Yun-Kyoung Ryu

Regulation of glycogen synthase kinase-3 by thymosin beta-4 is associated with gastric cancer cell migration.

Thymosin beta‐4 (Tβ4), actin‐sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK‐3 activity is regulated by Tβ4 and it is associated with Tβ4‐mediated migration in gastric cancer cells. Various expression level of Tβ4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. Although the level of phosphorylated(p)‐GSK‐3α (inactive), β‐catenin, E‐cadherin and E‐cadherin:β‐catenin complex was relatively higher, p‐GSK‐3β (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK‐3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK‐3α increased SNU638 cell migration in accordance with the reduction of E‐cadherin:β‐catenin complex formation through a decrease in β‐catenin and E‐cadherin. Expression level of GSK‐3α/β, β‐catenin and E‐cadherin in SNU668 and SNU638 was reversed by Tβ4‐siRNA and by the treatment with acetylated‐serine‐aspartic acid‐lysine‐proline (SDKP) tetrapeptide of Tβ4, respectively. E‐cadherin expression in SNU638 cells was decreased by β‐catenin‐siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p‐GSK‐3α, β‐catenin and E‐cadherin. Taken together, data indicated that the expression of GSK‐3α, β‐catenin and E‐cadherin could be negatively regulated by Tβ4‐induced ERK phosphorylation. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways.

Mouse Melanoma Cell Migration is Dependent on Production of Reactive Oxygen Species under Normoxia Condition.

Cell migration plays a role in many physiological and pathological processes. Reactive oxygen species (ROS) produced in mammalian cells influence intracellular signaling processes which in turn regulate various biological activities. Here, we investigated whether melanoma cell migration could be controlled by ROS production under normoxia condition. Cell migration was measured by wound healing assay after scratching confluent monolayer of B16F10 mouse melanoma cells. Cell migration was enhanced over 12 h after scratching cells. In addition, we found that ROS production was increased by scratching cells. ERK phosphorylation was also increased by scratching cells but it was decreased by the treatment with ROS scavengers, N-acetylcysteine (NAC). Tumor cell migration was inhibited by the treatment with PD98059, ERK inhibitor, NAC or DPI, well-known ROS scavengers. Tumor cell growth as judged by succinate dehydrogenase activity was inhibited by NAC treatment. When mice were intraperitoneally administered with NAC, the intracellular ROS production was reduced in peripheral blood mononuclear cells. In addition, B16F10 tumor growth was significantly inhibited by in vivo treatment with NAC. Collectively, these findings suggest that tumor cell migration and growth could be controlled by ROS production and its downstream signaling pathways, in vitro and in vivo.

Thymosin Beta-4, Actin-Sequestering Protein Regulates Vascular Endothelial Growth Factor Expression via Hypoxia-Inducible Nitric Oxide Production in HeLa Cervical Cancer Cells.

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 (Tβ4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tβ4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tβ4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(β-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tβ4 expression with Tβ4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with NG-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tβ4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tβ4 plasmids for Tβ4 overexpression. Taken together, these results suggest that Tβ4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.

γ‐Irradiated cancer cells promote tumor growth by activation of Toll‐like receptor 1‐mediated inducible nitric oxide synthase in macrophages

RT is commonly used to treat malignant tumors. However, tumor regrowth is a major limitation to RT as an antitumor treatment. In the present study, we investigated the tumor‐promoting effects of high‐dose (or ablative) RT treatments on tumor‐bearing mice. We focused on the role of macrophages that interact with IR‐CCs in the TME, which cause tumor regrowth. We observed that CT26(H‐2d) tumor growth was enhanced by i.v. injection of IR‐CT26 cells compared with NR control CT26 cells. The levels of iNOS gene expression and NO production from RAW264.7 macrophages (H‐2d) in response to the interaction with IR‐CT26 cells were higher than with NR‐CT26 cells. When CT26 tumor‐bearing mice were treated i.v. with L‐NMMA, a NOS inhibitor, the reduction in in vivo tumor growth was higher in the IR‐CT26‐injected group compared with the NR‐CT26‐injected control group. In vivo CT26 tumor growth was decreased after transplanting PEM extracted from L‐NMMA‐treated, tumor‐bearing mice. Although iNOS activity was reduced by inhibiting TLR1 expression with TLR1‐siRNA, it was enhanced by TLR1 overexpression. Transcriptional activation and protein expression levels of iNOS were also decreased in the presence of TLR1‐siRNA but increased as a result of TLR1 overexpression. These results demonstrate that postradiotherapeutic tumor regrowth may be caused by interaction of IR‐CCs with macrophages that induce TLR1‐mediated iNOS expression and NO production. Our data suggest that iNOS in macrophages could be a useful target to regulate postradiotherapeutic responses in hosts and subsequently limit tumor regrowth.

Cell motility is decreased in macrophages activated by cancer cell-conditioned medium.

Macrophages play a role in innate immune responses to various foreign antigens. Many products from primary tumors influence the activation and transmigration of macrophages. Here, we investigated a migration of macrophages stimulated with cancer cell culture-conditioned medium (CM). Macrophage activation by treatment with CM of B16F10 cells were judged by the increase in protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). The location where macrophages were at 4 h-incubation with control medium or CM was different from where they were at 5 h-incubation in culture dish. Percentage of superimposed macrophages at every 1 h interval was gradually increased by CM treatment as compared to control. Total coverage of migrated track expressed in coordinates was smaller and total distance of migration was shorter in CM-treated macrophages than that in control. Rac1 activity in CM-treated macrophages was also decreased as compared to that in control. When macrophages were treated with CM in the presence of dexamethasone (Dex), an increase in COX2 protein levels, and a decrease in Rac1 activity and total coverage of migration were reversed. In the meanwhile, biphasic changes were detected by Dex treatment in section distance of migration at each time interval, which was more decreased at early time and then increased at later time. Taken together, data demonstrate that macrophage motility could be reduced in accordance with activation in response to cancer cell products. It suggests that macrophage motility could be a novel marker to monitor cancer-associated inflammatory diseases and the efficacy of anti-inflammatory agents.

Dexamethasone inhibits in vivo tumor growth by the alteration of bone marrow CD11b⁺ myeloid cells.

Inflammation is closely associated with tumor growth, which is mediated by the activation of bone marrow-derived CD11b(+) cells. Here, we investigated whether anti-inflammatory dexamethasone (Dex), a synthetic glucocorticoid (GC), could regulate tumor growth and CD11b(+) myeloid bone marrow cells (BMCs) in lymphocyte (R1), monocyte (R2) and granulocyte (R3) regions of FSC-SSC dot plot. The growth of B16F10 mouse melanoma tumor was inhibited in Dex-injected group. Lung metastasis was decreased and the lifespan was elongated in Dex-injected mice with tumor resection. Intravenous injection of B16F10 cells increased the percentage of CD11b(+) myeloid BMCs in R1 and R2 regions from 3h to 72h. In contrast, little changes in the percentage of CD11b(+) myeloid BMCs were detected in R3 region. Among CD11b(+) myeloid BMCs, the percentage of CD11b(+)Gr-1(+) cells was increased in R1, R2 and R3 regions. Absolute number of CD11b(+) and CD11b(+)Gr-1(+) cells was enhanced in R1 region from 3h to 72 h. B16F10 tumor growth was significantly increased by intravenous injection of CD11b(+) BMCs. Tumor-bearing mice showed an increase in the percentage of CD11b(+) myeloid BMCs in R2 region and CD11b(+)Gr-1(+) cells in R2 and R3 regions, which are reduced by intravenous injection with Dex. Absolute number of CD11b(+)Gr-1(+) cells was enhanced in R2 and R3 regions. Tumor growth was significantly inhibited by intravenous injection of BMCs collected from Dex-treated tumor-bearing mice. Taken together, data demonstrate that tumor regression by Dex was resulted from the alteration of CD11b(+) myeloid BMCs and their inhibitory function to tumor growth. It suggests that CD11b(+) myeloid BMCs could regulate antitumor efficacy of GCs such as Dex.

The Actin-Sequestering Protein Thymosin Beta-4 Is a Novel Target of Hypoxia-Inducible Nitric Oxide and HIF-1α Regulation

The actin-sequestering protein thymosin beta-4 (Tβ4) is involved in various cellular and physiological processes such as proliferation, motility, growth and metastasis. Nitric oxide (NO) promotes tumor invasiveness and metastasis by activating various enzymes. Herein, we investigated whether hypoxia-inducible NO regulates Tβ4 expression and cancer cell migration using HeLa cervical cancer cells. NO production and Tβ4 expression were increased in a hypoxic condition. The treatment with N-(β-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, enhanced the transcription of Tβ4 and cancer cell migration. SNAP-1-induced cell migration was decreased by the inhibition of Tβ4 with small interference (si) RNA. In a hypoxic condition, treatment with NG-monomethyl-L-arginine (L-NMMA), nitric oxide synthase (NOS) inhibitor, reduced Tβ4 transcriptional activity, and hypoxia-inducible factor (HIF)-1α. Hypoxia-induced cancer cell migration was also decreased by L-NMMA treatment. In a normoxic condition, Tβ4 transcriptional activity was decreased in the cells incubated in the presence of L-NMMA after co-transfection with Tβ4 promoter and GST-conjugated HIF-1α. Collectively, these results suggest that NO could regulate the expression of Tβ4 by direct or indirect effect of HIF-1α on Tβ4 promoter.