Biosci. Biotechnol. Biochem.

, 73 (3), 683687, 2009

Multiple Functions of Short Synthetic Enantiomeric Peptides Based

on Beetle Defensins
Takashi I WASAKI,1;2 Jun I SHIBASHI,2 Masanori K UBO,3 DeMar T AYLOR,4 and Minoru Y AMAKAWA1;2; y
1

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Innate Immunity Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan
3
Diagnostic Section, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan
4
Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
2

Received October 15, 2008; Accepted December 2, 2008; Online Publication, March 7, 2009
[doi:10.1271/bbb.80735]

Four enantiomeric 9-mer peptides, D-peptides A

(RLYLRIGRR-NH2 ), B (RLRLRIGRR-NH2 ), C
(ALYLAIRRR-NH2 ), and D (RLLLRIGRR-NH2 ), were
designed and synthesized on the basis of a beetle
defensin antimicrobial peptide. These D-9-mer peptides
have been reported to exhibit multiple functions,
including antimicrobial and antiprotozoan activity,
without cytotoxicity on normal broblasts and leukocyte
cells. In this study, we found that the D-9-mer peptides
inhibited telomerase activity (IC100 = 40 M). A new
peptide, D-peptide C2 (ALYLAIRRRRRRRR-NH2 ),
designed from D-peptide C to translocate into the
cytoplasm by a penetrating sequence (octa-arginine),
showed extremely strong telomerase inhibitory activity
(IC100 = 0.1 M). D-Peptide C2 exhibited a great increase in cytotoxicity against various cancer cell lines
(IC50 = 3.426.4 M). However, the immediate death
of the cells suggested that the high cytotoxicity was not
an eect of telomerase inhibitory activity. Mitochondrial swelling assay and microscopical observations of
mitochondria indicated that the major target of the
D-peptide C2 was the mitochondrial membrane.
Key words:

antimicrobial peptide; octa-arginine; telomerase; mitochondria

Telomeres are nucleoprotein structures that protect

the ends of eukaryotic chromosomes.1) Telomerase is a
reverse transcriptase that adds nucleotide repeats to
telomeres with a RNA template, providing karyotype
stability and compensating for the loss of DNA that
occurs during replication.2,3) In the absence of telomerase, telomeres become shorter as cells divide, until
senescence occurs.4) Recent studies report that activation
of the telomerase enzyme is an important step in
tumorigenesis and conrm an unlimited capacity for
proliferation.5) Telomerase activity has been detected in
at least 8090% of human tumors and tumor-derived
cell lines, indicating the signicance of telomerase
activity in the immortalization of cancer cells.6,7) A
connection between telomerase activity and resistance to
apoptosis has also been established.8) Indeed, telomere
shortening following inhibition of telomerase triggers
apoptotic death in various cell types, whereas induction

of telomerase activity is associated with resistance to

apoptosis.9) Hence telomerase has been proposed as a
selective target in cancer therapy, and telomerase
inhibition reagents are considered to be good candidates
for novel anti-tumor drugs.
In this study, we investigated the telomerase inhibitory
activity and pharmacological potentials of four enantiomeric 9-mer peptides (all sequences were synthesized
as D-amino acids): D-peptides A (RLYLRIGRR-NH2 ),
B (RLRLRIGRR-NH2 ), C (ALYLAIRRR-NH2 ), and D
(RLLLRIGRR-NH2 ), derived from insect defensin antimicrobial peptides.1012) Previous studies have reported
the antimicrobial and antiprotozoan activities of these
cationic and amphipathic 9-mer peptides. They have
been reported to show membrane disruptive activity
against bacteria, including drug-resistant bacteria
(Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa),1317) and growth-inhibitory activity against pathogenic protozoa (Trypanosoma brucei
brucei) (Yamage, M., Yoshiyama, M., Dennis, J. G.,
Kubo, M., Iwasaki, T., Kitani, H., Ishibashi, J., and
Yamakawa, M., unpublished results), but more eective
inhibitory activities are required for the application of
D-9-mer peptides in clinical practice. Hence we focused
on telomerase as a novel target of D-9-mer peptides
for antitumor activity, because previous studies have
reported that some cationic compounds inhibited telomerase activity and that positive charges are important
to telomerase inhibitory activity,18) but telomerase is an
enzyme existing in the cytoplasm and D-9-mer peptides
cannot penetrate the cell membrane. To deliver D-9mer peptides into the cytoplasm, we designed and
synthesized a new enantiomeric peptide, D-peptide C2
(ALYLAIRRRRRRRR-NH2 ). D-Peptide C2 has a penetrating sequence (octa-arginine) to translocate it into
the cytoplasm to inhibit telomerase activity in cancer
cells. The octa-arginine sequence has been reported to
cross a variety of biological barriers readily, and it is
a promising transporter for intracellular delivery.19,20)
Hence, we report the telomerase inhibitory activity of
D-9-mer peptides and D-peptide C2. Moreover, mitochondria were also identied as a possible target of
D-peptide C2 in cancer cells.

y
To whom correspondence should be addressed. Fax: +81-29-838-6028; E-mail: yamakawa@nias.arc.go.jp
Abbreviations: AMP, antimicrobial peptide; TAMRA, tetra-methyl rhodamine; TRAP, telomerase repeat amplication protocol

684

T. IWASAKI et al.

Materials and Methods

Cell lines. Cos-1 African green monkey kidney cancer and U-251
human glioma were cultured in D-MEM (Dulbeccos Modied Eagle
Medium). VA-13 human lung cancer and MRC-5 human broblasts
were cultured in MEM-alpha, Jurkat human leukemia in RPMI1640,
and RERF-LC-AI human carcinoma in MEM. All the media contained
1,000 mg/ml of glucose, 584 of mg/ml glutamine, 10% FCS, 100 mg/ml
of streptomycin, and 100 units/ml of penicillin.
Peptide synthesis and purication. The enantiomeric 9-mer peptides
(D-peptides A, B, C, and D) were synthesized with D-amino acids and
an amide group at their C-termini by the multi-pin technique (Chiron
Mimotopes, Clayton, Victoria, Australia)21) according to methods of
our previous studies,10,12) and were puried to homogeneity by
SMART using a mRPC C2/C18 PC 3.2/3 (Pharmacia, Sweden)
reverse-phase column. The column was eluted for 20 min at 100
ml/min with a linear gradient from 0 to 30% (v/v) acetonitrile in water
containing 0.05% (v/v) trifuoroacetic acid. The nal purity of all the
peptides was >95 calculated from peak areas of the HPLC charts. The
peptide concentrations were determined, using a spectrometer (the UV
method). The amino acid sequences and molecular masses were
conrmed with a protein sequencer (Procise cLC 492, PE Biosystems,
California, USA) and matrix-assisted laser desorption/ionization
time-of-ight mass spectrometry (Autoex II, Bruker Daltonics,
Massachusetts, USA) respectively.
Telomerase activity assay. Telomerase activity was determined by
telomerase repeat amplication protocol (TRAP) assay, as previously
described.2) TRAP assay was done with a Quantitative Telomerase
Detection Kit (US Biomax, Rockville, USA) according to the
manufacturers protocol. In brief, 1:0
106 Jurkat cells were lysed
in 200 ml of lysis reagent and incubated on ice for 30 min. After
centrifugation at 12;000
g for 30 min, 160 ml of the supernatant was
transferred to a fresh tube, and was saved as the cell extract. The
telomeric substrate was allowed to extend by telomerase activity in the
cell extract with the TRAP reaction mixture in a total volume of 25 ml
at 25  C for 30 min. The telomeric product was amplied by 40 cycles
of real-time PCR (95  C for 30 s, 68  C for 30 s, and 72  C for 30 s).
Telomerase activity was determined by collecting the threshold cycle
after the cycles nished. The threshold cycle is the cycle at which a
statistically signicant increase in
Rn is rst detected. The
telomerase inhibitory concentration of each peptide was determined
in three experiments.
Assay of internalization. D-Peptide C2 was conjugated with tetramethyl rhodamine (TAMRA) (Invitrogen, California, USA) in 0.1 M
sodium tetraborate (pH 8.5) for 6 h at room temperature. After
incubation, D-peptide C2-TAMRA was puried by HP1100 using a
reverse-phase PEGASIL ODS column (Sensyu Pak, Tokyo, Japan).
The column was eluted for 30 min at 500 ml/min with a linear gradient
from 10 to 40% (v/v) acetonitrile in water containing 0.05% (v/v)
triuoroacetic acid. Jurkat human leukemia cells were seeded onto 96well plates at a density of 8:0
103 /well in a nal volume of 100 ml,
and incubated with 1 mM of D-peptide C2-TAMRA in 5% CO2 for 24 h
at 37  C. Internalization of D-peptide C2-TAMRA into the Jurkat
cytoplasm was conrmed by uorescence microscopy by rhodamine
detection (Ex/Em = 540/570 nm).
Cell proliferation and viability assay. The cancer cells were seeded
onto 96-well plates at a density of 8:0
103 cells in a nal volume of
100 ml, as follows: various concentrations of D-9-mer peptides (1, 2, 4,
8, 16, and 32 mM) were determined with a spectrophotometer, and 10 ml
aliquots added to each well. The plate was then incubated for 24 h at
37  C before the addition of 10 ml Cell Counting Kit-8 reaction solution
(Dojindo, Kumamoto, Japan) to each well. The absorbance was read at
450 nm with an enzyme-linked immunosorbent assay plate reader 4 h
after incubation. Cell viability relative to the control was determined
by three experiments.
JC-1 staining. Jurkat cells were seeded at a density of
5:0
104 cells/well in 96-well plates at a nal volume of 100 ml and

incubated with and without 10 mM D-peptide C2 in 5% CO2 for 24 h at

37  C. Then 10 ml of JC-1 staining solution (Cayman Chemical
Company, Michigan, USA) was added to each well. After incubation
for 30 min in a CO2 incubator, red uorescence (JC-1 J-aggregates)
indicating healthy mitochondria, was measured at an excitation
wavelength of 540 nm and an emission wavelength of 590 nm,
and green uorescence (JC-1 monomer), indicating damaged mitochondria, at an excitation wavelength of 485 nm and an emission
wavelength of 535 nm by uorescence microscopy (Olympus IX71,
Tokyo).
Electron microscopy. Jurkat cells treated with and without 10 mM
C2 were xed in 2.5% glutaraldehyde in 0.1 M potassium
phosphate buer, pH 7.4, for 30 min at room temperature. The cells
were packed into a plastic hematocrit tube and centrifuged at 700
g
for 3 min, and then postxed in 1% osmic acid. The xed specimens
were dehydrated with an ethanol series and embedded in epoxy resin.
Ultrathin sections were cut with glass knives and stained with uranyl
acetate and lead citrate, and then observed with a transmission electron
microscope (Hitachi 7500, Hitachi, Tokyo).
D-peptide

Isolation of mitochondria. All procedures were carried out at 4  C.

Mouse liver mitochondria were prepared, using a Cytochrome c
Releasing Apoptosis Assay Kit (Bio Vision, California, USA) according to the manufacturers protocol. Mouse liver was washed with 10 ml
of ice-cold PBS and hashed in 2 ml of 1X Cytosol Extraction Buer
Mix containing dithiothreitol (DTT) and protease inhibitors. After
incubation for 10 min, mouse liver cells were homogenated in a
Dounce tissue grinder (WHEATON, Millville, USA) with 3050
passes. Homogenate was transfered to a 1.5-ml microcentrifuge tube
and centrifuged at 700
g for 10 min at 4  C. The supernatant was
transferred to a fresh 1.5-ml tube and centrifuged at 10;000
g for
30 min at 4  C. The pellet was resuspended in 0.1 ml of Mitochondrial
Extraction Buer Mix containing DTT and protease inhibitors, and
was saved as a mitochondrial fraction.
Measurement of mitochondrial swelling. Mitochondria (200 mg of
mitochondrial protein) isolated from mouse liver were resuspended in
1 ml of swelling buer containing 125 mM KCl, 20 mM Tris, 1 mM
MgCl2 , 1 mM EGTA, 5 mM glutamate, and 5 mM malate (pH 7.2).
Mitochondrial swelling was monitored by measurement of light
scattering at 540 nm with a Appliskan microplate reader (Thermo
Fisher Scientic, Massachusetts, USA). Permeability transition was
induced by the addition of CaCl2 (200 mM) 2 min after starting the
experiment. D-Control peptide (AKGFAANHS-NH2 ) with no antimicrobial activity was used as a negative control.

Results
Telomerase inhibitory activity
The eects of D-peptides A, B, C, D, and C2 on
telomerase isolated from Jurkat cell extracts were
examined by TRAP assay. The D-9-mer peptides showed
similar inhibitory activities against telomerase
(IC100 = 40 mM). D-Peptide C2, with octa-arginine in
the C-terminus, showed 400-fold higher telomerase
inhibitory activity than D-peptide C (IC100 = 0.1 mM)
(Table 1).

Table 1. Telomerase Inhibitory Activity of Enantiomeric Peptides

Sequence
D-Control
D-Peptide
D-Peptide
D-Peptide
D-Peptide
D-Peptide

peptide
A
B
C
D
C2

AKGFAANHS-NH2
RLYLRIGRR-NH2
RLRLRIGRR-NH2
ALYLAIRRR-NH2
RLLLRIGRR-NH2
ALYLAIRRRRRRRR-NH2

IC100 (mM)
>100
40
40
40
40
0.1

Multiple Targets of D-Peptides Derived from Beetle Defensin

Table 2. Comparison of Inhibitory Potency between D-Peptide C and
C2
IC50 (mM)
Cos-1 Jurkat VA-13
D-Peptide
D-Peptide


C
C2

>640
3.4

574
11.0

>640
16.0

RERFU-251 MRC-5
LC-AI
>640
22.3

>640
26.4

>640
11.1

IC50 , 50% inhibitory concentration

Internalization of D-peptide C2-TAMRA

D-Peptides C and C2 were conjugated with TAMRA,
and internalization into Jurkat cells was examined by
detecting TAMRA by uorescence microscopy. DPeptide C-TAMRA showed no uorescence in the
Jurkat cytoplasm (Fig. 1c). On the other hand, as we
expected, Jurkat treated with D-peptide C2-TAMRA for
30 min showed strong red uorescence (Fig. 1d), indicating that D-peptide C2 with octa-arginine penetrating
sequences translocated into the Jurkat cytoplasm.
Inhibitory concentrations of enantiomeric peptides
The anticancer activity of D-peptide C2 against
various cancer cells was examined by counting live
cells. D-Peptide C2 inhibited the growth of all cancer
cell lines at lower concentrations than the original D-9mer peptides (D-peptide AD) (Table 2). In particular,
Cos-1 African green monkey kidney cancer was the cell
line most sensitive to D-peptide C2 (IC50 = 3.4 mM).
It showed a 200-fold increase in cytotoxicity. DPeptide C2 inhibited the growth of MRC-5 human
broblasts (IC50 = 11.1 mM) as well as other cancer cell
lines did even though MRC-5 has no activated telomerase. Furthermore, the immediate cytotoxicity of Dpeptide C2 was earlier than that which would indicate
an eect of telomerase inhibitory activity, because
telomerase inhibitory activity is normally detectable
weeks later during cancer cell proliferation. These
results indicate that the major target of D-peptide C2 is
not telomerase.
Eect of D-peptide C2 on mitochondrial potential in
Jurkat cells
Changes in mitochondrial potential were observed
in that JC-1 formation changed from J-aggregates (red
uorescence) to monomers (green uorescence) in the
Jurkat cells. Both the control cells and the D-peptide C
treated cells showed low green uorescence, indicating
the loss of mitochondrial potential and high accumulation of red uorescence characteristic of healthy
mitochondria (Fig. 2d and e). On the other hand, a
large increase in green uorescence intensity and a lack
of red uorescence was observed in the Jurkat cells
treated with D-peptide C2 (Fig. 2f). These results indicate that D-peptide C2 aects the mitochondrial
potential of Jurkat cells.
Electron microscopy
To assess the morphological damage induced by Dpeptide C2, the ultrastructure of mitochondria in Jurkat
human leukemia exposed to D-peptides C and C2 was
investigated by transmission electron microscopy. The
mitochondrial membrane and cristae remained intact in

685

the untreated cells and in the cells treated with Dpeptide C (Fig. 3a and b). However, the Jurkat cells
treated with 10 mM D-peptide C2 for 24 h exhibited
profound changes in the mitochondria. A loss of the
mitochondrial membrane was observed (Fig. 3c), and
the cristae structures were largely lost as a consequence
of the mitochondrial membrane loss, a typical feature
of mitochondrial lysis. Furthermore, mitochondria
were not observed in the cells with more advanced
damage (Fig. 3d). This profound disruption of the
mitochondria of Jurkat cells by D-peptide C2 perhaps
induces a thorough collapse of the mitochondrial
membrane potentials and irreversible inhibition of cell
growth.
Mitochondrial swelling assay
Mitochondrial swelling was evaluated by monitoring
light scattering (absorbance at 540 nm) immediately
after the mitochondria were isolated from the mouse
liver. The relative absorbances were calculated by
normalization with the negative control, the absorbance
of mitochondria treated with H2 O. A decrease in the
absorbance of mitochondrial swelling was quickly
initiated by the addition of 100 mM of D-peptide C2
(Fig. 4). However, 100 mM each of D-peptides A, B, C,
and D as well as the positive control, 200 mM of CaCl2 ,
also induced mitochondrial swelling. These results
suggest that the four D-9-mer peptides also disrupt the
mitochondrial membrane and exhibit cytotoxity similar
to D-peptide C2 when in the cytoplasm.

Discussion
In this study, we found that D-peptide C2, a cellpenetrating peptide, induced large increases in telomerase inhibitory activity (IC100 = 0.1 mM) as compared
to D-9-mer peptides (IC100 = 40 mM). It is unclear how
D-peptide C2 can strongly inhibit telomerase activity.
However, the activity of telomerase can be inhibited by
the addition of arginine residues into the 9-mer peptide,
inducing more positive charges to enhance the binding
anities of the compounds to DNA.18) As we expected,
D-peptide C2 killed cancerous cells at low concentrations, indicating strong antitumor activity attributable to
strong telomerase inhibitory activity. However, the cell
proliferation assay showed that D-peptide C2 induced
cell death in 24 h even though the telomerase inhibitory
eect was not detectable until at least 2 weeks later
during cancer cell proliferation. Moreover, the growth of
MRC-5 human broblasts was also highly inhibited
even though MRC-5 has no activated telomerase and the
nite cell lifetime diers from cancerous cells. These
two contradictions indicate that the major target of
D-peptide C2 is not telomerases in cancer cells and
broblasts. Three main points as to targets of Dpeptide C2 were illuminated by the present study: (i)
D-peptide C2 kills both normal and cancerous eukaryote
cells at lower concentrations than the original Dpeptide C, (ii) it maintains a sequence common with
D-peptide C, reported to disrupt negatively charged
bacterial membranes,1315) and (iii) it aects something
in the cytoplasm to induce the death of a cell. This
information led us to hypothesize that the major target of
D-peptide C2 is the mitochondria, because mitochondria

686

T. IWASAKI et al.
D-Peptide

C-TAMRA 1 M

D-Peptide

Bright-fieldl

C2-TAMRA 1 M

20m
500 nm

c
Dark-field

d
c

Fig. 1. Internalization of D-Peptide C2-TAMRA.

Jurkat cells were incubated in 1 mM of D-peptide C-TAMRA (a,
c) or D-peptide C2-TAMRA (b, d) at 37  C for 24 h. D-Peptide C2
was uptaken into the Jurkat cytoplasm, as shown by TAMRA reduorescence (d). Scale bar indicates 20 mm.
120%
D-control

peptide
Control(H2O)

100%

A540 (A/Acont )

Mitochondrial swelling

500 nm

D-peptide

80%

D-peptide
D-peptide

B
D
C

Ca2+
D-peptide A
D-peptide C2

60%

40%

10 (min)

2 min
Incubation

8 min
Absorbance determination

Fig. 4. Mitochondrial Swelling Assay.

Representative traces of changes in the light scattering of
isolated liver mitochondria were examined. Mitochondrial swelling indicates changes in mitochondrial volume due to changes in
mitochondrial membrane potential. In these experiments, mitochondrial swellings were monitored simultaneously by following
changes in light scattering at 540 nm for 2 min after reagent
treatment. Ca2 and D-control peptide were used as positive and
negative controls, respectively.

Red-green
fluorescence merge

Bright-field

Non-treated

500 nm

500 nm

Fig. 3. Electron Microscopy.

Jurkat cells were incubated with no peptides (a), 10 mM of Dpeptide C (b), or 10 mM of C2 (c, d) at 37  C for 24 h, and then
xed and examined by transmission electron microscopy. The
arrows indicate examples of mitochondria of normal density. The
open box indicates mitochondrial morphological changes with
ruptured cristae and unclear outlines (c). Panel d shows more
seriously damaged mitochondria. Ultrathin sections are shown.
Original magnications,
20;000 (a, b, c) and
50;000 (d).

exist in both normal and cancerous eukaryote cells, have

a negatively charged outer membrane derived from
aerobic bacteria,22) and are present in the cytoplasm.
Hence mitochondria were examined as a target of Dpeptide C2. Fluorescence and electron microscopic
observations of mitochondria in Jurkat human leukemia
treated with D-peptide C2 indicated a loss of mitochondrial membrane potential and disruption of the outer
membrane, providing direct evidence that D-peptide C2
aects the mitochondrial membrane and can induce the
death of the cell. Furthermore, mitochondrial swelling
assay showed that D-peptide C2 as well as D-9-mer
peptides induced mitochondrial swelling. This implies
that the original D-9-mer peptides also have mitochon-

D-Peptide

C 10 M

D-Peptide

C2 10 M

100m

Fig. 2. Eect of D-Peptide C2 on Mitochondrial Potential in Jurkat Cells.

Jurkat cells were incubated with no peptides (a, d), 10 mM of D-peptide C (b, e), or C2 (c, f) at 37  C for 24 h. After incubation, the cells were
stained with JC-1 dye for 30 min, and the eect of each peptide on the mitochondria was determined by detecting changes in JC-1 uorescence.
Red uorescence indicates healthy mitochondria and green uorescence indicates a loss of mitochondrial membrane potential. The scale bar
indicates 100 mm.

Multiple Targets of D-Peptides Derived from Beetle Defensin

drial membrane disruptive activity and exhibit eectiveness on cytotoxity similar to D-peptide C2 when they are
translocated into the cytoplasm by a drug delivery
system (DDS). In fact, an antimicrobial peptide analog
(KLAKLAKKLAKLAK) showing mitochondria disruptive potency has been reported to indicate extreme
increases in antitumor activity by conjugating with
tumor-specic antibodies.23) The anti-CD19 and antiCD33 antibodies that target surface antigens selectively
expressed on B lymphoid24) and myeloid25) cells are
known to internalize compounds linked to them into the
cytoplasm. A previous study reported that immunoconjugates, mitochondria disruptive peptides conjugated
with anti-CD19 or anti-CD33 antibody, killed lymphoid
and myeloid more eectively (IC50 = 2.16.2 nM) than
the peptide alone (IC50 = 1015 mM).23) D-Peptides A,
B, C, and D are also expected to exhibit substantial
increases in cytotoxicity and specicity with a tumorspecic DDS.
The results of this study, then, suggest that mitochondrial disruptive D-9-mer peptides are good warheads for
missile therapy against various tumors. The development of novel strategies for the application of D-9-mer
peptides with tumor-specic DDS is required.

11)

12)

13)

14)

15)

16)

Acknowledgments
This work was supported in part by a Grant-in-Aid for
Scientic Research (B) (20380039, to M.Y.), and by
Research Fellowships from the Japan Society for the
Promotion of Science for Young Scientists (19-1733,
to T.I.).

References
1)

2)

3)
4)

5)

6)
7)
8)

9)

10)

Bertuch, A. A., and Lundblad, V., The maintenance and

masking of chromosome termini. Curr. Opin. Cell Biol., 18,
247253 (2006).
Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B.,
West, M. D., Ho, P. L., Coviello, G. M., Wright, W. E.,
Weinrich, S. L., and Shay, J. W., Specic association of human
telomerase activity with immortal cells and cancer. Science,
266, 20112015 (1994).
Holt, S. E., and Shay, J. W., Role of telomerase in cellular
proliferation and cancer. J. Cell Physiol., 180, 1018 (1999).
Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu,
C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S.,
and Wright, W. E., Extension of life-span by introduction of
telomerase into normal human cells. Science, 279, 349352
(1998).
Harley, C. B., Futcher, A. B., and Greider, C. W., Telomeres
shorten during ageing of human broblasts. Nature, 345, 458
460 (1990).
Greider, C. W., Telomerase activation: one step on the road to
cancer? Trends Genet., 15, 109112 (1999).
Shay, J. W., and Bacchetti, S., A survey of telomerase activity
in human cancer. Eur. J. Cancer, 33, 787791 (1997).
Ramirez, R., Carracedo, J., Jimenez, R., Canela, A., Herrera, E.,
Aljama, P., and Blasco, M. A., Massive telomere loss is an early
event of DNA damage-induced apoptosis. J. Biol. Chem., 278,
836842 (2003).
Herbert, B., Pitts, A. E., Baker, S. I., Hamilton, S. E., Wright,
W. E., Shay, J. W., and Corey, D. R., Inhibition of human
telomerase in immortal human cells leads to progressive
telomere shortening and cell death. Proc. Natl. Acad. Sci.
USA, 96, 1427614281 (1999).
Miyanoshita, A., Hara, S., Sugiyama, M., Asaoka, A., Taniai,

17)

18)

19)

20)

21)

22)

23)

24)

25)

687

K., Yukuhiro, F., and Yamakawa, M., Isolation and characterization of a new member of the insect defensin family from
a beetle, Allomyrina dichotoma. Biochem. Biophys. Res.
Commun., 220, 526531 (1996).
Ishibashi, J., Saido-Sakanaka, H., Yang, J., Sagisaka, A., and
Yamakawa, M., Purication, cDNA cloning and modication of
a defensin from the coconut rhinoceros beetle, Oryctes
rhinoceros. Eur. J. Biochem., 266, 616623 (1999).
Saido-Sakanaka, H., Ishibashi, J., Sagisaka, A., Momotani, E.,
and Yamakawa, M., Synthesis and characterization of bactericidal oligopeptides designed on the basis of an insect antibacterial peptide. Biochem. J., 338, 2933 (1999).
Saido-Sakanaka, H., Ishibashi, J., Momotani, E., Amano, F., and
Yamakawa, M., In vitro and in vivo activity of antimicrobial
peptides synthesized based on the insect defensin. Peptides, 25,
1927 (2004).
Iwasaki, T., Saido-Sakanaka, H., Asaoka, A., Taylor, D.,
Ishibashi, J., and Yamakawa, M., In vitro activity of diastereomeric antimicrobial peptides alone and in combination with
antibiotics against Methicillin-resistant Staphylococcus aureus
and Pseudomonas aeruginosa. J. Insect Biotechnol. Sericol., 76,
2529 (2007).
Saido-Sakanaka, H., Ishibashi, J., Momotani, E., and
Yamakawa, M., Protective eects of synthetic antibacterial
oligopeptides based on the insect defensins on Methicillinresistant Staphylococcus aureus in mice. Dev. Comp. Immunol.,
29, 469477 (2005).
Yamada, M., Nakamura, K., Saido-Sakanaka, H., Asaoka, A.,
Yamakawa, M., Sameshima, T., Motobu, M., and Hirota, Y.,
Eect of modied oligopeptides from the beetle Allomyrina
dichotoma on Escherichia coli infection in mice. J. Vet. Med.
Sci., 66, 137142 (2004).
Yamada, M., Nakamura, K., Saido-Sakanaka, H., Asaoka, A.,
Yamakawa, M., Yamamoto, Y., Koyama, Y., Hikosaka, K.,
Shimizu, A., and Hirota, Y., Therapeutic eect of modied
oligopeptides from the beetle Allomyrina dichotoma on
Methicillin-resistant Staphylococcus aureus (MRSA) infection
in mice. J. Vet. Med. Sci., 67, 10051011 (2005).
Zhang, L., Huang, J., Ren, L., Bai, M., Wu, L., Zhai, B., and
Zhou, X., Synthesis and evaluation of cationic phthalocyanine
derivatives as potential inhibitors of telomerase. Bioorg. Med.
Chem., 16, 303312 (2008).
Futaki, S., Oligoarginine vectors for intracellular delivery:
design and cellular-uptake mechanisms. Biopolymers, 84, 241
249 (2006).
Rothbard, J. B., Kreider, E., VanDeusen, C. L., Wright, L.,
Wylie, B. L., and Wender, P. A., Arginine-rich molecular
transporters for drug delivery: role of backbone spacing in
cellular uptake. J. Med. Chem., 45, 36123618 (2002).
Maeji, N. J., Bray, A. M., and Geysen, H. M., Multi-pin peptide
synthesis strategy for T cell determinant analysis. J. Immunol.
Methods, 134, 2333 (1990).
Trapp, S., and Horobin, R. W., A predictive model for the
selective accumulation of chemicals in tumor cells. Eur.
Biophys. J., 34, 959966 (2005).
Marks, A. J., Cooper, M. S., Anderson, R. J., Orchard, K. H.,
Hale, G., North, J. M., Ganeshaguru, K., Steele, A. J., Mehta,
A. B., Lowdell, M. W., and Wickremasinghe, R. G., Selective
apoptotic killing of malignant hemopoietic cells by antibodytargeted delivery of an amphipathic peptide. Cancer Res., 65,
23732377 (2005).
Pulczynski, S., Boesen, A. M., and Jensen, O. M., Antibodyinduced modulation and intracellular transport of CD10 and
CD19 antigens in human B-cell lines: an immunouorescence
and immunoelectron microscopy study. Blood, 81, 15491557
(1993).
van Der Velden, V. H., te Marvelde, J. G., Hoogeveen, P. G.,
Bernstein, I. D., Houtsmuller, A. B., Berger, M. S., and van
Dongen, J. J., Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in
vivo and in vitro saturation and internalization by leukemic and
normal myeloid cells. Blood, 97, 31973204 (2001).