A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells
for the Efficient Delivery of an scFv into Cancer
Cells. PLoS ONE 8(6): e66084. doi:10.1371/journal.pone.0066084
A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells
Ki Jung Lim 0
Bong Hyun Sung 0
Ju Ri Shin 0
Young Woong Lee 0
Da Jung Kim 0
Kyung Seok Yang 0
Sun Chang Kim 0
Robert W. Sobol, University of Pittsburgh, United States of America
0 1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon , Korea , 2 Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology , Daejeon , Korea
Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.
Funding: This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and
Technology (2011-0031955). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The beneficial effects of many newly-discovered potential
therapeutic agents, such as proteins, nucleic acids, and hydrophilic
drugs, are limited because of their inability to reach the
appropriate intracellular targets [1,2]. Thus, numerous
approaches such as microinjection, eletroporation, liposomal formulation
and the use of viral vectors have been explored to promote
efficient drug delivery [3,4]. One major concern about these
techniques is their poor cell specificity [4,5]. Therefore, the
development of a target-specific drug delivery system is a primary
concern for improving the therapeutic efficacy of drugs while
reducing their effective doses and side effects [6,7].
Cell-penetrating peptides (CPPs), also referred to as protein
transduction domains, have drawn special attention as an
alternative intracellular drug delivery vehicle since the discovery
of the first CPP, Tat, in 1988 . CPPs are short peptides
consisting of fewer than 30 amino acids and composed mostly of
basic, positively charged amino acids (e.g. Arg, Lys and His) that
have the capacity to translocate through the cell membrane and to
deliver a variety of cell-impermeable cargoes across the cellular
membrane , including proteins , nucleic acids , siRNA
, peptide nucleic acids (PNAs) , small molecule
therapeutics , quantum dots , and MRI contrast agents .
Although the exact mechanism of CPPs is unknown, recent
mechanistic studies imply that their cellular uptake results from an
initial rapid electrostatic interaction with the plasma membrane
followed by endosomal uptake [17,18].
Using CPPs for the intracellular delivery of a wide range of
macromolecules is a powerful approach because of their versatility
paired with easy functionalization of linked cargoes and the high
delivery efficiency into various cell lines, overcoming challenges
often faced with other delivery methods [19,20]. Therefore, many
studies have focused on the development of novel CPPs; the
number of available CPPs with different characteristics, such as
increased stability and efficient cargo delivery, continues to
Although the potential of CPPs as delivery agents is large, their
lack of cell specificity, cytotoxic effects and unexpected side effects
are major concerns for their development as drug delivery vehicles
. For cancer therapy, CPP cell specificity is especially
important so that side effects on normal cells are minimized
[22,23]. Therefore, there is a strong need for the development of
cancer-specific and non-toxic CPPs for effective cancer treatments.
We have previously reported that a potent antimicrobial
peptide, buforin IIb (RAGLQFPVG[RLLR]3), has strong
cellpenetrating ability and anticancer activity against various cancer
cell lines [24,25]. Even though buforin IIb showed selective
cytotoxicity against cancer cells, it also affected the viability of
normal cells at high concentrations. To develop buforin IIb as an
efficient drug delivery vehicle, its cytotoxicity against normal cells
should be minimized while maintaining its cancer cell specificity.
In this study, we designed a novel cancer-specific and non-toxic
cell-penetrating peptide, BR2, based on the cell-penetrating motif
of buforin IIb and studied the potential as an efficient drug
delivery vehicle into cancer cells by fusing BR2 to a single-chain
variable fragment (scFv) antibody against mutated K-ras.
Materials and Methods
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 [Dulbeccos modified eagle medium] (DMEM)
supplemented with 10% fetal bovine serum (FBS), 100 units/ml
penicillin, 100 mg/ml streptomycin. Cells were grown in
humidified conditions at 37uC with 5% CO2.
Peptide Design and Synthesis
We designed several derivatives of buforin IIb (BR3) by stepwise
elimination of the C-terminal regular a-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 a-helical motif RLLR and named BR1 and
BR2 (Table 1).
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
Nterminus via an aminohexanoic acid spacer by treating a
resinbound 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
reversedphase 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 (26105) were plated on a glass coverslip placed in a
6well plate, grown overnight, and then incubated with
FITClabeled peptides (5 mM 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 [26,27]. The distribution of FITC-labeled
peptides was analyzed using a confocal scanning laser Zeiss LSM
RAGLQFPVGRLLRRLLRRLLR (21 aa)
Underline indicates the model a-helical sequence.
510 microscope (Jena, Germany) equipped with a 406 and 206
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 manufacturers instructions. In brief, cells were
plated onto 96-well microplates (16104 cells per well) in complete
DMEM supplemented with 10% FBS and incubated overnight at
37uC to allow for attachment and spreading of cells. After 24 h of
incubation, cells were treated with various concentrations of
peptides (0100 mM) and incubated for another 24 h at 37uC. The
extracellular medium from each well was transferred to a new
microplate and incubated for 10 min with 100 ml/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 ml), serially diluted in PBS, were added to
190 ml of the cell suspension in microcentrifuge tubes. Following
gentle mixing, the tubes were incubated at 37uC for 30 min and
then centrifuged at 4,0006g for 5 min. The supernatant (100 ml)
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 in PBS)]6100.
Characterization of Peptide Uptake
To evaluate the internalization of FITC-labeled peptides, HeLa
cells were seeded onto 12-well plates at a density of 26105 cells per
well and incubated for 24 h. FITC-labeled peptides, at various
concentrations ranging from 2 to 10 mM, were then incubated
with the cells for 30 min at 37uC. To compare the cellular uptake
of peptides, cancer and normal cells were treated with
FITClabeled peptides (each, 10 mM) and incubated for 30 min at 37uC.
Following the incubation, cells were washed three times with
icecold 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 (1,0006g for 5 min),
resuspended with 500 ml 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 4uC for 30 min prior to the addition of the
peptides. Next, cells were treated with FITC-labeled peptides
(each, 5 mM) at 4uC for 30 min. For the energy-depletion study,
HeLa cells were preincubated with sodium azide (NaN3, 10 mM)
at 37uC for 1 h and then incubated with FITC-labeled peptides
(each, 5 mM) at 37uC for 30 min.
To study the role of endocytosis in peptide uptake, cells were
pretreated with several endocytosis inhibitors at 37uC 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--cyclodextrin (MCD, 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 mg/ml)
for 30 min. For the inhibition of ganglioside biosynthesis, cells
were pretreated with D-threo-1-phenyl-2-hexadecanoyl
amino-3morpholino-1-propanol (PPMP, 5 mM) 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 Students t-test at a 95% confidence
Cloning and Expression of Peptide-fusion Proteins
To employ BR2 as a vehicle for the delivery of therapeutic
proteins, a cDNA of the Y13259 single-chain variable fragment
(scFv) gene was synthesized at Bioneer (Daejeon, Korea). The
Tator BR2-fused Y13259 scFv genes were obtained by recombinant
PCR. The recombinant cDNAs encoding the anti-Ras scFv,
TatscFv 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
BR2scFv fusion proteins were expressed in E. coli Origami (DE3) after
induction with 0.1 mM IPTG for 4 h at 37uC. Cells were
harvested by centrifugation at 3,0006g for 15 min at 4uC. The cell
pellet was resuspended in phosphate buffer (10 mM sodium
phosphate, 150 mM NaCl, pH 7.4); cells were disrupted by
sonication at 4uC (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 14,0006g for 15 min at 4uC.
The pellet containing the inclusion bodies was resuspended in
wash buffer (20 mM TrisHCl, 5 mM EDTA and 1% Triton
X100, pH 8.0) and centrifuged at 8,0006g for 10 min at 4uC.
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 14,0006g for
15 min at 4uC. 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%
Nlauroyl 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% SDSPAGE. 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 4uC for 12 h against 206 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 (16106) were treated with PBS,
purified scFv, Tat-scFv, or BR2-scFv fusion proteins (each, 2 mM)
for 2 h at 37uC. The cells were then washed twice with PBS (4uC),
scraped into 0.5 ml of PBS and centrifuged at 1,0006g at 4uC for
5 min. The cell pellets were resuspended in 100 ml of lysis buffer
(20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA,
1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
beta-glycerophosphate, 1 mM Na3VO4, 1 mg/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 12,0006g at
4uC 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 mg 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,
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 26104 cells/well in 100 mL
of DMEM supplemented with 10% FBS and cultured for 24 h at
37uC. After 24 h of incubation, cells were treated with scFv or
peptide-scFv fusion proteins (0, 0.5, 1 and 2 mM) and incubated
for another 24 h. Cell viability was measured with the
3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay using the CellTiter 96H Non-radioactive Cell Proliferation
assay kit (Promega, Madison, WI) according to the manufacturers
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 (16106/well in a 6-well plate) were treated with
peptide-fused scFvs (each, 2 mM) or a well-known apoptosis
inducer staurosporine (0.5 mM) for 24 h at 37uC 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-atubulin 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
In addition, apoptotic cells were identified by staining with
annexin V-FITC (150 ng/ml) and 7 amino-actinomycin D
(7AAD; BD Biosciences, San Diego, CA). 16106 HCT116 cells were
treated with PBS, staurosporine (0.5 mM), or peptide-fused scFvs
(each, 2 mM) as described above. At the designated time, cells were
washed twice with PBS (pH 7.4), harvested and resuspended in
500 ml of binding buffer supplemented with Annexin-V fluos
(1:100 diluted; BioBud, Seoul, Korea), and incubated for 15 min
at 25uC 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). 16104
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 manufacturers 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 ml of RBD protein fused to
glutathione transferase (GST) was coated on to a 96-well
glutathione-coated plate at 4uC 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 16Mg2+ Lysis/Wash
Buffer (Millipore). The protein concentration was calculated by
the Bradford assay. Cell lysates containing 50 mg of protein were
incubated for 1 h in the RBD-GST-coated wells at 25uC. After
washing three times with TBST, primary antibody solution was
added and incubated at 4uC for 1 h. After washing with TBST,
the secondary HRP-conjugated anti-mouse antibody was added
and incubated at 25uC for 1 h. After a final washing with TBS
(50 mM Tris, 150 mM NaCl, pH 7.6), 50 ml 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.
BR2 Efficiently Internalizes into Various Cancer Cell Lines
without Cytotoxicity to Normal Cells
The cellular uptake and intracellular distribution of the
designed peptides, BR1 and BR2, were studied using confocal
microscopy. BR2 was readily internalized into cancer cells (HeLa,
HCT116 and B16/F10) within 30 min and distributed throughout
the cytoplasm and nucleus of cancer cells (Fig. 1A). However, it
was very poorly internalized into normal cells (HaCat, BJ and NIH
3T3) under the same experimental conditions. In contrast, BR1
displayed negligible cellular uptake levels in both cancer and
normal cells, indicating that it could not translocate across the cell
membrane (Fig. 1A). Fluorescence from Tat-treated cells was also
clearly detected in both the nucleus and cytoplasm of cancer and
normal cells. More Tat accumulated in the nucleus than
cytoplasm, whereas most BR2 was evenly distributed in the
intracellular regions (Fig. 1A).
To assess the in vitro cytotoxicity of the designed peptides, the
LDH leakage was measured in cancer cell lines (HeLa, HCT116
and B16/F10) and normal cell lines (HaCat, BJ and NIH 3T3).
BR2 treatment (20100 mM) was associated with LDH leakage
from cancer cells; the leakage gradually increased with BR2
concentrations, reaching about 4060% at 100 mM (Fig. 1B ac).
However, LDH leakage from the BR2-treated normal cells was
not detected at BR2 concentrations below 70 mM, and a weak
leakage (,17%) was observed at 100 mM (Fig. 1B df). BR3
induced substantial LDH leakage from both cancer (approx. 80
90%) and normal cell lines (approx. 30%) even at 50 mM (Fig. 1B
To examine the cytotoxicity of peptides further, the hemolytic
activity of BR1, BR2, BR3 and Tat against human erythrocytes
was also examined. In accordance with the LDH leakage results,
no hemolysis was induced even after the erythrocytes were treated
with Tat, BR1 or BR2 at $200 mM, whereas BR3 triggered
hemolysis ($5%) at $25 mM (Fig. 1C). Subsequently about 34.3%
of erythrocytes were lysed after being treated with 200 mM BR3.
Taken together, these results indicate that BR2 efficiently
penetrates cancer cells without cytotoxic effects in normal cells,
whereas Tat showed similar penetration into both cell types.
BR2 Specifically Penetrates Cancer Cells in a
To compare the quantity of the internalized peptides in different
cell types, we performed flow cytometric analysis. BR2 was
internalized more efficiently into cancer cells (HeLa, HCT116 and
B16/F10) than into normal cells (HaCat, BJ and NIH 3T3); more
than 95% of BR2-treated cancer cells internalized BR2, whereas
only 2334% of BR2-treated normal cells did. However, Tat
penetrated both cancer and normal cells without much
discrimination (Fig. 2A). Specific penetration of BR2 and Tat into cancer
cells was also analyzed in the presence of both HeLa and BJ
fibroblast cells by confocal laser microscopy. BR2 was
preferentially uptaken by HeLa cells, whereas the cellular uptake of Tat
was similar in both HeLa and BJ fibroblast cells (Fig. S1). These
results clearly show that the buforin-derivative BR2 has cancer cell
specificity, whereas Tat does not.
The fluorescence intensity of peptide-treated cells was enhanced
with increasing concentrations of BR2 or Tat. At concentrations
above 5 mM, BR2 entered more than 80% of HeLa cells within
30 min (Fig. 2B). BR2 penetrated cells more efficiently than did
the Tat peptide at every tested concentration. Moreover, BR2
internalization for all cancer cell lines (HCT116 and B16/F10,
data not shown) appeared to be homogeneous in the whole cell
population as a single peak on the histogram. Among the peptides,
BR1 displayed the lowest uptake, indicating its inability to
penetrate the cell membrane even at 10 mM.
Initial Electrostatic Interaction with Positively Charged
BR2 and Negatively Charged Gangliosides on the Cancer
Cell Membrane is Essential for the Energy-dependent
Endocytosis of BR2
The cellular uptake mechanism of BR2 was analyzed with
HeLa, the most representative and widely used cancer cell line, to
compare with those of other cell-penetrating peptides. We first
examined the effect of temperature on BR2 penetration. Lowering
the temperature dramatically reduced peptide uptake in HeLa
cells (Fig. 3A); the cellular uptake of BR2 and Tat at 4uC was
decreased by 88.5% and 31.6%, respectively, compared to that
observed at 37uC. In addition, an energy-dependent uptake of
peptides was also observed when the cellular ATP pool was
depleted by preincubation of the cells with sodium azide (NaN3).
ATP depletion also reduced the uptake of BR2 and Tat in HeLa
cells, by 67.7% and 42.5%, respectively (Fig. 3A). These results
support the involvement of endocytosis for BR2 and Tat uptake.
We next determined whether the cellular uptake of BR2 occurs
through a specific endocytic pathway. Depletion of cholesterol
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 mM) 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 37uC, 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 mM for
30 min at 37uC. 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).
from the plasma membrane with methyl--cyclodextrin (MCD)
significantly inhibited the penetration of BR2 (44.6%) into HeLa
cells, suggesting that BR2 penetrates the cell membrane via lipid
raft-mediated endocytosis (Fig. 3B). Furthermore, pretreatment of
cells with amiloride, a specific inhibitor of a sodium channel
required for macropinocytosis, also prevented the cellular uptake
of BR2 (36.5%). In contrast, pretreatment with nocodazole, an
inhibitor of clathrin-mediated endocytosis, showed a negligible
effect on peptide transduction. This result indicates that lipid
raftmediated macropinocytosis is a major mode of BR2 transduction.
To determine if BR2 is degraded via the lysosomal pathway,
codistribution with LysoTracker, an agent that accumulates in late
endosomes/lysosomes, was examined. An overlay of respective
images showed this lysosomal marker (red) primarily in a random
punctuate distribution that was mostly separated from the BR2
signal (green) (Fig. 3C). BR2 was dispersed widely in the
cytoplasmic region and the majority of BR2 was not colocalized
with lysosomes. These results indicate that BR2 can escape from
endosomes into the cytosol, bypassing further steps in the
To identify the initial cell-surface binding targets for BR2
internalization into cancer cells, we examined whether exogenous
gangliosides, heparins or sialic acids affected this process.
Preincubation of gangliosides and sialic acids with BR2 partially
inhibited peptide penetration into HeLa cells, whereas Tat
penetration was not affected (Fig. 3D a and b). However, Tat
uptake was severely reduced when heparin was added to the
culture medium. Moreover, incubation of HeLa cells with the
ganglioside synthesis inhibitor PPMP, significantly reduced BR2
but not Tat uptake (Fig. 3D c and d). These results indicate that
gangliosides on cancer cell membranes are one of the main target
molecules for BR2 binding.
BR2-scFv Inhibits Cancer Cell Growth in a
The ability of BR2 to deliver proteins into cancer cells (HeLa
cells) was demonstrated by fusion with an EGFP. BR2 delivered
EGFP more efficiently into cancer cells than Tat (Fig. S2). These
results suggest that BR2 can be used to deliver proteins efficiently
into cancer cells by fusion with cargo proteins.
To further investigate the effects of BR2 on therapeutic protein
delivery into cancer cells, Tat and BR2 were each fused with
antiRas scFv (Fig. 4A), and the intracellular penetration of these
fusions was assessed by Western blotting. This analysis showed that
both Tat- and BR2-scFv fusion proteins were effectively delivered
into HCT116 cells and accumulated in the intracellular region
within 2 h, whereas the transduction of scFv itself was not
observed (Fig. 4B). The amount of BR2-scFv fusion protein
delivered into the cytoplasm was 32.6% more than that of the
intracellularly delivered Tat-scFv fusion protein under the same
Moreover, the intracellular localization of peptides and anti-Ras
scFv fusion proteins in HCT116 cells was examined by
immunocytochemistry analysis. The fluorescence was detected in
cytoplasmic regions of Tat- and BR2-scFv treated cells (Fig. S3).
Especially, BR2-scFv treated cells showed higher fluorescence
intensity than Tat-scFv treated cells, indicating that BR2 delivered
scFv more efficiently into cells than Tat did. Unlike Tat- or
BR2scFv fusion proteins, unconjugated scFv was not detected in the
Next, we investigated the anticancer activity of peptide-scFv
fusion proteins against Ras-mutated cancer cells by comparing it
with the activity of unconjugated scFv in vitro. A significant
reduction of cell viability was observed 24 h after the introduction
of BR2-scFv or Tat-scFv fusion proteins versus unconjugated scFv
(Fig. 4C), BR2 or Tat alone (Fig. S4). The viability of 2 mM
BR2scFv treated cancer cells was 39.3%, much lower than that of scFv
or Tat-scFv treated cells, which had viabilities of 87.8% and
50.5%, respectively. Inhibition of cell proliferation positively
correlated with protein concentration, suggesting that BR2-scFv
more efficiently suppresses cancer cell proliferation than Tat-scFv.
BR2-anti-Ras scFv Fusion Protein Promotes Apoptosis
and Inactivates Ras in Ras-mutated Cancer Cells
To examine how the BR2-scFv fusion protein inhibits cancer
cell proliferation, we investigated whether apoptosis was induced.
67.7% of BR2-scFv treated cells and 47.9% of Tat-scFv treated
cells were in the apoptotic stage, versus 5.6% and 25.3% of the
PBS- and scFv-treated cells, respectively (Fig. 5A). The percentage
of BR2-scFv treated cells that were apoptotic was higher than that
of staurosporine-treated cells (61.6%). The apoptotic pathway
induced by BR2-scFv was further investigated by Western blotting.
Treatment of HCT116 cells with Tat- or BR2-scFv fusion proteins
resulted in the cleavage of the 116-kDa PARP protein to the
apoptosis-specific 89-kDa fragment after 24 h. A larger amount of
the 89-kDa fragment was detected in the BR2-scFv treated cells as
compared to the staurosporine- or Tat-scFv treated cells.
However, the cleaved PARP fragment was not detectable in
control cells or scFv-treated cells (Fig. 5B).
Next, we performed Ras activation assays that specifically detect
activated GTP-bound Ras to obtain evidence for the suppression
of Ras activity by peptide-scFv fusion proteins. These peptide-scFv
fusion proteins and staurosporine reduced Ras activity similarly in
HCT116 cells. As shown in Fig. 5C, a significant decrease of Ras
activity in HCT116 cells was observed both 1 h and 2 h after
peptide-scFv fusion protein treatment. The activity of Ras-GTP in
cells was significantly suppressed by exogenous Tat- and BR2-scFv
fusion proteins (1, 2 mM) in a dose- and time-dependent manner.
Even though both Tat- and BR2-scFv fusion proteins induced a
remarkable decrease in the levels of GTP-bound Ras, BR2-scFv
was more effective than either staurosporine or Tat-scFv. The
decreased relative Ras activity in cells treated with 2 mM Tat-scFv
(about 34% and 53% after 1 h and 2 h, respectively) was roughly
similar to that treated with 1 mM BR2-scFv (about 27% and 52%
after 1 h and 2 h, respectively). Active Ras was decreased by about
67% when cells were treated with 2 mM BR2-scFv for 2 h
Here, we report that BR2, which is derived from the anticancer
peptide buforin IIb, has a potent ability to deliver therapeutic
proteins specifically into target cancer cells. Buforin IIb is known
for its efficient ability to penetrate cancer cells through an
electrostatic interaction with gangliosides on the cancer cell surface
. Buforin IIb, however, is also cytotoxic to normal cells at high
concentrations. Therefore, reducing this cytotoxicity is critical if
buforin IIb is to be used for drug delivery. It has been reported by
many researchers that hemolytic activity and cytotoxicity of
ahelical peptides are closely correlated with their helicity; stronger
helicity usually means the more complete non-polar face of an
ahelical peptide, which is correlated with its higher apparent
hydrophobicity when interacting with cell biomembrane,
subsequently contributing to cell membrane lysis . Therefore,
we stepwisely reduced the helicity of buforin IIb to minimize
hemolytic activity and cytotoxic effect of buforin IIb on normal
cells while maintaining its cancer cell specificity. The stepwise
elimination of the C-terminal RLLR repeats of buforin IIb results
in cancer cell specific peptides with reduced cytotoxicity, BR1 and
Unlike BR1, BR2 transduced across the plasma membrane of
various cancer cells and accumulated in the nucleus and cytoplasm
within 30 min. We confirmed the autonomous translocation of
BR2 into the cytoplasm with comparable efficiency in all cancer
cell types investigated. BR2 penetrated cancer cell membranes
more efficiently than the Tat peptide and in a
concentrationdependent manner. Moreover, BR1 and BR2 did not show
hemolytic activity even at 200 mM, whereas more than 30%
hemolysis was induced by 200 mM BR3. Among the peptides
tested, BR2 exhibited an efficient penetration into cancer cells
without cytotoxicity to normal cells, whereas BR1 displayed a
weak cell-penetrating ability and cytotoxicity. From these
observations, we can conclude that the number of RLLR repeats at
Cterminus affects the cell-penetrating ability and cytotoxicity of
peptides. Two RLLR repeats are required for the efficient
translocation of buforin-derivatives into cells; however, more than
3 repeats can cause gradual cell damage like that induced by
Furthermore, we observed a considerable difference between
the penetration of BR2 and Tat into normal cells. BR2 showed
about 4-fold higher transduction efficiency into cancer cells versus
normal cells whereas Tat showed similar penetration efficiency
regardless of cell type. Possible reasons for why BR2 displays
cancer cell specificity include distinctive features of the cancer cell
membrane, such as different membrane composition, altered
fluidity, more negative surface charges, higher transmembrane
potential and an increased level of acidic components on the
surface [34,35]. It is also known that a cancer cell membrane
typically contains a net negative charge due to a high expression of
anionic molecules such as phosphatidyl serine (PS) and
Oglycosylated mucins on the outer membrane leaflet . To
identify factors associated with cancer cell specificity of BR2 and
analyze the cellular uptake mechanism of BR2, a mechanistic
study was performed in HeLa cells, the representative cancer cell
line. When we added negatively charged cellular membrane
components, such as ganglioside or sialic acid, BR2 uptake was
partially inhibited, indicating that exogenous gangliosides and
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 mM) or staurosporine (0.5 mM) 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 V2/7AAD+) 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 mM) or staurosporine (0.5 mM) treatment, HCT116 cell
extracts were subjected to Western blot analysis with anti-PARP or anti-a-tubulin antibodies. The molecular sizes of the proteins are indicated with
arrows at the right. a-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 mM) were determined by an ELISA-based activity assay; results are expressed as relative Ras
sialic acids act as antagonists of the same molecules that bind BR2
and thereby hamper this binding. Depleting gangliosides with
PPMP also decreased BR2 uptake. However, heparin, another
negatively charged component, has almost no effect on BR2
binding to cancer cells. Prior to endocytosis, positively charged
BR2 specifically interacts with negatively charged gangliosides on
the cancer cell plasma membrane. In contrast, the high content of
zwitterionic phosphatidylcholine in the outer membrane leaflet of
normal cells confers an overall neutral charge to these cells,
resulting in a reduced capacity for electrostatic interactions with
BR2. This further decreases cell penetration of BR2 in normal
CPPs are known to be internalized into cells by two different
endocytic mechanisms , clathrin-dependent and
clathrinindependent endocytosis . Among several
clathrin-independent pathways, lipid raft-mediated macropinocytosis, which
gives rise to larger vesicles that do not necessarily fuse with early
endosomes , is attributed to the internalization of BR2, as
confirmed by the experiment using specific endocytosis inhibitors,
such as amiloride, methyl-b-cyclodextrin, and nocodazole (Fig. 3B).
Furthermore, the LysoTracker probes, which were used to track
acidic organelle like lysosomes, revealed that most of internalized
BR2s are present in the cytoplasm, but not in the lysosome,
escaping lysosomal degradation. These results clearly show that
BR2 can be an effective and long-lasting drug delivery vehicle.
Given the need for efficient therapeutic protein delivery systems,
we investigated the possibility of using BR2 for delivering an
anticancer therapeutic protein by fusing it with anti-p21 Ras
Y13259 scFv. Ras is a small GTP-binding protein that plays a critical
role in the regulation of cell proliferation, transformation and
differentiation. Over-expression or mutations in the Ras oncogene
have been identified in a large number of human tumors (,30%)
and therefore constitute a primary target for cancer treatment
. It was reported that microinjection of the neutralizing
antiRas monoclonal antibody Y13-259 into Ras-transformed rodent
fibroblasts induces transient phenotypic reversion the cells in vitro
and inhibits all biological responses that require Ras proteins
[43,44]. Moreover, intracellular expression of scFv, a fragment
derived from antibody Y13-259, specifically promoted apoptosis in
human cancer cells in vitro and led to tumor regression in a colon
carcinoma tumor model in vivo . Thus, in this study, we made
recombinant proteins in which BR2 and Tat peptides were fused
with anti-Ras scFv. These peptide-scFv constructs were efficiently
internalized into K-ras mutated colon cancer HCT116 cells within
2 h. BR2 delivered anti-Ras scFv into the cells 32.6% more
efficiently than Tat did, whereas peptide-unconjugated scFv was
not detectable in the intracellular region.
BR2- and Tat-anti Ras scFv fusion proteins clearly exerted
strong anti-proliferative activity in exponentially growing
Rasmutated cancer cells by inducing apoptosis, whereas BR2, Tat or
unconjugated scFv did not show any detectable inhibitions at the
same concentrations. It seems that BR2 and Tat contribute to the
enhancement of intracellular delivery of conjugated scFvs without
causing the cytotoxicity against HCT116. The better
internalization efficiency of BR2-fused scFv in cancer cells may be the basis
for the higher anticancer activity of this fusion protein as
compared with Tat-scFv. Furthermore, inactivation of GTP-Ras
proteins inside tumor cells might be mediated via neutralization by
specific binding between Ras and BR2-scFv fusion proteins. Our
results clearly suggest that BR2 promotes delivery and proper
localization of fused scFv antibody fragments to their target
antigen, the Ras protein. These results imply that after
peptidescFv fusion proteins penetrated the cell membranes, they
specifically bound and neutralized the target mutant GTP-Ras
proteins. All of these findings suggest that biologically active
antiRas scFv can be efficiently introduced into target cancer cells by
In conclusion, we have found a novel CPP, BR2, which
specifically penetrates cancer cells without causing cytotoxicity to
normal cells. BR2 can be used to transport therapeutic proteins
efficiently into target cells by covalent conjugation with the
cargoes, which retain bioactivity. Although further studies are
needed to assess the utility of using BR2 in vivo for delivering other
kinds of therapeutics into target cancer cells as well as the
feasibility of simple conjugations of BR2 to therapeutics for
practical cancer therapy, the cancer-specific cell-penetrating
peptide BR2 will provide valuable tools for the efficient cancer
Figure S1 Cancer cell specific penetration of BR2.
Specific penetration into cancer cells of FITC-labeled Tat and
BR2 were examined in the presence of both cancer and normal
cells by confocal laser microscopy. HeLa and BJ fibroblast cells
were seeded and co-cultured in the same well of a 6-well plate
1 day prior to the experiment to reach 70% confluence. Cells were
incubated with FITC-labeled Tat or BR2 (5 mM) for 30 min at
37uC and washed three times with phosphate buffered saline
(PBS). Nuclei were stained with Hoechst 33342 (blue). Peptide
internalization was then analyzed using a confocal laser
microscope. HeLa cells and BJ fibroblast cells were indicated with red
arrows and white arrows, respectively.
Figure S2 Efficient protein transduction mediated by
BR2. (A) Schematic representation of peptide-EGFP cDNA
constructs. DNA encoding peptides (Tat, BR1 or BR2) and EGFP
were fused as described in Supplementary Materials and Methods
in Information S1 and cloned into the BglII and NdeI sites of
pET16b. The BglII and NdeI restriction sites and factor Xa
cleavage site are indicated. (B,C) Cellular uptake of peptide-EGFP
fusion proteins was analyzed by confocal laser microscopy and
flow cytometry. Purified EGFP or peptide-EGFP fusion proteins
(10 mM) were incubated with HeLa cells at 37uC for 2 h.
Figure S3 Intracellular localization of peptides and
anti-Ras scFv fusion proteins using
immunocytochemistry. Intracellular localization of fusion proteins was analyzed in
HCT116 cells by immunocytochemistry. HCT116 cells were
incubated with scFv, Tat-scFv or BR2-scFv fusion protein (each,
2 mM) for 2 h at 37uC. Cells were washed with PBS, fixed and
permeabilized. FITC-conjugated anti-His antibody was used to
detect intracellular localization of scFv, Tat-scFv and BR2-scFv.
Nuclei were stained with DAPI (blue). Intracellular localization of
fusion proteins was then analyzed by confocal laser microscope.
Figure S4 Cytotoxic effect of BR2 and Tat against
HCT116 cells. HCT116 cells were treated with PBS, BR2 or
Tat (0, 1, 2 and 5 mM) and incubated for 24 h. Cell viability was
measured by MTT assay. Data represent the mean 6 s.d. of three
We would like to thank Prof. Ju Hyun Cho and Dr. Jin Huh for thoughtful
discussions, and Dr. Heather McDonald for valuable editorial assistance.
Conceived and designed the experiments: KJL BHS SCK. Performed the
experiments: KJL. Analyzed the data: KJL JRS BHS. Contributed
reagents/materials/analysis tools: KJL JRS BHS YWL DJK KSY SCK.
Wrote the paper: KJL BHS SCK.
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