Inhibition by the S1 peptide of AR/Src complex, Src activation and DNA synthesis triggered by EGF in HT1080 cells.

<p>Quiescent HT1080 cells were used. Cells were left un-stimulated or stimulated for 10 min with EGF (at 100 ng/ml) in the absence or presence of S1 or Ss peptide (both at 1 nM). Casodex (at 10 μM) was used for comparison with the S1 peptide. Upper section in <b>A</b>, Western blot of HT1080 cell lysates with anti-EGFR antibody. Tubulin (tubulin) was revealed by immunoblot, as a loading control. Lower section in <b>A</b>, lysates were immune-precipitated with anti-EGFR Ab and proteins in immune complexes were detected using antibodies against the indicated proteins. In <b>B</b>, lysates were immune-precipitated with the anti-Src MAb and Src activity in immune complexes was assayed using acidified enolase, as a substrate. In <b>C</b>, cells on coverslips were left untreated or treated for 18 h with the indicated compounds. After <i>in </i><i>vivo</i> pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total nuclei. Several independent experiments were performed in duplicate and the results were obtained from at least 500 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. (*) p value < 0,001; (°) p value< 0,005.</p

Casodex and S1 peptide inhibit EGF-stimulated BrdU incorporation and migration in HCT116 and KP-2 cells.

<div><p>Quiescent human colon cancer-derived HCT116 cells were used. In <b>A</b>, cells on coverslips were left un-stimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After <i>in </i><i>vivo</i> pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In <b>B</b>, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments. </p> <p>Quiescent human pancreatic cancer-derived KP-2 cells were used. In <b>C</b>, cells on coverslips were left unstimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After <i>in </i><i>vivo</i> pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In <b>D</b>, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments. </p> <p>In <b>A</b>, B, C and <b>D</b>, (*) <i>p</i> value < 0.001; (**) p value < 0,005. </p> <p>Panel <b>E</b> shows the Western blot of HCT116 or KP-2 cell lysates with the antibodies against the indicated proteins: tubulin, epidermal growth factor receptor (EGFR) and androgen receptor (AR).</p></div

Adoptive transfer of CD8+ T cells cultured with Cl-IB-MECA mediates enhanced tumor suppression in melanoma-bearing mice.

<p><b>A</b>) C57Bl6j mice were s.c. inoculated with 2.5×10⁵cells B16-F10 cells/mouse. 10 days after B16 injection, mice received a single p.t. injection of Cl-IB-MECA (20 ng/mouse) or 1×10⁶ CD8+ T cells/mouse treated with Cl-IB-MECA (0.1 µg/ml) or 1×10⁶ untreated CD8+ T cells/mouse or PBS (Ctr). <b>B</b>) Tumor volume (mm³) measured in control mice (Ctr, n = 11) and mice receiving a single dose of Cl-IB-MECA (n = 13) or adoptively transferred with Cl-IB-MECA-treated CD8+ T cells (n = 14) or with untreated CD8+ T cell (n = 10). <b>C</b>) Increased survival of melanoma-bearing mice receiving Cl-IB-MECA (p<0.05) or CD8+ T cell transfer cultured with Cl-IB-MECA (p<0.001) compared with control groups (n = 5/group). Data are from three independent experiments and represent mean ± SEM. Statistical differences were determined by one way ANOVA and Student's t test, as appropriate. ***p<0.001. Comparison of survival between groups was performed using long-rank test.</p

<i>In vivo</i> effectiveness of CD8+ T cell transfer after Cl-IB-MECA treatment is associated with increased apoptosis rate, granzyme B and TNF-α release into melanoma lesions.

<p><b>A</b>) and <b>B</b>) TNF-α and granzyme B levels, respectively, detected into tissue homogenates from mice adoptively transferred with Cl-IB-MECA-stimulated CD8+ T cells or Cl-IB-MECA-treated mice. <b>C</b>) Representative pictures of melanoma cryosections Tunel stained (FITC) and stained with PI (red). Positive and negative controls are also provided. <b>D</b>) Quantitative analysis of Tunel+ cells detected in melanoma sections. Results are expressed as mean ± SEM (n = 5/group). Data are from two independent experiments and represent mean ± SEM, n = 6 in each experiment. Statistical difference was determined by one way ANOVA. *p<0.05, **p<0.01, ***p<0.001.</p

Neutralization of TNF-α abrogated the anti-tumor effect of Cl-IB-MECA-treated CD8+ T cells.

<p><b>A</b>) Tumor volume (mm3) in mice receiving anti-TNF-α mAb (dashed lines) or isotype IgG control (continuous lines) and injected with Cl-IB-MECA or PBS or CD8+ T cells treated or not with Cl-IB-MECA. <b>B</b>) Percentage of CD11c+CD207 high cells in the tissue of mice described above. <b>C</b>) Representative dot plot is shown. <b>D</b>) and <b>E</b>) Expression of CD80 and MHC I, respectively, on CD11c+CD207 high cells in the tissue of mice described above. Data are from two independent experiments and represent mean ± SEM, n = 9 for each experiment. Statistical difference was determined by one way ANOVA. *p<0.05, **p<0.01, ***p<0.001.</p

Aptamer Functionalization of Nanosystems for Glioblastoma Targeting through the Blood–Brain Barrier

Polymeric nanoparticles (PNPs) may efficiently deliver in vivo therapeutics to tumors when conjugated to specific targeting agents. Gint4.T aptamer specifically recognizes platelet-derived growth factor receptor β and can cross the blood–brain barrier (BBB). We synthesized Gint4.T-conjugated PNPs able of high uptake into U87MG glioblastoma (GBM) cells and with astonishing EC₅₀ value (38 pM) when loaded with a PI3K-mTOR inhibitor. We also demonstrated in vivo BBB passage and tumor accumulation in a GBM orthotopic model

Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

<div><p>The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR_88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR^S90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR^S90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR^S90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR^S90P. In conclusion, our findings indicate that Ser⁹⁰ is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR^S90E and uPAR^S90P are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.</p> </div

uPAR^S90E and uPAR^S90P retain the ability to bind to ATF. A

<p>. Transfected 293 clones were grown adherent (2×10⁵cells/well) on 24-multiwell plates. ¹²⁵I-ATF (150,000 cpm/sample) was incubated with cells, in the presence of increasing concentrations of unlabeled ATF for 3 hours at 4°C and cell surface-associated radioactivity was determined. Data represent the mean of specific binding ± SD of three independent experiments performed in duplicate. <b>B</b>. Directional cell migration of the indicated transfected 293 clones toward 10 nM ATF. The extent of migration is expressed as percentage of cell migration of 293/mock cells assessed in the absence of ATF, considered as 100%. Data represent the mean ± SD of three independent experiments in triplicate. *: <i>p</i><0.001. <b>C–D</b>. Whole cell lysates(50 µg/sample) from cells exposed to ATF for the indicated times (C) or concentrations (D) immunoblotted with anti-phospho-AKT Ab (pAKT) and then with anti-Akt mAb (tAKT). Quantitative assessment of the pAKT and tAKT content of each sample was performed using NIH Image 1.62 software. Data are means of three experiments, with SD indicated by error bars.</p

Expression of uPAR^S90E and uPAR^S90P induces changes in 293 cell morphology, cytoskeletal organization and migration without affecting cell proliferation. A–B

<p>. Representative images of the indicated transfected 293 clones analyzed by phase contrast microscopy (A) or stained with rhodamine-phalloidin (B). <b>C</b>. Cell migration and invasion of stably transfected 293 cells toward 10%FBS. The extent of migration or invasion is expressed as percentage of migrating or invading 293/mock cells in the absence of chemoattractant, considered as 100% (none). Data represent the means ± SD of three experiments in duplicate. <b>D–E</b>. Cell tracking analysis of the steady-state transfected 293 cells. Cells were recorded for 70 minutes every 15 sec. while kept at 37°C, under a 5% CO2 atmosphere. Ten cells/field were followed in a total of 3 independent experiments. Straight distance (D) and tortuosity (E) were quantified using the Axiovision 4.8 software (Carl Zeiss). In F straight distance versus tortuosity is plotted for each cell analyzed. G. Time-dependent proliferation of transfected 293 cells in growth medium. Data represent the means ± SD of three experiments, performed in duplicate.</p

uPAR carrying S90E and S90P substitutions. A

<p>: Arg⁹¹ and Tyr⁹² superposition of the backbone atoms of the various 88–92 segments of SuPAR from x-ray structures. Residue 89 is not visible in the electron density map of 1YWH. Side chains are reported as stick, Arg residues are in blue. Backbones are reported as ribbon drawing: Yellow, 2I9B, chain E and F, residues 88–92; Red, 3BT2, chain U, residues 88–92; Mauve, 1YWH, chain E, residues 89–92; Cyan, 1YWH chain M, residues 89–92; Purple, 1YWH chain A, residues 89–92. <b>B</b>. Lysates (25 µg/sample) from 293 stably transfected either with pcDNA3 empty vector (293/mock) or pcDNA3 coding for human uPAR (293/uPAR^wt, uPAR^S90E, uPAR^S90P), were resolved on a 10% SDS-PAGE followed by Western blotting with R4 anti-uPAR or anti-αv mAbs. <b>C</b>. Cytofluorimetric analysis of the indicated stably transfected 293 clones with R4 anti-uPAR mAb or nonimmune immunoglobulins. <b>D</b>. Representative images of the indicated stably transfected 293 clones immuno-stained with anti-uPAR R4 mAb.</p