Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin

BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy

Hemodialysis Removes Uremic Toxins That Alter the Biological Actions of Endothelial Cells

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure

Cell cycle perturbations induced by HB-19 treatment.

<p>(A) Analysis of cell cycle parameters in HB-19 treated cells. MDA-MB-231 cells were cultured for 48 hours in medium without FBS (starvation) or in medium containing 10% FBS supplemented or not with 10 µM HB-19. DNA synthesis was quantified after BrdU incorporation and staining with anti-BrdU antibody and 7-AAD, by FACScan analysis. The histograms indicate the relative amount of cells in G1, S and G2/M cell phases. (B) HB-19 treatment inhibits serum-induced phosphorylation of ERK1/2. Serum starved MDA-MB-231 cells were stimulated with 10% FBS in the absence or presence of 2, 5, and 10 µM of HB-19. Five minutes after serum stimulation, cells were lysed directly in electrophoresis sample buffer and processed for immunoblotting using anti-phospho-p42/44 ERK1/2 and anti-p42/44 ERK antibodies. NS stands for non-stimulated cells.</p

HB-19 inhibits colony formation in soft agar by tumor cell lines.

<p>(A) MDA-MB-231 cells in culture medium were seeded in triplicate in the absence (histogram C) or presence of 0.1, 0.5 and 1 of µM HB-19, or 0.1 µM of the anti-nucleolin mAb MS-3 or control IgG, or 10 µM bisphosphonate (BisP). (B) Various tumor cell lines (as indicated) in culture medium were seeded in triplicate in the absence (histogram Control) or presence of 5 µM HB-19. After 10–21 days, colonies with diameters greater than 50 µm were scored as positive. Statistical significance: *0.01</p

HB-19 treatment has no effect on blood cell number.

<p>MDA-MB-231 tumor bearing mice untreated, and treated with 5-FU or HB-19 were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002518#pone-0002518-g008" target="_blank">Figure 8B</a> for 8 weeks. Blood samples collected in EDTA were processed using an automated flow cytometric blood cell counter (¹ ×10³/µl; ² ×10⁶/µl). The results are presented as the mean areas ±standard deviation for each mice group (n = 10). Data from untreated and treated group were compared and analyzed using the paired t-test to obtain <i>p</i> value: ^* p<0.05, ^** p<0.01, ^*** p<0.0001.</p

HB-19 inhibits tumor growth in the nude mice.

<p>(A) HB-19 inhibits the growth of MDA-MB-231 tumor-cell xenografts. Cells (2×10⁶) were injected subcutaneously into the right flank of female nude mice. Two weeks later, mice with a palpable tumor of approximately 40 mm³ in volume were randomly separated into three groups (n = 5) and were given peritumoral injections 3 times/week of 0.1 ml PBS alone (Control), HB-19 (5 mg/kg), or Tamoxifen (Tmx) 10 mg/kg) for 6 weeks. (B) HB-19 inhibits the growth of MDA-MB-435 tumor-cell xenografts. Cells (1×10⁶) were injected in the mammary fat pad of female nude mice. Two weeks later, mice with a palpable tumor were randomly separated into three groups (n = 10) and were given intraperitoneal injections 3 times/week of 0.1 ml PBS alone (Control), HB-19 (5 mg/kg), or 5-fluouracil (5-FU, 40 mg/kg) for 8 weeks. At the end of each experiment (in A and B), mice were sacrificed and the tumors were excised and weighed. The results are presented as the mean weight ±standard deviation (±S.D.) obtained from the number of mice in each group. (C, D) Inhibition of tumor development in mice treated by intraperitoneal (i.p.) and subcutaneous (s.c.) administration of HB-19. MDA-MB-231 tumor bearing mice in three groups (n = 10) were treated with HB-19 (10 mg/kg) by i.p. or s.c. injections, 3 times/week for 28 days. The arrow at day 0 shows initiation of HB-19 treatment. Panel D shows MDA-MB-231 tumor bearing mice, untreated control and HB-19 treated (i.p. injection). Statistical significance: *p<0.05, **p<0.01, ***p<0.001.</p

Reduced density of blood vessels in HB-19 treated tumor-bearing mice.

<p>Sections of tumors recovered from mice, untreated control and treated with either HB-19 or 5-FU (experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002518#pone-0002518-g008" target="_blank">Figure 8B</a>) were stained with antibodies against the CD31 endothelial marker and analyzed by fluorescence microscopy. Representative macroscopic image (magnification 200×) from each group of mice shows the marked reduction of blood vessels in the tumors recovered from HB-19 or 5-FU treated animals. Angiogenesis was quantified by image analysis of CD31-labeled endothelial cells. The graph shows the mean areas ±standard deviation obtained from control and treated mice. ***p<0.001.</p