Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.     

Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (WI 38) cells. The radical scavenging activity of homogentisic acid was found to protect WI 38 cells against hydrogen peroxide (H₂O₂) induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects WI 38 cells against H₂O₂ damage by enhancing the intracellular antioxidative activity.     

Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells

The role of dihydroceramide desaturase as a key enzyme in the de novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water-soluble analogs of dihydroceramide, dihydroceramidoids (D-erythro-dhCCPS analogs), was used to measure desaturase activity in situ. Conversion of D-erythro-2-N-[12'-(1'-pyridinium)-dodecanoyl]-4,5-dihydrosphingosine bromide (C(12)-dhCCPS) to its 4,5-desaturated counterpart, D-erythro-2-N-[12'-(1'-pyridinium)dodecanoyl]sphingosine bromide (C(12)-CCPS), was determined by liquid chromatography/mass spectrometry analysis. The validity of the assay was confirmed using C(8)-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homolog (DEGS-1) of the Drosophila melanogaster des-1 gene was recently identified and reported to have desaturase activity. Transfection of SMS-KCNR cells with small interfering RNA to DEGS-1 significantly blocked the conversion of C(12)-dhCCPS to C(12)-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth, with cell cycle arrest at G(0)/G(1). This was accompanied by a significant decrease in the amount of phosphorylated retinoblastoma protein. This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose-dependent manner. An increase in dihydroceramides (but not ceramides) paralleled this process as measured by liquid chromatography/mass spectrometry. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of the retinoblastoma protein observed upon fenretinide treatment. These findings suggest a novel biological function for dihydroceramides.     

Variable overoxidation of peroxiredoxins in human lung cells in severe oxidative stress

Peroxiredoxins (Prxs) are a group of thiol containing proteins that participate both in signal transduction and in the breakdown of hydrogen peroxide (H(2)O(2)) during oxidative stress. Six distinct Prxs have been characterized in human cells (Prxs I-VI). Prxs I-IV form dimers held together by disulfide bonds, Prx V forms intramolecular bond, but the mechanism of Prx VI, so-called 1-Cys Prx, is still unclear. Here we describe the regulation of all six Prxs in cultured human lung A549 and BEAS-2B cells. The cells were exposed to variable concentrations of H(2)O(2), menadione, tumor necrosis factor-alpha or transforming growth factor-beta. To evoke glutathione depletion, the cells were furthermore treated with buthionine sulfoximine. Only high concentrations (300 microM) of H(2)O(2) caused a minor increase (<28%, 4 h) in the expression of Prxs I, IV, and VI. Severe oxidant stress (250-500 microM H(2)O(2)) caused a significant increase in the proportion of the monomeric forms of Prxs I-IV; this was reversible at lower H(2)O(2) concentrations (< or =250 microM). This recovery of Prx overoxidation differed among the various Prxs; Prx I was recovered within 24 h, but recovery required 48 h for Prx III. Overall, Prxs are not significantly modulated by mild oxidant stress or cytokines, but there is variable, though reversible, overoxidation in these proteins during severe oxidant exposure.     

Transfer RNA(4lys) and cell cycle progression: characterization of ts-694 cells

Lysine tRNA modification has been studied in mammalian ts-694 cells with respect to cell cycle progression in temperature downshift and upshift experiments. The modification of tRNA(lys) measured in temperature downshift experiments showed that tRNA(4lys) levels start to increase 6 h following the temperature shift, approximately 10-12 h prior to the cells entry into S phase. Ts-694 cells showed a gradual decrease in the level of tRNA(4lys) and the rates of DNA synthesis following a temperature upshift. The cells became growth arrested following incubation for 36-45 h at the rt. Cell cycle mapping of the temperature restriction point suggests a G1 block prior to the serum deprivation restriction point. Depletion of cellular tRNA(4lys) by serum deprivation followed by simultaneously shifting cells to the rt and feeding medium containing 10% serum showed that cells with low tRNA(4lys) levels and no mechanism for the synthesis of tRNA(4lys) could not enter S phase and synthesize DNA. Blocking of ts-694 at the G1/S boundary with aphidicolin indicates that cells that have passed through G1 are capable of entering S phase and synthesizing DNA independent of the incubation temperature. These results indicate that tRNA(4lys) is not needed during S phase for DNA replication but suggests that tRNA(4lys) is required for cells to progress through G1.     

Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells. Strong evidence indicates that NPM is required for embryonic development and genomic stability. Here we report that NPM enhances the proliferative potential of hematopoietic stem cells (HSCs) and increases their survival upon stress challenge. Both short term liquid culture and clonogenic progenitor cell assays show a selective expansion of NPM-overexpressing HSCs. Interestingly, HSCs infected with NPM retrovirus show significantly reduced commitment to myeloid differentiation compared with vector-transduced cells, and majority of the NPM-overexpressing cells remains primitive during a 5-day culture. Bone marrow transplantation experiments demonstrate that NPM promotes the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation. NPM overexpression induces rapid entry of HSCs into the cell cycle and suppresses the expression of several negative cell cycle regulators that are associated with G(1)-to-S transition. NPM knockdown elevates expression of these negative regulators and exacerbates stress-induced cell cycle arrest. Finally, overexpression of NPM promotes the survival and recovery of HSCs and progenitors after exposure to DNA damage, oxidative stress, and hematopoietic injury both in vivo and in vitro. DNA repair kinetics study suggests that NPM has a role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC/progenitor cell cycle progression and stress response.     

Induction of cell cycle progression by adenovirus E1A gene 13S- and 12S-mRNA products in quiescent rat cells.

Rat 3Y1 cell lines that express either adenovirus type 12 E1A 13S mRNA or 12S mRNA in response to dexamethasone treatment were established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines were growth arrested, and the induction of cell cycle progression was analyzed by flow cytometry after switch on of the cDNA by the addition of dexamethasone. The results indicate that the 13S- or 12S-mRNA product alone has the ability to cause progression of the cell cycle at a similar rate. The simultaneous addition of epidermal growth factor accelerated the rate of cell cycle progression in the transition from the G0/G1 phase to the S phase.     

Apoptotic response and cell cycle transition in ataxia telangiectasia cells exposed to oxidative stress

The recently identified ATM gene plays a role in a signal transduction network activating multiple cellular functions in response to DNA damage. An attractive hypothesis is that the ATM protein is involved in a specialized antioxidant system responsible for detoxifying reactive oxygen intermediate and that the absence or dysfunction of this protein in AT cells would render them less capable of dealing with oxidative stress. In order to investigate the role of the ATM gene in cell cycle control and programmed cell death, Lymphoblastoid cell lines derived from four Ataxia-Telangiectasia (AT) patients and six controls have been analyzed. All cell lines were incubated with 2-deoxy-D-ribose (dRib), a reducing sugar that induces apoptosis through oxidative stress. The result showed an impaired response to dRib-induced apoptosis in AT cells, as well as a defect of cellular cycle arrest in G1/S phase and a normal expression of p53 protein. This indicate that the kinase activity of ATM gene product plays a very important role in the cellular response to oxidative stress. In conclusion the altered response of AT cells to oxidative stress and particularly their resistance to apoptotic cell death, could explain the high predisposition of these cells to progress toward malignant transformation.     

Silica induces cell cycle changes through PI-3K/AP-1 pathway in human embryo lung fibroblast cells

Exposure to silica is associated with progressive pulmonary inflammation and fibrosis. Our previous study had demonstrated silica exposure could cause cell cycle alternation and activator protein-1 (AP-1) activation. This study showed that silica exposure induced phosphorylation of p70S6 kinase (p70S6K) and Akt in human embryo lung fibroblasts (HELFs). These changes were blocked by overexpression of dominant-negative mutants of phosphatidylinositol-3 kinase (Δp85) or Akt (DN-Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70S6K inhibitor, could inhibit silica-induced cell cycle alteration, AP-1 activation, and phosphorylation of p70S6K, but had no effect on Akt phosphorylation. This suggested that phosphatidylinositol-3 kinase (PI-3K)/AP-1 pathway was likely responsible for cell cycle changes. Furthermore, we observed the effect of the pathway on cell cycle regulatory proteins. Our results indicated that inactivation of PI-3K, Akt, or p70S6K could inhibit silica-induced overexpression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) and decreased expression of E2F-4. Taken together, silica could induce cell cycle changes through PI-3K/ AP-1 pathway in HELFs.     

Pyocyanin induces oxidative stress in human endothelial cells and modulates the glutathione redox cycle

Pyocyanin is a redox active virulence factor produced by the human pathogen Pseudomonas aeruginosa. Treatment of endothelial cells with pyocyanin (1-50 microM) resulted in the dose-dependent formation of hydrogen peroxide that was detected in the extracellular medium. Total intracellular glutathione levels decreased in response to pyocyanin in a dose-dependent manner from a control value of 19.9 +/- 2.7 nmol/mg protein to 10.0 +/- 2.4 nmol/mg protein. Prior treatment of cells with catalase afforded complete protection against loss of glutathione. Total intracellular soluble thiols decreased from 95.0 +/- 6.2 nmol/mg protein to 78.6 +/- 2.3 nmol/mg protein at the highest test dose. Intracellular levels of NADPH increased up to 2.4-fold in response to pyocyanin exposure. It is concluded that pyocyanin exposes endothelial cells to oxidative stress by the generation of hydrogen peroxide, which subsequently depletes intracellular glutathione and increases intracellular levels of mixed disulfides.     

Delay of cell cycle progression after X-irradiation of synchronized populations of human cells (NHIK 3025) in culture.

The effect of X-irradiation on the cell cycle progression of synchronized populations of the human cell line NHIK 3025 has been studied in terms of the radiation-induced delay of DNA replication and cell division. Results were obtained by flow cytometric measurement of histograms of cellular DNA content and parallel use of conventional methods for cell cycle analysis, such as pulse labelling with [3H]thymidine and counting of cell numbers. The two sets of methods were generally in good agreement, but the advantages of employing two independent techniques are pointed out. Irradiation was found to have a minor influence on DNA replication. As compared with unirradiated populations, half-completed DNA replication was 20--30 min delayed in populations 580 rad in mid-G1 or 290 rad in early S. Cell cycle progression was markedly delayed in G2. The sensitivity induction of this delay was 0.6 min/rad for populations irradiated in mid-G1, and 1.4 min/rad for populations irradiated in early S.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

Lycopene prevents 7-ketocholesterol-induced oxidative stress,cell cycle arrest and apoptosis in human macrophages

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10–25 μM) alone and in combination with lycopene (0.5–2 μM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, β-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than β-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.     

Endoplasmic reticulum stress inhibits cell cycle progression via induction of p27 in melanoma cells

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a stress signaling pathway. The UPR coordinates the induction of ER chaperones with decreased protein synthesis and growth arrest in G1 phase of the cell cycle. However, the molecular mechanism underlying UPR-induced G1 cell cycle arrest remains largely unknown. Here we report that activation of the UPR response by tunicamycin (TM), an ER stress inducer, leads to accumulation of p27 and G1 cell cycle arrest in melanoma cells. This accumulation of p27 is due to the inhibition on its polyubiquitination and subsequent degradation upon TM treatment. Correlated with p27 stabilization, the levels of Skp2, an E3 ligase for p27, are decreased in response to TM treatment. More importantly, knockdown of p27 greatly reduces TM-induced G1 cell cycle arrest. Taken together, these data implicate p27 as a critical mediator of ER stress-induced growth arrest.     

Unique natural antioxidants (NAOs) and derived purified components inhibit cell cycle progression by downregulation of ppRb and E2F in human PC3 prostate cancer cells

Prostate cancer (PCA) is the leading cause of cancer mortality among older men in Western countries. Epidemiological studies have shown correlation between a lower risk of PCA and a higher consumption of antioxidants. However, the mechanism by which antioxidants exert their effects is still unknown. In the present study, we explored the signaling mechanism through which unique natural antioxidant derived from spinach extract (NAO) exerts their beneficial effects in the chemoprevention of PCA using human PC3 cells. Probing into the effect of NAO and its derived polyphenols on cell-cycle G1 arrest, we found that they cause cell-cycle prolongation. NAO and its two derived purified components exhibited a significant increase in the level of p21cip1 expression after 36 h of starvation, followed by 18 h of treatment with NAO in the presence of serum. In addition, under similar conditions, the expressed level of Cyclin A and CDK-2 in the PC3 cells was significantly reduced after treatment with NAO or its purified components. Immunoblot analysis demonstrated a significant increase in the hypophosphorylated form of pRb and a decrease in ppRb. NAO and its purified derived components were found to downregulate the protein expression of another member of the pRb family, p107, as well as that of E2F-1. These results suggest that NAO-induced G1 delay and cell cycle prolongation are caused by downregulation of the protein expression of ppRb and E2F in the human PCA cell line PC3.     

The NAD(P)H:Quinone Oxidoreductase 1 induces cell cycle progression and proliferation of melanoma cells

The oxidoreductase NQO1 plays a prominent role in maintaining the cellular homeostasis. NQO1 is mainly a cytosolic enzyme which catalyzes the metabolism of quinones and is present in almost all tissue types providing protection against different stresses including xenobiotics, oxidants, UV light, and ionizing radiation. This enzyme is overexpressed in many cancerous tissues and its function in carcinogenesis remains unclear. Due to the relative lack of information on the role of NQO1 in melanoma pathogenesis, we attempted to determine the expression and basic function of NQO1 in melanoma cell proliferation. We found that NQO1 is overexpressed in most melanoma cell lines with respect to melanocytes. Furthermore, the expression of this oxidoreductase significantly induces cell cycle progression by upregulating the expression of cyclins A2, B1 and D1, leading to the proliferation of melanoma cells. Our results also indicate that NQO1 is an upstream regulator of NF-κB p50, a factor linked to melanoma progression and poor patient prognosis. Interestingly, we found that NQO1 stabilizes the transactivator BCL3, which in turn upregulates NF-κB p50. More importantly, our results also indicate that NF-κB p50 correlates with the expression of NQO1 and mediates its role in the proliferation of melanoma cells.     

Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells)

Traffic is a major source of particulate matter (PM), and ultrafine particulates and traffic intensity probably contribute significantly to PM-related health effects. As a strong relationship between air pollution and motor vehicle-originated pollutants has been shown to exist, air pollution genotoxicity studies of urban cities are steadily increasing. In Korea, the death rate caused by lung cancer is the most rapidly increased cancer death rate in the past 10 years. In this study, genotoxicity of PM2.5 (<2.5μm in aerodynamic diameter particles) collected from the traffic area in Suwon City, Korea, was studied using cultured human lung bronchial epithelial cells (BEAS-2B) as a model system for the potential inhalation health effects. Organic extract of PM2.5 (CE) generated significant DNA breakage and micronucleus formation in a dose-dependent manner (1μg/cm(3)-50μg/cm(3)). In the acid-base-neutral fractionation of PM2.5, neutral samples including the aliphatic (F3), aromatic (F4) and slightly polar (F5) fractions generated significant DNA breakage and micronucleus formation. These genotoxic effects were significantly blocked by scavenging agents [superoxide dismutase (SOD), sodium selenite (SS), mannitol (M), catalase (CAT)]. In addition, in the modified Comet assay using endonucleases (FPG and ENDOIII), CE and its fractions (F3, F4, and F5) increased DNA breakage compared with control groups, indicating that CE and fractions of PM2.5 induced oxidative DNA damage. These results clearly suggest that PM2.5 collected in the Suwon traffic area has genotoxic effects and that reactive oxygen species may play a distinct role in these effects. In addition, aliphatic/chlorinated hydrocarbons, PAH/alkylderivatives, and nitro-PAH/ketones/quinones may be important causative agents of the genotoxic effects.