Young Myeong Kim

CD44-Epidermal Growth Factor Receptor Interaction Mediates Hyaluronic Acid-promoted Cell Motility by Activating Protein Kinase C Signaling Involving Akt, Rac1, Phox, Reactive Oxygen Species, Focal Adhesion Kinase, and MMP-2

Hyaluronic acid (HA) is known to play an important role in motility of tumor cells. However, the molecular mechanisms associated with HA-promoted melanoma cell motility are not fully understood. Treatment of cells with HA was shown to increase the production of reactive oxygen species (ROS) in a CD44-dependent manner. Antioxidants, such as N-acetyl-l-cysteine and seleno-l-methionine, prevented HA from enhancing cell motility. Protein kinase C (PKC)-α and PKCδ were responsible for increased Rac1 activity, production of ROS, and mediated HA-promoted cell motility. HA increased Rac1 activity via CD44, PKCα, and PKCδ. Transfection with dominant negative and constitutive active Rac1 mutants demonstrated that Rac1 was responsible for the increased production of ROS and cell motility by HA. Inhibition of NADPH oxidase by diphenylene iodonium and down-regulation of p47Phox and p67Phox decreased the ROS level, suggesting that NADPH oxidase is the main source of ROS production. Rac1 increased phosphorylation of FAK. FAK functions downstream of and is necessary for HA-promoted cell motility. Secretion and expression of MMP-2 were increased by treatment with HA via the action of PKCα, PKCδ, and Rac1 and the production of ROS and FAK. Ilomastat, an inhibitor of MMP-2, exerted a negative effect on HA-promoted cell motility. HA increased interaction between CD44 and epidermal growth factor receptor (EGFR). AG1478, an inhibitor of EGFR, decreased phosphorylation of PKCα, PKCδ, and Rac1 activity and suppressed induction of p47Phox and p67Phox. These results suggest that CD44-EGFR interaction is necessary for HA-promoted cell motility by regulating PKC signaling. EGFR-Akt interaction promoted by HA was responsible for the increased production of ROS and HA-promoted cell motility. In summary, HA promotes CD44-EGFR interaction, which in turn activates PKC signaling, involving Akt, Rac1, Phox, and the production of ROS, FAK, and MMP-2, to enhance melanoma cell motility.

Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-gamma-induced macrophage NO synthesis by PGE2.

Macrophage nitric oxide (NO) synthesis is an integral component of the host defense system. We have previously found that NO and prostaglandins interact in a variety of ways. NO modulates Kupffer cell prostaglandin E2 (PGE2) production and we have recently described the inhibitory effects of PGE2 on NO synthesis in both Kupffer cells and hepatocytes. Activated macrophages produce a number of prostaglandins but studies regarding the capacity of prostaglandins to regulate macrophage NO synthesis have yielded conflicting results. We found that exogenous PGE2 decreased lipopolysaccharide (LPS)‐induced NO synthesis in murine resident peritoneal macrophages and in the RAW 264.7 murine macrophage cell line. PGE2 also suppressed NO synthesis in response to interferon‐γ (IFN‐γ) alone and a combination of LPS + IFN‐γ. Inhibition of endogenous PGE2 synthesis with indomethacin or ibuprofen had no effect on NO synthesis. PGE2 added with the activating stimulus was most effective. PGE2 lost the capacity to block NO synthesis if added more than 180 min after LPS. PGE2 decreased inducible NO synthase (iNOS) mRNA and immunoreactive iNOS protein, consistent with the hypothesis that exogenous PGE2 inhibits macrophage iNOS expression but that the inhibition depends on the time and concentration of prostaglandin exposure. J. Leukoc. Biol. 61: 712–720; 1997.

Nitric Oxide Induces Thymocyte Apoptosis Via a Caspase-1-Dependent Mechanism

We previously showed that NO induces apoptosis in thymocytes via a p53-dependent pathway. In the present study, we investigated the role of caspases in this process. The pan-caspase inhibitor, ZVAD-fmk, and the caspase-1 inhibitor, Ac-YVAD-cho, both inhibited NO-induced thymocyte apoptosis in a dose-dependent manner, whereas the caspase-3 inhibitor, Ac-DEVD-cho, had little effect even at concentrations up to 500 μM. ZVAD-fmk and Ac-YVAD-cho were able to inhibit apoptosis when added up to 12 h, but not 16 h, after treatment with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Caspase-1 activity was up-regulated at 4 h and 8 h and returned to baseline by 24 h; caspase-3 activity was not detected. Cytosolic fractions from SNAP-treated thymocytes cleaved the inhibitor of caspase-activated deoxyribonuclease. Such cleavage was completely blocked by Ac-YVAD-cho, but not by Ac-DEVD-cho or DEVD-fmk. Poly(ADP-ribose) polymerase (PARP) was also cleaved in thymocytes 8 h and 12 h after SNAP treatment; addition of Ac-YVAD-cho to the cultures blocked PARP cleavage. Furthermore, SNAP induced apoptosis in 44% of thymocytes from wild-type mice; thymocytes from caspase-1 knockout mice were more resistant to NO-induced apoptosis. These data suggest that NO induces apoptosis in thymocytes via a caspase-1-dependent but not caspase-3-dependent pathway. Caspase-1 alone can cleave inhibitor of caspase-activated deoxyribonuclease and lead to DNA fragmentation, thus providing a novel pathway for NO-induced thymocyte apoptosis.

Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism.

Previously, we showed that NO induces thymocyte apoptosis via acaspase‐1‐dependent mechanism [ 1 ]. In the present study,we investigated the role of heme oxygenase, catalase, bax, and p53 inthis process. The NO donor, S‐nitroso‐N‐acetyl penicillamine (SNAP),induced DNA fragmentation in thymocytes in a time‐ andconcentration‐dependent way. SNAP (100 μM) induced 50–60%apoptosis; higher doses did not increase the rate of apoptosissignificantly. SNAP decreased catalase and heme iron (Fe) levelswithout affecting superoxide dismutase, glutathione, or total Fe storesin thymocytes. SNAP significantly increased the expression of hemeoxygenase 1 (HSP‐32), p53, and bax but notbcl‐2. Treatment with the heme oxygenase inhibitor, tinprotoporphyrin IX inhibited SNAP‐induced thymocyte apoptosis.Furthermore, thymocytes from p53 null mice were resistantto NO‐induced apoptosis. Our data suggest that NO may induce itscytotoxic effects on thymocytes by modulating heme oxygenase andcatalase activity as well as up‐regulating pro‐apoptotic proteinsp53 and bax.

Nitric oxide metabolism in canine sepsis: relation to regional blood flow

PURPOSE To investigate the role of nitric oxide (NO) in early endotoxemia on the systemic and regional blood flow by measuring the plasma nitrite/nitrate (NOx) and blood nitrosyl-hemoglobin (NO-Hb) levels. MATERIALS AND METHODS This was a prospective, controlled, experimental study conducted in an animal research laboratory on 15 male mongrel dogs. Escherichia coli endotoxin (1 mg/kg) was injected intravenously. RESULTS Hepatic, renal, and iliac blood flow and cardiac output (CO) were measured before and 15, 30, 45, 90 and 180 minutes after injection of Escherichia coli endotoxin (1 mg/kg) (n = 6). NOx efflux from the organs was calculated by measuring plasma NOx levels. The arterial blood levels of NO-Hb were also measured (n = 4). As control studies, blood samples from dogs (n = 5) without exposure to endotoxin were assayed at 180 minutes for NOx and NO-Hb. Following endotoxin injection, mean arterial pressure decreased and reached its lowest value at 90 minutes (baseline vs. 90 minutes: 119.1+/-5.8 vs. 82.5+/-16.7 mm Hg, P<.0001). Hepatic artery blood flow increased significantly (baseline vs. 180 minutes: 23.6+/-12.0 vs. 170.0+/-68.4 mL/ min, P<.0001). There were no significant changes in plasma levels of NOx, uptake or release of NOx across the measured vascular beds, NO-Hb levels at any time point. In the portal system, the portal vein flow correlated with NOx release (R = 0.69, P<.0001). CONCLUSION In the early phase of endotoxemia in the dog, the significant reduction in systemic vascular resistance and hepatic arterial resistance are not associated with any measurable NOx release in the systemic circulation or the liver.

The pentapeptide Gly-Thr-Gly-Lys-Thr confers sensitivity to anti-cancer drugs by inhibition of CAGE binding to GSK3β and decreasing the expression of cyclinD1

We previously reported the role of cancer/testis antigen CAGE in the response to anti-cancer drugs. CAGE increased the expression of cyclinD1, and pGSK3βSer9, an inactive GSK3β, while decreasing the expression of phospho-cyclinD1Thr286. CAGE showed binding to GSK3β and the domain of CAGE (amino acids 231–300) necessary for binding to GSK3β and for the expression regulation of cyclinD1 was determined. 269GTGKT273 peptide, corresponding to the DEAD box helicase domain of CAGE, decreased the expression of cyclinD1 and pGSK3βSer9 while increasing the expression of phospho-cyclinD1Thr286. GTGKT peptide showed the binding to CAGE and prevented CAGE from binding to GSK3β. GTGKT peptide changed the localization of CAGE and inhibited the binding of CAGE to the promoter sequences of cyclin D1. GTGKT peptide enhanced the apoptotic effects of anti-cancer drugs and decreased the migration, invasion, angiogenic, tumorigenic and metastatic potential of anti-cancer drug-resistant cancer cells. We found that Lys272 of GTGKT peptide was necessary for conferring anti-cancer activity. Peptides corresponding to the DEAD box helicase domain of CAGE, such as AQTGTGKT, QTGTGKT and TGTGKT, also showed anti-cancer activity by preventing CAGE from binding to GSK3β. GTGKT peptide showed ex vivo tumor homing potential. Thus, peptides corresponding to the DEAD box helicase domain of CAGE can be developed as anti-cancer drugs in cancer patients expressing CAGE.