An-Sik Chung

Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production

The Akt/protein kinase B (PKB) serine/ threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoino‐sitide 3‐kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/ PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase‐9 (MMP‐9) production, in a manner highly dependent on its kinase activity and membrane‐translocating ability. The increase in MMP‐9 production was mediated by activation of nuclear factor‐κB transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell‐cell or cell‐matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.

Inhibitory Effect of Selenite on Invasion of HT1080 Tumor Cells

Selenium, an essential biological trace element, has been shown to reduce and prevent the incidence of cancer. Our previous studies have shown that selenite is involved in the chemoprevention of cancer and induction of apoptosis of cancer cells. In this study, we demonstrate that selenite also inhibits the invasion of tumor cells. Cancer cell invasion requires coordinated processes, such as changes in cell-cell and cell-matrix adhesion, degradation of the extracellular matrix, and cell migration. We found that selenite inhibited invasion of HT1080 human fibrosarcoma cells. Adhesion of HT1080 cells to the collagen matrix was also inhibited by treatment with selenite, but cell-cell interaction and cell motility were not affected by selenite. Moreover, selenite reduced expression of matrix metalloproteinase-2 and -9 and urokinase-type plasminogen activator, which are involved in matrix degradation, but increased a tissue inhibitor of metalloproteinase-1. This inhibitory effect of selenite on the protease expressions was mediated by the suppression of transcription factors, NF-κB and AP-1. However, selenate showed no remarkable effect on all the steps of cancer cell invasion.

Sustained Production of H2O2Activates Pro-matrix Metalloproteinase-2 through Receptor Tyrosine Kinases/Phosphatidylinositol 3-Kinase/NF-κB Pathway

A rate-limiting step of tumor cell metastasis is matrix degradation by active matrix metalloproteinases (MMPs). It is known that reactive oxygen species are involved in tumor metastasis. Sustained production of H2O2 by phenazine methosulfate (PMS) induced activation of pro-MMP-2 through the induction of membrane type 1-MMP (MT1-MMP) expression in HT1080 cells. MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 and -2 levels were changed negligibly by PMS. A one time treatment with H2O2 did not induce activation of MMPs. It was also demonstrated that superoxide anions and hydroxyl radicals were not related to PMS action. PMS-induced pro-MMP-2 activation was regulated by the receptor tyrosine kinases, especially the receptors of platelet-derived growth factor and vascular endothelial growth factor, and downstream on the phosphatidylinositol 3-kinase/NF-κB pathway but not Ras, cAMP-dependent protein kinase, protein kinase C, and mitogen-activated protein kinases. PMS did not induce pro-MMP-2 activation in T98G and NIH3T3 cells. This may be related to a low level of MT1-MMP, indicating a threshold level of MT1-MMP is important for pro-MMP-2 activation. Furthermore, PMS increased cell motility and invasion but decreased cell-cell interaction. Cell-matrix interaction was not affected by PMS.

Effects of reactive oxygen species on proliferation of Chinese hamster lung fibroblast (V79) cells

Reactive oxygen species (ROS) have emerged as important signaling molecules in the regulation of various cellular processes. In our study, we investigated the effect of a wide range of ROS on Chinese hamster lung fibroblast (V79) cell proliferation. Treatment with H2O2 (100 microM), superoxide anion (generated by 1 mM xanthine and 1 mU/ml xanthine oxidase), menadione, and phenazine methosulfate increased the cell proliferation by approximately 50%. Moreover, a similar result was observed after partial inhibition of superoxide dismutase (SOD) and glutathione peroxidase. This upregulation of cell proliferation was suppressed by pretreatment with hydroxyl radical scavengers and iron chelating agents. In addition to ROS, treatment with exogenous catalase and SOD mimic (MnTMPyP) suppressed the normal cell proliferation. Short-term exposure of the cells to 100 microM H2O2 was sufficient to induce proliferation, which indicated that activation of the signaling pathway is important as an early event. Accordingly, we assessed the ability of H2O2 to activate mitogen-activated protein kinases (MAPK). Jun-N-terminal kinase (JNK) and p38 MAPK were both rapidly and transiently activated by 100 microM H2O2, with maximal activation 30 min after treatment. However, the activity of extracellular signal-regulated kinase (ERK) was not changed. Pretreatment with SB203580 and SB202190, specific inhibitors of p38 MAPK, reduced the cell proliferation induced by H2O2. The activation of both JNK and p38 MAPK was also suppressed by pretreatment with hydroxyl radical scavenger and iron chelating agents. Our results suggest that the trace metal-driven Fenton reaction is a central mechanism that underlies cell proliferation and MAPK activation.

Selenite suppresses hydrogen peroxide-induced cell apoptosis through inhibition of ASK1/JNK and activation of PI3-K/Akt pathways.

The relationship between selenium and signal molecules has not been well elucidated. It was found that physiological concentration of selenite, >3 μM, reduced ASK1 activity and induced PI3‐kinase (PI3‐K)/Akt pathways in HT1080 cells. Duration of these signal molecules by selenite was much longer than that by growth factors and other stresses. The longer duration time of these signal molecules may be important to maintain normal functions against stresses. Selenite increased cell proliferation through up‐regulation of Bcl‐2 expression, mitochondrial membrane potential, adenosine triphosphate (ATP) generation, and glucose uptake mediated by PI3‐K pathway. High concentration of H2O2 increased an apoptotic signal molecule, ASK1, which resulted in Bcl‐2 down‐regulation, membrane potential disruption, decreased ATP and glucose uptake, and activation of caspases. However, an antiapoptotic signal molecule, Akt, was activated also by H2O2, but duration of its activation was much shorter. Selenite blocked apoptosis induced by H2O2, which was related to blocking ASK1 and further stimulating PI3‐kinase/Akt activities. Selenite blocked mitochondrial membrane potential disruption by 400 μM H2O 2. Selenite also blocked caspase‐9 and ‐3 activities and apoptosis induced by 500 μM H2O2, even after mitochondrial membrane potential disruption. These observations demonstrate that selenite increases cell proliferation and maintains cell survival by activating the antiapoptotic signal and blocking the apoptotic signal.

Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells.

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.

Rainfall, phycocyanin, and N:P ratios related to cyanobacterial blooms in a Korean large reservoir

Nutrient concentrations and other environmental factors were measured in the Daechung Reservoir for 25 weeks from spring until autumn in 1999. The high irradiance after heavy rainfall provided optimal meteorological conditions for bloom formation during summer, therefore, rain would also appear to forecast imminent bloom. The bloom formation was largely governed by cyanobacteria, in particular, Microcystis spp. and Anabaenaspp. Phycocyanin showed higher correlation with cyanobacteria (r = 0.744, P < 0.001) compared to chlorophyll-a(r = 0.599, P < 0.01). Therefore, phycocyanin was more accurate and useful than chlorophyll-a in quantitatively measuring cyanobacterial blooms. The atomic N:P ratio of the particulate form also showed a high correlation with cyanobacteria (r = 0.541, P < 0.01), increasing from 4.3 to 14.6 during bloom formation, while that of the dissolved form decreased from 25.5 to 8.7. These results indicated that the algae assimilated N significantly without comparable P uptake during the blooming season, which was in sharp contrast to the excessive storage of P during the spring.

Studies on the biosorption of heavy metals onto Chlorella vulgaris

Abstract The metal binding characteristics of Chlorella vulgaris were investigated. Metal uptake by intact algal cells was found to consist of two processes; (1) a fast, metabolism‐independent surface reaction and (2) metabolism‐dependent slow uptake. Results of both the pH and the ionic strength effects on the metal adsorption indicate that the specific interaction accounts for the majority of metal adsorption. Carboxyl and amine surface functional groups of Chlorella vulgaris were modified to assess the role of the surface charge site as the metal binding one. The carboxyl‐modified algae showed major decreases in the adsorption capacity of Cd(II) and Zn(II) binding. The amine‐modified algae also displayed some decreases in metal adsorptions.

Type D Retrovirus Gag Polyprotein Interacts with the Cytosolic Chaperonin TRiC

ABSTRACT The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1γ, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1γ also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.

Induction of apoptosis by selenite and selenodiglutathione in HL‐60 cells: Correlation with cytotoxicity

Effects of selenite and selenodiglutathione, an initial metabolite of selenite, on the induction of apoptosis and cytotoxicity were investigated in human promyelocytic leukemia HL‐60 cells. Treatment of selenite or selenodigtutathione resulted in concentration‐dependent cytotoxicity, measured by lactate dehydrogenase leakage assay, and by tetrazolium salt reduction assay. Selenodiglutathione has been shown to exert more cytotoxic effect than selenite in both assay systems. Time‐course study of cellular selenium uptake suggests that the higher cytotoxicity of selenodiglutathione be largely due to faster and greater selenium uptake rate. Treatment with selenite or selenodiglutathione also induced apoptosis in a dose‐dependent manner, as detected by enzyme‐linked immunosorbent assay and by DNA fragmentation assay. The dose‐response data of apoptosis induced by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating a relationship between the induction of apoptosis and cytotoxicity. Zn, which is a well‐known inhibitor of apoptosis, dose‐dependently blocked not only the induction of apoptosis, but also the membrane damage induced by selenium, corroborating this hypothesis. It was noted that the inhibition of apoptosis by Zn exerted little protective effect on cytotoxicity at higher concentrations of selenium, compared with a perfect protective effect at low concentration of selenium. These results suggest that cytotoxicity induced by selenium may be partially correlated with apoptosis.