Efficient delivery of small interfering RNA to
bone-metastatic tumors by using atelocollagen in vivo
Fumitaka Takeshita*, Yoshiko Minakuchi†, Shunji Nagahara†, Kimi Honma‡, Hideo Sasaki*, Kotaro Hirai*,
Takumi Teratani*, Nachi Namatame§, Yusuke Yamamoto§, Koji Hanai†, Takashi Kato§, Akihiko Sano†,
and Takahiro Ochiya*¶
*Section for Studies on Metastasis, National Cancer Center Research Institute, Tokyo 104-0045, Japan;†Formulation Research Laboratories, Sumitomo
Pharmaceutical, Osaka 567-0878, Japan;‡Koken Bioscience Institute, Tokyo 115-0051, Japan; and§Department of Biology, School of Education,
Waseda University, Tokyo 169-0051, Japan
Edited by Inder M. Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved July 12, 2005 (received for review March 3, 2005)
Silencing of gene expression by small interfering RNAs (siRNAs) is
rapidly becoming a powerful tool for genetic analysis and repre-
sents a potential strategy for therapeutic product development.
However, there are no reports of systemic delivery for siRNAs
toward treatment of bone-metastatic cancer. Accordingly, we
report here that i.v. injection of GL3 luciferase siRNA complexed
with atelocollagen showed effective reduction of luciferase ex-
pression from bone-metastatic prostate tumor cells developed in
mouse thorax, jaws, and?or legs. We also show that the siRNA?
atelocollagen complex can be efficiently delivered to tumors 24 h
after injection and can exist intact at least for 3 days. Furthermore,
atelocollagen-mediated systemic administration of siRNAs such as
enhancer of zeste homolog 2 and phosphoinositide 3?-hydroxy-
kinase p110-?-subunit, which were selected as candidate targets
for inhibition of bone metastasis, resulted in an efficient inhibition
of metastatic tumor growth in bone tissues. In addition, up-
regulation of serum IL-12 and IFN-? levels was not associated with
the in vivo administration of the siRNA?atelocollagen complex.
Thus, for treatment of bone metastasis of prostate cancer, an
atelocollagen-mediated systemic delivery method could be a reli-
able and safe approach to the achievement of maximal function of
bone metastasis ? prostate cancer
is capable of suppressing expression of individual genes with a
high degree of specificity (1). The technique has been used for
studies of gene function in vivo, primarily in mice. The first
demonstration of RNAi-mediated repression in an adult animal
showed effective repression of a luciferase reporter gene after
hydrodynamic transfection of siRNA expression plasmids into
mouse liver (2, 3). Subsequent studies have delivered siRNA by
various methods, including viral vector-mediated delivery (4, 5)
and lipid-based delivery (6, 7). A more recent study showed that
chemically modified siRNAs can silence an endogenous gene
after i.v. injection in mice (8). These findings provide hope for
using RNAi technology in disease control.
Many studies have used siRNAs as an experimental tool to
dissect the cellular pathways that lead to uncontrolled cell
proliferation and cancer. To develop siRNAs for cancer therapy,
several researchers have investigated them in animal models
(9–13). However, reports of RNAi-delivery strategies for bone-
metastatic cancer are very limited. For example, in advanced
prostate cancer, the sites most frequently affected by metastasis
are the bones and regional lymph nodes. Patients with these
metastases suffer pain and low limb edema, making it extremely
important to explore avenues of treating such bone metastases.
We previously demonstrated the efficacy of atelocollagen for
delivery of nucleotides, such as plasmid DNA and antisense
oligonucleotides, in vitro and in vivo (14–19). Recently, we also
NA interference (RNAi) induced by small interfering RNA
(siRNA) has recently emerged as a powerful technique that
nucleases and is efficiently transduced into cells, thereby allow-
ing long-term gene silencing (20). Furthermore, intratumor
injection of atelocollagen complexed with siRNA against fibro-
growth in an orthotopic xenograft model of a human nonsemi-
nomatous germ cell tumor (20). Another group reported that
radiolabeled siRNA mixed with atelocollagen existed in the
tumors for at least a week and remained intact and that the
vascular endothelial growth factor siRNA with atelocollagen
dramatically suppressed tumor angiogenesis and tumor growth
in a PC-3 s.c. xenograft model (21). Thus, for local administra-
tion of siRNA, an atelocollagen-based nonviral delivery method
could be a reliable approach to achieve the maximal function of
siRNA in vivo. In addition, an atelocollagen complex can be
delivered for i.v. injection as nanoparticles, making systemic
delivery of siRNA possible. A recent report showed the potential
for atelocollagen-mediated systemic antisense therapeutics for
treating inflammatory disease (19).
In this study, noninvasive optical imaging technologies were
used to facilitate the detection of metastatic lesions and the
effects of synthetic siRNAs on tumor regression. The results
indicate that systemic administration of atelocollagen com-
plexed with siRNA into a mouse model of bone metastasis
demonstrated effective gene silencing and tumor regression in
bone-metastatic lesions. Furthermore, we also showed that
atelocollagen-mediated systemic delivery of siRNA did not
cause any side effects. Thus, systemic delivery of a siRNA?
atelocollagen complex may have therapeutic potential in the
treatment of advanced prostate cancer with bone metastasis.
Materials and Methods
Atelocollagen. Atelocollagen is a highly purified type I collagen
of calf dermis with pepsin treatment (Koken, Tokyo). A collagen
N and C termini, which confers most of the collagen’s antige-
nicity. Atelocollagen obtained by pepsin treatment is low in
immunogenicity because it is free from telopeptides (22), and it
is used clinically for a wide range of purposes, including wound
healing and vessel prosthesis and as a bone cartilage substitute
and haemostatic agent (16).
Cell Lines. The bioluminescent human prostate carcinoma cell
line PC-3M-luc-C6 (Xenogen, Alameda, CA) was cultured in
Eagle’s minimum essential medium (Invitrogen) supplemented
This paper was submitted directly (Track II) to the PNAS office.
Freely available online through the PNAS open access option.
2; p110-?, phosphoinositide 3?-hydroxykinase p110-?-subunit.
¶To whom correspondence should be addressed at: Section for Studies on Metastasis,
Japan. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
August 23, 2005 ?
vol. 102 ?
no. 34 ?
with 10% heat-inactivated FBS (Equitech-Bio, Kerrville, TX),
nonessential amino acids (Sigma-Aldrich), L-glutamine (ICN), 1
mM sodium pyruvate (Sigma-Aldrich), MEM vitamin solution
(Sigma-Aldrich), and 200 ?g?ml zeocin (Invitrogen). The cells
were maintained in vitro at 37°C in a humidified atmosphere of
siRNA Preparation. Synthetic 21-nt RNAs were purchased from
Dharmacon Research (Lafayette, CO) in deprotected, desalted,
and annealed form. The sequence for GL3 siRNA is reported in
ref. 23. The sequence of human enhancer of zeste homolog 2
(EZH2) siRNA was 5?-GGA AAG AAC GGA AAU CUU
and human phosphoinositide 3?-hydroxykinase p110-?-subunit
(p110-?) siRNA was 5?-GGU UAA AGA UCC AGA AGU
The nonspecific control siRNA duplex was also purchased from
In Vivo Imaging of siRNA Delivery in Mice with Bone-Metastatic
Tumors. Animal experiments in the present study were per-
formed in compliance with the guidelines of the Institute for
Laboratory Animal Research at the National Cancer Center
(CLEA Japan, Osaka) were anesthetized by exposure to 1–3%
isoflurane on day 0 and subsequent days. On day 0 of the
experiments, to generate an experimental metastasis model, the
anesthetized animals were injected with 3 ? 106PC-3M-luc-C6
cells suspended in 100 ?l of sterile Dulbecco’s PBS into the left
heart ventricle (24, 25). For in vivo imaging, the mice were
administered D-luciferin (150 mg?kg, Promega) by i.p. injection.
Ten minutes later, photons from animal whole bodies were
counted by using the IVIS imaging system (Xenogen) according
to the manufacturer’s instructions. Data were analyzed by using
LIVINGIMAGE 2.50 software (Xenogen). A successful intracardiac
injection was indicated by day 0 images that showed a systemic
bioluminescence distributed throughout the animal, and only
those mice evidencing a satisfactory injection were continued in
the experiment. The development of subsequent metastasis was
monitored twice a week in vivo by bioluminescent imaging.
For preparing the siRNA?atelocollagen complex, equal vol-
umes of atelocollagen (0.1% in PBS at pH 7.4) and siRNA
solution were combined and mixed by rotating for 20 min at 4°C.
The final concentration of atelocollagen was 0.05%. Four weeks
after tumor injection, individual mice (from cohorts containing
five animals) were injected with 200 ?l of atelocollagen con-
taining 25 ?g of luciferase GL3 siRNA, atelocollagen alone,
siRNA alone, or nonspecific siRNA?atelocollagen by i.v. tail
vein injection. Tumor growth was not affected by these treat-
ments. To control for mouse-to-mouse variability, the biolumi-
nescence ratio for each mouse was normalized by dividing by the
1-day-posttreatment?pretreatment ratio of luciferase intensity
for that mouse.
Detection of siRNA in Tumor Tissues or Normal Tissues by RNase
Protection Assay. To show siRNA delivery in tumor tissues,
10-week-old male athymic nude mice were inoculated s.c. with
3 ? 106PC-3M-luc-C6 cells suspended in 50 ?l of sterile
Dulbecco’s PBS. After 8 days, when a tumor reached a volume
of 50–100 mm3, tumor-bearing mice (from cohorts containing
three animals) were injected with 200 ?l of 0.05% atelocollagen
containing 25 ?g of luciferase GL3 siRNA, atelocollagen alone,
or siRNA alone by i.v. tail vein injection. The mice were killed
1 and 3 days after treatment of siRNA?atelocollagen complexes,
and total RNA was extracted from a tumor and selected mice
tissues by using ISOGEN (Nippon Gene, Tokyo). The RNase
protection probe was made with a mirVana microRNA Probe
Construction Kit (Ambion, Austin, TX). The cRNA probe
specific for the antisense strand of GL3 siRNA was generated by
using T7 RNA polymerase and32P-labeled UTP. Total RNAs
were used in an RNase protection assay using the mirVana
miRNA Detection Kit (Ambion) per the manufacturer’s proto-
col. Protected fragments were separated by electrophoresis in
15% polyacrylamide 8 M urea gels. The gels were exposed to
x-ray films for 30 min, and the films were then scanned and
analyzed by using NIH IMAGE software. GL3 siRNA levels were
corrected for wet tissue weights.
Atelocollagen-Mediated siRNA Transfection and Tumor Growth Assay
in Vitro. The EZH2 siRNA or p110-? siRNA complexed with
atelocollagen (final concentration ? 0.008%) was prefixed to a
six-well plate (37.5 pmol of siRNA?250 ?l per well) according to
the method described in refs. 15 and 20. The cultured PC-3M-
luc-C6 cells were plated into the complex-prefixed plate at 5 ?
104cells per well. Bioluminescence from PC-3M-luc-C6 cells
highly correlated to the total number of cells (26). For moni-
toring the inhibition of cell growth, the cells were lysed (n ? 3)
on days 2, 4, and 6 and then analyzed for luciferase activity
(Bright-Glo Luciferase Assay System, Promega). Inhibition of
luciferase production was normalized to the level of vehicle-
Quantitative RT-PCR. Total RNA was extracted from PC-3M-
luc-C6 cells by using ISOGEN and treated with DNase I (Takara
Shuzo, Otsu, Japan). Five micrograms of total RNA was used to
produce cDNAs with oligo(dT) 12 primer by superscript III
RNA polymerase (Invitrogen). cDNA was diluted 5-fold and
used for quantitative PCR. For quantitation, aliquots of 5 ?l of
cDNA samples were subjected to quantitative PCR in 50-?l
reactions by using Platinum Quantitative PCR SuperMix-UDG
(Invitrogen) and Assays-on-Demand TaqMan primers?probe
GAPDH. Reactions were carried out by using the Applied
Biosystems PRISM 7700 Sequence Detection System. The re-
actions were incubated at 50°C for 2 min and then heated to 95°C
for 2 min, followed by 45 cycles of 30 s at 95°C, 15 s at 60°C, and
20 s at 72°C. Human EZH2 and p110-? expression levels were
normalized to GAPDH levels.
Analysis of siRNA?Atelocollagen Treatment for Bone-Metastatic Pros-
tate Cancer. Mice were inoculated with PC-3M-luc-C6 cells into
the left cardiac ventricle on day 0 as described above. The EZH2,
p110-?, and nonspecific control siRNA (50 ?g) with or without
0.05% atelocollagen in a 200-?l volume were injected into the
mouse tail vein on days 3, 6, and 9 postinoculation. Each
experimental condition included eight animals per group. The
development of subsequent metastasis was monitored twice a
week in vivo by bioluminescent imaging for 4 weeks. To control
for mouse-to-mouse variability, the bioluminescence ratio for
each mouse was normalized by dividing by the before?after
treatment ratio of luciferase intensity for that mouse. At the end
of the experiment on day 28, to confirm the presence of
neoplastic cells, selected tissues were excised from the mice at
necropsy. Tissues were fixed in 4% formaldehyde-PBS(?), em-
bedded in paraffin, cut into 5-mm sections, and stained with
Monitoring of IFN Induction in Mice Treated with Atelocollagen-
Mediated siRNA. Eight-week-old male athymic nude mice were
injected with nonspecific control siRNA (50 ?g) with 0.05%
atelocollagen in a 200-?l volume by i.v. tail vein injection. Each
experimental condition included four animals per group. The
positive control group was injected with poly(I:C) (Amersham
Pharmacia Biosciences). To measure serum cytokine levels, blood
was harvested from mice 2 h after injection by cardiac puncture.
www.pnas.org?cgi?doi?10.1073?pnas.0501753102Takeshita et al.
IL-12 (p40) and IFN-? levels (R & D Systems) were measured by
ELISA according to the manufacturer’s instructions.
analysis was conducted by using the analysis of variance with the
Bonferroni correction for multiple comparisons. P ? 0.05 was
considered a significant difference.
Efficient Delivery of Atelocollagen-Mediated Luciferase siRNA in
Bone-Metastatic Regions. To increase the potential for bone
metastasis from PC-3M-luc-C6 cells, we injected the cells into
the left ventricle of the heart (25). Mice with successful intra-
cardiac injection of PC-3M-luc-C6 cells on day 0 were imaged
twice a week for up to 4 weeks. In all mice, early indications of
metastasis to various tissues were observed within 1 week after
the observed patterns of metastasis indicated lesions developing
in the thorax, jaws, and?or legs of the mice (Fig. 1A). To test
whether atelocollagen-mediated siRNA systemic delivery is
valid for a gene silencing effect on the metastatic sites, the
animals were treated with atelocollagen alone, a nonspecific
control siRNA?atelocollagen complex, a luciferase GL3 siRNA
alone, or a luciferase GL3 siRNA?atelocollagen complex i.v. In
mice receiving the luciferase siRNA?atelocollagen complex,
bioluminescence was inhibited by 80–90% in the whole body,
including the bone metastases, when compared with before
treatment (Fig. 1). In contrast, the bioluminescent signals of
most of the metastatic sites in the mice treated with atelocol-
lagen alone or the control siRNA?atelocollagen complex had
increased. Treatment with luciferase siRNA alone either had no
effect or slightly suppressed photon emission from the tumor
cells. After the imaging analysis, tissues expressing biolumines-
cence were excised from the mice at necropsy. Subsequent
histopathology analysis confirmed micrometastases in the lung,
dental pulp, tibia, femur, and other soft tissues (data not shown).
Thus, our results indicate that siRNA can be delivered by using
atelocollagen and can thereby inhibit gene expression in a
specific manner in metastatic sites, including bone metastases.
Enhanced Delivery of siRNA into Tumors by Atelocollagen. The
efficacy of delivery of siRNA into tumors was evaluated. Athy-
mic nude mice were inoculated s.c. with 3 ? 106PC-3M-luc-C6
cells and injected i.v. with luciferase siRNA?atelocollagen, lu-
ciferase siRNA alone, or atelocollagen alone. We assessed the
delivery of siRNA 1 day after the i.v. administration. As shown
in Fig. 2A and also in Fig. 7, which is published as supporting
information on the PNAS web site, a significant amount of
siRNA was detected in tumors with atelocollagen-mediated
delivery (4.3 ng of siRNA?mg of tumor weight). In contrast, i.v.
injection of siRNA alone (0.7 ng?mg of tumor weight) was less
efficient compared with atelocollagen-mediated delivery. We
also assessed the delivery of luciferase siRNA in several tissues,
such as liver, lung, spleen, and kidney. As shown in Fig. 2B, a
relatively high amount of siRNA was detected in tissues from
mice administered with the siRNA?atelocollagen complex com-
ing. (A) Representative images of nude mice injected with 3 ? 106PC-3M-
luc-C6 cells suspended in 100 ?l of sterile Dulbecco’s PBS into the left ventricle
of the heart. Four weeks after tumor injection, each animal was administered
GL3 siRNA?atelocollagen complex, or nonspecific siRNA?atelocollagen com-
plex. (B) Normalized fold change (1 day posttreatment?pretreatment) of
bioluminescence emitted from whole body of mice. Data represent the
mean ? SD (n ? 4).*, P ? 0.001 versus other experimental groups.
Monitoring luciferase inhibition in vivo with bioluminescent imag-
and normal tissues. The nude mice were inoculated s.c. with 3 ? 106PC-3M-
treatment with siRNA?atelocollagen complexes, and total RNA was extracted
from a tumor (A) and selected tissues (B). Detection of luciferase GL3 siRNA
wet tissue weights.
Distribution of siRNA delivered with atelocollagen in tumor tissues
Takeshita et al.
August 23, 2005 ?
vol. 102 ?
no. 34 ?
pared with mice with siRNA alone. In addition, siRNA delivered
with atelocollagen existed intact for at least 3 days (data not
shown). Taken together, these results suggest that the systemic
injection of the siRNA?atelocollagen complex allows a more
efficient delivery of siRNA into tumors than siRNA alone and
causes siRNA to be retained for a longer period therein.
Atelocollagen-Mediated siRNA Transfer Allows Efficient Inhibition of
PC-3M-luc Cell Growth in Vitro. To screen target genes for showing
growth inhibition of PC-3M-luc cells, EZH2 and p110-? were
selected as target genes. The atelocollagen-mediated siRNA
reverse cell transfection method was used. The cultured PC-3M-
luc-C6 cells were plated into a siRNA?atelocollagen complex-
prefixed plate. For monitoring cell growth, we analyzed lucif-
erase activity. Inhibition of cell growth was observed on PC-
3M-luc cells treated with EZH2 and p110-? siRNA?
atelocollagen complexes (Fig. 3A). Inhibition of mRNA levels of
targets was also shown (Fig. 3B). These results revealed that
EZH2 and p110-? may be the target of inhibition of the
metastasis of PC-3M-luc cells.
Inhibition of Metastatic Tumor Growth in Bone Tissues in Animals with
by the atelocollagen-mediated siRNA delivery system, EZH2
and p110-? siRNA?atelocollagen complexes were administered
i.v. into mice on days 3, 6, and 9 of postintracardiac ventricle
injection of PC-3M-luc cells. The development of bone metas-
tasis was monitored in vivo by bioluminescent imaging. At the
end of the experiment on day 28, mice treated with atelocollagen
alone and the control siRNA?atelocollagen complex-treated
group showed high metastasis in the thorax, jaws, and?or legs
(Figs. 4A and 5A). Total luminescence from all tumors was
determined at different times posttreatment for each mouse. As
seen in Fig. 4B, there was an increase in luminescence in mice
treated with atelocollagen alone, the control siRNA?
atelocollagen complex, EZH2 siRNA alone, and p110-? siRNA
alone, whereas the EZH2 siRNA?atelocollagen-treated and
p110-? siRNA?atelocollagen-treated groups had no increase in
luminescence during the same observation period. There were
significant differences between the EZH2- and p110-? siRNA?
atelocollagen-treated groups and the other three experimental
groups on day 28 (P ? 0.05). Histopathological analysis revealed
that metastasis of PC-3M-luc-C6 cells in the dental pulp was
significantly inhibited by the EZH2 and p110-? siRNA?
atelocollagen complexes (Fig. 5B). Therefore, the atelocollagen-
mediated systemic delivery of siRNA could be a unique strategy
for inhibition of bone-metastatic prostate tumor growth in vivo.
Absence of IFN Response to Atelocollagen-Mediated siRNA Delivery
System. To test whether the atelocollagen-mediated siRNA
systemic delivery has the possibility of inducing IFN responses in
mice, the plasma levels of IFN-? and IL-12 in mice exposed to
the siRNA?atelocollagen complex or poly(I:C) by i.v. injection
were measured by ELISA (Fig. 6). As observed with IFN-? and
IL-12, the siRNA?atelocollagen complex failed to elicit IFN-?
and IL-12 responses, whereas poly(I:C) induced a strong re-
sponse. These results show that it is possible to administer a
siRNA?atelocollagen complex without inducing nonspecific
turning on of genes, leading to an immune response.
Our findings indicate that siRNA can be delivered to bone-
metastatic lesions by atelocollagen-mediated systemic injection.
Furthermore, we also showed that an atelocollagen-mediated
siRNA delivery system can be used to silence endogenous genes
involved in metastatic tumor cell growth.
proliferation. For monitoring the inhibition of cell growth, cells were lysed on days 2, 4, and 6 and then analyzed for luciferase activity. (B) The effects of siRNA
SD (n ? 3).*, P ? 0.05 versus cells treated with atelocollagen alone.
The EZH2 and p110-? siRNA inhibit PC-3M-luc-C6 cell proliferation and suppress EZH2 and p110-? expression. (A) Inhibition of PC-3M-luc-C6 cell
www.pnas.org?cgi?doi?10.1073?pnas.0501753102 Takeshita et al.
At present, the main obstacle to the development of thera-
peutic products using RNAi technologies is a suitable delivery
method. Viral delivery systems are efficient but cause concerns
over serious side effects (27). Cationic lipid complexes also can
be effective siRNA delivery agents (6). However, lipid delivery
of synthetic siRNAs can reportedly induce immune activation
in vivo (28). An important consideration for siRNA-mediated
inhibition of gene expression is whether the observed effects are
specific and not due to nonspecific ‘‘off-target’’ effects (29) and
are free from potential IFN responses (30). Heidel et al. (31)
showed that it is possible to administer naked, synthetic siRNAs
to mice and down-regulate an endogenous or exogenous target
without inducing an IFN response. In our experiments, in
agreement with Heidel et al. (31), injection of siRNA?
atelocollagen did not induce an IFN response or IL-12. There-
fore, our atelocollagen-based siRNA delivery method can be
varied to minimize the potential for an off-target effect of
Successful application of RNAi as a therapeutic method
requires an efficient and suitable delivery system that can target
a restricted cell population in vivo. The prolonged circulation
time of high-molecular-weight macromolecules enables them to
use the vascular abnormalities of solid tumor tissues, a phenom-
enon called the enhanced permeability and retention (EPR)
effect (32, 33). This EPR effect is attributed to anatomical and
pathophysiological alterations such as increased vascular density
due to neoangiogenesis, impaired lymphatic recovery, and lack
of smooth muscle layer in solid tumor vessels. The EPR effect
facilitates extravasation of polymeric drugs more selectively at
tumor tissues, and this selective targeting to solid tumor tissues
may lead to superior therapeutic benefits with fewer systemic
adverse effects. In our experiments, siRNA?atelocollagen com-
plexes showed greater selective accumulation in tumor tissues,
compared with normal tissues, possibly due to an EPR mecha-
nism. Although further analysis is required, our atelocollagen-
mediated siRNA delivery method could possess the potential for
selective targeting to tumor tissues.
The major risk faced by patients with prostate cancer is the
development of metastatic disease. Although genes associated
with metastatic prostate cancer can be identified readily by
collagen-mediated siRNA delivery system. Mice were inoculated with PC-3M-
luc-C6 cells into the left cardiac ventricle on day 0. The EZH2, p110-?, and
nonspecific control siRNAs (50 ?g) with or without 0.05% atelocollagen in a
200-?l volume were injected into the mouse tail vein on days 3, 6, and 9
postinoculation. Each experimental regimen comprised eight animals. (A)
Representative images of nude mice at the end of the experiment on day 28.
(B) Normalized fold change (posttreatment?pretreatment) of biolumines-
cence emitted from whole body of mice. Data represent the mean ? SD (n ?
8).*, P ? 0.05 versus other experimental groups.
Inhibition of metastatic tumor growth in bone tissues by the atelo-
leg of mice treated with atelocollagen alone, EZH2 siRNA?atelocollagen, and
p110-? siRNA?atelocollagen, respectively. (B) Hematoxylin?eosin-stained sec-
marks the carcinomatous micrometastasis. (Scale bars, 100 ?m.)
Confirmation of prostate cancer bone metastasis by ex vivo imaging
Takeshita et al.
August 23, 2005 ?
vol. 102 ?
no. 34 ?
screening techniques [e.g., gene arrays (34)], the validation and Download full-text
characterization of these genes will require sophisticated animal
identified EZH2 as a gene overexpressed in hormone-refractory
metastatic prostate cancer, and it has been found that patients
with clinically localized prostate cancers that express EZH2 have
In addition, knocking down the EZH2 protein in PC3 invasive
prostate cancer cells by using RNAi technology inhibited pro-
liferation of cells in vitro. In contrast, catalytic subunits of the
phosphatidylinositol 3-kinase p110-? regulate a variety of cel-
lular responses such as survival, proliferation, and cell migration
(35). In this report, our data demonstrate that expression of
EZH2 and p110-? are involved in tumor growth in metastatic
are also elevated in breast cancer (36, 37). Therefore, EZH2 and
p110-? siRNA?atelocollagen complexes may also have thera-
peutic potential for inhibiting the growth of breast cancer in
bone-metastatic sites. Although EZH2 and p110-? siRNA effi-
ciently inhibited proliferation of PC-3M-luc cells, further work
will be required to develop a siRNA therapy that induces the
cytocidal effect specific to prostate cancer cells.
In conclusion, we have developed a technique to efficiently
and safely deliver siRNA to a bone-metastatic lesion by an
atelocollagen-mediated systemic injection and demonstrated
specific inhibition of target gene expression. To our knowledge,
of systemic delivery of siRNA?atelocollagen complexes may
have therapeutic potential in the treatment of advanced prostate
cancer with bone metastasis.
We thank Ms. Ayako Inoue and Ms. Maho Kodama for their excellent
technical work. This work was supported in part by a grant-in-aid for the
Third-Term Comprehensive 10-Year Strategy for Cancer Control; Health
Science Research grants for Research on the Human Genome and Gene
Therapy from the Ministry of Health, Labour, and Welfare of Japan; a
grant-in-aid for Scientific Research on Priority Areas (Cancer) from the
Ministry of Education, Culture, Sports, Science, and Technology; and the
Program for Promotion of Fundamental Studies in Health Sciences of the
Organization for Pharmaceutical Safety and Research of Japan.
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ery system. Nude mice were injected with nonspecific control siRNA (50 ?g)
with 0.05% atelocollagen in a 200-?l volume by i.v. tail vein injection. The
positive control group was injected with poly(I:C). Serum was collected 2 h
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www.pnas.org?cgi?doi?10.1073?pnas.0501753102Takeshita et al.