The role of redox environment in neurogenic development.

The dynamic changes of cellular redox elements during neurogenesis allow the control of specific programs for selective lineage progression. There are many redox couples that influence the cellular redox state. The shift from a reduced to an oxidized state and vice versa may act as a cellular switch mechanism of stem cell mode of action from proliferation to differentiation. The redox homeostasis ensures proper functioning of redox-sensitive signaling pathways through oxidation/reduction of critical cysteine residues on proteins involved in signal transduction. This review presents the current knowledge on the relation between changes in the cellular redox environment and stem cell programming in the course of commitment to a restricted neural lineage, focusing on in vivo neurogenesis and in vitro neuronal differentiation. The first two sections outline the main systems that control the intracellular redox environment and make it more oxidative or reductive. The last section provides the background on redox-sensitive signaling pathways that regulate neurogenesis.

Directed differentiation of embryonic P19 cells and neural stem cells into neural lineage on conducting PEDOT-PEG and ITO glass substrates.

Differentiation of pluripotent and lineage restricted stem cells such as neural stem cells (NSCs) was studied on conducting substrates of various nature without perturbation of the genome with exogenous genetic material or chemical stimuli. Primary mouse adult neural stem cells (NSCs) and P19 pluripotent embryonal (P19 EC) carcinoma cells were used. Expression levels of neuronal markers ß-III-tubulin and neurofilament were evaluated by immunochemistry and flow cytometry. It was shown that the ability of the substrate to induce differentiation directly correlated with its conductivity. Conducting substrates (conducting oxides or doped π-conjugated organic polymers) with different morphology, structure, and conductivity mechanisms all promoted differentiation of NSC and P19 cells into neuronal lineage to a similar degree without use of additional factors such as poly-L-ornithine coating or retinoic acid, as verified by their morphology and upregulation of the neuronal markers but not astrocyte marker GFAP. However, substrates with low conductance below ca. 10(-4) S cm(-2) did not show this ability. Morphology of differentiating cells was visualized by atomic force microscopy. NSCs cells increased ß-III-tubulin expression by 95% and P19 cells by over 30%. Our results suggest that the substrate conductivity is a key factor governing the cell fate. Differentiation of P19 cells into neuronal lineage on conducting substrates was attributed to downregualtion of Akt signaling pathway and increase in expression of dual oxidase 1 (DUOX 1).

Homocysteine and age-associated disorders.

There are numerous theories of aging, a process which still seems inevitable. Aging leads to cancer and multi-systemic disorders as well as chronic diseases. Decline in age- associated cellular functions leads to neurodegeneration and cognitive decline that affect the quality of life. Accumulation of damage, mutations, metabolic changes, failure in cellular energy production and clearance of altered proteins over the lifetime, and hyperhomocysteinemia, ultimately result in tissue degeneration. The decline in renal functions, nutritional deficiencies, deregulation of methionine cycle and deficiencies of homocysteine remethylation and transsulfuration cofactors cause elevation of homocysteine with advancing age. Abnormal accumulation of homocysteine is a risk factor of cardiovascular, neurodegenerative and chronic kidney disease. Moreover, approximately 50% of people, aged 65 years and older develop hypertension and are at a high risk of developing cardiovascular insufficiency and incurable neurodegenerative disorders. Increasing evidence suggests inverse relation between cognitive impairment, cerebrovascular and cardiovascular events and renal function. Oxidative stress, inactivation of nitric oxide synthase pathway and mitochondria dysfunction associated with impaired homocysteine metabolism lead to aging tissue degeneration. In this review, we examine impact of high homocysteine levels on changes observed with aging that contribute to development and progression of age associated diseases.

Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53.

Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.

Oxidative stress in rheumatoid arthritis leukocytes: suppression by rutin and other antioxidants and chelators.

The enhanced production of superoxide ion and peroxynitrite by bloodstream neutrophils and of superoxide ion by monocytes from rheumatoid arthritis (RA) patients was registered. It was suggested that NADPH oxidase together with NO synthase were the major sources of superoxide ion in RA neutrophils, while in RA monocytes superoxide ion was produced by NADPH oxidase and mitochondria. Among the different free radical inhibitors studied (antioxidant enzymes, SOD and catalase; free radical scavengers, bioflavonoid rutin and mannitol; and the iron chelator desferrioxamine), SOD and rutin were the most efficient suppressors of oxygen radical overproduction by RA neutrophils, while mannitol and desferrioxamine were inactive. Thus, in contrast to Fanconi anemia (FA) leukocytes (Korkina LG et al., J Leukocyte Biol 1992;52:357-62), iron-catalyzed hydroxyl radical formation was unimportant in RA leukocytes, which mainly produced superoxide ion. Natural non-toxic bioflavonoid rutin (vitamin P) inhibited oxygen radical overproduction in both RA and FA in an equally efficient manner and therefore may be considered as a useful supporting pharmaceutical agent for the treatment of "free radical" pathologies.

Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals.

The antioxidant and anti-inflammatory activities of two transition metal complexes of bioflavonoid rutin, Fe(rut)Cl(3) and Cu(rut)Cl(2), were studied. It was found that Cu(rut)Cl(2) was a highly efficient in vitro and ex vivo free radical scavenger that sharply decreased (by 2-30 times compared to the parent rutin): oxygen radical production by xanthine oxidase, rat liver microsomes, and rat peritoneal macrophages; the formation of thiobarbituric acid-reactive products in microsomal lipid peroxidation; and the generation of oxygen radicals by broncho-alveolar cells from bleomycin-treated rats. The copper-rutin complex was also a superior inhibitor of inflammatory and fibrotic processes (characterized by such parameters as macrophage/neutrophil ratio, wet lung weight, total protein content, and hydroxyproline concentration) in the bleomycin-treated rats. The antioxidant activity of Fe(rut)Cl(3) was much lower and in some cases approached that of rutin. Fe(rut)Cl(3) also stimulated to some degree spontaneous oxygen radical production by macrophages. We suggested that the superior antioxidant and anti-inflammatory activity of the copper-rutin complex is a consequence of its acquiring the additional superoxide-dismuting copper center. The inhibitory activity of Fe(rut)Cl(3) was lower, probably due to the partial reduction into Fe(rut)Cl(2) in the presence of biological reductants; however, similarly to the copper-rutin complex, this complex efficiently suppressed lung edema.

Redox environment and its meaning for breast cancer cells fate.

This review attempts to provide a broad overview of the changes in the cellular redox environment in breast cancer cells. The regulatory power of the redox environment lies in its capacity to control the growth behavior, spread, and differentiation. Neoplastic cells adapt to a wide variety of environmental conditions, including persistent oxidative stress and genomic instability by shifting their redox environment to more reductive conditions, which in its turn triggers upregulation of various redox sensitive prosurvival pathways. This review also examines the interactions between prosurvival signaling pathways, metallothioneins, and hydrogen peroxide generating dual oxidase, and presents a hypothesis to explain their relevance for therapeutic response.

Interplay between Numb and Notch in epithelial cancers: role for dual oxidase maturation factor.

Numb and Notch signalling pathways are vitally important in cell fate and differentiation. The outcome of these signalling processes is determined by a delicate balance between opposing effects of Notch and Numb. Imbalance in Numb/Notch regulation was implicated in aberrant differentiation programme and epithelial cancer progression and metastasis. Recent identification of Numb-interacting protein (NIP), which is also known as dual oxidase maturation factor, and was shown to associate with Numb and DUOX and promote their translocation, sheds a new light on how Numb/Notch network may be coordinated in epithelial cancers. Here, a possible link between Numb, Notch and Dual oxidase maturation factor is examined.

Role of p53 and reactive oxygen species in apoptotic response to copper and zinc in epithelial breast cancer cells.

Previous studies revealed that cells may differ in their response to metal stress depending on their p53 status; however, the sequence of events leading to copper-induced apoptosis is still unclear. Exposure of copper (10 and 25 microM) and zinc (10 and 25 microM) caused activation of p53 in ER+/p53+ human epithelial breast cancer MCF7 cells and resulted in up-regulation of p21. Transactivation of p53 in MCF7 cells also led to increase in expression of Bax, proapototic Bcl-2 family member, triggering mitochondrial pore opening, and PIG3 (p53-induced gene 3 product), and also generation of intracellular reactive oxygen species (ROS). The treatment of MCF7 cells with either copper or zinc for 4 h also caused decrease in mitochondrial membrane potential (Delta psi(m)), accompanied by an elevation in the ROS production and redistribution of p53 into mitochondria. The loss of Delta psi(m) was correlated with accumulation of Annexin V positive apoptotic cells. However, the release of apoptosis inducing factor (AIF) and its translocation into nucleus was observed only in MCF7 cells treated with copper. In MDA-MB-231 (ER-/p53-) and MCF7-E6 (ER+/p53-) cells, both p53 and p21 protein levels were not altered in the presence of metals. These cells were resistant to metals, and there was no alteration in Delta psi(m). Copper treatment did not result in accumulation of ROS in these cell lines with an inactive p53 even after exposure to 50 microM of copper for 6 h, indicating a key role for p53 in the ROS generation. Pretreatment of MCF7 cells with p53 inhibitor, pifithrin-alpha, resulted in decrease of copper and zinc induced ROS production to the control level, suppression of both Bax expression and AIF release. Therefore, the activation of p53 seems to play a crucial role in copper and zinc induced generation of ROS in epithelial breast cancer cells, and expression of downstream targets of p53, such as PIG3 and Bax, responsible for increased generation of the intracellular ROS, as well as disruption of mitochondrial integrity. Our data suggest that copper induces apoptosis in MCF-7 cells with no caspases through the depolarization of mitochondrial membrane with release of AIF and its translocation into the nucleus. The results demonstrate that a functional p53 is required for the execution of apoptosis in epithelial cells.

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc.

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.

Direct enzymatic reduction of lucigenin decreases lucigenin-amplified chemiluminescence produced by superoxide ion.

The interaction of superoxide ion with lucigenin produces chemiluminescence (CL), which is widely used for the detection of this radical anion. However, in many biological systems lucigenin may be directly reduced to its semiquinone by some enzymes. We found that if the direct reduction of lucigenin takes place, it decreases superoxide production due to the competition with one-electron reduction of dioxygen to superoxide ion. Comparison of two methods of superoxide detection (lucigenin-amplified CL and cytochrome c reduction) showed that there are excellent correlations between the results obtained by the two methods. Hence, lucigenin-amplified CL remains a sensitive and reliable assay of superoxide detection.