Human cervical cancer cell line HeLa, human colon cancer cell line HCT116, mouse melanoma cell line B16/F10, mouse fibroblast cell line NIH 3T3, human keratinocyte cell line HaCat and human fibroblast cell line BJ were all obtained from American Type Culture Collection (ATCC; Manassas, VA) and cultured in a complete medium [Dulbecco’s modified eagle medium] (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin. Cells were grown in humidified conditions at 37°C with 5% CO2.
Peptide Design and Synthesis
We designed several derivatives of buforin IIb (BR3) by stepwise elimination of the C-terminal regular α-helical motif RLLR repeats of buforin IIb to create a cancer cell specific and non-toxic CPP. The designed peptides consisted of different numbers of the C-terminal regular α-helical motif RLLR and named BR1 and BR2 ().
Amino acid sequences of peptides used in this study.
CPPs Tat, BR1, BR2, and BR3 were chemically synthesized (Anygen, Kwangju, Korea) on a MilliGen 9050 peptide synthesizer. The fluorescein moiety (FITC) was attached to the N-terminus via an aminohexanoic acid spacer by treating a resin-bound peptide (0.1 mM) with FITC (0.1 mM) and diisopropyl ethyl amine (0.5 mM) in N, N-Dimethylformamide (DMF) for 12 h. All crude peptides were purified and analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) on a C18 column, and the purified peptides were characterized by electrospray ionization mass spectrometry (ESI-MS).
Confocal Laser Scanning Microscopy
To investigate the cell-penetrating ability and the intracellular distribution of the internalized peptides, live confocal microscopy was performed on three cancer lines (HeLa, HCT116 and B16/F10) and three normal cell lines (HaCat, BJ and NIH 3T3). Briefly, cells (2×105
) were plated on a glass coverslip placed in a 6-well plate, grown overnight, and then incubated with FITC-labeled peptides (5 µM for each cell line) for 30 min. The cells were then rinsed three times with phosphate buffered saline (PBS, pH 7.4), and mounted on microscope slides with fluorescence mounting solution (Dako Corp, Carpinteria, CA). Colocalization of BR2 with lysosomes was observed by using LysoTracker Red DND-99 (Molecular Probe, Eugene, OR). To avoid the effects of fixation artifacts, involving both methanol and paraformaldehyde, cells were not fixed 
. The distribution of FITC-labeled peptides was analyzed using a confocal scanning laser Zeiss LSM 510 microscope (Jena, Germany) equipped with a 40× and 20× objective. Fluorophores were excited with an argon laser (488 nm) for FITC and a HeNe laser (543 nm) for LysoTracker Red.
In vitro Cytotoxicity Assay
The cytotoxicity of peptides to mammalian cells was investigated by assessing the release of lactate dehydrogenase (LDH) from cancer and normal cells. The amount of LDH released from damaged cells into the supernatant was measured using the Cytotoxicity Detection Kit (Roche Applied Science, Germany) according to the manufacturer’s instructions. In brief, cells were plated onto 96-well microplates (1×104 cells per well) in complete DMEM supplemented with 10% FBS and incubated overnight at 37°C to allow for attachment and spreading of cells. After 24 h of incubation, cells were treated with various concentrations of peptides (0–100 µM) and incubated for another 24 h at 37°C. The extracellular medium from each well was transferred to a new microplate and incubated for 10 min with 100 µl/well reaction mixture, followed by a stop solution. Absorbance was measured at 490 nm by using an ELISA plate reader. LDH release from cells lysed with 0.2% Triton X-100 in PBS was defined as 100% leakage and LDH release from untreated cells as 0% leakage.
Hemolytic activity was assayed as described by Aboudy et al.
with slight modifications 
; 3 ml of freshly prepared human erythrocytes was washed with isotonic PBS, pH 7.4, until the color of the supernatant turned clear. The washed erythrocytes were then diluted to a final volume of 20 ml with the same buffer. Peptide samples (10 µl), serially diluted in PBS, were added to 190 µl of the cell suspension in microcentrifuge tubes. Following gentle mixing, the tubes were incubated at 37°C for 30 min and then centrifuged at 4,000×g for 5 min. The supernatant (100 µl) was removed to a new tube and the absorbance at 567 nm was determined. The relative optical density, as compared with that of the cell suspension treated with 0.2% Triton X-100, was defined as percentage of hemolysis. The hemolysis percentage was calculated using the following equation: percentage hemolysis
[(Abs 567 nm
in the peptide solution – Abs 567 nm
in PBS)/(Abs 567 nm
in 0.2% Triton X-100 – Abs 567 nm
Characterization of Peptide Uptake
To evaluate the internalization of FITC-labeled peptides, HeLa cells were seeded onto 12-well plates at a density of 2×105 cells per well and incubated for 24 h. FITC-labeled peptides, at various concentrations ranging from 2 to 10 µM, were then incubated with the cells for 30 min at 37°C. To compare the cellular uptake of peptides, cancer and normal cells were treated with FITC-labeled peptides (each, 10 µM) and incubated for 30 min at 37°C. Following the incubation, cells were washed three times with ice-cold PBS to remove excess extracellular complexes. Next, the cells were treated with trypsin (1 mg/ml) for 10 min to remove any remaining peptides bound to the cell surface. After trypsinization, the cells were collected by centrifugation (1,000×g for 5 min), resuspended with 500 µl ice-cold 2% FBS/PBS containing propidium iodide (PI), and then immediately analyzed (10,000 events/sample) by fluorescence activated cell sorting (FACS).
To understand further the cell-penetrating mechanism of peptides, the effects of temperature and metabolic inhibitors were examined. To elucidate the temperature dependency, HeLa cells were incubated at 4°C for 30 min prior to the addition of the peptides. Next, cells were treated with FITC-labeled peptides (each, 5 µM) at 4°C for 30 min. For the energy-depletion study, HeLa cells were preincubated with sodium azide (NaN3, 10 mM) at 37°C for 1 h and then incubated with FITC-labeled peptides (each, 5 µM) at 37°C for 30 min.
To study the role of endocytosis in peptide uptake, cells were pretreated with several endocytosis inhibitors at 37°C for 1 h: (i) amiloride (5 mM), which is known to block macropinocytosis by inhibiting a sodium channel; (ii) nocodazole (100 ng/ml), which inhibits the clathrin-mediated pathway; and (iii) methyl-ß-cyclodextrin (MßCD, 5 mM), which inhibits lipid raft-mediated processes by depleting cholesterol from the plasma membrane 
. All inhibitors were purchased from Sigma (St. Louis, MO).
To examine the effects of negatively charged components on the cell surface for peptide internalization, HeLa cells were pretreated with gangliosides (monosialoganglioside GM3 from canine blood), heparin sulfate, or sialic acid (Neu5Ac, all from Sigma) (20 µg/ml) for 30 min. For the inhibition of ganglioside biosynthesis, cells were pretreated with D-threo-1-phenyl-2-hexadecanoyl amino-3-morpholino-1-propanol (PPMP, 5 µM) for 48 h.
For all of these experimental conditions, flow cytometry analyses were performed with live cells using a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). In each case, the fluorescence of 10,000 viable cells was acquired. Viable cells were gated based on a sideward and forward scatter. For data analysis, WinMDI software (Joe Trotter, Scripps Research Institute, La Jolla, CA) was used. The statistical significance was evaluated by Student’s t-test at a 95% confidence interval.
Cloning and Expression of Peptide-fusion Proteins
To employ BR2 as a vehicle for the delivery of therapeutic proteins, a cDNA of the Y13–259 single-chain variable fragment (scFv) gene was synthesized at Bioneer (Daejeon, Korea). The Tat- or BR2-fused Y13–259 scFv genes were obtained by recombinant PCR. The recombinant cDNAs encoding the anti-Ras scFv, Tat-scFv and BR2-scFv fusions were digested with NcoI and EcoRI (both from New England Biolabs, Beverly, MA), and cloned into the NcoI and EcoRI sites of pET21c, producing pscFv, pTat-scFv and pBR2-scFv, respectively. Anti-Ras scFv, Tat-scFv and BR2-scFv fusion proteins were expressed in E. coli Origami (DE3) after induction with 0.1 mM IPTG for 4 h at 37°C. Cells were harvested by centrifugation at 3,000×g for 15 min at 4°C. The cell pellet was resuspended in phosphate buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4); cells were disrupted by sonication at 4°C (B. Braun instruments, Allentown, PA). The protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 1 mM) was added prior to sonication. The soluble and insoluble fractions were separated by centrifugation at 14,000×g for 15 min at 4°C. The pellet containing the inclusion bodies was resuspended in wash buffer (20 mM Tris–HCl, 5 mM EDTA and 1% Triton X-100, pH 8.0) and centrifuged at 8,000×g for 10 min at 4°C.
The washed inclusion bodies were denatured and solubilized in lysis buffer (0.3% N-lauroyl sarcosine, 50 mM CAPS buffer, and 0.3 M NaCl, pH 11.0) for 3 h and centrifuged at 14,000×g for 15 min at 4°C. All proteins were affinity-purified by using the Ni–IDA agarose resin (ELPIS biotech, Daejeon, Korea). In brief, the Ni-IDA His-Bind Resin was packed into a column equilibrated with binding buffer (identical to lysis buffer). The supernatant was slowly applied to the column, after which wash buffer (0.3% N-lauroyl sarcosine, 50 mM CAPS buffer, 150 mM NaCl, and 30 mM imidazole, pH 11.0) was applied. The proteins were eluted with elution buffer (0.3% N-lauroyl sarcosine, 50 mM CAPS buffer, 150 mM NaCl, and 250 mM imidazole, pH 11.0) and analyzed by 10% SDS–PAGE. The eluted proteins were refolded by dialysis in PBS containing 200 mM NaCl, 10% glycerol, 1 mM GSH, 0.2 mM GSSG and 0.4 M arginine with gradual pH reduction (pH 10, pH 9, pH 8 and pH 7.4). Each dialysis step was performed at 4°C for 12 h against 20× sample volume to remove the detergent completely.
To monitor the peptide-mediated intracellular uptake of scFv proteins, the internalized fusion proteins were examined by Western blotting. HCT116 cells (1×106
) were treated with PBS, purified scFv, Tat-scFv, or BR2-scFv fusion proteins (each, 2 µM) for 2 h at 37°C. The cells were then washed twice with PBS (4°C), scraped into 0.5 ml of PBS and centrifuged at 1,000×g at 4°C for 5 min. The cell pellets were resuspended in 100 µl of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2
EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3
, 1 µg/ml leupeptin and 1 mM PMSF (Cell Signaling Tech., Danvers, MA) and kept on ice for 30 min. The cell lysates were then centrifuged at 12,000×g at 4°C for 15 min and the supernatants were collected. Protein concentrations in the cell extracts were determined by the Bradford method 
(Bio-Rad, Hercules, CA). 50 µg of protein from cell extracts was fractionated on a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred onto a nitrocellulose membrane in transfer buffer (192 mM glycine, 25 mM Tris-HCl, pH 8.8, and 20% methanol [v/v]) by electroblotting. After blocking with 5% skim milk for 1 h, the membrane was incubated with a 1
1,000-diluted anti-His primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), washed three times with TTBS (50 mM Tris, 150 mM NaCl, and 0.5% Tween-20), and subsequently incubated with a peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Uppsala, Sweden) in milk containing TTBS for 1 h. After final washing, the membrane was then exposed and protein bands were detected using Enhanced Chemiluminescence (WESTSAVE GOLD; AbFrontier, Seoul, Korea).
Cell Proliferation Assays (MTT Assay)
To assess the anti-proliferative activity of peptides and anti-Ras scFv fusion proteins, HCT116 cells (K-ras mutated cells) were seeded in 96-well plates at a density of 2×104 cells/well in 100 µL of DMEM supplemented with 10% FBS and cultured for 24 h at 37°C. After 24 h of incubation, cells were treated with scFv or peptide-scFv fusion proteins (0, 0.5, 1 and 2 µM) and incubated for another 24 h. Cell viability was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using the CellTiter 96® Non-radioactive Cell Proliferation assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. The absorbance of the solution was measured at 570 nm using a Microplate Reader (Bio-Rad). Cell viability was expressed as the percentage of viable cells treated with scFv or peptide-scFv fusion proteins compared to the PBS-treated control (100%). All experiments were done in triplicate.
Detection of Apoptosis
HCT116 cells (1×106
/well in a 6-well plate) were treated with peptide-fused scFvs (each, 2 µM) or a well-known apoptosis inducer staurosporine (0.5 µM) for 24 h at 37°C and cell extracts were prepared as described above. Cleaved poly (ADP ribose) polymerase (PARP), an indicator of apoptosis, was detected by Western blotting as described above. Anti-PARP and anti-α-tubulin antibodies (Cell Signaling Tech.) were used at a 1
1,000 dilution as the primary antibodies, whereas horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, Uppsala, Sweden) was used at a 1
10,000 dilution as the secondary antibody.
In addition, apoptotic cells were identified by staining with annexin V-FITC (150 ng/ml) and 7 amino-actinomycin D (7-AAD; BD Biosciences, San Diego, CA). 1×106
HCT116 cells were treated with PBS, staurosporine (0.5 µM), or peptide-fused scFvs (each, 2 µM) as described above. At the designated time, cells were washed twice with PBS (pH 7.4), harvested and resuspended in 500 µl of binding buffer supplemented with Annexin-V fluos (1
100 diluted; BioBud, Seoul, Korea), and incubated for 15 min at 25°C in the dark. To detect necrosis, 7AAD was added prior to measurement. Samples were immediately subjected to FACS analysis with a FACSCalibur flow cytometer (FL1 and FL3) and WinMDI software (Joe Trotter, Scripps Research Institute). 1×104
cells per sample were analyzed by flow cytometry.
Ras Activation Assay
To study the peptide-scFv fusion protein-dependent suppression of Ras activity, the level of Ras-GTP (active form) was measured using a Ras activation assay kit (Millipore, Billecia, MA) according to the manufacturer's instructions. This method is based on a selective binding of Ras-GTP using the Ras binding domain (RBD) of Raf-1 (a kinase downstream of Ras) that fails to bind Ras-GDP (inactive form). Briefly, 50 µl of RBD protein fused to glutathione transferase (GST) was coated on to a 96-well glutathione-coated plate at 4°C for 1 h and washed with TBST (50 mM Tris, 150 mM NaCl, and 0.05% Tween-20, pH 7.6). HCT116 cells were treated with scFv, Tat-scFv, or BR2-scFv for 1 or 2 h, after which cells were lysed using 1×Mg2+ Lysis/Wash Buffer (Millipore). The protein concentration was calculated by the Bradford assay. Cell lysates containing 50 µg of protein were incubated for 1 h in the RBD-GST-coated wells at 25°C. After washing three times with TBST, primary antibody solution was added and incubated at 4°C for 1 h. After washing with TBST, the secondary HRP-conjugated anti-mouse antibody was added and incubated at 25°C for 1 h. After a final washing with TBS (50 mM Tris, 150 mM NaCl, pH 7.6), 50 µl of the Chemiluminescent substrates was added to each well. Chemiluminescence signals from each well were monitored using the Berthold luminometer (MicroLumat LB96P; Berthold Technologies, Oak Ridge, TN). Results were expressed as relative Ras activity.