on the urinary excretion of 15-F2alpha-isoprostane in the treated patients with Chronic Kidney Disease (CKD). MATERIAL AND METHODS: 84 patients (32 females and 52 males); age 18-65 years (average 39.06 +/- 4.92), with chronic non-diabetic proteinuric nephropathy, normal or slightly impaired stable renal function expressed as estimated creatinine cleamace above 30 mi/min, were selected from the cohort that attended our renal outpatients department. Clinical evaluation and laboratory tests were performed at the randomization point and after each period of the study A commercial ELISA Kit (Cayman Chemical Co) was used to measure the urinary excretion of 15-F2alpha-isoprostane in the treated patients. RESULTS: It was found that the blockade of renin-angiotensin-aldosteron system (with aliskiren, cilazapril, perindopril, spironolakton) and the treatment with atorwastatin significantly reduced urinary levels of 15-F2alpha-isoprostane relatively to the control group. It was not observed for pentoxyfilline treatment. CONCLUSIONS: Urine levels of isoprostanes are significantly decreased in patients with Chronic Kidney Disease during nephroprotective treatments.

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.

Protein tyrosine phosphatase CD45 as a molecular biosensor of hydrogen peroxide generation in cell culture media.

We have designed a useful method of assessing reactive oxygen species generation in biological fluids. The novel assay utilizes tyrosine phosphatase CD45 as a biosensor of oxidative stress. Applying this new method, we examined oxygen species generation in the following cell culture media: RPMI 1640, DMEM, DMEM enriched with pyruvate and MEM. We discovered that the media (especially RPMI 1640) significantly reduced the activity of protein tyrosine phosphatase. The media-caused inactivation of CD45 was reversible after treatment with dithiothreitol being a powerful reducing agent. Interestingly, the media supplemented with catalase did not exhibit any inhibitory effect on CD45 activity which suggests a hydrogen peroxide-mediated mechanism of the enzyme inactivation. In addition to that, we assessed the impact of oxidative stress level on the activity of CD45 as measured in Jurkat cells cultured in RPMI 1640 either exposed or not exposed to the light of laminar flow cabinet fluorescent lamp. We found that Jurkat cells that were exposed to light displayed ca. 20% lower activity of CD45 than the cells protected against the light. The obtained results indicate that production of hydrogen peroxide in the medium leading to inhibition of CD45 was light-dependent, and that careful protection of cell culture media from the light may help to prevent the artifact in cell studies. Hydrogen peroxide, responsible for CD45 inactivation, can be generated in cell culture media after exposition to light due to photoreactive amino acids present in the media.

A novel biosensor for evaluation of apoptotic or necrotic effects of nitrogen dioxide during acute pancreatitis in rat.

The direct and accurate estimation of nitric dioxide levels is an extremely laborious and technically demanding procedure in the molecular diagnostics of inflammatory processes. The aim of this work is to demonstrate that a stop-flow technique utilizing a specific spectroscopic biosensor can be used for detection of nanomolar quantities of NO(2) in biological milieu. The use of novel compound cis-[Cr(C(2)O(4))(AaraNH(2))(OH(2))(2)](+) increases NO(2) estimation accuracy by slowing down the rate of NO(2) uptake. In this study, an animal model of pancreatitis, where nitrosative stress is induced by either 3g/kg bw or 1.5 g/kg bw dose of L-arginine, was used. Biochemical parameters and morphological characteristics of acute pancreatitis were monitored, specifically assessing pancreatic acinar cell death mode, NO(2) generation and cellular glutathione level. The severity of the process correlated positively with NO(2) levels in pancreatic acinar cell cytosol samples, and negatively with cellular glutathione levels.

[Protein tyrosine phosphatases--endogenous markers of oxidative stress]. / Bialkowe fosfatazy tyrozynowe--endogenne markery stresu oksydacyjnego.

The reversible phosphorylation of structural and regulatory proteins in eucaryotic cells is one of the most important regulatory mechanisms. Protein tyrosine phosphatases (PTP) regulate a wide range of signal transduction pathways that control many cellular processes such as cell proliferation, differentiation and growth. Disorder in PTP gene expression is implicated in the development of cancer, autoimmune and neurodegenerative diseases. The active sites of these enzymes are characterized by the consensus sequence containing cysteine which is essential for enzyme activity and highly susceptible to oxidation. Reversible oxidation of the catalytic cysteine is becoming recognized as a general mechanism for regulation of PTP enzymatic activity. These findings suggest that protein tyrosine phosphatases may be considered as very sensitive markers of oxidative stress. Many studies have demonstrated that the production of reactive oxygen species during oxidative stress can inactivate protein tyrosine phosphatases.

Modification of DNA structure by reactive nitrogen species as a result of 2-methoxyestradiol-induced neuronal nitric oxide synthase uncoupling in metastatic osteosarcoma cells.

2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17ß-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death. The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME. The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide (•NO) with concurrent measurements of peroxynitrite (ONOO-) in real time in a single cell of 143B cell line by using •NO/ONOO- sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide (•NO2) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay. Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction - a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO- and •NO2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of •NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO- > â€¢NO2 > â€¢NO. Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy.

Dynamics of oxidative damage at early stages of estrogen-dependant carcinogenesis.

The objective of this study was to assess the dynamics of oxidative damage to cellular macromolecules such as proteins, lipids, and DNA under conditions of oxidative stress triggering early stages of estrogen-dependent carcinogenesis. A rodent model of carcinogenesis was used. Syrian hamsters were sacrificed after 1, 3, 5 h and 1 month from the initial implantation of 17beta-estradiol (E2). Matching control groups were used. Kidneys as target organs for E2-mediated oxidative stress were excised and homogenized for biochemical assays. Subcellular fractions were isolated. Carbonyl groups (as a marker of protein oxidation) and lipid hydroxyperoxides were assessed. DNA was isolated and 8-oxodGuo was assessed. Electron paramagnetic resonance spectroscopy was used to confirm the results for lipid peroxidation. Exposition to E2 in rodent model leads to a damage of macromolecules of the cell, including proteins and DNA, but not lipids. Proteins appear to be primary target of the damage but are shortly followed by DNA. It has previously been speculated that protein peroxides can increase DNA modifications. This time sequence was observed in our study. Nevertheless, direct relation between protein and DNA damage still remains unsolved.

hmSOD1 gene mutation-induced disturbance in iron metabolism is mediated by impairment of Akt signalling pathway.

BACKGROUND: Recently, skeletal muscle atrophy, impairment of iron metabolism, and insulin signalling have been reported in rats suffering from amyotrophic lateral sclerosis (ALS). However, the interrelationship between these changes has not been studied. We hypothesize that an impaired Akt-FOXO3a signalling pathway triggers changes in the iron metabolism in the muscles of transgenic animals. METHODS: In the present study, we used transgenic rats bearing the G93A hmSOD1 gene and their non-transgenic littermates. The study was performed on the muscles taken from animals at three different stages of the disease: asymptomatic (ALS I), the onset of the disease (ALS II), and the terminal stage of the disease (ALS III). In order to study the molecular mechanism of changes in iron metabolism, we used SH-SY5Y and C2C12 cell lines stably transfected with pcDNA3.1, SOD1 WT and SOD1 G93A, or FOXO3a TM-ER. RESULTS: A significant decrease in P-Akt level and changes in iron metabolism were observed even in the group of ALS I animals. This was accompanied by an increase in the active form of FOXO3a, up-regulation of atrogin-1, and catalase. However, significant muscle atrophy was observed in ALS II animals. An increase in ferritin L and H was accompanied by a rise in PCBP1 and APP protein levels. In SH-SY5Y cells stably expressing SOD1 or SOD1 G93A, we observed elevated levels of ferritin L and H and non-haem iron. Interestingly, insulin treatment significantly down-regulated ferritin L and H proteins in the cell. Conversely, cells transfected with small interfering RNA against Akt 1, 2, 3, respectively, showed a significant increase in the ferritin and FOXO3a levels. In order to assess the role of FOXO3a in the ferritin expression, we constructed a line of SH-SY5Y cells that expressed a fusion protein made of FOXO3a fused at the C-terminus with the ligand-binding domain of the oestrogen receptor (TM-ER) being activated by 4-hydroxytamoxifen. Treatment of the cells with 4-hydroxytamoxifen significantly up-regulated ferritin L and H proteins level. CONCLUSIONS: Our data suggest that impairment of insulin signalling and iron metabolism in the skeletal muscle precedes muscle atrophy and is mediated by changes in Akt/FOXO3a signalling pathways.

Dynamics of estrogen-induced oxidative stress.

The objective of this study was to assess the dynamics of oxidative damage to cellular macromolecules such as proteins, lipids and DNA under conditions of oxidative stress triggering early stages of estrogen-dependent carcinogenesis. A rodent model of carcinogenesis was used. Syrian hamsters were sacrificed after 1, 3, 5 h and one month from the initial implantation of estradiol. Matching control groups were used. Kidneys as target organs for estradiol-mediated oxidative stress were excised and homogenized for biochemical assays. Subcellular fractions were isolated. Carbonyl groups (as a marker of protein oxidation) and lipid hydroxyperoxides were assessed. DNA was isolated and 8-oxodGuo was assessed. Electron paramagnetic resonance spectroscopy was used to confirm the results for lipid peroxidation. Exposition to estradiol in the rodent model leads to damage of macromolecules of the cell, including proteins and DNA, but not lipids. Proteins appear to be the primary target of the damage but are closely followed by DNA. It has previously been speculated that protein peroxides can increase DNA modifications. This time sequence was observed in our study. Nevertheless, the direct relation between protein and DNA damage still remains unsolved.

Protein tyrosine phosphatases in pathological process.

Protein tyrosine phosphatases (PTPs) modulate the cellular level of tyrosine phosphorylation under normal and pathological conditions, and thus exert either stimulatory or inhibitory effect on signal transduction. Hence, PTPs are potential pharmacological targets for novel drugs being developed in order to treat numerous pathologies including cancer. For example, PTPs have been found to play a key role in pathogenesis of autoimmune disorders, allergic response, cardiovascular or neurodegenerative diseases, among others Alzheimer\'s disease. Moreover, since many PTPs fine-tune subtle regulation of microbial biochemistry controlling the viability and virulence, they can be candidates for new therapies of infection diseases. In this review, authors summarize the current knowledge on PTPs implication in etiopathogenesis of most common human diseases focusing on PTPs as potential therapeutical targets.

Dual effect of 2-methoxyestradiol on cell cycle events in human osteosarcoma 143B cells.

We have demonstrated for the first time that the steroid metabolite, 2-methoxyestradiol (2-ME) is a powerful growth inhibitor of human osteosarcoma 143 B cell line by pleiotropic mechanisms involving cell cycle arrest at two different points and apoptosis. The ability of 2-ME to inhibit cell cycle at the respective points has been found concentration dependent. 1 microM 2-ME inhibited cell cycle at G1 phase while 10 microM 2-ME caused G2/M cell cycle arrest. As a natural estrogen metabolite 2-ME is expected to perturb the stability of microtubules (MT) in vivo analogously to Taxol--the MT binding anticancer agent. Contrary to 2-ME, Taxol induced accumulation of osteosarcoma cells in G2/M phase of cell cycle only. The presented data strongly suggest two different mechanisms of cytotoxic action of 2-ME at the level of a single cell.