Intraperitoneal administration of a small interfering RNA targeting nuclear
factor-kappa B with paclitaxel successfully prolongs the survival of
xenograft model mice with peritoneal metastasis of gastric cancer
Masashi Inoue1, Sachiko Matsumoto2, Hiroaki Saito1*, Shunichi Tsujitani1and Masahide Ikeguchi1
1Division of Surgical Oncology, Department of Surgery, Faculty of Medicine, Yonago, Japan
2Division of Pathological Biochemistry, Department of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Japan
Activation of nuclear factor-kappa B (NF-jB) has been detected
in various malignant tumors, including gastric carcinoma, and is
associated with tumor growth, metastasis, resistance to chemo-
therapeutic agents and poor prognosis. Therefore, NF-jB is a
potential target for antitumor therapy. In this study, we used a
small interfering RNA (siRNA) to knockdown NF-jB p65 expres-
sion and determined whether intraperitoneal administration of
NF-jB p65 siRNA and paclitaxel was effective for treating perito-
neal metastasis of gastric cancer. Western blot analysis revealed
that NF-jB p65 expression was diminished by NF-jB p65 siRNA.
Apoptotic cells were increased after transfection of NF-jB p65
siRNA compared with control siRNA in the treatment with pacli-
taxel. In a murine xenograft model, abundant fluorescence was
observed on the surface of intraperitoneal nodules of gastric can-
cer after siRNA administration. Moreover, intraperitoneal admin-
istration of NF-jB p65 siRNA reduced NF-jB expression in intra-
peritoneal nodules of gastric cancer. Finally, mice treated by in-
traperitoneal administration of NF-jB p65 siRNA and paclitaxel
survived for a significantly longer time than mice treated by intra-
peritoneal administration of paclitaxel alone (p 5 0.0002). Taken
together, the present results demonstrate that intraperitoneal
administration of NF-jB p65 siRNA and paclitaxel inhibited can-
cer growth in mice with peritoneal metastasis of gastric cancer.
Therefore, intraperitoneal administration of NF-jB p65 siRNA
and paclitaxel may provide a breakthrough in the treatment of
peritoneal metastasis of gastric cancer.
' 2008 Wiley-Liss, Inc.
Key words: chemosensitivity; gastric cancer; nuclear factor-kB p65;
peritoneal metastasis; siRNA
Gastric cancer is one of the most common malignancies. De-
spite the recently reduced mortality rates due to both earlier detec-
tion and improved therapy, death from gastric cancer still ranks
second among all cancer deaths worldwide.1The most frequent
site of metastasis in gastric cancer is the peritoneum.2Peritoneal
carcinomatosis is a common manifestation of digestive-tract can-
cer and has been regarded as a terminal disease with a short me-
dian survival. There are currently no effective treatments for peri-
toneal metastasis of gastric cancer. The development of new treat-
ments is therefore crucial for improving the survival rates of
gastric cancer patients.
Apoptosis induction is an important mechanism in chemother-
apy-induced killing of tumors. On the other hand, many anticancer
agents that induce apoptosis also activate the nuclear factor-kappa
B (NF-jB) transcription factor pathway. NF-jB is a critical regu-
lator of the expression of genes involved in immune and inflam-
matory responses and is also involved in the control of cell
growth.3,4When activated, NF-jB increases the expression of
genes that promote cell survival and block apoptosis.5,6As a con-
sequence, activation of NF-jB induces resistance toward anti-
cancer agents. Importantly, systemic use of NF-jB inhibitors,
such as the proteasome inhibitor PS-341, in conjunction with iri-
notecan (CPT-11) enhances chemotherapy-induced cell killing by
stimulating apoptosis.6,7Therefore, controlling NF-jB may be
extremely important for enhancing the efficacy of chemotherapeu-
tic agents in the treatment of cancer patients.
RNA interference (RNAi) is a newly discovered cellular path-
way for silencing genes at the mRNA level in a sequence-specific
manner via the introduction of cognate double-stranded small
interfering RNA (siRNA). A major breakthrough in the applica-
tion of RNAi technology in mammalian cells came from the de-
velopment of 21- to 22-nucleotide synthetic siRNAs for silencing
target genes in mammalian cells.8,9siRNA is a highly specific and
efficient tool for diminishing the expression of a target gene and
also has the potential to be used as a targeted therapeutic agent. In
fact, siRNAs have recently been experimentally introduced into
cancer therapy (24, 25). For instance, successful transfection of
PLK-1 siRNA into cancer cells using intravesical siRNA/cationic
liposomes reduced PLK-1 expression, thereby preventing bladder
cancer growth in the murine bladder.10Furthermore, siRNA was
used to inhibit the chemokine receptor CXCR4, which is one of
the critical factors for breast cancer metastasis through interac-
tions with its ligand, stromal cell-derived factor-1, and this inhibi-
tion of CXCR4 expression significantly impaired the invasion of a
breast cancer cell line and blocked lung metastasis in an animal
model.11These results indicate that siRNA administration may be
a promising treatment for cancer patients.
Intraperitoneal chemotherapy combined with general chemo-
therapy was recently shown to significantly improve survival in
ovarian cancer patients with peritoneal metastasis.12In our study,
paclitaxel was administered by intraperitoneal injection. Pacli-
taxel, with its high molecular weight and bulky chemical structure,
has an extremely low clearance rate from the peritoneal cavity,
perhaps lower than the clearance rates of any other known chemo-
therapeutic agents. This low clearance rate increases exposure
in the peritoneal cavity and reduces systemic exposure and
toxicity.13Moreover, the antimicrotubule and cytotoxic effects of
paclitaxel are dependent on the concentration and exposure time,
and these factors may be optimized by regional drug delivery. The
pharmacological and clinical characteristics of paclitaxel indicate
that it may be an ideal drug for intraperitoneal administration.
Although some papers have indicated the efficacy of intraperito-
neal chemotherapy in gastric cancer,14the efficacy of this treat-
ment seems to be limited at the present time. In this study, we
investigated the efficacy of intraperitoneal chemotherapy using
paclitaxel with NF-jB p65 siRNA for the treatment of peritoneal
metastasis from gastric cancer.
Material and methods
Cell lines and reagents
Human gastric cancer cell lines, MKN1 and MKN45, were pur-
chased from the Riken Cell Bank (Tsukuba, Japan). The cells
were maintained in 5% CO2at 37?C in RPMI-1640 (Cambrex Bio
Science Walkersville, Walkersville, MD) supplemented with 10%
Masashi Inoue and Sachiko Matsumoto contributed equally to this work.
Grant sponsor: Ministry of Education, Japan; Grant number: 17591399.
*Correspondence to: Division of Surgical Oncology, Department of
Surgery, Faculty of Medicine, Tottori University 36-1 Nishi-cho, Yonago
683-8504, Japan. Fax: 181-859-38-6569.
Received 9 February 2008; Accepted after revision 4 July 2008
Int. J. Cancer: 123, 2696–2701 (2008)
' 2008 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
heat-inactivated fetal bovine serum (Thermo Trace, Melbourne,
Australia) and 1% penicillin/streptomycin (Invitrogen, Carlsbad,
CA). Paclitaxel was provided by Bristol-Myers Squibb (New
York, NY). Recombinant human tumor necrosis factor (TNF)-a
(Genzyme, Cambridge, MA) was used to activate NF-jB.
siRNAs for human NF-jB p65 (Rela/NF-jB siRNA SMART-
pool reagents; sequences: 50-GAUGAGAUCUUCCUACUGU-30,
and 50-GGCUAUAACUCGCCUAGUG-30) and
mismatch control siRNA were designed and synthesized by Dhar-
macon (Lafayette, CO).
Approximately 1 3 106cells were plated per 10-cm plate in
media containing 10% fetal bovine serum to give 30–50% conflu-
ency. Transfection of siRNAs was performed using Dharma-
FECT1 (Dharmacon) to result in a final RNA concentration of 50
or 100 nM in vitro.
Western blot analysis
Cells were lysed in sample buffer (Tris-HCl pH 6.8, SDS, glyc-
erol and bromophenol blue) containing protease inhibitors (Com-
plete Mini; Roche Molecular Biochemicals, Mannheim, Ger-
many). Nucleic and cytoplasmic extracts were prepared using a
ParisTMKit (Ambion, Austin, TX) according to the manufac-
turer’s protocol. Western blot analysis was performed as described
previously.15An anti-human NF-jB p65 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) and anti-b-actin antibody
(Sigma, St Louis, MO) were used as primary antibodies.
Quantification of NF-jB p65 activation
The nuclear extracts were assayed using an enzyme-linked im-
munosorbent assay (ELISA) kit (TransAMTMNF-jB; Active
Motif, Carlsbad, CA) to detect and quantify the NF-jB p65 activ-
ity according to the manufacturer’s instructions. Briefly, 2 lg of
nuclear extract protein was incubated for 1 h at 25?C in micro-
wells coated with an oligonucleotide containing an NF-jB p65-
binding consensus sequence. Next, the wells were incubated with
rabbit anti-NF-jB p65 antibodies (1:1,000 dilution) for 1 h at
25?C, followed by incubation with peroxidase-conjugated goat
anti-rabbit IgG (1:1,000 dilution) for 1 h at 25?C. The peroxidase
activity was visualized by the tetramethylbenzidine reaction, and
the optimal density was measured at 450 nm. TNF-a, which was
used to activate NF-jB, was used as a positive control.
Cell cycle analysis
The cell cycle was determined using propidium iodide (PI;
Calbiochem, San Diego, CA,) and fluorescence-activated cell sort-
ing (FACS; Becton Dickinson, Franklin Lakes, NJ). Briefly, after
treatment with various concentrations of each drug for 24 h,
the cells were harvested, fixed in 70% ethanol, incubated with
2 mg/ml RNase and stained with PI solution (50 lg/ml). The pro-
FIGURE 1 – (a) Localization of NF-jB p65 protein in MKN-1 and MKN-45 cells by western blot analysis. NF-jB p65 normally resides in the
cytoplasm, but not in the nucleus. W, whole cell; N, nucleus; C, cytoplasm. (b) NF-jB p65 activity in the nucleus is upregulated in response to
either paclitaxel (black columns) or TNF-a (open columns).
FIGURE 2 – Western blot analysis revealing that NF-jB p65 expres-
sion is diminished by NF-jB p65 siRNA. Mock, cells were treated
using DharmaFECT1without siRNA; control, cells were treated using
DharmaFECT1with control siRNA.
INTRAPERITONEAL CHEMOTHERAPY WITH NF-kB p65 siRNA
portions of cells in the G0/G1, S and G2/M phases were analyzed
by FACS and a DNA software program.
Trypsinized adherent cells and floating cells were washed with
PBS and centrifuged into a pellet. After fixation with Clarke fixa-
tive (ethanol:acetic acid, 3:1) and staining with Hoechst 33258,
apoptotic cells were assessed morphologically under an ultraviolet
laser microscope (Optiophot-2; Nikon, Tokyo, Japan).
Murine xenograft model
Six-week-old female BALB/c nude mice were purchased from
CLEA Japan (Tokyo, Japan) and housed under specific pathogen-
free conditions. The mice received an intraperitoneal injection of a
single suspension of 3 3 106MKN45 or 1 3 107MKN1 cells
resuspended in 1 ml of PBS, followed by intraperitoneal injections
of siRNA (mixture of 100 lg of siRNA, 4 ll of DharmaFECT1
and 896 ll of RPMI) and paclitaxel on Days 7 and 9, respectively.
The mice received an intraperitoneal injection every 7 days
in total of 8 times. With regard to the amount of paclitaxel used
in our study, paclitaxel was administered intraperitonealy at doses
of 25 mg/kg /week and maximal total dose of paclitaxel was
200 mg/kg according to previous results.16Approval for these
experiments was obtained from the Animal Care Committee of
Immunohistochemical staining was performed as described
previously.15An anti-NF-jB antibody (1:50 dilution; Santa Cruz
Biotechnology) was used as the primary antibody.
Xenograft model mice received an intraperitoneal injection of
fluorescein-conjugated siRNA (100 lM; siGLO Cyclophilin B
siRNA; Dharmacon). Following sacrifice of the mice at 48 h after
the injection, peritoneal nodules were resected and assessed by flu-
FIGURE 3 – Enhancement of paclitaxel-induced apoptosis by treatment with NF-jB p65 siRNA in vitro. (a) The frequencies of apoptosis of
MKN-1 and MKN-45 gastric cancer cells are significantly higher after treatment with both paclitaxel and NF-jB p65 siRNA than after treatment
with paclitaxel alone. All results are representative of 3 experiments. (b) Hoechst 33258 staining reveals many apoptotic cells after treatment
with both paclitaxel and NF-jB p65 siRNA.
INOUE ET AL.
orescence microscopy to confirm successful transfection of
Survival curves were calculated according to the Kaplan-Meier
method. Differences between survival curves were examined with
the log rank test. The accepted level of significance was p < 0.05.
Stat View software (Abacus Concepts, Berkeley, CA) was used
for all statistical analyses.
NF-jB p65 siRNA efficiently suppresses NF-jB p65 expression in
gastric cancer cell lines
First, we determined the localization of NF-jB p65 protein in
MKN-1 and MKN-45 cells by Western blot analysis. NF-jB p65
normally resided in the cytoplasm, and not in nucleus, but was
upregulated in the nucleus in response to paclitaxel (Figs. 1a and
1b). Next, we examined whether NF-jB p65 siRNA could dimin-
ish NF-jB p65 expression. Western blot analysis revealed that
NF-jB p65 expression was diminished by treatment with NF-jB
p65 siRNA (Fig. 2).
NF-jB p65 siRNA enhances the chemosensitivity of MKN-1 and
MKN-45 cells to paclitaxel
To investigate whether NF-jB p65 siRNA could enhance the
chemosensitivity of gastric cancer cells, we examined the fre-
quency of apoptosis of MKN-1 and MKN-45 cells after treatment
with paclitaxel. Pre-G0G1 cells, corresponding to apoptotic cells,
increased after transfection of NF-jB p65 siRNA compared with
control siRNA in the treatment with paclitaxel, although antitumor
effect of NF-jB p65 siRNA itself was low (Fig. 3a). In terms of
NF-jB p65 siRNA concentration, on the other hand, preG0G1
population of gastric cancer cell lines (MKN45) after the treat-
ment of NF-jB siRNA with paclitaxel showed no differences at
the concentration of between 50 and 100 nM (data not shown). To
morphologically confirm the induction of apoptosis, we performed
Hoechst 33258 staining and found exclusive apoptosis of MKN-
45 cells after treatment with paclitaxel and NF-jB p65 siRNA
(Fig. 3b). These results indicate that NF-jB siRNA increases
paclitaxel-mediated apoptosis in MKN-1 and MKN-45 cells
In vivo inhibition of peritoneal metastasis of gastric cancer in a
murine xenograft model by intraperitoneal administration
of NF-jB p65 siRNA with paclitaxel
Before we examined the anticancer effects of NF-jB p65
siRNA and paclitaxel in vivo, we determined whether the siRNA
could be delivered to peritoneal metastasis in xenograft model
mice. Abundant fluorescence was detected on the surface of intra-
peritoneal nodules of gastric cancer after siRNA/DharmaFECT1
complex administration (Fig. 4a). Next, we examined whether the
NF-jB p65 siRNA/DharmaFECT1 complex could reduce NF-jB
expression in intraperitoneal nodules of gastric cancer. Abundant
expression of NF-jB was found in intraperitoneal nodules in xen-
ograft model mice. In contrast, NF-jB expression was mainly
reduced on the surface of intraperitoneal nodules where the siRNA
was transfected in mice treated with the NF-jB p65 siRNA/Dhar-
maFECT1 complex (Fig. 4b).
To extend our in vitro studies, we established peritoneal metas-
tasis by intraperitoneal injection of MKN-45 cells into nude mice.
The survival rates of the xenograft model mice are shown in Fig-
ure 5. Mice treated by intraperitoneal administration of NF-jB
p65 siRNA and paclitaxel survived for a significantly longer time
FIGURE 4 – Transfection of siRNA in vivo. (a) Abundant fluorescence is detected on the surface of intraperitoneal nodules of gastric cancer af-
ter intraperitoneal siGLO administration to xenograft model mice with peritoneal metastasis. (b) Abundant expression of NF-jB is detected in
the inner part of intraperitoneal nodules of xenograft model mice, whereas NF-jB expression is mainly reduced on the surface of intraperitoneal
nodules where siRNA was transfected in mice treated with mice with NF-jB p65 siRNA. Left panel, hematoxylin and eosin staining; right
panel, immunohistochemistry using an anti-NF-jB p65 antibody.
INTRAPERITONEAL CHEMOTHERAPY WITH NF-kB p65 siRNA
than mice treated by intraperitoneal administration of paclitaxel
alone (p 5 0.0002).
In this study, we targeted NF-jB to enhance the chemosensitiv-
ity of gastric cancer cells, because NF-jB is closely related to the
chemoresistance of cancer cells.17In fact, gastric cancer cells
treated with paclitaxel showed upregulated NF-jB activation in
the nucleus. Our in vitro studies demonstrated that apoptotic cells
were increased after transfection of NF-jB p65 siRNA compared
with control siRNA in the treatment with paclitaxel, indicating
that NF-jB p65 siRNA can enhance the chemosensitivity of gas-
tric cancer cells.
As an in vivo therapeutic model, we focused on peritoneal me-
tastasis, because the peritoneum is the most frequent site of gastric
cancer recurrence. Of importance is that the metastatic cancer
grows in a closed cavity. In this environment, local administration
of the siRNA/DharmaFECT1 complex could produce a high con-
centration of the agent for attachment to the target cancer cells
during a limited time period. We demonstrated that NF-jB p65
siRNA could be transfected into intraperitoneal nodules of gastric
cancer in xenograft model mice by intraperitoneal injection with
DharmaFECT1. Finally, we showed that mice treated with NF-jB
p65 siRNA and paclitaxel survived for a significantly longer time
than mice treated with paclitaxel alone, indicating that NF-jB p65
siRNA enhanced the chemosensitivity of cancer cells in vivo as
well as in vitro. The procedure of intraperitoneal administration is
quite simple and repeatable in the clinic. Moreover, the estimated
side effects of intraperitoneal administration would be fewer than
those of general administration. Therefore, intraperitoneal admin-
istration of NF-jB p65 siRNA and paclitaxel may cause a break-
through in the treatment of peritoneal metastasis of gastric cancer.
Our in vivo experiments indicated that transfection of NF-jB
p65 siRNA into gastric cancer cells was only detected on the sur-
face of intraperitoneal nodules. Recently, expression of collagen
type I, which is mainly produced by fibroblasts, was reported to be
inversely correlated with intratumoral uptake of compounds of
varying molecular weights, such as IgG,18bovine serum albu-
min19or chemotherapeutic agents.20This mechanism may be cor-
related with the limited uptake of siRNA into intraperitoneal nod-
ules. Recently, Loeffler et al.21constructed an oral DNA vaccine
targeting fibroblast activation protein (FAP), which is specifically
overexpressed by fibroblasts in the tumor stroma, and found that
tumor tissue of FAP-vaccinated mice exhibited markedly
decreased collagen type I expression and up to 70% greater uptake
of chemotherapeutic drugs.21Therefore, targeting the stromal
compartment may be useful for enhancing the uptake of siRNA
and paclitaxel into intraperitoneal nodules.
The biggest obstacle in the development of siRNA-based thera-
pies is the delivery of the siRNA molecules to the target tissue.
There have been numerous reports of in vivo gene silencing with
siRNA,22,23delivery using lipid-based agents24–26and various car-
riers, such as atelocollagen,27a protamine-antibody fusion protein28
and polyethyleneimine,29as well as local administration.30–32On
the other hand, to conceive and optimize experimental treatment
strategies, a method for noninvasive assessment of siRNA delivery
to the tissues of interest using clinically relevant imaging paradigms
is required. At present, reports of in vivo imaging of siRNA in target
tissues are limited to bioluminescence imaging of siRNA-mediated
silencing.27,33Furthermore, the assessment of siRNA delivery is
currently based only on ex vivo studies. Recently, a new dual-pur-
pose probe for simultaneous noninvasive imaging and delivery of
siRNAs to tumors was developed.34This probe consisted of mag-
netic nanoparticles (for magnetic resonance imaging) labeled with
the Cy5.5 dye (for near-infrared in vivo optical imaging) and conju-
gated to a synthetic siRNA duplex targeting a gene of interest. In
addition, the probe was modified with myristoylated polyarginine
peptides serving as a membrane translocation module. In a series of
experiments targeting model (green fluorescent protein) and thera-
peutic (survivin) genes, it was found that the delivery of the probe
could be monitored in vivo by magnetic resonance imaging and op-
tical imaging. In addition, it was possible to follow the silencing
process by optical imaging and correlate it with histological data.
Overall, this new approach may be able to advance siRNA targeting
and potential therapy in the treatment of peritoneal metastasis of
In conclusion, to the best of our knowledge, we have demon-
strated for the first time that intraperitoneal administration of NF-
jB p65 siRNA and paclitaxel inhibited cancer growth in mice
with peritoneal metastasis of gastric cancer. The efficacy and
safety of intraperitoneal administration of siRNA and paclitaxel
should be verified by early phase clinical trials.
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