TMSB10, a potential prognosis prediction biomarker, promotes the invasion and angiogenesis of gastric cancer.

BACKGROUND AND AIM: The thymosin beta 10 (TMSB10) was originally identified from the thymus, which plays a key role in the development of many cancers. However, the underlying molecular mechanisms of TMSB10 involved in GC have not been understood. METHODS: We sought to determine the expression of TMSB10 in human GC tissues and illustrate whether it is correlated with the clinical pathologic characteristics and prognosis in GC patients. Its roles and potential mechanisms in regulating tumor growth, invasion, and angiogenesis were evaluated by TMSB10 knockdown/overexpression of GC cells in vitro and ex vivo. RESULTS: Marked overexpression of TMSB10 protein expression was observed in GC cells and tissues, which was associated with the advanced tumor stage and lymph nodes (LN) metastasis of GC patients. Furthermore, prognostic analysis showed that GC patients with high TMSB10 expression had a remarkably shorter survival and acted as an important factor for predicting poor overall survival in GC patients. Moreover, TMSB10 overexpression promoted, while TMSB10 knockdown the proliferation, EMT process, and angiogenesis of GC cells. CONCLUSION: The study highlights that TMSB10 may hold promise as potential prognosis prediction biomarker for the diagnosis of GC and a potential therapeutic target, which will facilitate the development of a novel therapeutic strategy against GC.

Thymosin ß10 promotes tumor-associated macrophages M2 conversion and proliferation via the PI3K/Akt pathway in lung adenocarcinoma.

BACKGROUND: Thymosin ß10 (TMSB10) has been reported to play a protumorigenic role in a majority of solid cancers. However, the existence of TMSB10 in immune microenvironment may contribute to the pathogenesis of lung adenocarcinoma has not been previously explored. METHOD: TAMs-associated TMSB10 expression was evaluated by immunohistochemistry (IHC) in 184 lung adenocarcinomas. Xenograft mice model was established to investigate the effect of TMSB10 shRNA on TAMs phenotypes. The macrophages phenotype associated cytokines IL-6, IL-8, IL-12 and TNF-α were detected by ELISA after treated with TMSB10 shRNA or scramble. Furthermore, the target proteins were detected by immunoblotting. RESULTS: We found that high TAMs-associated TMSB10 expression was significantly correlated with the advanced TNM stage and T3/T4 tumor size. And high TAMs-associated TMSB10 expression was significantly correlated with poor overall and progression-free survival of lung adenocarcinoma, acting as an independent prognostic factor for lung adenocarcinoma. Furthermore, we investigated the biological functions of TMSB10 in macrophages in vivo and in vitro. TMSB10 knockdown dramatically reduced TAMs, THP-1 and RAW264.7 cell proliferation, and promoted macrophages phenotype conversion of M2 to M1, and TMSB10 knockdown reduced the levels of p-Akt (Sec473), p-mTOR (Sec2448) and p-p70S6K (Thr389) without effect on Akt, mTOR and p70S6K expression. CONCLUSIONS: These results demonstrate that TAMs-associated TMSB10 promotes tumor growth through increasing TAMs M2 conversion and proliferation via PI3K/Akt signaling pathway, providing a promising tumor biomarker for predicting prognosis and a potential therapeutic target for lung adenocarcinoma.

Thymosin beta-4 overexpression correlates with high-risk groups in gastric gastrointestinal stromal tumors: A retrospective analysis by immunohistochemistry.

BACKGROUND: Thymosin beta-4 (Tß4) is a protein that is linked to a number of important biological actions and recently tumor progression and poor prognosis of some tumors. The aim of this study was to evaluate Tß4 expression in gastric GISTs and correlate with some clinicopathological characteristics related with prognosis and clinical outcome in order to add further data to the current literature. METHODS: Tß4 antibody was applied to the 4µm-thick paraffin sections of 57 gastric GISTs by immunohistochemistry. RESULTS: Tß4 expression was found to be directly corrrelated with higher risk groups, tumor size, mitotic count, cellularity, and necrosis while it was inversely correlated with overall survival (OS) by univariate analysis (p=0.000, p=0.001, p=0.000, p=0.025, p=0.023, and p=0.042, respectively). The direct association between Tß4 expression and risk groups were also supported by multivariate analysis (p=0.000, ß=0.497, t=4.374). CONCLUSION: Overexpression of Tß4 was found to be related with predictive characteristics for tumor progression and adverse prognosis. Thus, we suggest that overexpression of Tß4 might play a role in the progression of gastric GISTs and might be used as a potential prognostic tool as well as a target for novel therapies.

Thymosin ß4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum.

Thymosin ß4 (Tß4), the principal G-actin regulating entity in eukaryotic cells, has also multiple intra- and extracellular functions related to tissue regeneration and healing. While its effect in adult organs is being widely investigated, currently, little is known about its influence on embryonic tissues, i.e., in the developing nervous system. The importance of Tß4 for neural stem cell proliferation in the embryonic chicken optic tectum (OT) has previously been shown by us for the first time. In the present study, using in ovo electroporation, we carried out a quantification of the effects of the Tß4-overexpression on the developing chicken OT between E4 and E6 at the hemisphere as well as cellular level. We precisely examined tissue growth and characterized cells arising from the elevated mitotic activity of progenitor cells. By using spinning-disk confocal laser scanning microscopy, we were able to visualize these effects across whole OT sections. Our experiments now demonstrate more clearly that the overexpression of Tß4 leads to a tangential expansion of the treated OT-hemisphere and that, under these circumstances, overall density of tectal and in particular of postmitotic neuronal cells is increased. Thanks to this new quantitative approach, the present results extend our previous findings that Tß4 is important for the proliferation of progenitor cells, neurogenesis, tangential expansion, and tissue growth in the young embryonic chicken optic tectum. Taken together, our results further illustrate and support the current idea that Tß4 is widely implicated in shaping and maintenance of the nervous system.

A Tobacco-Derived Thymosin ß4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice.

Thymosin ß4 (Tß4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tß4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tß4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bp Tß4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×Tß4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tß4 protein concentrations as high as 3 µg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tß4 was more effective than Tß4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.

Effect of Thymosin beta4 on the Differentiation and Mineralization of MC3T3-E1 Cell on a Titanium Surface.

Osteoblasts are responsible for the synthesis of bone matrix through the secretion of collagenous and non-collagenous proteins with mineralization. Thymosin beta4 (Tbeta4) is an actin-sequestering peptide that is involved in the regulation of cell proliferation, differentiation and motility. A recent study reported that the inhibition of Tbeta4 mRNA synthesis strongly decreases the level of gene expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), osteonectin (ON) and collagen type I (Col I) with mineralization during differentiation in odontoblasts. Titanium (Ti) is used commonly as an implant material for dental implants, which have strong mechanical potential and good biocompatibility with bone. This study examined whether Tbeta4 can be a potential molecule for promoting the differentiation and mineralization of MC3T3-E1 cells on a Ti surface. Tbeta4 increased the viability of MC3T3-E1 cells during differentiation on Ti discs compared to that of the control. The expression of Tbeta4 mRNA and protein in the Tbeta4-treated MC3T3-E1 cells was higher than the control during differentiation on the Ti discs. In addition, Tbeta4 increased the formation of mineralization nodules and the mRNA expression of alkaline phosphatase (ALP), DSPP, dentin matrix protein1 (DMP1), BSP and Col I compared to that of the control in MC3T3-E1 cells during differentiation on Ti discs. From the results, Tbeta4 increased the viability and promoted the differentiation and mineralization of MC3T3-E1 cells on Ti discs. This highlights the potential use of Tbeta4 for increasing osseointegration through osteoblast differentiation and mineralization on Ti discs.

Thymosin ß4 promotes hepatoblastoma metastasis via the induction of epithelial-mesenchymal transition.

Hepatoblastoma (HB) is the most common malignant hepatic tumor in children and complete surgical resection offers the highest possibility for cure in this disease. Tumor metastasis is the principle obstacle to the development of efficient treatments for patients with HB. The present study aimed to measure the expression levels of thymosin ß4 (Tß4) in liver samples from patients with HB and to investigate the involvement of Tß4 in HB metastasis. The expression of Tß4 was significantly higher in liver samples from patients with metastatic HB and in the HepG2 metastatic HB cell line, compared with that in adjacent healthy liver samples and in the L02 healthy hepatic cell line. By contrast, the expression levels of epithelial-cadherin (E-cadherin) and cytosolic accumulation of ß-catenin, the two most prominent markers involved in epithelial-mesenchymal transition (EMT), were reduced in liver specimens from patients with metastatic HB compared with that of healthy adjacent control tissue. HepG2 cells were transfected with small interfering-RNA in order to downregulate Tß4 gene expression. This resulted in a reduced cell migratory capacity compared with control cells. Tß4 gene expression knockdown significantly inhibited transforming growth factor ß1-mediated-EMT in vitro by upregulating the expression of E-cadherin. The results of the present study suggested that Tß4 may promote HB metastasis via the induction of EMT, and that Tß4 may therefore be a target for the development of novel treatments for patients with HB.

Increased expression of prothymosin-α, independently or combined with TP53, correlates with poor prognosis in colorectal cancer.

Human prothymosin-α (PTMA) plays an important role in tumorigenesis, and its overexpression triggers a TP53 response. In this study, we identified that PTMA expression was up-regulated at both the transcriptional and translational level in tumor tissue compared to that in adjacent normal tissue. PTMA overexpression was significantly associated with the depth of tumor invasion, lymph node metastasis (LNM), distant metastasis, advanced AJCC stage, and tumor differentiation. There was also a significant association between PTMA over-expression and mutant TP53 expression (r=0.515, P < 0.001). Survival analysis revealed that the disease-free survival (DFS) and overall survival (OS) rates were significantly lower among patients with PTMA- and TP53-positive tumors. Hence, PTMA might play an important role in the progression of CRC, and the assessment of both PTMA and mutant TP53 expression can help predict colon cancer prognosis.

Thymosin ß4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.

Thymosin ß4 (Tß4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tß4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tß4 on the TLR pathway. Cells were grown in the presence of Tß4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tß4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tß4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tß4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tß4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tß4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.

Loss of nuclear prothymosin-α expression is associated with disease progression in human superficial bladder cancer.

In this paper, we report a study on the clinical relevance of prothymosin-α expression and its correlation with intratumoral Foxp3(+) and CD8(+) lymphocytes (Foxp3(+)TIL and CD8(+)TIL) in bladder cancer patients. We used immunohistochemical staining for prothymosin-α, Foxp3, and CD8 on 101 tumor specimens harvested by endoscopic resection. The results were correlated with clinicopathological variables and clinical outcome in bladder cancer patients, particularly in 73 patients with superficial disease, using the log-rank test and Cox proportional hazard model. Overall, of the tumors, 30 % were negative, 34 % showed nuclear, and 37 % showed cytoplasmic prothymosin-α expression. Foxp3(+)TILs were detected in 11 % of patients (nonnuclear vs. nuclear, p = 0.096). Patients with a history of urothelial carcinoma have a higher frequency of nonnuclear prothymosin-α expression than those without (p = 0.016, chi-square test). By univariate and multivariate analyses of cases with superficial disease, grade and stage were identified as independent predictors for recurrence-free survival (p = 0.016 and 0.016, respectively). Higher stage and nonnuclear prothymosin-α expression independently predict shorter progression-free survival (p = 0.006 and 0.043, respectively). The presence of Foxp3(+)TILs was significantly associated with disease progression by univariate analysis (p = 0.022), but not by multivariate analysis (p = 0.147). In vitro assays showed that J82 cells which express ectopically nuclear prothymosin-α exhibit higher growth rate and secrete less TGF-ß1 than those with cytoplasmic expression or control cells. Altogether, prothymosin-α expression is a determinant of disease progression in superficial bladder cancer. Foxp3(+)TILs tend to be found more often in bladder cancer with nonnuclear prothymosin-α expression. Future study is required to unravel their interaction.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma.

Thymosin beta 4 (Tß4) and thymosin beta 10 (Tß10) are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tß4 and Tß10 in hepatocellular carcinoma (HCC). To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tß4 and Tß10. Immunoreactivity for Tß4 and Tß10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tß4 reactivity was detected in 7/23 cases (30%) and Tß10 reactivity in 22/23 (97%) cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tß10 showed a strong homogeneous expression, was Tß4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tß10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tß10 in HCC progression.

Thymosin ß 4 gene silencing decreases stemness and invasiveness in glioblastoma.

Thymosin beta 4 is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. Thymosin beta 4 gene silencing promotes differentiation of neural stem cells whereas thymosin beta 4 overexpression initiates cortical folding of developing brain hemispheres. A role of thymosin beta 4 in malignant gliomas has not yet been investigated. We analysed thymosin beta 4 staining on tissue microarrays and performed interrogations of the REMBRANDT and the Cancer Genome Atlas databases. We investigated thymosin beta 4 expression in seven established glioma cell lines and seven glioma-initiating cell lines and induced or silenced thymosin beta 4 expression by lentiviral transduction in LNT-229, U87MG and GS-2 cells to study the effects of altered thymosin beta 4 expression on gene expression, growth, clonogenicity, migration, invasion, self-renewal and differentiation capacity in vitro, and tumorigenicity in vivo. Thymosin beta 4 expression increased with grade of malignancy in gliomas. Thymosin beta 4 gene silencing in LNT-229 and U87MG glioma cells inhibited migration and invasion, promoted starvation-induced cell death in vitro and enhanced survival of glioma-bearing mice. Thymosin beta 4 gene silencing in GS-2 cells inhibited self-renewal and promoted differentiation in vitro and decreased tumorigenicity in vivo. Gene expression analysis suggested a thymosin beta 4-dependent regulation of mesenchymal signature genes and modulation of TGFß and p53 signalling networks. We conclude that thymosin beta 4 should be explored as a novel molecular target for anti-glioma therapy.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo.

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

Construction, expression, and characterization of thymosin alpha 1 tandem repeats in Escherichia coli.

Thymosin alpha 1 (T α 1), which is composed of 28 amino acids, has been commercialized worldwide for its immune-modulatory and antitumor effects. T α 1 can stimulate T cell proliferation and differentiation from bone marrow stem cells, augment cell-mediated immune responses, and regulate homeostasis of immune system. In this study, we developed a novel strategy to produce T α 1 concatemer (T α 1③) in Escherichia coli and compared its activity with chemically synthesized T α 1. Results showed that T α 1③ can more effectively stimulate T cell proliferation and significantly upregulate IL-2 receptor expression. We concluded that the expression system for T α 1 concatemer was constructed successfully, which could serve as an efficient tool for the production of large quantities of the active protein.

Expression patterns of Thymosin ß4 and cancer stem cell marker CD133 in ovarian cancers.

Thymosin ß4 (Tß4), a small acidic actin binding peptide, is overexpressed in a side population of cancer stem cells and CD133-positive colorectal cancer stem cells. In order to understand the relationship between Tß4 and CD133, we studied the expression patterns of Tß4 and CD133 in ovarian cancers. The expression patterns of Tß4 and CD133 were studied in normal ovaries, primary ovarian cancers, metastatic ovarian cancers, primary stomach cancers, and normal stomachs by Western blot and immunohistochemistry. Expression patterns and co-localization of Tß4 and CD133 were examined by immunofluorescence and confocal laser-scanning microscopy. Tß4 is overexpressed in primary ovarian cancers, but not in primary stomach cancers, when compared with normal controls. However, Tß4 levels in metastatic stomach cancers to the ovary are significantly upregulated compared with levels in normal stomachs and primary stomach cancers. These results suggest that Tß4 levels are related to tumorigenesis in ovarian cancers and metastasis in stomach cancers. The expression of Tß4 in normal ovaries and normal stomachs was weak, but was co-localized with CD133 expression. Tß4 expression was also co-localized with CD133 expression in primary ovarian carcinomas, metastatic ovarian cancers from stomach cancers and primary stomach cancers. These data suggest that Tß4 expression is strongly related to CD133 expression and is a characteristic of stem cells or cancer stem cells.

Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis.

Little is known about the cellular mechanisms modulating the shift in balance from a state of survival to cell death by caspase-mediated apoptosis in response to a lethal stress. Here we show that the RNA-binding protein HuR has an important function in mediating this switch. During caspase-mediated apoptosis, HuR is cleaved to generate two cleavage products (CPs). Our data demonstrate that the cleavage of HuR switches its function from being a prosurvival factor under normal conditions to becoming a promoter of apoptosis in response to a lethal stress. In the absence of an apoptotic stimuli, HuR associates with and promotes the expression of caspase-9 and prothymosin α (ProT) mRNAs, and pro- and antiapoptotic factors, respectively, both of which have been characterized as important players in determining cell fate. During the early steps of caspase-mediated apoptosis, however, the level of caspase-9 protein increases, while ProT remains unchanged. Under these conditions, the two HuR-CPs selectively bind to and stabilize caspase-9 mRNA, but do not bind to ProT. Hence, taken together, our data show that by maintaining a threshold of expression of proapoptotic factors such as caspase-9 in response to a lethal stress, the HuR-CPs help a cell to switch from resisting death to undergoing apoptosis.

[The influence of pulsed infrared laser radiation on the hormone production in the thymus (an experimental study)].

Local irradiation with pulsed (1500 Hz) low-energy infrared laser light of the thymus and thyroid gland region caused well-apparent stimulation of alpha-1-thymosin production in the healthy animals and normalized its level in the stressed ones. Similar stimulation of alpha-1-timosine biosynthesis was observed in an experiment with direct laser irradiation of the cultured HTSC epitheliocytes from the human thymus.

Thymosin ß4 expression correlates with lymph node metastasis through hypoxia inducible factor-α induction in breast cancer.

Intratumoral hypoxia has been correlated with distant metastatic potential. Two hypoxia inducible factors (HIFs), HIF-1α and HIF-2α, are induced by hypoxia, and high expression of these proteins has been correlated to angiogenesis and distant metastasis. Thymosin ß4 (Tß4) is frequently highly expressed in cancer, and this overexpression correlates with malignant progression. The objective of this study was to investigate the clinical correlation of HIF-α with Tß4 and the intracellular functional roles of Tß4 on HIF-α activation. We examined HIF-1α, HIF-2α and Tß4 expressions in clinical human breast carcinoma (n=70) by immunohistochemistry. We show that high expression of HIF-1α and HIF-2α strongly correlates with Tß4 expression (P≤0.0001) and overexpression of Tß4 correlates significantly with patients with lymph node metastasis (P<0.05) of human breast cancer. Additionally, we demonstrate that hypoxia up-regulates intracellular Tß4 protein, which then affects HIF-α activity, which is the key in regulating VEGF expression. We confirmed that hypoxia-induced intracellular Tß4 and HIF-α activities were reduced by interference of Tß4 expression using Tß4 shRNA lentivirus. Vascular epidermal growth factor (VEGF)-A, a well-recognized lymphangiogenic cytokine, was also down-regulated, but VEGF-C and VEGF-D expressions were not affected. These findings suggest that the overexpression of Tß4 is strongly associated with HIF-1α and HIF-2α expression and is also clinicopathologically involved with lymph node metastatic potential of breast cancer through the modulation of HIF-αactivation and induction of VEGF-A. Ultimately, these results highlight Tß4 as a potentially therapeutic target in malignant cancers.

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.