Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.
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.
Background/AimsPatients with diabetes are prone to coronary artery disease (CAD); however, the majority of diabetic patients show normal coronary arteries. We examined differences in the clinical aspects of diabetic patients with insignificant and with significant stenosis of the coronary artery.MethodsA total of 418 consecutive diabetic patients with stable angina who had undergone coronary angiography from January 2004 to March 2007 were included in this study. Patients were subdivided into control and CAD groups and then clinical characteristics and CAD-associated factors were evaluated.ResultsA total of 92 (22%) patients were assigned to the control group and 326 (78%) patients were assigned to the CAD group. Using univariate regression analysis, we found that patients with CAD were significantly older (control vs. CAD; 59±21 vs. 64.7±33.7, years, p<0.001), had a longer duration of diabetes (8.2±21.8 vs. 10.2±29.8, years, p=0.027), higher titers of high sensitivity C-reactive protein (hsCRP; 0.3±6.79 vs. 0.9±12.6, mg/dL, p=0.015), and increased hemoglobin A1c (HbA1c) levels (7.1±3.8 vs. 7.5±4.8, %, p=0.007) compared to control patients. Multivariate regression analysis showed that only differences in age, hsCRP, and HbA1c were statistically significant. When patients were subdivided into groups based on hsCRP levels (208 patients in the low group [49.8%], 210 patients in the high group [50.2%]), we found that patients with higher hsCRP levels showed more frequent multivessel disease.ConclusionsIn diabetic patients, age, hsCRP, and HbA1c were associated with stable CAD. Among these factors, hsCRP levels were significantly correlated with multivessel involvement in diabetic CAD. Therefore, high hsCRP levels may be a strong predictor for atherosclerotic progression of the coronary arteries in diabetic patients, suggesting that regular screening tests should be performed.
Abstract. 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-1· 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 cooperatively 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-1·, VEGF-A isoform 164/120 and Tß4 was increased by scratching cells. Cell migration was decreased by the inhibition of Tß4 or HIF-1· expression with lentiviral shRNA of Tß4 or siRNA of HIF-1·, respectively. In contrast, cell migration was increased by the treatment with Tß4 proteins. The inhibitory effect of Tß4-shRNA or HIF-1·-siRNA was also attenuated by treatment with Tß4 proteins. Collectively, these findings suggest that Tß4 and HIF-1· cooperatively enhance tumor cell migration. IntroductionThysmosin-ß-4 (TB4), a small, naturally occurring 43-amino acid peptide, was initially isolated in 1981 from the thymus. TB4 is the most abundant member of the ß-thymosins, a family of highly conserved polar 5-kDa peptides (1). TB4 is the major G actin-sequestering molecule in mammalian cells (2,3) and can be also cross-linked to some proteins including fibrin and collagen increased in the sites of tissue damage (1,4,5). TB4 led to paclitaxel-resistance (6,7), tumor growth and metastasis (8,9).Cell migration plays a role in many physiological and pathological processes, including tumor metastasis (10). Cell migration requires the integration and temporal coordination of many different processes that occur in spatially distinct locations in the cell (11). Migration can be demonstrated as a multistep cycle including extension of a protrusion, formation of stable attachments near the leading edge of the protrusion, translocation of the cell body forward, release of adhesions and retraction at the cell rear (12,13). Spatially controlled polymerization of actin is at the origin of cell motility and actin networks continuously generated at the leading edge (14,15). The cellular factor that generates new filaments in a site-directed, signaling-controlled fashion is the Arp2/3 complex (16). Profilin, G-actin-binding protein is involved in motile processes mediated by actin polymerization. Actinsequestering protein, TB4, do not modify filament dynamics or the rate of movement but it is thought to buffer the free ATP-G-actin concentration (17). Tß4 proteins induce HIF-1· stabilization, through Erk activation (18). In the meanwhile, hypoxic condition enhances tumor cell migration through the sequential induction of . However, little is known about whether tumor cell migration could be cooperatively controlled by HIF-1· stabilization and Tß4 expression under normoxia.Here, we investigated wheth...
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