Ectopic Expression of Human Thymosin ß4 Confers Resistance to Legionella pneumophila during Pulmonary and Systemic Infection in Mice.

Thymosin beta-4 (Tß4) is an actin-sequestering peptide that plays important roles in regeneration and remodeling of injured tissues. However, its function in a naturally occurring pathogenic bacterial infection model has remained elusive. We adopted Tß4-overexpressing transgenic (Tg) mice to investigate the role of Tß4 in acute pulmonary infection and systemic sepsis caused by Legionella pneumophila Upon infection, Tß4-Tg mice demonstrated significantly lower bacterial loads in the lung, less hyaline membranes and necrotic abscess, with lower interstitial infiltration of neutrophils, CD4+, and CD8+ T cells. Bronchoalveolar lavage fluid of Tß4-Tg mice possessed higher bactericidal activity against exogenously added L. pneumophila, suggesting that constitutive expression of Tß4 could efficiently control L. pneumophila Furthermore, qPCR analysis of lung homogenates demonstrated significant reduction of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), which primarily originate from lung macrophages, in Tß4-Tg mice after pulmonary infection. Upon L. pneumophila challenge of bone marrow-derived macrophages (BMDM) in vitro, secretion of IL-1ß and TNF-α proteins was also reduced in Tß4-Tg macrophages, without affecting their survival. The anti-inflammatory effects of BMDM in Tß4-Tg mice on each cytokine were affected when triggering with tlr2, tlr4, tlr5, or tlr9 ligands, suggesting that anti-inflammatory effects of Tß4 are likely mediated by the reduced activation of Toll-like receptors (TLR). Finally, Tß4-Tg mice in a systemic sepsis model were protected from L. pneumophila-induced lethality compared to wild-type controls. Therefore, Tß4 confers effective resistance against L. pneumophila via two pathways, a bactericidal and an anti-inflammatory pathway, which can be harnessed to treat acute pneumonia and septic conditions caused by L. pneumophila in humans.

Fetuin-B regulates vascular plaque rupture via TGF-ß receptor-mediated Smad pathway in vascular smooth muscle cells.

Fetuin-B is a serum protein linked to the regulation of physiological or pathophysiological events such as fertility, energy metabolism, and liver disease. Recently, fetuin-B has been reported to be involved in the modulation of the rupture of atherosclerotic plaques associated with acute myocardial infarction. However, the exact mechanism involved in the modulation of atherosclerotic plaque rupture event by fetuin-B is not fully elucidated yet. In the present study, we investigated whether fetuin-B could influence atherosclerotic plaque rupture through vascular smooth muscle cells (VSMCs). Immunoprecipitation assay using membrane proteins from VSMCs revealed that fetuin-B tightly bound to transforming growth factor-ß receptor (TGF-ßR). Fetuin-B treatment elevated TGF-ßR signals (e.g., phosphorylation of Smad2 and Smad3) in VSMCs. Fetuin-B also stimulated nuclear translocation of phosphorylated Smads. Phosphorylation of Smad and its nuclear translocation by treatment with fetuin-B were inhibited in VSMCs by treatment with SB431542, a selective inhibitor of TGF-ßR. Fetuin-B enhanced expression levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in VSMCs through its epigenetic modification including recruitments of both histone deacetylase 1 and RNA polymerase II. These epigenetic alterations in VSMCs were also inhibited by treatment with SB431542. In vivo administration of fetuin-B protein increased expression levels of PAI-1 and MMP-2 in the vascular plaque. However, these increases in expression were inhibited by the administration of SB43154. These results indicate that fetuin-B may modulate vascular plaque rupture by promoting expression of PAI-1 and MMP-2 in VSMCs via TGF-ßR-mediated Smad pathway.

A monoclonal antibody derived by inoculation of human umbilical vein endothelial cells is a potential inhibitor of acetylcholine receptor-linked vasorelaxation.

In a previous study, we produced antibodies from rats immunized with human umbilical vein endothelial cells (HUVECs) and determined the vascular function of the monoclonal antibodies. However, unanswered question remains still about their role in vascular function. The current study explored vasoreactivity, in particular, focusing on the vascular contractility of a functional antibody against proteins expressed on the plasma membrane of HUVECs developed in a previous study. Among the antibodies developed, A-7 significantly attenuated endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to sodium nitroprusside or histamine. In addition, the A-7 antibody did not affect norepinephrine-stimulated contraction in both endothelium-intact and -denuded aorta. Immunocytochemical and immunoblotting analyses showed that A-7 attenuated ACh-increased expression of ACh receptor on the plasma membrane of HUVECs. These findings suggest that the monoclonal A-7 antibody may act as an inhibitor of endothelium-dependent vasorelaxation, probably in part via downregulation of ACh receptor expression.

Sabinene Prevents Skeletal Muscle Atrophy by Inhibiting the MAPK-MuRF-1 Pathway in Rats.

Chrysanthemum boreale Makino essential oil (CBMEO) has diverse biological activities including a skin regenerating effect. However, its role in muscle atrophy remains unknown. This study explored the effects of CBMEO and its active ingredients on skeletal muscle atrophy using in vitro and in vivo models of muscle atrophy. CBMEO reversed the size decrease of L6 myoblasts under starvation. Among the eight monoterpene compounds of CBMEO without cytotoxicity for L6 cells, sabinene induced predominant recovery of reductions of myotube diameters under starvation. Sabinene diminished the elevated E3 ubiquitin ligase muscle ring-finger protein-1 (MuRF-1) expression and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations in starved myotubes. Moreover, sabinene decreased the increased level of reactive oxygen species (ROS) in myotubes under starvation. The ROS inhibitor antagonized expression of MuRF-1 and phosphorylation of MAPKs, which were elevated in starved myotubes. In addition, levels of muscle fiber atrophy and MuRF-1 expression in gastrocnemius from fasted rats were reduced after administration of sabinene. These findings demonstrate that sabinene, a bioactive component from CBMEO, may attenuate skeletal muscle atrophy by regulating the activation mechanism of ROS-mediated MAPK/MuRF-1 pathways in starved myotubes, probably leading to the reverse of reduced muscle fiber size in fasted rats.

Inhibitory effects of scoparone from chestnut inner shell on platelet-derived growth factor-BB-induced vascular smooth muscle cell migration and vascular neointima hyperplasia.

BACKGROUND: Compounds of the inner shell of chestnut (Castanea crenata) have diverse biological activities, including anti-cancer and anti-oxidant activities. Here we explored the effects of an extract of chestnut inner shells and of its bioactive component scoparone on vascular smooth muscle cell migration and vessel damage. RESULTS: The ethanol extract of chestnut inner shells, containing 11 major compounds, inhibited platelet-derived growth factor (PDGF)-BB-induced migration of rat aortic smooth muscle cells (RASMCs). Among these compounds, scoparone (6,7-dimethoxycoumarin) suppressed RASMC migration and wound healing in response to PDGF-BB but did not affect RASMC proliferation. In RASMCs, scoparone inhibited the PDGF-BB-induced rat aortic sprout outgrowth and attenuated the PDGF-BB-mediated increase in phosphorylation of mitogen-activated protein kinases (MAPKs), p38 MAPK and extracellular signal-regulated kinase 1/2. The in vivo administration of scoparone resulted in the attenuation of neointima formation in balloon-injured carotid arteries of rats. CONCLUSION: These findings demonstrate that scoparone, found in chestnut inner shells, may inhibit cell migration through suppression of the phosphorylation of MAPKs in PDGF-BB-treated RASMCs, probably contributing to the reduction of neointimal hyperplasia induced after vascular injury. Therefore, scoparone and chestnut inner shell may be a potential agent or functional food, respectively, for the prevention of vascular disorders such as vascular restenosis or atherosclerosis. © 2019 Society of Chemical Industry.

Low-power laser irradiation inhibits PDGF-BB-induced migration and proliferation via apoptotic cell death in vascular smooth muscle cells.

Vascular restenosis after injury of blood vessel has been implicated in various responses including apoptosis, migration, and proliferation in vascular smooth muscle cells (VSMCs) stimulated by diverse growth factors underlying platelet-derived growth factor (PDGF). Previous studies evaluated the effects of low-power laser (LPL) irradiation over various wavelength ranges on VSMC events in normal and pathologic states. However, whether VSMC responses are affected by LPL irradiation remains unclear. The purpose of this study is to explore the effects of LPL (green diode laser 532-nm pulsed wave of 300 mW at a spot diameter of 1 mm) irradiation on the responses, apoptosis, migration, and proliferation of VSMCs. The effect of LPL irradiation was tested on VSMCs through cytotoxicity, proliferation, migration, and apoptotic assays. Aortic ring assay was used to assess the effect of LPL irradiation on aortic sprout outgrowth. Protein expression levels were determined by western blotting. LPL irradiation did not affect VSMC viability but slightly attenuated PDGF-BB-induced proliferation in VSMCs. In addition, LPL irradiation inhibited PDGF-BB-evoked migration of VSMCs. Aortic sprout outgrowth in response to PDGF-BB was diminished in cells treated with LPL. In contrast, LPL irradiation evoked apoptosis in VSMCs in the presence of PDGF-BB. Similarly, activation of caspase-3 and Bax, as well as p38 mitogen-activated protein kinase (MAPK), in VSMCs treated with PDGF-BB was enhanced by exposure to LPL. These findings indicate that LPL irradiation induces vascular apoptosis via p38 MAPK activation and simultaneously inhibits VSMC proliferation and migration in response to PDGF-BB.

Adipose Mesenchymal Stem Cell-Derived Exosomes Promote the Regeneration of Corneal Endothelium Through Ameliorating Senescence.

Purpose: Human corneal endothelial cells (hCECs) have been considered unable to regenerate in vivo, resulting in corneal decompensation after significant loss of hCECs. adipose-derived mesenchymal stem cell (ASC)-derived exosomes can regenerate tissues and organs. In this study, we investigated whether ASC-derived exosomes could protect and regenerate CECs. Methods: We performed cell viability and cell-cycle analyses to evaluate the effect of ASC-derived exosomes on the regeneration capacity of cultured hCECs. Transforming growth factor-ß (TGF-ß) and hydrogen peroxide (H2O2) were used to induce biological stress in CECs. The effect of ASC-derived exosomes on CECs was investigated in vivo. ASC-derived exosomes were introduced into rat CECs using electroporation, and rat corneas were injured using cryoinjury. Next-generation sequencing analysis was performed to compare the differentially expressed microRNAs (miRNAs) between ASC-derived and hCEC-derived exosomes. Results: ASC-derived exosomes induced CEC proliferation and suppressed TGF-ß- or H2O2-induced oxidative stress and senescence. ASC-derived exosomes protect hCECs against TGF-ß- or H2O2-induced endothelial-mesenchymal transition and mitophagy. In an in vivo study, ASC-derived exosomes promoted wound healing of rat CECs and protected the corneal endothelium against cryoinjury-induced corneal endothelium damage. Next-generation sequencing analysis revealed differentially expressed miRNAs for ASC-derived and hCEC-derived exosomes. They are involved in lysine degradation, adherens junction, the TGF-ß signaling pathway, the p53 signaling pathway, the Hippo signaling pathway, the forkhead box O (FoxO) signaling pathway, regulation of actin cytoskeleton, and RNA degradation based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Conclusions: ASC-derived exosomes promoted wound healing and regeneration of endothelial cells by inducing a shift in the cell cycle and suppressing senescence and autophagy.

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

Thymosin beta-4 (Tß4), 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 Tß4 and it is associated with Tß4-mediated migration in gastric cancer cells. Various expression level of Tß4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tß4. Cell migration was higher in SNU668 with a higher expression level of Tß4 than that in SNU638 with a lower Tß4. Although the level of phosphorylated(p)-GSK-3α (inactive), ß-catenin, E-cadherin and E-cadherin:ß-catenin complex was relatively higher, p-GSK-3ß (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/ß 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:ß-catenin complex formation through a decrease in ß-catenin and E-cadherin. Expression level of GSK-3α/ß, ß-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tß4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tß4, respectively. E-cadherin expression in SNU638 cells was decreased by ß-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, ß-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, ß-catenin and E-cadherin could be negatively regulated by Tß4-induced ERK phosphorylation. It suggests that Tß4 could be a novel regulator to control Wnt signaling pathways.

Bioluminescence Imaging of Matrix Metalloproteinases-2 and -9 Activities in Ethanol-injured Cornea of Mice.

BACKGROUND/AIM: This study aimed to investigate the usefulness of in vivo bioluminescence imaging (BLI) to examine the role of matrix metalloproteinases (MMP)-2 and MMP-9 activation in the development and healing of ethanol-induced damage in the cornea of mice. MATERIALS AND METHODS: Mouse corneal injury was induced by topical treatment with 20% ethanol. BLI was obtained from the ocular region of mice intravenously injected with an active-MMP-2/9 probe. In vivo results were validated in primary corneal epithelial cells. RESULTS: BLI indicated that treatment of the eye with 20% ethanol elevated MMP-2/9 activity, which was inhibited by the application of eye drops (hyaluronic acid and serum). Treatment of corneal epithelial cells with 20% ethanol-increased the activities of MMP-2 and MMP-9, which were also inhibited by eye drops. CONCLUSION: BLI can be applied in vivo in mice with corneal injury to examine the activity of MMPs and clarify the efficacy of eye drops.

In the Setting of Negative Mammogram, Is Additional Breast Ultrasound Necessary for Evaluation of Breast Pain?

PURPOSE: To evaluate whether in the setting of negative diagnostic mammogram for breast pain additional ultrasound is necessary. METHODS: Retrospective IRB-approved review of our database identified 8085 women who underwent ultrasound evaluation for breast pain from 1/1/2013-12/31/2013. Of 8085 women, 559 women had mammogram evaluation preceding the ultrasound and these women comprise the basis of this study. The patient's age, type of mammogram examination (screening or diagnostic), Breast Imaging-Reporting and Data System (BI-RADS) breast density (BD), type of breast pain (focal, diffuse, cyclical, unilateral, bilateral), additional breast symptoms (palpable concern, nipple discharge, skin changes, others), mammogram or ultrasound findings and final BI-RADS assessment, follow-up imaging, and follow-up biopsy results were reviewed and recorded. RESULTS: The median age of patients was 46 years old (range: 27-97). Patients recalled from negative screening mammogram were 29.8% (167/559). Patients with preceding negative diagnostic mammogram were 70.2% (392/559). The BI-RADS BD distribution was BD1: 5.5%, BD2: 39.9%, BD3: 46.0%, BD4: 8.6%. Final BI-RADS assessments were BI-RADS 1/2 (79%), BI-RADS 3 (12.9%), BI-RADS 4 (8.1%), BI-RADS 5 (0%). Majority (66.9%, 374/559) of the patient had breast pain alone. Additional breast symptoms were also noted as follows: palpable concern (24%), nipple discharge (3.9%), skin changes or other (5.2%). On follow-up evaluation, 26 findings were recommended for tissue sampling yielding 2 malignancies (0.4%, 2/559) in 2 patients. In the setting of negative mammogram and clinical symptom of breast pain alone yielded no malignances (NPV, 100%, 374/374) and was not impacted by BD. In patients with additional symptoms accompanying pain, malignancies were present despite negative mammogram in 2 patients; nipple discharge (4.5%, 1/22), and palpable concern (0.7%, 1/134). CONCLUSION: In the setting of negative mammogram and breast pain alone, additional evaluation with ultrasound is likely low yield and may be unnecessary. However, with additional symptoms such as palpable concern or nipple discharge, ultrasound is likely an important adjunct modality for identifying mammographically occult tumors.

Correction: Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4.

[This corrects the article DOI: 10.18632/oncotarget.3218.].

Desalted Salicornia europaea extract attenuated vascular neointima formation by inhibiting the MAPK pathway-mediated migration and proliferation in vascular smooth muscle cells.

Salicornia europaea L. (SE) has been used as folk medicine for the treatment of various diseases such as obesity, diabetes, and cancer. However, its effects on atherosclerotic events in vascular smooth muscle cells (VSMCs) remain unknown. The present study explored the effects of the ethyl acetate fraction of desalted SE hot water extract (SEWEAF) on atherosclerotic responses (especially migration and proliferation) in VSMCs and vascular neointima formation. Treatment with the SEWEAF significantly suppressed the platelet-derived growth factor (PDGF)-BB-induced VSMC migration and proliferation as well the phosphorylation of mitogen-activated protein kinases (MAPKs) such as the p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2. Moreover, oral administration of the SEWEAF resulted in the attenuation of neointima formation in balloon-injured rat carotid arteries. Additionally, HPLC analysis showed that the major components in the two subfractions of the SEWEAF were five phenolic acids and four flavonols. In the SEWEAF components, for which atherosclerosis-linked responses in VSMCs have not been known, p-coumaric acid, quercetin-3-ß-d-glucoside, and isorhamnetin-3-ß-d-glucoside inhibited both PDGF-BB-induced migration and proliferation and isorhamnetin attenuated only PDGF-BB-stimulated VSMC proliferation. These results suggest that the SEWEAF may suppress PDGF-BB-induced VSMC migration by downregulating the phosphorylation of p38 MAPK and ERK1/2, thus leading to the reduction of neointimal hyperplasia during vascular remodeling. Therefore, the desalted SE extract, SEWEAF may be a potential ingredient for dietary supplements or nutraceuticals to ameliorate and/or prevent vascular remodeling-related disorders.

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 (Tß4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tß4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tß4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(ß-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tß4 expression with Tß4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tß4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tß4 plasmids for Tß4 overexpression. Taken together, these results suggest that Tß4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.

Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4.

Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tß4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tß4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tß4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tß4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tß4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tß4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

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-2(d)) 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-2(d)) 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.

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 (Tß4) 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 Tß4 expression and cancer cell migration using HeLa cervical cancer cells. NO production and Tß4 expression were increased in a hypoxic condition. The treatment with N-(ß-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, enhanced the transcription of Tß4 and cancer cell migration. SNAP-1-induced cell migration was decreased by the inhibition of Tß4 with small interference (si) RNA. In a hypoxic condition, treatment with N(G)-monomethyl-L-arginine (L-NMMA), nitric oxide synthase (NOS) inhibitor, reduced Tß4 transcriptional activity, and hypoxia-inducible factor (HIF)-1α. Hypoxia-induced cancer cell migration was also decreased by L-NMMA treatment. In a normoxic condition, Tß4 transcriptional activity was decreased in the cells incubated in the presence of L-NMMA after co-transfection with Tß4 promoter and GST-conjugated HIF-1α. Collectively, these results suggest that NO could regulate the expression of Tß4 by direct or indirect effect of HIF-1α on Tß4 promoter.

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.

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.

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.

Cooperation of actin-sequestering protein, thymosin ß-4 and hypoxia inducible factor-1α in tumor cell migration.

Cell migration plays an important role in many physiological and pathological processes, including tumor metastasis. Tumor cell migration is increased through the sequential induction of HIF-1α and VEGF under hypoxic conditions. Thymosin ß-4 (Tß4) is an actin-sequestering protein which controls cytoskeletal reorganization. Here, we investigated whether tumor cell migration could be co-operatively controlled by hypoxia and Tß4. Cell migration was measured by wound healing assay with scratching confluent monolayers of tumor cells. Cell migration was enhanced 18 h after scratching cells. In addition, we found that the expression of HIF-1α, VEGF-A isoform 164/120 and Tß4 was increased by scratching cells. Cell migration was decreased by the inhibition of Tß4 or HIF-1α expression with lentiviral shRNA of Tß4 or siRNA of HIF-1α, respectively. In contrast, cell migration was increased by the treatment with Tß4 proteins. The inhibitory effect of Tß4-shRNA or HIF-1α-siRNA was also attenuated by treatment with Tß4 proteins. Collectively, these findings suggest that Tß4 and HIF-1α cooperatively enhance tumor cell migration.