Table 1.
Names and amino acid sequences of DRS-B2 analogs.
Table 2.
Concentration of DRS-B2 that inhibits 50% cell growth (GI50) extracted from S2 Fig.
Proliferation of each cell type was performed in plastic 24-well plates (1.91 cm2; cell density of 1x104 cells/well/0.5 mL). On the first and third day after plating, the cells were treated with DRS-B2 at different concentrations ranging from 0 to 10 μM. Twenty four hours after the last treatment cell counting was performed with crystal violet staining as described in Materials and Methods section. Results are represented in peptide concentration inhibiting 50% of the cell growth GI50 ± SD of at least three determinations.
Fig 1.
Immunostaining of the membrane of PC3 cells after treatment with [Alexa594]—(Cys0) -DRS-B2.
PC3 cells were treated with 2.5 μM [Alexa594]—(Cys0) -DRS-B2 (red) for either 5 min. (1A-C) or 1 hour (2A-D). After fixation, the membrane was visualized by the anti-CD147 antibody labeled with an anti-mouse antibody A488 (green) and nuclei by DAPI (blue). The confocal images were obtained using an Olympus IX81 inverted microscope (60x oil immersion objective NA 1.25) with a spinning disk confocal system DSU (Olympus), coupled to a CCD camera R2 Orca (Hamamatsu Corporation, Japan). Image processing was performed using the ImageJ software, and the program Zoom in Images and Stack. z is the distance from the base to the top of the cell in 0.5 μm steps. The boxes are zoom lenses, the zoom factor = 2.
Fig 2.
Immunostaining of the membrane of U87MG cells after treatment by [Alexa594]—(Cys0) -DRS-B2.
U87MG cells were treated with 2.5 μM of [Alexa594]—(Cys0) -DRS-B2 (red) for either 5 min. (1A-D) or 1 hour (2A-C). After fixation, the membrane was visualized using anti CD147 antibody labeled with an anti-mouse antibody A488 (green) and nuclei by DAPI (blue). The confocal images were acquired using Olympus IX81 inverted microscope (60x oil immersion objective NA 1.25) with a spinning disk confocal system DSU (Olympus), coupled to a CCD camera R2 Orca (Hamamatsu Corporation, Japan). Image processing was performed using the program Zoom in Images and Stack. z is the distance from the base to the top of the cell in 0.5 μm steps. The boxes are zoom lenses, the zoom factor = 2.
Fig 3.
PC3 and U87MG cell viability after DRS-B2 treatment.
PC3 and U87MG cells were treated with DRS-B2 (2.5 μM) for one hour. Then cells where double stained with FITC-Annexin-V (A-V) and 7-AAD and analyzed by flow cytometry. The cell viability was observed by measuring the amount of A-V and 7-AAD negative and positive cells. Counter diagrams show an example of A-V/7-AAD double staining of PC3 cell control (A), PC3 treated with DRS-B2 (2.5 μM) (B), U87MG cell control (C) and U87MG treated with DRS-B2 (2.5 μM) (D).
Table 3.
Mean amount of cells per staining in % extracted from Fig 3.
PC3 or U87MG cells were treated with DRS B2 (2.5 μM). One hour after treatment cells where double stained with FITC-Annexin-V and 7-AAD and analyzed by flow cytometry. The cell viability of cells was observed by measuring the amount of Annexin-V and 7-AAD negative and positive cells. Results represent the mean ± SEM of 3 determinations of A-V-/7-AAD- = FITC-Annexin-V and 7-ADD negative; A-V-/7-AAD+ = FITC-Annexin-V negative and 7-AAD positive; A-V+/7-AAD- = FITC-Annexin-V positive and 7-AAD negative; A-V+/7-AAD+ = FITC-Annexin-V and 7-AAD positive.
Fig 4.
(A) Saturation binding curves of increasing concentration of biotinylated DRS-B2 on PC3 cells. In the presence (black triangles) or absence (black squares) of excess DRS-B2 (100 times more concentrated) with representation of the resulting specific binding (black dots). (B) Inhibition of biotinylated DRS-B2 (1 μM) binding to PC3 cells with increasing concentrations of DRS-B2.
Fig 5.
(A) In vitro anti-proliferative effect of PC3 cells. In presence of DRS-B2 (black bars) or in presence of its analog [all. D]-DRS-B2 (white bars). On the first and third day after plating, the cells were treated with different concentrations of peptides. Twenty-four hours after the last treatment cell counting was performed with crystal violet staining. Results are expressed in percent of cell viability per well. Results represent the mean ± SEM of three determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (untreated cells). (B) In vitro anti-proliferative effect of U87MG cells. In presence of DRS-B2 (black bars) or in presence of its analog [all. D]-DRS-B2 (white bars). Cells were treated as described for Fig 5A.
Fig 6.
Percentage of living PC3 cells in the presence of different concentrations of sodium chlorate (0–80 mM).
PC3 cells were first seeded in a 24-well plate as previously described and on the second day of incubation, increasing concentrations of sodium chlorate (0 to 80 mM) were added and crystal violet staining was performed on the fourth day. Results are expressed in percent of cell viability per well. Results represent the mean ± SEM of three determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (untreated cells).
Fig 7.
Percentage of living PC3 cells in presence of DRS-B2 and sodium chlorate.
Cells were seeded and treated on the second day with 10 mM sodium chlorate as described for Fig 6. Experiments were performed in the absence or presence of 3 different concentrations of DRS-B2 (2.5, 5 or 10 μM) in the absence (light gray bars) or presence (dark gray bars) of sodium chlorate (10 mM) crystal violet staining was performed on the fourth day after seeding. Results are expressed in percent of cell viability per well. Results represent the mean ± SEM of three determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (untreated cells).
Fig 8.
Percentage of living PC3 cells in presence of DRS-B2 and CS-C.
Cells were seeded as previously described in Fig 5A; experiments were performed in presence of different concentrations of CS-C (0–3.3 nM) alone or in presence of DRS-B2 at different concentrations (0–10 μM). Crystal violet staining was performed on the fourth day after seeding. Results are expressed in percent of cell viability per well. Results represent the mean ± SEM of three determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (untreated cells).
Fig 9.
Percentage of cytotoxicity of PC3 cells in presence of DRS-B2, sodium chlorate and CS-C.
Experiments were performed in presence of DRS-B2 (2.5 μM) alone or in the presence of sodium chlorate (10 mM) and with or without different concentrations of CS-C (0–3.3 nM). LDH release was measured with CytoTox 96 kit 3 hours after treatment. Results are expressed in percentage of cytotoxicity versus time of treatment (see Materials and methods section). Results represent the mean ± SEM of 3 determinations. * p < 0.05; ** p < 0.01 ***; p < 0.001 versus treatment with DRS-B2 alone.
Fig 10.
Fluorescence emission spectra of Trp of DRS-B2 (2 μM).
In the absence or in the presence of increasing concentrations of CS-C (0.2–10 μM).
Fig 11.
Circular dichroism spectrum of the DRS-B2 (10 μM).
In water or in the presence of increasing concentration of CS-C: (1–20 μM).
Fig 12.
Effect of increasing concentrations of DRS-B2 or [W/F]3-DRS-B2 on proliferation of PC3 cells.
On the first and third day after plating, the cells were treated with different concentrations of peptides. Twenty-four hours after the last treatment cell counting was performed with crystal violet staining. Results are expressed in percent of cell viability per well. Results represent the mean ± SEM of three determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (untreated cells).
Table 4.
Concentration of DRS-B2 and [W/F]3-DRS-B2 that inhibits 50% of cell growth (GI50) extracted from Fig 12.
Fig 13.
Percentage of living PC3 cells in the presence of different concentrations of CS-C (0–3.3 nM).
Experiments were performed without peptide or in the presence of DRS-B2 (1 μM) or [W/F]3DRS-B2 (5 μM). On the first and third day after plating, the cells were treated with or without peptides and different concentrations of CS-C. Twenty-four hours after the last treatment cell counting was performed with crystal violet staining. Results are expressed in percent of cell viability per well. Results represent the mean ± SEM of three determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (untreated cells).
Table 5.
Quantitative analysis of DRS-B2 and [W/F]3-DRS-B2 in a Far UV circular dichroism spectra by Neural Networks (16).