Shih-Wei Wang

The promotion of human mesenchymal stem cell proliferation by superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide (SPIO) nanoparticles are very useful in cell imaging; meanwhile, however, biosafety concerns associated with their use, especially on therapeutic stem cells, have arisen. Most studies of biosafety issues focus on whether the nanoparticles have deleterious effects. Here, we report that Ferucarbotran, an ionic SPIO, is not toxic to human mesenchymal stem cells (hMSCs) under the conditions of these experiments but instead increases cell growth. Ferucarbotran-promoted cell growth is due to its ability to diminish intracellular H2O2 through intrinsic peroxidase-like activity. Also, Ferucarbotran can accelerate cell cycle progression, which may be mediated by the free iron (Fe) released from lysosomal degradation and involves the alteration of Fe on the expression of the protein regulators of the cell cycle.

Interleukin-6 induces vascular endothelial growth factor expression and promotes angiogenesis through apoptosis signal-regulating kinase 1 in human osteosarcoma.

Osteosarcoma is characterized by a high malignant and metastatic potential. Angiogenesis is essential for the caner metastasis. Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the relationship between IL-6 and vascular endothelial growth factor (VEGF) expression in human osteosarcoma is mostly unknown. Here we found that the IL-6 and VEGF expression was correlated with tumor stage and significantly higher than that in normal bone. Incubation of osteosarcoma cells with IL-6 increased VEGF mRNA and protein expression. Pretreatment of cells with IL-6R antibody reduced IL-6-mediated VEGF production. The apoptosis signal-regulating kinase 1 (ASK1)/p38/AP-1 pathway was activated after IL-6 treatment, and IL-6-induced VEGF expression was abolished by the specific inhibitor and siRNA of ASK1, p38, and AP-1 cascades. Importantly, knockdown IL-6 reduced VEGF expression and abolished osteosarcoma conditional medium-mediated angiogenesis. Taken together, these results indicate that IL-6 occurs through ASK1 and p38, which in turn activates AP-1, resulting in the activations of VEGF expression and contributing the angiogenesis of human osteosarcoma cells.

CCL5 and CCR5 Interaction Promotes Cell Motility in Human Osteosarcoma

Background Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown. Methodology/Principal Findings Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells. Conclusions/Significance CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells.

Endothelin-1 promotes vascular endothelial growth factor-dependent angiogenesis in human chondrosarcoma cells

Chondrosarcoma is the second most common sarcoma in bone malignancy and is characterized by a high metastatic potential. Angiogenesis is essential for the cancer metastasis. Endothelin-1 (ET-1) has been implicated in tumor angiogenesis and metastasis. However, the relationship of ET-1 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells is mostly unknown. Here, we found that the expression of ET-1 and VEGF were correlated with tumor stage and were significantly higher than that in the normal cartilage. Exogenous ET-1 with chondrosarcoma cells promoted VEGF expression and subsequently increased migration and tube formation in endothelial progenitor cells. ET-1 increased VEGF expression and angiogenesis through ETAR, integrin-linked kinase (ILK), Akt and hypoxia-inducible factor-1α (HIF-1α) signaling cascades. Knockdown of ET-1 decreased VEGF expression and also abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. In addition, in the xenograft tumor angiogenesis model, knockdown of ET-1 significantly reduced tumor growth and tumor-associated angiogenesis. Taken together, these results indicate that ET-1 occurs through ETAR, ILK and Akt, which in turn activates HIF-1α, resulting in the activation of VEGF expression and contributing to the angiogenesis and tumor growth of human chondrosarcoma cells.

CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression.

Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts.Cell Death and Disease advance online publication, 23 October 2014; doi:10.1038/cddis.2014.453

CHM-1, a novel synthetic quinolone with potent and selective antimitotic antitumor activity against human hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma is highly chemoresistant to currently available chemotherapeutic agents. In this study, 2′-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1), a synthetic 6,7-substituted 2-phenyl-4-quinolone, was identified as a potent and selective antitumor agent in human hepatocellular carcinoma. CHM-1 induced growth inhibition of HA22T, Hep3B, and HepG2 cells in a concentration-dependent manner but did not obviously impair the viability of normal cells at the IC50 for liver cancer cells. CHM-1-induced apoptosis was also characterized by immunofluorescence microscopy. CHM-1 interacted with tubulin at the colchicine-binding site, markedly inhibited tubulin polymerization both in vitro and in vivo, and disrupted microtubule organization. CHM-1 caused cell cycle arrest at G2-M phase by activating Cdc2/cyclin B1 complex activity. CHM-1-induced cell death, activation of Cdc2 kinase activity, and elevation of MPM2 phosphoepitopes were profoundly attenuated by roscovitine, a specific cyclin-dependent kinase inhibitor. CHM-1 did not modulate the caspase cascade, and the pan-caspase-inhibitor z-VAD-fmk did not abolish CHM-1-induced cell death. However, CHM-1 induced the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. Small interfering RNA targeting of AIF substantially attenuated CHM-1-induced AIF translocation. Importantly, CHM-1 inhibited tumor growth and prolonged the lifespan in mice inoculated with HA22T cells. In conclusion, we show that CHM-1 exhibits a novel antimitotic antitumor activity against human hepatocellular carcinoma both in vitro and in vivo via a caspase-independent pathway. CHM-1 is a promising chemotherapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially hepatocellular carcinoma. [Mol Cancer Ther 2008;7(2):350–60]

CCL5/CCR5 axis induces vascular endothelial growth factor-mediated tumor angiogenesis in human osteosarcoma microenvironment

Chemokines modulate angiogenesis and metastasis that dictate cancer development in tumor microenvironment. Osteosarcoma is the most frequent bone tumor and is characterized by a high metastatic potential. Chemokine CCL5 (previously called RANTES) has been reported to facilitate tumor progression and metastasis. However, the crosstalk between chemokine CCL5 and vascular endothelial growth factor (VEGF) as well as tumor angiogenesis in human osteosarcoma microenvironment has not been well explored. In this study, we found that CCL5 increased VEGF expression and production in human osteosarcoma cells. The conditioned medium (CM) from CCL5-treated osteosarcoma cells significantly induced tube formation and migration of human endothelial progenitor cells. Pretreatment of cells with CCR5 antibody or transfection with CCR5 specific siRNA blocked CCL5-induced VEGF expression and angiogenesis. CCL5/CCR5 axis demonstrably activated protein kinase Cδ (PKCδ), c-Src and hypoxia-inducible factor-1 alpha (HIF-1α) signaling cascades to induce VEGF-dependent angiogenesis. Furthermore, knockdown of CCL5 suppressed VEGF expression and attenuated osteosarcoma CM-induced angiogenesis in vitro and in vivo. CCL5 knockdown dramatically abolished tumor growth and angiogenesis in the osteosarcoma xenograft animal model. Importantly, we demonstrated that the expression of CCL5 and VEGF were correlated with tumor stage according the immunohistochemistry analysis of human osteosarcoma tissues. Taken together, our findings provide evidence that CCL5/CCR5 axis promotes VEGF-dependent tumor angiogenesis in human osteosarcoma microenvironment through PKCδ/c-Src/HIF-1α signaling pathway. CCL5 may represent a potential therapeutic target against human osteosarcoma.

ERK Activation Globally Downregulates miRNAs through Phosphorylating Exportin-5.

MicroRNAs (miRNA) are mostly downregulated in cancer. However, the mechanism underlying this phenomenon and the precise consequence in tumorigenesis remain obscure. Here we show that ERK suppresses pre-miRNA export from the nucleus through phosphorylation of exportin-5 (XPO5) at T345/S416/S497. After phosphorylation by ERK, conformation of XPO5 is altered by prolyl isomerase Pin1, resulting in reduction of pre-miRNA loading. In liver cancer, the ERK-mediated XPO5 suppression reduces miR-122, increases microtubule dynamics, and results in tumor development and drug resistance. Analysis of clinical specimens further showed that XPO5 phosphorylation is associated with poor prognosis for liver cancer patients. Our study reveals a function of ERK in miRNA biogenesis and suggests that modulation of miRNA export has potential clinical implications.

Moscatilin, a bibenzyl derivative from the India orchid Dendrobrium loddigesii, suppresses tumor angiogenesis and growth in vitro and in vivo

Attacking angiogenesis is considered an effective strategy for controls the expansion and metastasis of tumors and other related-diseases. The aim of this study was to assess the effects of moscatilin, a bibenzyl derivative, on VEGF and bFGF-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Moscatilin significantly inhibited growth of lung cancer cell line A549 (NSCLC) and suppressed growth factor-induced neovascularization. In addition, VEGF- and bFGF-induced cell proliferation, migration, and tube formation of HUVECs was markedly inhibited by moscatilin. Western blotting analysis of cell signaling molecules indicated that moscatilin inhibited ERK1/2, Akt, and eNOS signaling pathways in HUVECs. These results suggest that inhibition of angiogenesis by moscatilin may be a major mechanism in cancer therapy.

CCL5 promotes vascular endothelial growth factor expression and induces angiogenesis by down-regulating miR-199a in human chondrosarcoma cells

Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis. Angiogenesis is a critical step in tumor growth and metastasis. Chemokine CCL5 (previously called RANTES) has been shown to facilitate tumor progression and metastasis. However, the relationship of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study, CCL5 increased VEGF expression and also promoted chondrosarcoma medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. MicroRNA analysis was performed in CCL5-treated chondrosarcoma cells versus control cells to investigate the mechanism of CCL5-mediated promotion of chondrosarcoma angiogenesis. Among the miRNAs regulated by CCL5, miR-199a was the most downregulated miRNA after CCL5 treatment. In addition, co-transfection with miR-199a mimic reversed the CCL5-mediated VEGF expression and angiogenesis in vitro and in vivo. Moreover, overexpression of CCL5 increased tumor-associated angiogenesis and tumor growth by downregulating miR-199a in the xenograft tumor angiogenesis model. Taken together, these results demonstrated that CCL5 promotes VEGF-dependent angiogenesis in human chondrosarcoma cells by downregulating miR-199a.