Table 1.
Amino acid sequences of peptides used in this study.
Figure 1.
BR2 efficiently translocates into various cancer cells without cytotoxicity to normal cells.
(A) Intracellular distribution of FITC-labeled peptides examined by confocal laser microscopy. Both cancer cells (HeLa, HCT116 and B16/F10) and normal cells (HaCat, BJ and NIH 3T3) were seeded in 6-well plates 1 day prior to the experiment to reach 70% confluence. Cells were incubated with FITC-labeled peptides (5 µM) for 30 min at 37°C and washed three times with phosphate buffered saline (PBS). Peptide distribution was then analyzed using a confocal scanning LSM 510 laser microscope equipped with a 40× objective. (B,C) In vitro cytotoxicity of peptides. (B) Membrane disturbance was measured by lactate dehydrogenase (LDH) leakage from the indicated cell lines 24 h after peptide treatment. LDH leakage from cells seeded in a 96-well plate at 10,000 cells/well was measured after exposure to 1, 2, 5, 10, 20, 50 or 100 µM peptides for 24 h. LDH release from PBS treated cells was regarded as 0% leakage and LDH released from 0.2% Triton X-100 treated cells as 100% leakage. (C) The hemolytic activity of each peptide against human erythrocytes was analyzed at graded concentrations (0–200 µM) and compared to a 0.2% Triton X-100 positive control, for which hemolysis was defined as 100%. Error bars in all figures represent the standard errors of the means (n = 3).
Figure 2.
BR2 specifically penetrates cancer cell membranes in a concentration-dependent manner.
(A) Analysis of the cell-penetrating efficiency of each peptide in different cell types by flow cytometry. FITC-labeled Tat, BR1 and BR2 peptides (10 µM) were added to different cell types: HeLa, HCT116 and B16/F10 cancer cells and HaCat, BJ and NIH 3T3 normal cells. After 30 min of incubation at 37°C, the FITC-positive cells were counted by flow cytometry. Values represent the percentage of fluorescence-positive cells in the total cell population. (B) Quantitative assessment of cell penetration of each peptide by flow cytometry. HeLa cells were incubated with FITC-labeled peptides at concentrations of 1, 2, 5, or 10 µM for 30 min at 37°C. Afterwards the cells were washed with cold PBS and harvested and cellular fluorescence was analyzed by flow cytometry. Control cells did not receive peptide treatment. Prior to analysis, extracellular fluorescence of surface bound peptides was removed by a trypsin treatment (1 mg/ml for 10 min).
Figure 3.
Contribution of energy-dependent pathways and negatively charged molecules on cancer cell membranes to peptide internalization.
(A) Effects of low temperature and energy depletion on the internalization of FITC-labeled peptides into HeLa cells. HeLa cells were either preincubated at 4°C or pretreated with sodium azide (NaN3) to deplete ATP for 1 h, and then incubated with 5 µM Tat or BR2 for 30 min under the same conditions, as described in Materials and Methods. Peptide uptake was determined by flow cytometry. (B) Effects of endocytic inhibitors on the entry of BR2 and Tat. The influence of inhibitory drugs on peptide uptake was determined by preincubation of HeLa cells with the endocytosis inhibitors nocodazole, amiloride or methyl-ß-cyclodextrin for 1 h prior to the addition of FITC-labeled peptides. After peptide treatment for 30 min at 37°C, FITC-positive cells were counted by flow cytometry. Values represent the percentage of fluorescence-positive cells in the total cell population. Data represent the mean ± s.d. of three independent experiments. (C) Colocalization of BR2 with the lysosomal marker LysoTracker red DND-99 in living HeLa cells. After 30 min incubation of BR2 (5 µM) with LysoTracker, live HeLa cell images were obtained by confocal microscopy. (D) Effects of negatively charged molecules (gangliosides, heparins, and sialic acids) on peptide uptake. (a,b) HeLa cells were treated with BR2 and Tat in the presence of gangliosides, heparins, or sialic acids (each, 20 µg/ml) for 30 min. In (c,d), HeLa cells were pretreated with PPMP (5 µM) to deplete gangliosides. Cellular uptake of BR2 and Tat was determined by flow cytometry. All experiments were performed in triplicate.
Figure 4.
Intracellular uptake of peptides and anti-Ras scFv fusion proteins and their anti-proliferative activity.
(A) Schematic representation of peptide-anti-Ras scFv cDNA constructs. DNA encoding peptides and the Y13-259-scFv cDNA were fused as described in Materials and Methods and cloned into the NcoI and EcoRI sites of pET21c. The white boxes represent VH and VL of the Y13-259 scFv sequence. The round box indicates the sequence encoding the peptides: Tat or BR2. The NcoI and EcoRI restriction sites and stop codon positions are also indicated. (B) Protein uptake was analyzed by Western blotting of fractionated lysates from HCT116 cells treated with PBS, anti-Ras scFv, BR2- or Tat-scFv fusion protein (each, 2 µM) at 37°C for 2 h. An anti-His antibody was used to detect intracellular Tat-scFv and BR2-scFv (28-kDa). (C) The anti-proliferative activity of peptides and anti-Ras scFv fusion proteins. HCT116 cells were exposed to the indicated concentrations of anti-Ras scFv, Tat- or BR2-scFv fusion protein at 37°C for 24 h. Cell proliferation was determined using the MTT assay. Data represent the mean ± s.d. of three independent experiments.
Figure 5.
The BR2-anti-Ras scFv fusion protein promotes apoptosis by blocking Ras signaling in Ras-mutated HCT116 cells.
(A) HCT116 cells were treated with anti-Ras scFv, Tat- or BR2-scFv fusion protein (each, 2 µM) or staurosporine (0.5 µM) for 24 h. Cells were stained with Annexin V-FITC and 7AAD to allow detection of apoptotic cell fractions by flow cytometry. The lower left quadrant contains the live cell (double negative) population; the lower right contains the apoptotic (annexin V+/7AAD-) population; the upper right contains the late apoptotic/necrotic (annexin V+/7AAD+) population; and the upper left contains the pre-necrotic (annexin V−/7AAD+) population. The numbers on the top of the quadrants indicate the percentage of apoptotic and late apoptotic/necrotic cells counted from dot plots taken from one representative experiment, performed in triplicate. (B) 24 h after PBS, anti-Ras scFv, Tat- or BR2-scFv fusion protein (each, 2 µM) or staurosporine (0.5 µM) treatment, HCT116 cell extracts were subjected to Western blot analysis with anti-PARP or anti-α-tubulin antibodies. The molecular sizes of the proteins are indicated with arrows at the right. α-tubulin is shown as a control. (C) Ras activation assay. Changes in Ras activity level in HCT116 cells treated with anti-Ras scFv and BR2- and Tat-scFv fusion proteins (each, 1 and 2 µM) were determined by an ELISA-based activity assay; results are expressed as relative Ras activity (%).