Tartrate Density Gradient Assay
Approximately 106 FFU of dengue virus 2 produced in LLCMK2 cells and purified as described above for the cryo-electron microscopy studies, was treated with 100 mM DN59 or 1% (v/v) triton X-100 for 30 min at 37uC. Treated virus was loaded onto a 10-35% (w/v) potassium sodium tartrate step gradient and centrifuged at 175,1176g for 2 hrs. Individual fractions were collected and assayed for virus genome and E protein. Genome quantitation was carried out by qRT-PCR as described above for the RNase sensitivity assay using the 10503F/10599R primer set [33]. E protein detection was carried out using modified ELISA. High bind 96-well plates (Costar, Corning, NY) were coated with concavalin A (Vector Laboratories, Burlingame, CA) at 25 mg/ml in 0.01 M HEPES (for 1 hr and washed with PBS containing 0.1% (v/v) Tween-20. Equal aliquots of each gradient fraction were added for 1 hr to allow binding of E to the concavalin A and then washed again. Captured E protein was detected using a human anti-E monoclonal antibody, followed by goat antihuman HRP conjugate. After a final wash, color was developed with tetramethylbenzidine-peroxide (TMB)-H2O2 stopped by adding 1% (v/v) phosphoric acid. Optical density was measured at 450 nm.
Hemoglobin Release Assay
Sheep red blood cells (RBC) (Lampire Biological Products, Pipersville, PA) in anti-coagulant K2-EDTA, were washed and resuspended in PBS to a final concentration of 10% (v/v). Peptide was added to 2% RBC, incubated at 37uC for 1 hr and centrifuged at 13,000 rpm. Supernatants were collected and the absorbance at 560 nm was measured. Results were normalized against treatment with 1% (v/v) triton X-100 as a control for 100% hemolysis.
Vesicular Stomatitis Virus Plaque Reduction Assays
Plaque reduction assays in LLC-MK2 cells were carried out in a similar manner as above, except that a 1.2% solution of methylcellulose (FMC, Philadelphia, PA) in complete medium was used in place of agarose. Vesicular stomatitis virus eGFP-P was incubated for 24 hrs at 37uC before overlays were aspirated, rinsed with PBS, and plaques were visualized for GFP expression [35].
Abstract
The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement membrane collagen IV a1 chain. As the mere prevention of tumor angiogenesis leads to hypoxia that can result in selection of more aggressive cell types and reduces the efficacy of chemotherapy, we aimed here to elucidate how arresten influences the aggressive human carcinoma cells. Arresten efficiently inhibited migration and invasion of HSC-3 tongue carcinoma cells in culture and in an organotypic model. Subcutaneous Arr-HSC xenografts grew markedly more slowly in nude mice and showed reduced tumor cell proliferation, vessel density and local invasiveness. In the organotypic assay, HSC-3 cells overproducing arresten (Arr-HSC) showed induction of cell death. In monolayer culture the Arr-HSC cells grew in aggregated cobblestone-like clusters and, relative to the control cells, showed increased expression and localization of epithelial marker E-cadherin in cell-cell contacts. Application of electric cell-substrate impedance sensing (ECIS) further supported our observations on altered morphology and motility of the Arr-HSC cells. Administration of a function-blocking a1 integrin antibody abolished the impedance difference between the Arr-HSC and control cells suggesting that the effect of arresten on promotion of HSC-3 cell-cell contacts and cell spreading is at least partly mediated by a1b1 integrin. Collectively, our data suggest novel roles for arresten in the regulation of oral squamous carcinoma cell proliferation, survival, motility and invasion through the modulation of cell differentiation state and integrin signaling.
Citation: Aikio M, Alahuhta I, Nurmenniemi S, Suojanen J, Palovuori R, et al. (2012) Arresten, a Collagen-Derived Angiogenesis Inhibitor, Suppresses Invasion of Squamous Cell Carcinoma. Editor: Christina Lynn Addison, Ottawa Hospital Research Institute, Canada Received May 11, 2012; Accepted October 30, 2012; Published December 5, 2012 Copyright: ?2012 Aikio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported by Academy of Finland (1126783, 1130140, 115256, 128259, 132051, 138866), Centre of Excellence of Academy of Finland Grant 2012?017 (251314, Finnish Cancer Organizations, Cancer Foundation of Northern Finland, Sigrid Juselius Foundation, Oulu and Helsinki University Hospital KEVO-support, Oulu University Scholarship Foundation, and Finnish Dental Society Apollonia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Tumor growth does not just depend on carcinoma cells, as interactions between cancer cells, extracellular matrix (ECM) and various cell types in the tumor stoma have a major impact on the disease outcome. The remodeling of tumor stroma during tumorigenesis and the cleavage of basement membrane components results in molecules with novel biological activities [1,2]. Particularly, collagens IV and XVIII contain cryptic fragments, named arresten, canstatin, hexastatin, tetrastatin, tumstatin and endostatin, which inhibit angiogenesis and tumor growth via integrin binding [3?5]. Arresten is a 26-kDa fragment derived from the non-collagenous NC1 domain of the basement membrane collagen IV a1 chain [a1(IV)NC1] that efficiently inhibits the proliferation, migration and tube formation of different types of endothelial cells [3,16?8]. In vivo, arresten inhibits Matrigel neovascularization [18] and the growth of subcutaneous tumors in mice [3,16,18]. It has recently been shown that it also increasesapoptosis of endothelial cells by regulating intracellular signaling events. The pro-apoptotic effect of arresten is mediated by reducing the expression of the anti-apoptotic signaling molecules Bcl-2 and Bcl-xL and activating caspase-3/poly (ADP-ribose) polymerase via FAK/p38-MAPK signaling [2,19]. The production of arresten has recently been linked to the p53 tumor suppressor pathway. p53 was shown to induce an anti-angiogenic program whereby expression of a1(IV) chain is upregulated, stabilized by prolyl-4-hydroxylase and efficiently processed by MMPs to an arresten-containing peptide. This p53-dependent ECM remodeling was suggested to destabilize the vascular collagen IV network and thereby prevent endothelial cell adhesion and migration leading to reduced angiogenesis and tumor growth in vivo and in vitro. [20].
