External or internal localization of Gm and analogues.

<p>B16 cells were incubated with 2 µM of the biotin labeled peptides for different times. Images correspond to a single focal plane performed by confocal microscopy. External membrane peptides were labeled with streptavidin-Alexa Fluor 488 conjugated while internal membrane peptides were labeled with streptavidin-Alexa Fluor 647 conjugated. Unviable cells were excluded using PI stain. Nuclei were stain with DAPI. (<b>A</b>) Typical image of labeled Gm is showed. The images XY, XZ and YZ correspond to a single plane. Typical images obtained with (<b>B</b>) Gm, (<b>C</b>) [D-Thr^2,6,11,15]-D-Gm, (<b>D</b>) [D-Thr^2,6,11,15, Pro⁹]-D-Gm, (<b>E</b>) [Thr^2,6,11,15, D-Pro⁹]-Gm, (<b>F</b>) [Ser^2,6,11,15]-Gm and (<b>G</b>) [Thr^2,6,11,15]-Gm are shown.</p

Substitution of some amino acid residues in the Gm structure reduces the cytotoxic ability, but did not modify the cell death mechanism.

<p>B16 cells were stimulated by Gm and analogues for 24 h. (<b>A</b>) Cytotoxic activities of Gm and its analogues were quantified by the MTT reduction test. (<b>B</b>) Cell death type identification caused by Gm was evaluated using annexin-V and 7-AAD assay by flow cytometry using the IC₅₀ values. (<b>C-H</b>) Apoptosis (Z-VAD) and necroptosis (necrostatin) inhibitors were unable to reduce cell death induced by Gm and analogues. Cells were incubated with inhibitors for 1 h before to stimulation with the peptides (IC₅₀ values for each peptide was used) and the viability was assessed by the MTT. Results are the means ± SEM of three independent experiments preformed in duplicate.</p

Potencies and efficacies of gomesin and its analogues.

*<p>To quantify internal or external amount fluorescence biotinylated peptides were used.</p><p># Starting peptide concentration (10⁻⁴ M) was considered as 100%.</p

Primary structure of gomesin and its analogues.

a<p>Z  =  pyroglutamic acid, lowercase letters denote D-amino acids, X  =  Rh-Lys, Rh =  rhodamin; B  =  B₁₂-Lys, B₁₂ =  biotin. ^bThe observed m/z of the unresolved peak was compared with the calculated [M + H]⁺ average mass in Da.^cPercent purity as determined by HPLC analysis performed on a Waters Nova-Pak C₁₈ (2,1×150 mm, 60 Å, 3,5 µm); UV detection at 214 nm; 0.4 mL/min flow rate; [A]  =  0.1% TFA in H₂O and [B]  =  0.1% TFA in 60% MeCN/H2O; gradient  =  5–95%B in 30 min.</p

Lysosomal enzymes participate in the cytotoxic reduction activities of the Gm analogues.

<p>(<b>A, C and D</b>) Cell viability was assessed by the MTT reduction test. (<b>A</b>) Cells were incubated with cytoskeletal inhibitor (cytochalasin D), and lysosomal inhibitor (chloroquine) for 1 h before to stimulation by Gm, and its analogues for 24 h. Chloroquine potentiated the cytotoxicity of peptides. (<b>B</b>) Since chloroquine was able to potentiate the cytotoxicity of the peptides, resistance to degradation of lysosomal enzymes was evaluated by LC/ESI-MS. Peptides were incubated at 37°C for different times. (<b>C</b>) Cells were incubated with the IC₅₀ concentration for each peptide. Cytotoxic activities after 24 and 72 h of the peptide incubation were compared.</p

Effect of steroid hormones (E₂ and P₄) on the viability of leiomyoma and myometrial adjacent cells.

<p>(A) Leiomyoma cells (5 × 10³) and myometrial adjacent cells were treated with E₂ (100 nmol/L) and P4 (100 nmol/L) for 24 h in 96-well microtiter plates. Cell viability was assessed by the MTT reduction test. (B) Phase contrast–confluent culture of leiomyoma and myometrial adjacent cells after treat with E₂ (100 nmol/L) and P₄ (100 nmol/L). The statistical significance was evaluated using one-way ANOVA followed by the Tukey's test. A p-value of ≤0.05 was considered to indicate significance (*). These experiments were performed with cultured primary cells from specimens collected from patients.</p

Influence of Protein Corona on the Transport of Molecules into Cells by Mesoporous Silica Nanoparticles

Although there are several studies reporting the promising biological efficiency of mesoporous silica nanoparticles (loaded with antitumoral drugs) against cancer cells and tumors, there are no reports on the influence of the bio–nano interface interactions on the molecular diffusion process occurring along their pores. In this context, we show here that the protein coating formed on multifunctionalized colloidal mesoporous silica nanoparticles (MSNs) dispersed in a cell culture medium decreases the release of camptothecin (CPT, a hydrophobic antitumoral drug) from the pores of MSNs. This effect is related to the adsorption of biomolecules on the nanoparticle surface, which partially blocks the pores. Parallely, the hydrophobic functionalization inside the pores can offer suitable sites for the adsorption of other molecules present in the cell culture medium depending on the hydrophobicity, size, and conformation aspects of these molecules and adsorption sites of MSNs. Thus, the molecular cargo loaded in the pores (i.e. CPT) can be replaced by specific molecules present in the dispersion medium. As a consequence, we show that a non-permeable cellular staining molecule such as SYTOX green can be incorporated in MSNs through this mechanism and internalized by cells in an artificial fashion. By extrapolating this phenomenon for applications in vivo, one has to consider now the possible manifestation of unpredicted biological effects from the use of porous silica nanoparticles and others with similar structure due to these internalization aspects

Characteristics of Samples Used in the Study.

<p>Characteristics of Samples Used in the Study.</p

Detection of signaling phosphoproteins by Immunoblot analysis in leiomyoma and myometrial adjacent cells.

<p>Leiomyoma and myometrial adjacent cells (5 x 10⁵) were treated with E₂ (100 nmol/L) and P₄ (100 nmol/L) for 16 h in 6-well microtiter plates; lysate proteins were separated by 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked and incubated with rabbit primary antibodies, (A and C) anti-phospho-Src (Tyr-416), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti- phospho-Erk1/2 MAPK, anti-Erk1/2 MAPK, (E) anti-p130Cas Y165, (F) anti-p130Cas Y410, (H and J) anti-phospho-Akt, anti-Akt, and anti-β-actin. (B, D, G, I, and L) Graph bars represent the densitometric analyses of the immunoblotting results. The results are represented as band intensities in arbitrary units relative to the respective total phopho-proteins load and total control (β-actin) load. Antibody binding was visualized by chemiluminescence, and the relative levels of these proteins were determined by the densitometric analyses. These experiments were performed with cultured primary cells from specimens collected from patients (*p<0.01).</p

Immunophenotype of leiomyoma and myometrial cells from women with myoma uterine.

<p>Adhered and spread cells after tissue disaggregation in collagenase following with primary explants. Cells are shown under phase contrast microscopy and indirect immunofluorescence for phalloidin, fibronectin-integrin β1/CD29, vimentin, and DAPI (blue, for nuclei). (A-B) Phase contrast in the confluent culture of leiomyoma cells after three days. (A) Low density (B) high density (magnification, ×400). Mycoplasma contamination was not observed in any of the processed tissues. (C) Log-phase growth rate by cell counting–growth characteristics of leiomyoma cells, myometrial adjacent cells, and co-cultured myometrial adjacent (as feeders) with leiomyoma cells (on plastic surface). (D-F) Analysis of myometrial markers by confocal microscopy; vimentin and fibronectin integrin β1/CD29. (D) Cytoskeletal organization (Phalloidin, Alexa-594-red); (E) integrin β1/CD29 (FITC-488, green); and (F) Co-localization integrin β1(FITC-488, green) and vimentin (Alexa-594-red). Bar, 10 μm.</p