Novel Anti-Cancer Peptides Comprising Three Amino Acids

Abstract

Background: The NPxY motif common to all β integrin cytoplasmic domains forms part of a canonical recognition sequence for phosphotyrosine-binding domains which are protein modules present in a wide variety of signaling and cytoskeletal proteins. We have recently reported that a non-naturally occurring peptide, RSKAKNPLYR, derived from the β6 integrin cytoplasmic domain inhibits cancer cell growth in vitro and proposed that this may be due, at least in part, to the inhibition of c-Src activity [1]. In the present study we examined the role of the NPLY motif within RSKAKNPLYR in terms of its requirement for inhibition of cancer cell growth. Materials and Methods: The effects of peptide modifications to RSKAKNPLYR on in vitro proliferation of human cancer cell lines (colorectal HT29, prostate DU145, breast MCF-7 and ovarian A2780) were evaluated using the MTT cell growth assay. Passage of peptide across the plasma membrane was assessed by means of confocal microscopy using FITC-labelled peptide. The effect of peptide on kinase activity was assessed in cell-free in vitro kinase assays. Results: The NPLY motif within RSKAKNPLYR was found to be essential for the growth inhibitory effect of this peptide. However, modified forms of this peptide in which all amino acids except the charged residues arginine and lysine were replaced by single non-polar amino acids such as alanine or valine were equally effective at inhibiting cancer cell proliferation. Moreover, these peptides inhibited not only c-Src activity as seen for RSKAKNPLYR but also the activity of members of the PKB/Akt kinase family. Conclusion: Novel decapeptides comprising only three amino acids have anti-cancer effects without the requirement for an integrin-based NPLY motif. These peptides inhibit the activity of not only c-Src but also members of the Akt family of kinases and may be useful as potential anti-cancer agents when used either alone or in combination with compounds previously reported to inhibit c-Src kinase activity.

Full text

Journal of Cancer Therapy, 2012, 3, 230-236
http://dx.doi.org/10.4236/jct.2012.34033 Published Online August 2012 (http://www.SciRP.org/journal/jct)
Novel Anti-Cancer Peptides Comprising Three Amino
Acids*
Michael Agrez1, Madhu Garg2,3, Stephen Ackland2,3,4
1Division of Surgery, John Hunter Hospital, Newcastle, Australia; 2Department of Medical Oncology, Calvary Mater Hospital,
Waratah, Australia; 3Hunter Medical Research Institute, John Hunter Hospital, Newcastle, Australia; 4Faculty of Health, The Univer-
sity of Newcastle, Callaghan, Australia.
Email: michael.agrez@hnehealth.nsw.gov.au
Received May 21st, 2012; revised June 25th, 2012; accepted July 9th, 2012
ABSTRACT
Background: The NPxY motif common to all β integrin cytoplasmic domains forms part of a canonical recognition
sequence for phosphotyrosine-binding domains which are protein modules present in a wide variety of signaling and
cytoskeletal proteins. We have recently reported that a non-naturally occurring peptide, RSKAKNPLYR, derived from
the β6 integrin cytoplasmic domain inhibits cancer cell growth in vitro and proposed that this may be due, at least in
part, to the inhibition of c-Src activity [1]. In the present study we examined the role of the NPLY motif within
RSKAKNPLYR in terms of its requirement for inhibition of cancer cell growth. Materials and Methods: The effects
of peptide modifications to RSKAKNPLYR on in vitro proliferation of human cancer cell lines (colorectal HT29, pros-
tate DU145, breast MCF-7 and ovarian A2780) were evaluated using the MTT cell growth assay. Passage of peptide
across the plasma membrane was assessed by means of confocal microscopy using FITC-labelled peptide. The effect of
peptide on kinase activity was assessed in cell-free in vitro kinase assays. Results: The NPLY motif within RSKAKN-
PLYR was found to be essential for the growth inhibitory effect of this peptide. However, modified forms of this pep-
tide in which all amino acids except the charged residues arginine and lysine were replaced by single non-polar amino
acids such as alanine or valine were equally effective at inhibiting cancer cell proliferation. Moreover, these peptides
inhibited not only c-Src activity as seen for RSKAKNPLYR but also the activity of members of the PKB/Akt kinase
family. Conclusion: Novel decapeptides comprising only three amino acids have anti-cancer effects without the re-
quirement for an integrin-based NPLY motif. These peptides inhibit the activity of not only c-Src but also members of
the Akt family of kinases and may be useful as potential anti-cancer agents when used either alone or in combination
with compounds previously reported to inhibit c-Src kinase activity.
Keywords: Peptides; Cancer Cell Lines; MTT Assay; Kinase Activity; c-Src; Akt
1. Introduction
Integrins comprise a family of cell adhesion receptors com-
posed of alpha/beta heterodimeric subunits that provide a
functional and structural bridge between the extracellular
matrix and intracellular signaling molecules [2]. The im-
portance of integrins as components of extra- and intra-
cellular signaling pathways in cancer is only just emerg-
ing. Expression of the αvβ6 integrin in ovarian cancers
may contribute to the invasive potential of ovarian can-
cers [3] and in colon cancer it has been identified as an
independent prognostic indicator for worse outcome in
patients suffering from this disease [4]. We have previ-
ously reported that a sequence of 15 amino acids, RSK-
AKWQTGTNPLYR, located within the cytoplasmic tail
of the β6 integrin subunit binds to extracellular signal-
regulated kinase 2 (ERK2) and proposed that this con-
tributes to tumor growth [5]. More recently, we reported
that a novel peptide, RSKAKNPLYR, derived from the
β6 binding sequence inhibits cancer cell growth in vitro
and proposed that this may be due, in part at least, to the
inhibition of c-Src activity [1].
Notably, within the RSKAKNPLYR sequence there is
an NPxY motif common to all beta integrin cytoplasmic do-
mains that forms part of a canonical recognition sequence
for phosphotyrosine—binding (PTB) domains which are
protein modules present in a wide variety of signaling
and cytoskeletal proteins. Accordingly, it has been sug-
gested that phosphorylation of the tyrosine (Y) residue in
the NPxY motif may represent a mode of regulating in-
tegrin interactions with other proteins at the cytoplasmic
face of the plasma membrane [6]. The fundamental role
for the highly conserved NPxY motif in regulating in-
*Conflict of Interest: None.
Copyright © 2012 SciRes. JCT

Novel Anti-Cancer Peptides Comprising Three Amino Acids 231
tegrin-mediated function has been emphasized by Filardo
and colleagues who showed that the NPxY motif within
the β3 cytoplasmic tail is essential for αvβ3-dependent
post-ligand binding events involved in cell migration and
the metastatic phenotype of melanoma cells [7].
In the present study we examined the role of the NP-
LY motif within the β6-derived anti-cancer decapeptide,
RSKAKNPLYR, in terms of its requirement for inhibi-
tion of cancer cell growth. To our surprise, novel small
anti-cancer peptides that lacked the NPLY motif were
identified comprising only three amino acids, i.e., argin-
ine, lysine and a non-polar amino acid such as alanine or
valine. Moreover, these peptides inhibited not only c-Src
activity in a manner similar to RSKAKNPLYR but also
the activity of members of the PKB/Akt kinase family.
2. Materials & Methods
2.1. Peptides, Cell Lines and Culture Conditions
All peptides were synthesized by Auspep, Melbourne,
Australia. The peptides were RSKAKNPLYR, RKRK,
RKKR, ASAAANPLYA, RSKAKR, RSKAKNPLAR,
RSKAKNALYR, RAKAKAAAAR, RAAKAARAAK,
KAKAKAAAAK, RARAKAAAAK, RARARAAAAR,
RAKARAAAAK, KARARAAAAK, RβAKβAKβAβAβ-
AβAR, RAKAK, RAKAKAAAR, RAKAKAAAAAR,
RSKSKSSSSR, RGKGKGGGGR and RVKVKVVVVR.
For immune-fluorescent studies an extra lysine residue
was coupled to the amino-terminus of RAKAKAAAAR
to provide attachment for FITC (designated FITC-K10(4)
Ala).
The human colon cancer cell line HT29, an ovarian can-
cer cell line, A2780, a breast cancer cell line MCF-7 and a
prostate cancer cell line DU145 were bought from the
American Type Culture Collection (ATCC). The cell lines
were cultured at 37˚C, under air containing 5% CO2 and
passaged regularly for optimal growth. Cells were main-
tained in DMEM medium (HyClone Laboratories, Utah,
USA) containing 10% fetal bovine serum (FBS) (SAFC
Biosciences, Kansas, USA). All culture medium prepara-
tions were further supplemented with penicillin/strepto-
mycin (100 µg/ml) and glutamine (2 mM) (Gibco, Life
Technologies, Australia).
2.2. In vitro Growth Inhibition MTT Assay
Cells in logarithmic growth were transferred to 96-well
plates (Costar, Corning Incorporated, NY, USA) in 100
µl of serum-containing medium at a density of 4000 cells
per well. After 24 hours the previously added serum-con-
taining medium was removed and 200 µl of serum-free
medium with or without peptide added to each of tripli-
cate wells. Drug exposure experiments were carried
out on cell lines using varying concentrations of peptides
(50 nM - 100 µM) and cells were exposed to peptides for
72 hours. Growth-inhibitory effects were evaluated using
the MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl-te-
trazolium bromide) cell growth assay and absorbance
read at 540 nm. Growth of control cells was exponential
during the whole incubation period. Mean surviving frac-
tions ± SEM values (minimum of 3 separate experiments)
were determined for each peptide concentration.
2.3. c-Src Kinase Activity Assay
In vitro c-Src kinase activity assays were performed by
Upstate Kinase Profiling, Dundee, Scotland according to
the manufacturer’s instructions. In brief, in a final reac-
tion volume of 25 µL, c-Src(5 - 10 mU) was incubated with
8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 µM KVE-
KIGEGTYGVVYK (Cdc2 peptide), 10 mM Mg Acetate
and [ϒ-33P-ATP] (specific activity approximately 500
cpm/pmol). The reaction was initiated by the addition of
the Mg-ATP mix. After incubation for 40 minutes at room
temperature, the reaction was stopped by the addition of
5 µL of a 3% phosphoric acid solution. 10 µL of the re-
action was then spotted onto a P30 filtermat and washed
three times for 5 minutes in 75 mM phosphoric acid and
once in methanol prior to drying and scintillation count-
ing.
2.4. PKB (Akt) Kinase Activity Assay
In vitro PKB kinase activity assays were performed by
Upstate Kinase Profiling, Dundee, Scotland according to
the manufacturer’s instructions. In final reaction volumes
of 25 µL, 5 - 10 mU of either PKBβ (Akt2) or PKBϒ
(Akt3) were incubated with 8 mM MOPS pH 7.0, 0.2
mM EDTA, 30 µM of GRPRTSSFAEGKK, 10 mM Mg
Acetate and (ϒ-33P-ATP) (specific activity approximately
500 cpm/pmol, concentration as required). The reaction
was initiated by the addition of the Mg-ATP mix. After
incubation for 40 minutes at room temperature the reac-
tion was stopped by the addition of 5 µL of a 3% phos-
phoric acid solution. 10 µL of the reaction mix was then
spotted on to P30 filtermat and washed three times for 5
minutes in 75 mM phosphoric acid and once in methanol
prior to drying and scintillation counting.
2.5. Peptide Internalization into Cells
HT29 cells were seeded into four 35 mm optically clear
cell culture dishes at a density of 1 × 106 cells per dish in
1 mL of RPMI cell culture medium with supplements
(penicillin/streptomycin and glutamine). The following
day, media was removed from the four dishes and the
adherent cells gently washed twice with 1 mL PBS fol-
lowed by addition of 1 mL of RPMI cell culture medium
with supplements minus FBS. FITC compounds (FITC
alone and FITC-labelled peptide designated FITC-K10(4)
Copyright © 2012 SciRes. JCT

Novel Anti-Cancer Peptides Comprising Three Amino Acids
Copyright © 2012 SciRes. JCT
232
Ala (FITC-KRAKAKAAAAR)) were resuspended in DM-
SO to give a final concentration of 1 mM. 10 µL of FITC
was added to two dishes and 10 µL of FITC-K10(4) Ala
was added to the other two dishes to give a final com-
pound concentration of 10 µM per dish. The dishes were
incubated at 37˚C for 24 hours.
substitution of the charged residues (R and K) by alanine
as well as 4-mer peptides comprising only the charged
residues (RKRK and RKKR). As shown in Figure 1(a),
both the NPLY motif and the charged residues R and K
appeared necessary but not sufficient to inhibit prolifera-
tion of HT29 cells in vitro to the same degree as seen for
the full length β6-derived 10-mer peptide (see Figure
1(a)). Notably, the scrambled version of the 4-mer pep-
tide RKKR (i.e., RKRK) was less effective at inhibiting
cell proliferation at the highest peptide concentration of
100 µM (Figure 1(a)).
Confocal microscopy was performed using a Nikon C1-
Z laser-scanning confocal system equipped with a Nikon
E-2000 inverted microscope and three solid laser lines
(Sapphire 488 nm, Compass 532 nm, Compass 405 nm).
A Nikon 60× water-immersion lens (NA = 1.2) objective
was used. Green fluorescence was excited with Ar 488
nm laser line and the emission viewed through BA 495 -
520 nm narrow band barrier filter. Nikon C1Z software
was used to process the images.
Given the putative importance of the NPxY motif in
regulating integrin function we specifically examined the
effect of a deletion variant of RSKAKNPLYR that lacked
the NPLY sequence (i.e., RSKAKR) on cell prolixfera-
tion. As shown in Figure 1(b), RSKAKR failed to inhibit
proliferation of HT29 cells even at the highest concentra-
tion of 100 µM. Moreover, substitution of the specific
residues proline and tyrosine by alanine within the NPLY
motif abrogated the growth inhibitory effect seen in the
presence of the parent 10-mer RSKAKNPLYR as shown
in Figure 1(b).
3. Results
3.1. Effect of Peptide Modifications on Cell
Proliferation in vitro
We initially examined the effects of peptide modifications
to the β6-derived peptide RSKAKNPLYR that included
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RKRK
RKKR
ASAAANP LYA
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.0 0 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RSKAKR
RSKAKNPLAR
RSKAKNALYR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RKRK
RKKR
ASAAANP LYA
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.0 0 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RSKAKR
RSKAKNPLAR
RSKAKNALYR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RKRK
RKKR
ASAAANP LYA
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RKRK
RKKR
ASAAANP LYA
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.0 0 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RSKAKR
RSKAKNPLAR
RSKAKNALYR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.0 0 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RSKAKR
RSKAKNPLAR
RSKAKNALYR
(a) (b)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RAKAKAAAAR
RAAKAAR AAK
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RAKAKAAAAR
KAKAKAAAAK
RARAKAAAAK
RARARAAAAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RAKAKAAAAR
RAAKAAR AAK
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RAKAKAAAAR
KAKAKAAAAK
RARAKAAAAK
RARARAAAAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RAKAKAAAAR
RAAKAAR AAK
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RSKAKNPLYR
RAKAKAAAAR
RAAKAAR AAK
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RAKAKAAAAR
KAKAKAAAAK
RARAKAAAAK
RARARAAAAR
(c) (d)
Figure 1. (a) HT29 colon cancer cells cultured under serum-free conditions and exposed to peptides for 72 hours (RSKAKN-
PLYR versus RKKR versus ASAAANPLYA versus RKRK); (b) HT29 colon cancer cells cultured under serum-free condi-
tions and exposed to peptides for 72 hours (RSKAKNPLYR versus RSKAKR versus RSKAKNPLAR versus RSKAKNAL-
YR); (c) HT29 colon cancer cells cultured under serum-free conditions and exposed to peptides for 72 hours (RSKAKNPL-
YR versus RAKAKAAAAR versus RAAKAARAAK scrambled RAKAKAAAAR); (d) HT29 colon cancer cells cultured un-
der serum-free conditions and exposed to peptides for 72 hours (RAKAKAAAAR versus KAKAKAAAAK versus RARAK-
AAAAK versus RARARAAAAR). All data represent the means ± SEM of the means for at least three separate experiments.

Novel Anti-Cancer Peptides Comprising Three Amino Acids 233
To further confirm that the NPLY motif was necessary
to inhibit cancer cell growth, all non-charged residues
within RSKAKNPLYR were replaced with alanine and the
resulting peptide RAKAKAAAAR tested for its effect on
proliferation of HT29 cells. Surprisingly, as shown in Fig-
ure 1(c), the ability of both peptides to inhibit cell grow-
th was similar whereas a scrambled version of RAKA-
KAAAAR (i.e., RAAKAARAAK) was ineffective (Fig-
ure 1(c)).
The 10-mer peptides derived from the β2, β3 and β5
integrin cytoplasmic domains (KEKLKNPLFK, RARA-
KNPLYK and RSRARNPLYR, respectively) share sig-
nificant homology with the β6-derived peptide RSKA-
KNPLYR and have been shown to inhibit proliferation of
colon cancer cells in vitro [8].We therefore compared the
growth inhibitory effect seen for RAKAKAAAAR with
10-mer peptides in which all but the charged residues of
the β2, β3 and β5-integrin-derived peptides were substi-
tuted by alanine (KAKAKAAAAK, RARAKAAAAK
and RARARAAAAR, respectively). As shown in Figure
1D, RAKAKAAAAR was more effective at inhibiting
proliferation of HT29 cells in vitro than the three alanine-
substituted peptides derived from the β2, β3 and β5 in-
tegrin cytplasmic domains.
In order to determine whether the positions of arginine
and lysine within RAKAKAAAAR affected the ability of
this peptide to inhibit cancer cell growth, HT29 cells
were cultured in the presence of peptides in which the
positions of one or both arginine and lysine residues had
been inverted (RAKARAAAAK and KARARAAAAK,
respectively). As shown in Figure 2(a), this reduced the
ability of the peptides to inhibit cell growth. Moreover,
conversion of all alanine residues within RAKAKAAA-
AR to the isomeric form, β-alanine, similarly reduced the
ability of the peptide to inhibit growth of HT29 cells as
shown in Figure 2(a).
The specific requirement for four alanine residues within
the carboxy-terminal component of RAKAKAAAAR for
optimal growth-inhibitory effects is shown in Figure 2(b).
As seen in Figure 2(b) shorter variants of RAKAK-
AAAAR, ie, RAKAK and RAKAKAAAR, had minimal
effect on the proliferation of HT29 cells whereas an 11-
mer peptide containing an extra alanine residue was more
effective than the shorter peptide variants but not as effec-
tive as RAKAKAAAAR at inhibiting cell growth (Figure
2(b)).
To determine if the presence of alanine was a specific
requirement for the growth inhibitory effect, the alanine
residues within RAKAKAAAAR were replaced with
either valine, serine or glycine. As shown in Figure 2(c),
replacement of alanine with glycine rendered the peptide
ineffective except at the highest concentration, whereas
the serine substitute was slightly more effective, albeit
less than the alanine derivative. However, replacement of
alanine residues with valine resulted in similar inhibition
of growth of colon cancer cells to that observed for RA-
KAKAAAAR. The inhibitory effect of RAKAKAAAAR
on cell growth was also examined for other cancer cell
types. As shown in Figure 2(d), the peptide was equally
effective at inhibiting growth of human prostate, breast
and ovarian cancer cell lines in vitro.
3.2. Peptide Uptake by Cancer Cells
The ability of the peptide to cross the plasma membrane
under serum-free conditions was assessed by means of
confocal microscopy of HT29 cells exposed to KRAKA-
KAAAAR conjugated to FITC at its amino-terminus. As
shown in Figure 3, cytoplasmic localization of peptide
was observed after 24 hours in culture in contrast to the
absence of uptake of FITC alone.
3.3. Effect of Peptides on Kinase Activity
The ability of 50 µM of RSKAKNPLYR, RAKAKA-
AAAR, RAAKAARAAK (scrambled RAKAKAAAAR)
and RVKVKVVVVR to inhibit kinase activity was de-
termined by means of cell-free in vitro kinase assays. As
shown in Figure 4, the β6-derived peptide, RSKAKN-
PLYR, inhibited c-Src but not PKBβ or PKBϒ activity
whereas the alanine-substituted peptide, RAKAKAA-
AAR, inhibited both c-Src and PKBϒ but not PKBβ ac-
tivity. The scrambled version of the alanine-substituted
peptide, RAAKAARAAK, was ineffective at inhibiting
the activity of any of the three kinases tested. In contrast,
substitution of alanine by valine (RVKVKVVVVR) re-
sulted in inhibition of the activity of all three kinases
(Figure 4).
Inhibition of c-Src activity did not differ significantly
between RSKAKNPLYR and RAKAKAAAAR (p =
0.132) and neither between RSKAKNPLYR and RVKV-
KVVVVR (p = 0.422). However, RAKAKAAAAR was
significantly more effective at inhibiting c-Src and PKBϒ
compared with the scrambled peptide RAAKAARAAK
(p < 0.05 and p < 0.01 respectively, Anova Test) as shown
in Figure 4.
4. Discussion
Given the necessity for the NPLY motif and, in particular,
the tyrosine and proline residues within that motif to
render the β6-derived peptide, RSKAKNPLYR, effective
at inhibiting cancer cell proliferation, it was completely
unexpected to find that substitution of all non-charged
residues within this 10-mer peptide with either alanine or
valine, i.e., RAKAKAAAAR and RVKVKVVVVR, re-
spectively, resulted in inhibition of cell proliferation to
the same degree as seen for RSKAKNPLYR. This was
all the more surprising because alanine and valine are
Copyright © 2012 SciRes. JCT

Novel Anti-Cancer Peptides Comprising Three Amino Acids
234
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0. 10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RAKAKAAAAR
RAKARAAAAK
KARARAAAAK
RβAKβAKβAβAβAβAR
A
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10.00 100.00
Conc (µM)
Surviving Fraction
RAKAKAAAAR
RAKAK
RAKAKAAAR
RAKAKAAAAAR
B
(a) (b)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10. 00 100.00
Conc (µM)
Surviving Fraction
RAKAKAAAAR
RSKSKSS SSR
RGKGKGGG GR
RVKVKVV VVR
C
Cell lines exposed to RAKAKAAAAR in serum-free medium
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.01 0.10 1.00 10. 00 100.00
Conc (µM)
Surviving Fraction
DU145
MCF-7
A2780
D
(c) (d)
Figure 2. (a) HT29 colon cancer cells cultured under serum-free conditions and exposed to peptides for 72 hours (RA-
KAKAAAAR versus RAKARAAAAK versus KARARAAAAK versus RβAKβAKβAβAβAβAR); (b) HT29 colon cancer cells
cultured under serum-free conditions and exposed to peptides for 72 hours (RAKAKAAAAR versus RAKAK versus RA-
KAKAAAR versus RAKAKAAAAAR); (c) HT29 colon cancer cells cultured under serum-free conditions and exposed to
peptides for 72 hours (RAKAKAAAAR versus RSKSKSSSSR versus RGKGKGGGGR versus RVKVKVVVVR); (d) Pros-
tate (DU145), breast (MCF-7) and ovarian (A2780) cancer cell lines cultured under serum-free conditions and exposed to
RAKAKAAAAR for 72 hours. All data represent the means ± SEM of the means for at least three separate experiments.
FITC Transmission Merge
FITC‐K10(4)Ala FITC
Channel
Treatment
FITC Transmission Merge
FITC‐K10(4)Ala FITC
Channel
Treatment
FITC Transmission Merge
FITC‐K10(4)Ala FITC
Channel
Treatment
Figure 3. Uptake of FITC-conjugated KRAKAKAAAAR (designated FITC-K10(4)Ala) by HT29 cells exposed to peptide or
FITC alone after 24 hours in culture under serum-free conditions.
Copyright © 2012 SciRes. JCT

Novel Anti-Cancer Peptides Comprising Three Amino Acids 235
PKB β
C-S r c
C-S r c
C-S r c
C-S r c
PKB β
PKB β
PKB β
PKB ϒ
PKB ϒ
PKB ϒ
PKB ϒ
0
10
20
30
40
50
60
70
80
90
100
RSKAKNPL YR RAKAKAAAAR RAAKAARAAK RVKV KVVVVR
% Inhibition of Kinase Activity
*
**
Figure 4. Percentage inhibition of kinase activity by peptides at a concentration of 50 µM when tested by means of in vitro
kinase assays. All data represent the means ± SEM of the means for at least three separate experiments. c-Src-RAKA-
KAAAAR vs RAAKAARAAK *p < 0.05 and PKBϒ-RAKAKAAAAR vs RAAKAARAAK **p < 0.01 (Anova Test).
unlikely to bind strongly to a receptor given that they are
non-polar, hydrophobic amino acids with no opportunity
for electrostatic interactions. In contrast, a scrambled ver-
sion of RAKAKAAAAR was ineffective as shown in
Figure 1(c) and indicated that the inhibitory effect of
RAKAKAAAAR on cell proliferation was determined
by the correct spacing and sequence of the two arginine
and two lysine residues (Figure 2(a)).
Moreover, the length of RAKAKAAAAR appears to
be critical. For example, while a β-alanine still retains the
same tetrahedral structure as alanine, the effect of replac-
ing alanine with β-alanine residues in the peptide RA-
KAKAAAAR is to lengthen the backbone of the peptide
which rendered it less effective at inhibiting cancer cell
growth as shown in Figure 2(a). In addition, the peptides
RAKAKAAAR (9-mer) and RAKAKAAAAAR (11-mer)
that contain either 3 or 5 alanine residues between the
lysine and the c-terminal arginine, respectively, were both
less effective at inhibiting cell growth compared with RA-
KAKAAAAR (10-mer) as shown in Figure 2(b), Sub-
stitution of the alanine residues within RAKAKAAAAR
with either glycine, serine or valine residues rendered the
peptides increasingly effective at inhibiting cell prolifera-
tion, respectively, when compared with RAKAKAAAAR
as shown in Figure 2(c).
The lack of a relationship to integrin structure was fur-
ther highlighted by the finding that replacement of the
NPLY motif with alanine residues in the β6-derived se-
quence, RSKAKNPLYR, resulted in effective inhibition
of colon cancer cell proliferation whereas alanine substi-
tution of the NPLY motif within the respective integrin
cytoplasmic domains of β2, β3 and β5 generated com-
pounds that were relatively ineffective at inhibiting cell
growth (Figure 1(d)). The novel peptide RAKAKAA-
AAR was also found to be similarly effective at inhibit-
ing proliferation of prostate, breast and ovarian cancer cell
lines in vitro (Figure 2(d)).
The PKB subfamily comprises three mammalian iso-
forms, PKBα, PKBβ and PKBϒ (Akt1, Akt2 and Akt3,
respectively) that are frequently mutated or over-expressed
in human cancer [9]. The Akt family of kinases is impor-
tant not only for cell survival but also in malignant trans-
formation [10]. Elevated Akt2 levels have been identified
in 32 of 80 primary breast carcinomas [11] as well as in
some ovarian and pancreatic carcinomas [12]. Akt3 has
also been found to be over-expressed in breast and pros-
tate cancers [13] and in prostate cancer basal enzymatic
activity of Akt3 has been found to be constitutively ele-
vated and represent the major Akt isoform [14]. Interest-
ingly, inhibition of Akt3 has also recently been shown to
result in reduction of VEGF resulting in less vascularised
tumors in an ovarian xenograft mouse model [15]. Our
findings that modifications of a non-naturally occurring
decapeptide derived from the β6 integrin cytoplasmic
domain can lead to Akt kinase inhibition suggests these
molecules may be developed towards new targeted the-
rapies.
Targeting Src kinases is also relevant in cancer therapy
given that Src family kinases are required for the endo-
membrane activation of the growth-promoting Ras-MAPK
pathway and c-Src activation has been documented in up-
wards of 50% of tumors derived from the colon, liver,
lung, breast and pancreas [16-18]. While none of the
peptides described in the present study inhibits Akt1
(data not shown) RAKAKAAAAR inhibited Akt3 activ-
ity and RVKVKVVVVR inhibited the activity of both
Akt2 and Akt3 as well as c-Src. However, a limitation of
our study is that we have not shown that inhibition of
colon cancer cell growth by these peptides is a conse-
quence of inhibition of one or more of the kinases against
Copyright © 2012 SciRes. JCT

Novel Anti-Cancer Peptides Comprising Three Amino Acids
236
which inhibitory activity has been identified in the in
vitro kinase assays.
In summary, we have described novel anti-cancer pep-
tides comprising only three amino acids, i.e., arginine, ly-
sine and a non-polar, hydrophobic amino acid such as
alanine or valine that inhibit Src and members of the Akt
kinase family that are critical for growth of colorectal and
other cancers. The role of Akt kinases as central players in
regulation of cell survival and proliferation make them
attractive therapeutic targets for treatment of cancer. A
major challenge in cancer therapy is the ability of tumor
cells to escape the growth constraints imposed on a cell
when targeting a single kinase. The peptides described
herein warrant further investigation as potential anti-
cancer agents when used either alone or in combination
with previously reported anti-Src kinase-inhibiting pep-
tides.
REFERENCES
[1] M. Agrez, M. Garg, D. Dorahy and S. Ackland, “Syner-
gistic Anti-Tumor Effect of Cisplatin when Combined
with an Anti-Src Kinase Integrin-Based Peptide,” Journal
of Cancer Therapy, Vol. 2, No. 3, 2011, pp. 295-301.
doi:10.4236/jct.2011.23039
[2] R. O. Hynes, “Integrins: Versatility, Modulation, and Sig-
naling in Cell Adhesion,” Cell, Vol. 69, No. 1, 1992, pp.
11-25. doi:10.1016/0092-8674(92)90115-S
[3] N. Ahmed, F. Pansino, R. Clyde, P. Murthi, M. A. Quinn,
G. E. Rice, et al., “Overexpression of αvβ6 Integrin in
Serous Epithelial Ovarian Cancer Regulates Extracellular
Matrix Degradation via the Plasminiogen Activation Cas-
cade,” Carcinogenesis, Vol. 23, No. 2, 2002, pp. 237-244.
doi:10.1093/carcin/23.2.237
[4] R. C. Bates, D. I. Bellovin, C. Brown, E. Maynard, B. Wu,
H. Kawakatsu, et al., “Transcriptional Activation of In-
tegrin β6 during the Epithelial-Mesenchymal Transition
Defines a Novel Prognostic Indicator of Aggressive Co-
lon Carcinoma,” Journal of Clinical Investigation, Vol.
115, No. 2, 2005, pp. 339-347
[5] N. Ahmed, J. Niu, D. J. Dorahy, X. H. Gu, S. Andrews, C.
J. Meldrum, et al., “Direct Integrin αvβ6-ERK Binding:
Implications for Tumour Growth,” Oncogene, Vol. 21,
No. 9, 2002, pp. 1370-1380. doi:10.1038/sj.onc.1205286
[6] Y. Takada, X. Ye and S. Simon, “Protein family Review—
The Integrins,” Genome Biology, Vol. 8, No. 5, 2007, p. 215.
[7] E. J. Filardo, P. C. Brooks, S. L. Deming, C. Damsky and
D. A. Cheresh, “Requirement of the NPXY Motif in the
Integrin Beta 3 Subunit Cytoplasmic Tail for Melanoma
Cell Migration in Vitro and in Vivo,” Journal of Cell Bi-
ology, Vol. 130, 1995, pp. 441-450.
doi:10.1083/jcb.130.2.441
[8] M. V. Agrez, “Methods and Agents for the Treatment of
Cancer,” International Patent Application, No. PCT/AU-
2004/001416, Publication No. WO 2005/037308.
[9] L. Bozulic, P. J. Morin, T. Hunter and B. A. Hemmings,
“Meeting Report: Targeting the Kinome—20 Years Ty-
rosine Kinase Research in Basel,” Science STKE, Vol.
374, 2007, p. 8. doi:10.1126/stke.3742007pe8
[10] J. Q. Cheng, C. W. Lindsley, G. Z. Cheng, H. Yang and S.
V. Nicosia, “The AKT/PKB Pathway: Molecular Target
for Cancer Drug Discovery,” Oncogene, Vol. 24, 2005,
pp. 7482-7492. doi:10.1038/sj.onc.1209088
[11] M. Sun, J. E. Paciga, R. I. Feldman, Z. Yuan, D. Coppola,
Y. Y. Lu, et al., “Phosphotidylinositol-3-OH (PI3K)-
AKT2, Activated in Breast Cancer, Regulates and Is In-
duced by Estrogen Receptor α (ERα) via Interaction be-
tween ERα and PI3K,” Cancer Research, Vol. 61, 2001,
pp. 5985-5991.
[12] J. Q. Cheng, B. Ruggeri, W. M. Klein, G. Sonoda, D. A.
Altomare, D. K. Watson, et al., “Amplification of AKT2
in Human Pancreatic Cancer Cells and Inhibition of AKT2
Expression and Tumorigenicity by Antisense RNA,” Pro-
ceedings of the National Academy of Science USA, Vol.
93, No. 8, 1996, pp. 3636-3641.
doi:10.1073/pnas.93.8.3636
[13] K. E. Anderson, J. Coadwell, L. R. Stephens and P. T.
Hawkins, “Translocation of PDK-1 to the Plasma Mem-
brane Is Important in Allowing PDK-1 to Activate Pro-
tein Kinase B,” Current Biology, Vol. 8, No. 12, 1998, pp.
684-691. doi:10.1016/S0960-9822(98)70274-X
[14] K. Nakatani, D. A. Thompson, A. Barthel, S. Hiroshi, W.
Liu, R. J. Weigel, et al., “Upregulation of Akt3 in Estro-
gen Receptor-Deficient and Androgen Independent Pros-
tate Cancer Cell Lines,” Journal of Biological Chemistry,
Vol. 274, 1999, pp. 21528-21532.
doi:10.1074/jbc.274.31.21528
[15] T. A. Liby, P. Spyropoulos, H. B. Lindner, J. Eldridge, C.
Beeson, T. Hsu, et al., “Akt3 Controls Vascular Endothe-
lial Growth Factor Secretion and Angiogenesis in Ovarian
Cancer Cells,” International Journal of Cancer, Vol. 130,
No. 3, 2012, pp. 532-543 doi:10.1002/ijc.26010
[16] T. G. Bivona, I. Perez De Castro, I. M. Ahearn, T. M.
Grana, V. K. Chin, P. J. Lockyer, et al., “Phospholipase C
Gamma Activates Ras on the Golgi Apparatus by Means
of RasGRP1,” Nature, Vol. 424, No. 6949, 2003, pp. 694-
698. doi:10.1038/nature01806
[17] S. C. Dehm and K. Bonham, “SRC Gene Expression in
Human Cancer: The Role of Transcriptional Activation,”
Biochemistry and Cell Biology, Vol. 82, No. 2, 2004, pp.
263-274. doi:10.1139/o03-077
[18] J. B. Bolen, A. Veillett, A. M. Schwartz, V. DeSeau and
N. Rosen, “Activation of pp60c-src Protein Kinase Activ-
ity in Human Colon Carcinoma,” Proceedings of the Na-
tional Academy of Science USA, Vol. 84, No. 8, 1987, pp.
2251-2255. doi:10.1073/pnas.84.8.2251
Copyright © 2012 SciRes. JCT