Acta Med. Okayama, 2012
Vol. 66, No. 1, pp. 7ﾝ16
CopyrightⒸ 2012 by Okayama University Medical School.

http://escholarship.lib.okayama-u.ac.jp/amo/

Preclinical Safety and Efficacy of in Situ REIC/Dkk-3 Gene
Therapy for Prostate Cancer
Keiichiro Kawauchia, Masami Watanabea,b,c＊, Haruki Kakua,b,c, Peng Huangb,
Kasumi Sasakia, Masakiyo Sakaguchid, Kazuhiko Ochiaic, Nam-ho Huhd,
Yasutomo Nasua,c, and Hiromi Kumona,b,c
a

b

c

-

d

‑

The preclinical safety and therapeutic eﬃcacy of adenoviral vectors that express the REIC/Dkk-3
tumor suppressor gene (Ad-REIC) was examined for use in prostate cancer gene therapy. The
Ad-human (h) and mouse (m) REIC were previously demonstrated to induce strong anti-cancer eﬀects
and, and we herein report the results of two
studies. First, intra-tumor
Ad-hREIC administration was examined for toxicity and therapeutic eﬀects in a subcutaneous tumor
model using the PC3 prostate cancer cell line. Second, intra-prostatic Ad-mREIC administration wastested for toxicity in normal mice. The whole-body and spleen weights, hematological and serum
chemistry parameters, and histological evaluation of tissues from throughout the body were analyzed.
Both experiments indicated that there was no signiﬁcant diﬀerence in the examined parameters
between the Ad-REIC-treated group and the control (PBS- or Ad-LacZ-treated) group. In theanalysis using PC3 cells, a signiﬁcant apoptotic eﬀect was observed after Ad-hREIC treatment.
Conﬁrming this observation, the robust anti-tumor eﬃcacy of Ad-hREIC was demonstrated in the
subcutaneous prostate cancer model. Based on the results of these preclinical experiments, weconsider the adenovirus-mediated REIC/Dkk-3
gene therapy to be safe and useful for the clinical treatment of prostate cancer.
Key words: REIC, Dickkopf-3, gene therapy, prostate cancer, preclinical studyP

rostate cancer is a common and signiﬁcant disease in the male population of the United Statesand many other developed countries [1]. Cancer
progression accelerates death in cancer patients, andis often accompanied by a decrease in quality of life.
The current therapeutic strategies, such as androgendeprivation therapy, radiation, and cytotoxic chemotherapy, often fail to stop the disease progression.
Received June 10, 2011 ; accepted September 9, 2011.
Corresponding author. Phone :＋81ﾝ86ﾝ235ﾝ7287; Fax :＋81ﾝ86ﾝ231ﾝ3986
E-mail : mwcorrespondence@gmail.com (M. Watanabe)＊

Therefore, novel and eﬀective therapies against prostate cancer are urgently needed.
Cancer progression is often accompanied by thedown-regulation of apoptosis [2] and the increased
invasive and motile activity of the cancer cells [3].
Cancer cells modulate the apoptotic and metastatic
processes by producing both positive and negativeeﬀectors [4, 5]. REIC/Dkk-3, a member of the
Dickkopf (Dkk) gene family known to interfere with
Wnt signaling via Wnt receptors [6, 7], was previously reported to play a distinct role in the inductionof apoptosis and inhibition of metastasis [8, 9].

8Kawauchi et al.

Acta Med. Okayama Vol. 66, No. 1

REIC/Dkk-3 induced robust anti-tumor eﬀects
and cancer-speciﬁc apoptosis in a variety of cancer
types when expressed by adenovirus-mediated genetransfer [8‑13]. Moreover, our recent study disclosed that the REIC/Dkk-3 protein also has a role inmonocyte diﬀerentiation and tumor regression [14].
Intratumoral administration of the REIC/Dkk-3 protein signiﬁcantly suppressed tumor growth, with
CD11c＋ and CD8＋ (dendritic and killer T cell marker,
respectively) cell accumulation, and enhanced the anticancer cytolytic activity of splenocytes. These data
indicated a cytokine-like role of the REIC/Dkk-3
protein in monocyte diﬀerentiation that might be
exploited therapeutically.
Gene therapy has been applied to treat human diseases in clinical trials and is considered to have therapeutic potential. Our group has previously demonstrated the feasibility of using adenoviral vectors forthe treatment of prostate cancer in the clinic [15].
Based on the background information noted above, weare promoting the translation of
REIC/Dkk-3
gene therapy from the lab to the clinic for patientswith prostate cancer. To perform the necessary preclinical study as part of the progression to clinical useof the strategy, we herein examined the toxicity andtherapeutic eﬀects of the adenoviral vectors encoding
the novel tumor suppressor REIC/Dkk-3.

treatment strategy of using adenoviral vectors for thetreatment of prostate cancer. The mice were purchasedfrom Japan SLC, Inc. (Hamamatsu, Japan) and maintained in a speciﬁc pathogen-free environment withfree access to food and water at the laboratory animal
center of Okayama University. They were allowed toadapt to their environment for more than one week
before beginning the experiments. The animals werehoused and handled in accordance with the Okayama
University Animal Research Committee Guidelines.
/
The full-length cDNA of mouse or
human REIC/Dkk-3 was integrated into the cosmid
vector, pAxCAwt, and then transferred into an adenoviral vector by the COS-TPC method (Takara Bio,
Shiga, Japan) [8]. An adenoviral vector carrying the
LacZ gene (Ad-LacZ) was used as a control. The
adenoviral vectors are a replication-defective adenovirus of serotype 5 that contain the indicated gene under
the control of the CMV early enhancer/chicken β
actin (CAG) promoter. For the Ad-human REIC,
transduction ability after intratumoral vector
injection was conﬁrmed by detecting the REIC protein
expression [14].
The experimental designand doses of virus administered are shown in Fig. 1.
The ﬁrst experiment was done using the prostate
tumor mouse model. To generate this model, PC3
human prostate cancer cells (2.5 × 106 in 50 l phosphate buﬀered saline (PBS)) were subcutaneously
injected into the right thigh of BALB/C nu/nu mice.
The tumors were permitted to grow to approximately
5mm in diameter. The mice were then randomly

Materials and Methods
We used adult malemice (C57BL/6: 30‑39g, BALB/C nu/nu: 24‑31g)
to evaluate the toxicity and therapeutic eﬃcacy of theExperiment
No.1

PC3,
subcutaneous
tumor

ExperimentNo.2(No tumor)

Mouse

BALB/Cnu/nu

Mouse

C57BL/6

Treatment
Dose
Injectiongroup(viral particle)
site
PBS
Ad-LacZ
Ad-hREICTime frame(Days)
PC3 vector

5 × 1010-140Treatment
Dose
Injectiongroup(viral particle)
site
PBS
Ad-LacZ
Ad-mREIC

32

Time frame(Days)
vector

5 × 1010

harvestIntra-tumor

Intra-prostate0

harvest21

Fig. 1The experimental design of the preclinical study is shown. Toxicological analysis of mice administered Ad-hREIC and Ad-mREIC
was performed in both experiment 1 and 2 using the indicated mouse models. A study of the eﬃcacy of intratumoral administration of
Ad-hREIC in a subcutaneous PC3 prostate cancer model was performed in experiment 1.

February 2012

assigned into the treatment group with (1) intratumoral injection of Ad-LacZ, (2) intratumoral injectionof Ad-human REIC (Ad-hREIC) at the dose of 5 ×1010 viral particles/tumor in 50 l buﬀer, or (3) intratumoral injection of 50 l PBS. The selection of thedose was based on previous clinical studies of prostatecancer gene therapy in which adenovirus-mediatedgene delivery was performed using a dose between
1010 and 1011 viral particles/prostate [16‑18]. The
injections were targeted to the center and periphery ofeach mass to deliver the agent diﬀusely. The size ofthe tumor was measured with vernier calipers until 25
days after each treatment. Tumor volume was calculated using the following formula: 1/2 × (the shortest
diameter)2 × (the longest diameter). In the second
experiment, the same number of Ad-mouse REIC
(Ad-mREIC) viral particles in 50 l buﬀer was inoculated into the bilateral lobes of the dorsolateral prostate in normal C57BL/6 mice without any tumors.
The body weight of each mouse was monitored
using a sensitive balance during the entire study
period. All the animals were observed daily forphysical appearances, activity levels, and mortality
patterns. The ﬁrst day of vector administration was
taken as Day 0, while the day of sacriﬁce was designated as Day 32 and Day 21 in the ﬁrst and second
experiments, respectively. In both experiments, thespleen weight was measured at the time of sacriﬁce.
For the histological analysis of the potential side
eﬀects of the Ad-REIC treatment, the dorsolateral
prostate (the injection site) and selected organs (brain,lungs, heart, liver, stomach, spleen, kidneys, bladder, and rectum) were dissected at the sacriﬁce ofanimals in the second experiment. The tissues werethen ﬁxed in formalin and embedded in paraﬃn sections. The sections (5 m) were stained with hematoxylin and eosin and were examined for histopathological and cytotoxic changes.
Blood was drawn from theinferior vena cava of each animal under deep anesthesia with sodium pentobarbital. The blood samples
were collected in a plastic tube with ethylenediaminetetra-acetic acid (EDTA). The measurements ofwhite blood cells (WBC), hemoglobin, hematocrit, andplatelets were carried out with the EDTA-treatedanti-coagulated blood using a hematological instrument(MEK-6358, Celltac-alpha, Nihon Kohden, Tokyo,
Japan) within hours of collection. To analyze the

REIC/Dkk-3 Gene Therapy

9biochemical parameters, we centrifuged the anticoagulated blood at 3,000 rpm for 10min, and bloodplasma was obtained and stored at −80℃. Albumin
concentrations were determined by the Bromo Cresol
Green (BCG) assay, using a commercially available
kit (Wako Pure Chemical Industries Osaka, Japan)
and following the manufacturerʼs instructions. The
plasma levels of alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase(ALP), and lactate dehydrogenase (LDH) were determined by the JSCC transferable method by employing
the standard ready-to-use kits (Wako Pure Chemical
Industries). Total bilirubin was evaluated by the azobilirubin method, using a commercial kit (Daiichi
Pure Chemicals Co. Tokyo, Japan).
The PC3 cells
were washed twice with PBS and then lysed with lysis
buﬀer (50mM HEPES, pH7.4, 250mM NaCl, 1mM
EDTA, 1ｵ NP-40, 1mM DTT, 1mM PMSF,
5 g/ml leupeptin, 5 g/ml aprotinin, 2mM Na3VO4,
1mM NaF, 10mM ß-GP), and the proteins wereextracted. After centrifugation, the supernatants
were adjusted to achieve equal protein concentrations
in each sample and then diluted with the same volumeof 2 × SDS sample buﬀer and heated for 5min at
95℃. Samples (10 g of protein) were separated on
7.5ｵ SDS-PAGE gels and electroblotted onto a
polyvinylidene ﬂuoride (PVDF) membrane. The blots
were blocked for 1h with 10ｵ nonfat milk powder,
6ｵ Glycine, and 0.1ｵ Tween-20 in Tris buﬀeredsaline (TBS) at room temperature. The proteins werethen identiﬁed with the use of the primary antibody, arabbit anti-human REIC/Dkk-3 polyclonal antibody
raised in our laboratory [8] (1 : 1,000 dilution). Afterextensive washing with 0.1ｵ Tween-20 in TBS(T-TBS), the blots were exposed to the appropriate
horseradish peroxidase-conjugated secondary antibody.
After another round of extensive washing with T-TBS,
membranes were developed using the enhanced chemiluminescence detection method (ECL kit, Amersham
Pharmacia Biotech, Chandler, AZ, USA).
To examine
apoptosis
induction after the treatments, we seeded cells in ﬂatbottom 6-well plates and incubated them for 24h.
The cells were then treated with Ad-LacZ and
Ad-REIC at the indicated multiplicity of infection(MOI) in serum-free medium (500 l) for 2h, and the
medium was exchanged for fresh complete medium

10

Kawauchi et al.

Acta Med. Okayama Vol. 66, No. 1(2ml). After an additional 48h of incubation, Hoechst
33342 stock solution was added into the medium at a
2 g/ml concentration, and the cells were incubated in
the dark for 10min. Hoechst 33342 is an intercalating
dye that allows the determination of variations in the
total chromatin quantity and the degree of chromatincondensation [11]. Using ﬂuorescence microscopy, we
identiﬁed apoptotic cells by the presence of highly
condensed or fragmented nuclei. Apoptotic cells werecounted at 5 diﬀerent ﬁelds under microscopic observation. One hundred cells were judged under eachﬁeld.
The data are expressedas the means ± standard error (S.E.). An unpaired
Studentʼs -test was performed for the statisticalanalysis between the 2 groups. The diﬀerences wereconsidered statistically signiﬁcant at ＜ 0.05.

Results
In the ﬁrst experiment, we examined the
toxicity and eﬃcacy of the Ad-hREIC vector using the
subcutaneous human prostate tumor mouse xenograftmodel. The experiment was designed as a preclinicalstudy to support the future clinical use of Ad-hREIC,
and we therefore evaluated its toxicological proﬁle andanti-tumor eﬀects. We also conducted a second experiment in non-tumor-bearing mice to examine the safetyof Ad-mREIC, to ensure that speciﬁcally overexpressing REIC/Dkk3 via intra-prostatic injection does
not result in toxicity.
None of the mice died during the ﬁrst experimentin any of the treatment groups (n ＝ 5). In the second
experiment, 1 or 2 mice died in each group during theexperiment [PBS (n ＝ 15): one mouse died on day
18; Ad-LacZ (n ＝ 16): 1 mouse each died on day 7
and day 18; Ad-mREIC (n ＝ 16): 1 mouse died on day
18 and one on day 19]. At necropsy, there was no
obvious cause of death such as extensive inﬂammation,
abnormal bladder distension, trauma of various organs,or abundant ascites.
In both the ﬁrst and second experiments, there wasno statistically signiﬁcant diﬀerence in the survival
between the Ad-REIC-treated group and the control
groups by Mantel-Cox log-rank analysis (data notshown). The physical appearances and activities wereobserved carefully, and there were no deﬁnite diﬀerences among the groups in either experiment.

Inﬂammation-related ﬁndings such as abscesses were
not detected at the time of sacriﬁce in any of the animals. The time courses of body weight loss in bothexperiments are shown in Fig. 2A. The average bodyweight at the start point was as follows: PBS 27.0g,
Ad-LacZ 26.1g, Ad-hREIC 29.3g in experiment 1
and PBS 34.2g, Ad-LacZ 34.3g, Ad-mREIC 33.7g
in experiment 2. Although the average body weight of
Ad-mREIC-treated mice tended to decrease more
rapidly in experiment 2, no signiﬁcant diﬀerence wasobserved in the body weight at any time point between
the Ad-mREIC- and Ad-LacZ-treated groups. The
body weight change between pre-treatment and thetime of sacriﬁce was calculated for each treatmentgroup, and no signiﬁcant changes were observed in anyof the groups during either experiment (Fig. 2B).
To assess the systemic toxicology, we collectedblood and performed hematological (WBC, hemoglobin, hematocrit, and platelets) and plasma chemistry(albumin, ALT, AST, ALP, total bilirubin) evaluations (Table 1). There were no statistically signiﬁcantdiﬀerences between the Ad-LacZ and Ad-REIC groups
in any of the parameters for either experiment.
Because the LDH levels were aﬀected by hemolysis of
the collected blood samples, the diﬀerences in LDH
activity could not be attributed to the toxicity of
Ad-REIC treatment (data not shown).
To conﬁrm the protein expression and apoptotic
eﬀect induced by Ad-REIC, we treated PC3 prostatecancer cells with Ad-hREIC. A Western blotting
analysis demonstrated signiﬁcant expression of the
REIC/Dkk-3 protein in PC3 cells on day 2 after thetreatment (Fig. 3A). In an apoptosis assay using the
Hoechst 33342 stain, apoptotic cells were frequently
observed in Ad-hREIC-treated PC3 cells (Fig. 3B,
upper panel). The mean rate of apoptosis at 10, 20,
and 100 MOI was 13.7ｵ, 30.1ｵ, and 67.5ｵ, respectively, and signiﬁcant apoptotic induction was observedfor each Ad-hREIC concentration in comparison to thecontrol Ad-LacZ treatment (Fig. 3B, lower panel).
In the ﬁrst
experiment, we further demonstrated the anti-tumor eﬀects of Ad-hREIC using a
subcutaneous PC3 tumor model (Fig. 4). In the PBSand Ad-LacZ-treated groups, the tumor size progressively increased during the observation period. In
contrast, the tumor growth in the Ad-REIC-treatedgroup was signiﬁcantly suppressed. In fact, the tumor
had macroscopically disappeared at the time of sacri-February 2012

REIC/Dkk-3 Gene Therapy

A

No.2

No.1

35

11

35

Body weight (g)Body weight (g)

30
25
PBS
Ad-LacZ
Ad-hREIC

20
15

0

4

30

25

20

7 11 14 18 21 25 32

AdLacZ

AdhREIC

10

20

n.s.

No.1
30

0
Body weight decrease (%)

Body weight decrease (%)

0

PBS

0

3

7

10

14

17

21

Days after treatment

Days after treatment

B

PBS
Ad-LacZ
Ad-mREICPBS

AdLacZ

AdmREIC

10
n.s.

20
No.2
30

Fig. 2
A, The mouse body weight is shown on the indicated days after each treatment. Five to 9 mice were analyzed for eachgroup; B, The body weight changes between pre-treatment and the time of sacriﬁce are shown for each treatment group for experiments
1 and 2. Five to 9 mice were analyzed for each group. No signiﬁcant (n.s.) diﬀerence was observed in any group in terms of body weightchange.

ﬁce in one of the mice in the Ad-hREIC-treated group.
A statistically signiﬁcant reduction of the tumor volume was observed on days 21 and 25 in the Ad-REICtreated group compared to the Ad-LacZ group.
To further examine the systemic toxicology of the
Ad-mREIC treatment, we performed histologicalanalyses using sections of the various organs (brain,lungs, heart, liver, stomach, spleen, kidneys, bladder,and rectum) in the second experiment. There were no
histopathological abnormalities in the observed sections in any of the treatment groups (data not shown).
We also investigated the local eﬀects of Ad-mREIC
injection by a histopathological analysis of the dorsolateral prostate, the site where the vector was
injected. In the Ad-LacZ and Ad-mREIC treatmentgroups, inﬁltration of mononuclear cells typical of

lymphocytes was observed in the interstitial area (Fig.
5A). In the Ad-LacZ-injected prostate, some degenerated prostatic glands containing fewer epithelial cells
than normal were observed, indicating cytotoxic
changes after Ad-LacZ treatment. Epithelial cellproliferation was also observed in the prostatic glandsof the Ad-LacZ group, indicating some degree ofrelated inﬂammation. We could not detect any histological eﬀects in the prostatic sections of the PBStreated group.
Because the REIC/Dkk-3 gene product is reported
to possess immunological activity [14], we also
assessed the spleen weight to determine whether thetreatment induces the side eﬀect of splenomegaly. The
spleen weight of each mouse was measured at the timeof sacriﬁce in bothexperiments, and there12

Kawauchi et al.

Acta Med. Okayama Vol. 66, No. 1

Table 1
The hematological (WBC, hemoglobin, hematocrit and platelets) and plasma chemistry (albumin, ALT, AST, ALP, total
bilirubin) evaluations were performed with the blood samples collected at the time of sacriﬁce. Shown are the means and standard errorsof values for 4 to 5 animals per group.
No.1
Treatment

WBC

Hemoglobin

Hematocrit

Platelets

Alb

ALT

AST

ALP

Total Bilirubin(10 cells/µl)(g/dl)(%)

4(10 /µl)(g/dl)(IU/l)(IU/l)(IU/l)(mg/dl)53.0 ± 11.1
71.0 ± 5.8
53.0 ± 5.1

11.6 ± 1.5
10.2 ± 0.5
10.2 ± 0.3

43.5 ± 4.7
38.7 ± 1.8
37.7 ± 1.0

66.7 ± 21.6
98.3 ± 9.0
87.6 ± 10.9

2.2 ± 0.11
2.1 ± 0.10
2.0 ± 0.08

36.0 ± 1.9
36.6 ± 1.4
42.4 ± 7.3

100.2 ± 9.9
102.8 ± 13.1
85.6 ± 12.5

107.6 ± 4.2
76.6 ± 7.4
94.2 ± 8.8

0.04 ± 0.01
0.03 ± 0.00
0.02 ± 0.01

WBC

Hemoglobin

Hematocrit

Platelets

Alb

ALT

AST

ALP

Total Bilirubin(10 cells/µl)(g/dl)(%)

4(10 /µl)(g/dl)(IU/l)(IU/l)(IU/l)(mg/dl)50.5 ± 7.4
67.0 ± 6.7
69.5 ± 9.6

9.6 ± 0.2
9.6 ± 0.5
9.6 ± 0.9

35.4 ± 0.7
35.6 ± 1.6
35.7 ± 3.0

75.6 ± 16.6
60.8 ± 9.9
73.9 ± 14.8

2.2 ± 0.05
2.1 ± 0.08
1.9 ± 0.05

44.8 ± 26.5
26.5 ± 8.942.3 ± 19.0

52.8 ± 18.9
41.3 ± 7.4
47.5 ± 11.2

124.8 ± 2.9
136.3 ± 7.6
125.3 ± 2.9

0.03 ± 0.00
0.04 ± 0.01
0.03 ± 0.00

2

PBS
Ad-LacZ
Ad-hREIC
No.2
Treatment

2

PBS
Ad-LacZ
Ad-mREIC

WBC, white blood cells; Alb, Albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

A

B

Ad-hREIC
Bright ﬁeld

(kDa)

AdLacZ

Hoechst staining

AdhREIC

175
83
62
47.5

16.5
6.5

Percentage of apoptotic cells

25

Bar: 10 µm

80

32

70

＊

60
50
＊

40
30
＊

20
10
0

10

20
Ad-LacZ

100

10

20
Ad-hREIC

100 (MOI)

Fig. 3
A, REIC/Dkk-3 expression after Ad-LacZ and Ad-hREIC treatment at 100 MOI was examined in PC3 prostate cancer cells byWestern blotting analysis. The total protein was extracted on day 2 after the treatment, and 10 µg of protein was loaded per lane; B, The
induction of apoptosis after in vitro Ad-hREIC treatment was examined in PC3 cells by Hoechst 33342 staining. The upper panel indicatesthe appearance of apoptotic cells after Ad-hREIC treatment. The lower panel shows the apoptotic rate of PC3 cells after the indicatedtreatments. A total of 5 diﬀerent ﬁelds were examined under a microscope to determine the apoptotic rate. A signiﬁcant diﬀerence wasobserved (＊p ＜ 0.05) between the Ad-hREIC and the control Ad-LacZ treatment.

February 2012

REIC/Dkk-3 Gene Therapy
mouse No.
2.

3.

1200
4.

5.

PBS
Ad-LacZ
Ad-hREIC

Fig. 4The inhibitory eﬀect of the Ad-hREIC treatment on PC3 tumor growth innude mice is indicated. The left photograph shows the appearance of subcutaneous xenograft tumors at the time of sacriﬁce after treatment with PBS, Ad-LacZ,
and Ad-hREIC. The tumor could not be detected macroscopically on the sacriﬁceof one mouse in the Ad-hREIC-treated group. The mean volume of tumors wascalculated for 5 mice of each group, and the tumor growth curves are shown in theright panel. A signiﬁcant diﬀerence (＊p ＜ 0.05) was observed between the resultsof Ad-hREIC and the control Ad-LacZ treatment.
PBS

800
600
400
＊

200

Bar: １cm

A

PBS
Ad-LacZ
Ad-hREIC

1000
Tumor volume (mm)3

1.

13

0

＊

0

7

11

14

18

21

25

Days after treatment

Ad-LacZ†

4Ad-mREIC†＊

Bar: 50 µm

Spleen weight (mg)

B 300

No.1
n.s.

200

No.2

150

n.s.

100
50
0

PBS

Ad- AdLacZ hREIC

PBS

Ad- AdLacZ mREIC

Fig. 5
A, To examine the local eﬀects of Ad-mREIC treatment, we performed histological analysis of the vector injection site during
the second experiment. The dorsolateral prostates (the injection site) were dissected at the time of sacriﬁce and ﬁxed in formalin. The
sections, stained with hematoxylin and eosin, were analyzed for histopathological and cytotoxic changes. The asterisk shows a degenerated prostatic gland that contains fewer epithelial cells than normal. The daggers indicate the aﬀected prostatic glands with epithelial cellproliferation. The selected area (black square) is magniﬁed in the inset panel, and the arrowhead indicates a mononuclear cell typical of
lymphocytes; B, To assess the eﬀects on the spleen after Ad-REIC treatment, we measured the spleen weight of each mouse at the timeof sacriﬁce in both experiments. Four to 5 mice were analyzed for each group. No signiﬁcant (n.s.) diﬀerence was observed in the spleensof any of the mice in any of the treatment groups.

14

Kawauchi et al.

Acta Med. Okayama Vol. 66, No. 1

were no statistically signiﬁcant diﬀerences in spleen
weight following treatment in either treatment group(Fig. 5B).

Discussion
We previously cloned the REIC (Reduced
Expression in Immortalized Cells) gene and reported
that its expression is down-regulated in a variety ofcancer cell lines and tumors [8, 10‑12, 19‑21]. The
REIC gene is identical to the Dickkopf-3 (Dkk-3) gene[20] and REIC/Dkk-3 seems to be one of the mostinnovative tumor suppressor genes used for cancer
gene therapy [8, 9, 12, 21]. We have extensively
utilized an adenoviral vector agent, Ad-hREIC, containing the human REIC/Dkk-3 gene under the controlof the CAG promoter in preclinical cancer models inmice. The adenoviral vector carrying human REIC/
Dkk-3 selectively induced apoptosis in prostate cancer
and malignant mesothelioma cells through the activation of c-Jun-NH2-kinase (JNK) and c-Jun [8, 12].
The adenovirus-mediated expression of the REIC/
Dkk-3 gene eﬃciantly induces endoplasmic reticulum(ER) stress-mediated apoptosis in a cancer cell-speciﬁc manner [12, 13]. The current study also demonstrated that the Ad-hREIC treatment induced signiﬁcant apoptosis in a prostate cancer cell line. We
further demonstrated the robust therapeutic utility of
Ad-hREIC in a subcutaneous prostate cancer model.
Thus, the adenoviral agent appears to be eﬀective
against human prostate tumors in terms of the cancerspeciﬁc targeting and a more radical response toexpression of the gene.
The therapeutic eﬀects of Ad-REIC were previously observed in a variety oftumor models
generated with prostate, breast, and mesothelioma
cancer cells [8, 9, 11, 12], but the toxicity of the
Ad-REIC treatment remained unknown. Thus, wecurrently focused on the safety of local injection of
Ad-REIC for the treatment of prostate cancer. Nonhuman primates have been used in some studies of
adenoviral vector toxicity [22, 23], and mice have
many advantages for preclinical evaluations prior tothese primate studies [24]. To evaluate the toxicityof the Ad-REIC vector, we ﬁrst analyzed the lethality
and survival in 2 experiments. There was no toxicity
when tumor-bearing mice were administered the humanREIC vector, although some lethality was observed in

the mice during the second experiment. However,since the survival was not statistically diﬀerent
between the groups (1 or 2 mice died in both the control and treatment groups), it is likely that the
Ad-REIC treatment itself did not speciﬁcally induce
the deaths. The most conceivable reason for thelethality is that the mice in the second experiment died
due to post-operative complications of the open surgery for the prostatic injections. The other possibility
is that adenovirus toxicity may be the cause of thedeaths. Although deﬁnite histological damage was notobserved in the examined mice, we cannot exclude thispossibility. To conﬁrm the reproducibility of thedeaths and to exclude the adenovirus-related death, we
did similar additional experiments using C57BL/6
mice in which there was no death during the experimental schedule until day 28. Thus, it seems that thedeath in experiment 2 was not reproducible and notadenovirus-related. Based on these results, anotherpossibility is that the mice deaths may have beenprocedure-related.
We next investigated the systemic toxicity of the
Ad-REIC treatment. The liver has been widely recognized as the primary target organ of adenoviral vectors after an intravenous adenoviral administration[25]. Although the Ad-REIC was locally administered by intratumoral or intra-prostate injection in our
experiments, the possibility could not be excluded thatthe viral particles might disseminate into the generalcirculation and aﬀect the liver functions. However, inthe current studies using Ad-hREIC and Ad-mREIC,
we did not detect any diﬀerences in the liver enzymes
or other biochemical parameters in any of the 3 treatment groups. None of the mice showed histopathological liver abnormalities such as cytotoxicity or
inﬂammation in the PBS-, Ad-LacZ-, or Ad-mREICtreated groups during the second experiment (data notshown), indicating the consistency of the results ofbiochemical evaluations. As for other distant organs,no histological abnormalities were observed (data notshown) in the screening, although there were somehistological changes at the injection site, the dorsolateral prostate. In addition, there was no eﬀect of
Ad-REIC treatment on hematological parameters or
spleen weight. It therefore seems that Ad-REIC andthe gene product, secreted REIC protein, do not
induce serious systemic toxicity or non-speciﬁc inﬂammation under the indicated conditions.

February 2012

The second step of our work was to analyze thelocal side eﬀects associated with an intra-prostaticdelivery of the Ad-REIC vector by direct injection.
To investigate the probable local toxicity due to the
adenovirus or the gene products, including thesecreted REIC protein, we examined the histopathological changes of the dorsolateral prostate after the
Ad-mREIC injection. We could not detect any histological damage to the prostate gland after PBS or
Ad-REIC treatment. In the Ad-LacZ group, there wassome damage to the prostate gland, which may havebeen due to the toxicological proﬁle of the adenoviral
vectors [26]. The lack of local toxicity in the
Ad-mREIC-injected prostate glands was consistent
with the previous reports showing that Ad-hREIC
induces cytotoxic or apoptotic eﬀects only in cancer
cells and not in normal cells [8, 11]. As for thelymphocyte inﬁltration in the prostatic interstitialspace, the phenomenon was observed in both the
Ad-LacZ and Ad-mREIC treatment groups. It is
likely that the adenoviral vector may have caused theinﬂammatory reaction. In addition, a recent study
reported that the REIC protein induced a chemokinelike action that caused the accumulation of immune
cells when directly injected into subcutaneous tumors
in C57BL/6 mice [14]. Thus, the other possibility isthat the gene product, the REIC protein, might havebeen involved in lymphocyte inﬁltration in the
Ad-mREIC-treated group. Further studies will clarify
the mechanisms of the Ad-REIC-induced immunomodulatory eﬀects in locally injected normal and tumor
tissues.
Our results show that the adenoviral vector encoding the REIC/Dkk-3 gene and the CAG promoter has
no signiﬁcant toxicity and is therapeutically eﬀective
when directly injected into the tumors of mice withexperimental prostate cancer or into the prostates ofnormal mice at the indicated doses. We believe thatthe current study provides valuable information for
future clinical trials of Ad-REIC-mediated gene therapy against human prostate cancer.
Acknowledgments. This work was supported by a grant from the
Ministry of Education, Culture, Sports, Science and Technologyʼs
FY2006 “Creation of Innovation Centers for Advanced Interdisciplinary
Research Areas” Scheme in Japan. We thank Hideo Ueki and Shun-Ai Li(Okayama University) for their valuable assistance.

REIC/Dkk-3 Gene Therapy

15

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