Proteomic profiling of endorepellin angiostatic activity on human endothelial cells

<p>Abstract</p> <p>Background</p> <p>Endorepellin, the C-terminal domain V of the heparan sulfate proteoglycan perlecan, exhibits powerful and targeted anti-angiogenic activity on endothelial cells. To identify proteins involved with endorepellin anti-angiogenic action, we performed an extensive comparative proteomic analysis between vehicle- and endorepellin-treated human endothelial cells.</p> <p>Results</p> <p>Proteomic analysis of endorepellin influence on human umbilical vein endothelial cells identified five differentially expressed proteins, three of which (β-actin, calreticulin, and chaperonin/Hsp60) were down-regulated and two of which (vimentin and the β subunit of prolyl 4-hydroxylase also known as protein disulfide isomerase) were up-regulated in response to endorepellin treatment—and associated with a fold change (endorepellin/control) ≤ 0.75 and ≥ 2.00, and a statistically significant p-value as determined by Student's <it>t </it>test.</p> <p>Conclusion</p> <p>The proteins identified represent potential target areas involved with endorepellin anti-angiogenic mechanism of action. Further elucidation as such will ultimately provide useful in utilizing endorepellin as an anti-angiogenic therapy in humans.</p

Basement membrane proteoglycans: modulators Par Excellence of cancer growth and angiogenesis.

Proteoglycans located in basement membranes, the nanostructures underling epithelial and endothelial layers, are unique in several respects. They are usually large, elongated molecules with a collage of domains that share structural and functional homology with numerous extracellular matrix proteins, growth factors and surface receptors. They mainly carry heparan sulfate side chains and these contribute not only to storing and preserving the biological activity of various heparan sulfate-binding cytokines and growth factors, but also in presenting them in a more active configuration to their cognate receptors. Abnormal expression or deregulated function of these proteoglycans affect cancer and angiogenesis, and are critical for the evolution of the tumor microenvironment. This review will focus on the functional roles of the major heparan sulfate proteoglycans from basement membrane zones: perlecan, agrin and collagen XVIII, and on their roles in modulating cancer growth and angiogenesis

A central function for perlecan in skeletal muscle and cardiovascular development

Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity. Live videomicroscopy confirms the abnormal swimming pattern caused by the myopathy and anomalous head and trunk vessel circulation. The phenotype is partially rescued by microinjection of human perlecan or endorepellin. These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities

Proteomic profiling of endorepellin angiostatic activity on human endothelial cells-1

Lin and β-actin levels. Notice that the major actin isoforms also show reduced staining intensity in response to endorepellin treatment. (B) Immunoblotting of control and endorepellin-treated endothelial cell lysates using an antibody against human calreticulin. Equal amounts of total proteins were loaded. (C) Immunoblot analysis of β-actin levels following treatment with ~200 nM endorepellin for the designated time intervals. (D) The kinetic data for β-actin expression levels were derived from immunoblotting data as presented in C. Essentially HUVEC β-actin expression levels were examined by Western blot following exposure to endorepellin for the indicated time points. β-actin levels were quantified over similar amounts of protein loading and graphed as percent β-actin levels (based upon time point 0) versus exposure to endorepellin treatment (hours). The data were graphed in SigmaPlot v9 and fit by non-linear regression analysis. Each data point represents the mean ± S.E.M. from three experiments. The T1/2 of ~45 min. represents the time to reduce β-actin levels by 50% as compared to the control. (E) Immunoblot analysis of vimentin and prolyl 4-hydroxylase, β subunit protein levels following endorepellin treatment for the indicated time points. GAPDH is shown as loading control. All verification experiments presented in panels C-E utilized new samples of endorepellin-treated HUVEC lysates, separate from those used in proteomic analysis.<p><b>Copyright information:</b></p><p>Taken from "Proteomic profiling of endorepellin angiostatic activity on human endothelial cells"</p><p>http://www.proteomesci.com/content/6/1/7</p><p>Proteome Science 2008;6():7-7.</p><p>Published online 12 Feb 2008</p><p>PMCID:PMC2275231.</p><p></p

Erratum: Niche-localized tumor cells are protected from HER2-targeted therapy via upregulation of an anti-apoptotic program in vivo

A correction to this article has been published and is linked from the HTML version of this article

Niche-localized tumor cells are protected from HER2-targeted therapy via upregulation of an anti-apoptotic program in vivo

Several lines of evidence suggest that components of the tumor microenvironment, specifically basement membrane and extracellular matrix proteins, influence drug sensitivities. We previously reported differential drug sensitivity of tumor cells localized adjacent to laminin-rich extracellular matrix in three-dimensional tumor spheroid cultures. To evaluate whether differential intra-tumor responses to targeted therapy occur in vivo, we examined the sensitivity of human epidermal growth factor receptor 2-positive tumors to lapatinib using a previously described ductal carcinoma in situ-like model characterized by tumor cell confinement within ductal structures surrounded by an organized basement membrane. Here we show that tumor cells localized to a ‘niche’ in the outer layer of the intraductal tumors adjacent to myoepithelial cells and basement membrane are resistant to lapatinib. We found that the pro-survival protein BCL2 is selectively induced in the niche-protected tumor cells following lapatinib treatment, and combined inhibition of HER2 and BCL-2/XL enhanced targeting of these residual tumor cells. Elimination of the niche-protected tumor cells was achieved with the HER2 antibody–drug conjugate T-DM1, which delivers a chemotherapeutic payload. Thus, these studies provide evidence that subpopulations of tumor cells within specific microenvironmental niches can adapt to inhibition of critical oncogenic pathways, and furthermore reveal effective strategies to eliminate these resistant subpopulations

Clinical evaluation of BCL-2/XL levels pre- and post- HER2-targeted therapy.

Our previous pre-clinical work defined BCL-2 induction as a critical component of the adaptive response to lapatinib-mediated inhibition of HER2. To determine whether a similar BCL-2 upregulation occurs in lapatinib-treated patients, we evaluated gene expression within tumor biopsies, collected before and after lapatinib or trastuzumab treatment, from the TRIO-B-07 clinical trial (NCT#00769470). We detected BCL2 mRNA upregulation in both HER2+/ER- as well as HER2+/ER+ patient tumors treated with lapatinib or trastuzumab. To address whether mRNA expression correlated with protein expression, we evaluated pre- and post-treatment tumors for BCL-2 via immunohistochemistry. Despite BCL2 mRNA upregulation within HER2+/ER- tumors, BCL-2 protein levels were undetectable in most of the lapatinib- or trastuzumab-treated HER2+/ER- tumors. BCL-2 upregulation was evident within the majority of lapatinib-treated HER2+/ER+ tumors and was often coupled with increased ER expression and decreased proliferation. Comparable BCL-2 upregulation was not observed within the trastuzumab-treated HER2+/ER+ tumors. Together, these results provide clinical validation of the BCL-2 induction associated with the adaptive response to lapatinib and support evaluation of BCL-2 inhibitors within the context of lapatinib and other HER2-targeted receptor tyrosine kinase inhibitors