28 research outputs found
Effect of EGFt on the dimerization of EGFR.
<p>Cell lysate from MDA-MB-468 cells were treated with the indicated concentrations of hEGF, EGFt or a mixture of both for 30 min. Then the samples were cross-linked by addition of 40 mM of glutaraldehyde and analyzed by Western blotting using an anti-EGFR antibody. The position of the EGFR monomers and dimmers is indicated.</p
Effect of EGFt on the growth of two human cancer cells.
<p>MCF-7 and Caco-2 cells were incubated for 72 h or 96 h h in a culture medium without FBS supplemented with 10, 20 and 150 nM hEGF or EGFt. The cell proliferation was assessed by MTT assays. Each column in the graph represents the relative cell proliferation versus untreated cells (control) and was the mean Ā± SEM of three independent experiments (*P<0.05 vs. control cells).</p
Effect of EGFt on the phosphorylation of EGFR.
<p><b>A</b> Analysis of the total phosphorylation of EGFR. MDA-MB-468 cells were treated with 3 nM, 150 nM hEGF or 150 nM EGFt for 10 min. The whole cell lysates were analyzed in parallel by Western blotting with an antibody against total-phosphotyrosine residues (PY20) and with an antibody against EGFR (EGFR 1005). <b>B</b> Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the internalization of the receptor. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM nM EGFt for the indicated periods of time. The whole cell lysates were analyzed by Western blotting with site-specific antibodies for phospho-EGFR tyrosine 1045 and serines 1046/47. <b>C</b> Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the proliferation signaling pathway. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM EGFt for the indicated periods of time. The whole cell lysates were analysed by Western blotting with site-specific antibodies for phospho-EGFR tyrosines 1068 and 1173. Untreated cells were used as a negative control (Ctl) and Ī²-actin levels were used as the loading control in Western blotting.</p
Purification and characterization of hEGF and EGFt.
<p>A Protein analysis of the different purification steps by Coomassie blue-stained SDS-PAGE. Molecular weights (M); Lane 1: commercial recombinant hEGF. Lane 2: supernatant of the <i>E.coli</i> fermentor culture before concentration. Lane 3: 30x concentrated supernatant by tangential flow filtration. Lane 4: product of the first purification step (anionic exchange chromatography). Lane 5: purified product after the final purification step (gel filtration chromatography). B Determination of the molecular weight of hEGF and EGFt by mass spectrometry (MALDI-TOF). The analysis confirmed the corresponding molecular weight of the hEGF (6216.6 Da) and EGFt (5087.9 Da). C Analysis of the state of folding of purified hEGF and EGFt by RP-HPLC. The peaks on the chromatogram correspond to the elution time of well-folded hEGF.</p
Ribbon diagram of the crystal structure of an EGFR homodimer in complex with two EGF ligands (from pdb code 1IVO).
<p>A The subdomains I, II, III and IV of both receptors are colored pink, orange, cyan and purple, respectively. Held between domains I and III of each receptor are the EGF ligands colored red and their disulfide bridges yellow. The three main interactions sites between EGF and EGFR are outlined (1, 2 and 3). The interaction site 3 between C-terminal part of EGF and domain III of EGFR is magnified. This binding site is composed by two different kinds of interactions: hydrophobic interactions between Leu47 (EGF) and Leu382, Phe412, and Ile438 (EGFR), and the formation of hydrogen bonds with Gln384 side chain of EGFR and carbonyl and amide groups of Gln43 and Arg45, respectively, of EGF. All side chains of residues responsible for these interactions are shown in different colors depending on the type of interaction. B Ribbon diagram and amino acid sequence of EGF. The C-terminal part of the molecule is highlighted in black and the side chain of Leu47 is shown in orange in the ribbon diagram. The nucleotides that encode these 8 amino acids were removed from the encoding sequence to construct the EGF truncated form (EGFt).</p
Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells
A straightforward
methodology for the synthesis of conjugates between
a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted
anticancer compounds, is described. Under microwave irradiation, the
imidazole ligand incorporated on the aminoglycoside moiety (neamine
or neomycin) was found to replace one triphenylphosphine ligand from
the ruthenium precursor [(Ī·<sup>6</sup>-<i>p</i>-cym)ĀRuClĀ(PPh<sub>3</sub>)<sub>2</sub>]<sup>+</sup>, allowing the assembly of the target
conjugates. The guanidinylated analogue was easily prepared from the
neomycin-ruthenium conjugate by reaction with <i>N</i>,<i>N</i>ā²-di-Boc-<i>N</i>ā³-triflylguanidine,
a powerful guanidinylating reagent that was compatible with the integrity
of the metal complex. All conjugates were purified by semipreparative
high-performance liquid chromatography (HPLC) and characterized by
electrospray ionization (ESI) and matrix-assisted laser desorptionāionization
time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy.
The cytotoxicity of the compounds was tested in MCF-7 (breast) and
DU-145 (prostate) human cancer cells, as well as in the normal HEK293
(Human Embryonic Kidney) cell line, revealing a dependence on the
nature of the glycoside moiety and the type of cell (cancer or healthy).
Indeed, the neomycināruthenium conjugate (<b>2</b>) displayed
moderate antiproliferative activity in both cancer cell lines (IC<sub>50</sub> ā 80 Ī¼M), whereas the neamine conjugate (<b>4</b>) was inactive (IC<sub>50</sub> ā 200 Ī¼M). However,
the guanidinylated analogue of the neomycināruthenium conjugate
(<b>3</b>) required much lower concentrations than the parent
conjugate for equal effect (IC<sub>50</sub> = 7.17 Ī¼M in DU-145
and IC<sub>50</sub> = 11.33 Ī¼M in MCF-7). Although the same
ranking in antiproliferative activity was found in the nontumorigenic
cell line (<b>3</b> ā« <b>2</b> > <b>4</b>), IC<sub>50</sub> values indicate that aminoglycoside-containing
conjugates are about 2-fold more cytotoxic in normal cells (e.g.,
IC<sub>50</sub> = 49.4 Ī¼M for <b>2</b>) than in cancer
cells, whereas an opposite tendency was found with the guanidinylated
conjugate, since its cytotoxicity in the normal cell line (IC<sub>50</sub> = 12.75 Ī¼M for <b>3</b>) was similar or even
lower than that found in MCF-7 and DU-145 cancer cell lines, respectively.
Cell uptake studies performed by ICP-MS with conjugates <b>2</b> and <b>3</b> revealed that guanidinylation of the neomycin
moiety had a positive effect on accumulation (about 3-fold higher
in DU-145 and 4-fold higher in HEK293), which correlates well with
the higher antiproliferative activity of <b>3</b>. Interestingly,
despite the slightly higher accumulation in the normal cell than in
the cancer cell line (about 1.4-fold), guanidinoneomycināruthenium
conjugate (<b>3</b>) was more cytotoxic to cancer cells (about
1.8-fold), whereas the opposite tendency applied for neomycināruthenium
conjugate (<b>2</b>). Such differences in cytotoxic activity
and cellular accumulation between cancer and normal cells open the
way to the creation of more selective, less toxic anticancer metallodrugs
by conjugating cytotoxic metal-based complexes such as rutheniumĀ(II)
arene derivatives to guanidinoglycosides
Effect of EGFt treatment on EGFR degradation.
<p>A MCF-7 and Caco-2 cycloheximide treated cells were incubated with 3 nM, 150 nM hEGF or 150 nM EGFt at 37Ā°C for different time periods as indicated. Cells were lysed, and the amount of EGFR was determined by Western blotting. Actin levels were used as loading control. B Levels of EGFR were determined by densitometry and normalized versus actin levels. Each column represents the mean of two replicates Ā± SEM.</p
Effect of EGFt on the internalization and localization of EGFR compared to hEGF.
<p>A MCF-7 and Caco-2 cells were treated with 3 nM, 150 nM hEGF or 150 nM EGFt for 30 min at 4Ā°C and then incubated at 37Ā°C for 15 min. The detection of EGFR in the cell membrane was determined by performing a cell-ELISA assay with specific antibodies. Each column in the graph represents the relative EGFR expression in the cell membrane versus untreated control cells (CTL) and was the mean Ā± SEM of three independent experiments (*P<0.05 vs. control cells). B MDA-MB-468 cells were exposed for various times to 150 nM hEGF or 150 nM EGFt and stained for EGFR using FITC anti-EGFR antibody (green). The nucleus and its membrane were stained using Hoescht (blue) and Cy3 anti-lamin B1 antibody (red), respectively. Confocal images were acquired. C The merge images corresponding to 180 min of treatment were magnified and some slices of a merged <i>xz</i> reconstruction of the stack (slices at 0.3 microns on z axis) are shown. Arrows indicate green signals within the cell nucleus. Scale bar, 10 Ī¼m.</p
Representative binding curve for <sup>111</sup>In-DTPA-EGFt to MDA-MB-468 cells.
<p>The total binding curve was obtained by incubating cells with increasing concentration of <sup>111</sup>In-labeled EGFt (0ā300 nM). The non-specific binding curve was obtained by adding increasing concentrations of the <sup>111</sup>In-DTPA-EGFt in the presence of 100-fold excess of unlabeled EGFt (30 Ī¼M). The specific binding curve was obtained by subtracting non-specific binding from total binding. The dissociation constant (K<sub>d</sub>) for <sup>111</sup>In-labeled EGFt measured in this assay was 60.05Ā±2.03 nM. Each point represents the mean Ā± SEM of 3 assays performed in triplicate.</p
Design, Preparation, and Characterization of Zn and Cu Metallopeptides Based On Tetradentate Aminopyridine Ligands Showing Enhanced DNA Cleavage Activity
The conjugation of redox-active complexes
that can function as chemical nucleases to cationic tetrapeptides
is pursued in this work in order to explore the expected synergistic
effect between these two elements in DNA oxidative cleavage. Coordination
complexes of biologically relevant first row metal ions, such as ZnĀ(II)
or CuĀ(II), containing the tetradentate ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane
(<sup>Me2</sup>PyTACN) and (2<i>S</i>,2<i>S</i>ā²)-1,1ā²-bisĀ(pyrid-2-ylmethyl)-2,2ā²-bipyrrolidine
((<i>S,S</i>ā²)-BPBP) have been linked to a cationic
LKKL tetrapeptide sequence. Solid-phase synthesis of the peptide-tetradentate
ligand conjugates has been developed, and the preparation and characterization
of the corresponding metallotetrapeptides is described. The DNA cleavage
activity of Cu and Zn metallopeptides has been evaluated and compared
to their metal binding conjugates as well as to the parent complexes
and ligands. Very interestingly, the oxidative Cu metallopeptides <b>1</b><sub><b>Cu</b></sub> and <b>2</b><sub><b>Cu</b></sub> show an enhanced activity compared to the parent complexes,
[CuĀ(PyTACN)]<sup>2+</sup> and [CuĀ(BPBP)]<sup>2+</sup>, respectively.
Under optimized conditions, <b>1</b><sub><b>Cu</b></sub> displays an apparent pseudo first-order rate constant (<i>k</i><sub>obs</sub>) of ā¼0.16 min<sup>ā1</sup> with a supercoiled
DNA half-life time (<i>t</i><sub>1/2</sub>) of ā¼4.3
min. On the other hand, <i>k</i><sub>obs</sub> for <b>2</b><sub><b>Cu</b></sub> has been found to be ā¼0.11
min<sup>ā1</sup> with <i>t</i><sub>1/2</sub> ā
6.4 min. Hence, these results point out that the DNA cleavage activities
promoted by the metallopeptides <b>1</b><sub><b>Cu</b></sub> and <b>2</b><sub><b>Cu</b></sub> render ā¼4-fold
and ā¼23 rate accelerations in comparison with their parent
Cu complexes. Additional binding assays and mechanistic studies demonstrate
that the enhanced cleavage activities are explained by the presence
of the cationic LKKL tetrapeptide sequence, which induces an improved
binding affinity to the DNA, thus bringing the metal ion, which is
responsible for cleavage, in close proximity