Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors

BACKGROUND: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. METHODS: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1 = CT-CM, RT112/TP = RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT-PCR, ELISA and blocking antibodies. RESULTS: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 mu M TPI and 100 mu M L-dR, blocked migration and reduced the invasion by 50-70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-alpha, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. CONCLUSION: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy

Cancer Cell Invasion Is Enhanced by Applied Mechanical Stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor

A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein

Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication

Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression Model

BACKGROUND: Ovarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment. METHODOLOGY/PRINCIPAL FINDINGS: Mouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII. CONCLUSIONS/SIGNIFICANCE: Our studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression

Antiangiogenic Activity of Sterically Stabilized Liposomes Containing Paclitaxel (SSL-PTX): In Vitro and In Vivo

The purpose of this present study was to evaluate the antiangiogenic activity of sterically stabilized liposomes containing paclitaxel (SSL-PTX). The SSL-PTX was prepared by the thin-film method. The release of paclitaxel from SSL-PTX was analyzed using a dialysis method. The effect of SSL-PTX on endothelial cell proliferation and migration was investigated in vitro. The antitumor and antiangiogenic activity of SSL-PTX was evaluated in MDA-MB-231 tumor xenograft growth in BALB/c nude mice. The release of paclitaxel from SSL-PTX was 22% within 24 h. Our in vitro results indicated that SSL-PTX could effectively inhibit the endothelial cell proliferation and migration at a concentration-dependent manner. We also observed that metronomic SSL-PTX induced marked tumor growth inhibition in MDA-MB-231 xenograft model via the antiangiogenic mechanism, unlike that in paclitaxel injection (Taxol) formulated in Cremophor EL (CrEL). Overall, our results suggested that metronomic chemotherapy with low-dose, CrEL-free SSL-PTX should be feasible and effective