U.S. patent application number 11/886338 was filed with the patent office on 2009-01-01 for apoptosis-inducing agent for prostate cancer cells.
Invention is credited to Nam-ho Huh, Hiromi Kumon, Yasutomo Nasu, Masakiyo Sakaguchi.
Application Number | 20090005538 11/886338 |
Document ID | / |
Family ID | 36991430 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090005538 |
Kind Code |
A1 |
Kumon; Hiromi ; et
al. |
January 1, 2009 |
Apoptosis-Inducing Agent for Prostate Cancer Cells
Abstract
According to the present invention, an apoptosis-inducing agent
for prostate cancer comprising REIC/Dkk-3 DNA or an REIC/Dkk-3
protein, and a therapeutic agent for prostate cancer and an agent
for inhibiting prostate cancer metastasis that comprise such
apoptosis-inducing agent are provided. An apoptosis-inducing agent
for prostate cancer, comprising, as an active ingredient, an
REIC/Dkk-3 protein (a) or (b) or REIC/Dkk-3 DNA (c) or (d): (a) a
protein consisting of the amino acid sequence represented by SEQ ID
NO: 2; (b) a protein consisting of an amino acid sequence derived
from the amino acid sequence represented by SEQ ID NO: 2 by
substitution or deletion of 1 or more amino acids; (c) DNA
consisting of the nucleotide sequence represented by SEQ ID NO: 1;
or (d) DNA that hybridizes under stringent conditions to DNA
consisting of a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO: 1 and encodes a protein having
apoptosis activity; and a therapeutic agent for prostate cancer
comprising such apoptosis-inducing agent are provided
Inventors: |
Kumon; Hiromi; (Okayama,
JP) ; Huh; Nam-ho; (Okayama, JP) ; Sakaguchi;
Masakiyo; (Okayama, JP) ; Nasu; Yasutomo;
(Okayama, JP) |
Correspondence
Address: |
FITCH, EVEN, TABIN & FLANNERY
P. O. BOX 18415
WASHINGTON
DC
20036
US
|
Family ID: |
36991430 |
Appl. No.: |
11/886338 |
Filed: |
January 10, 2006 |
PCT Filed: |
January 10, 2006 |
PCT NO: |
PCT/JP2006/300411 |
371 Date: |
September 14, 2007 |
Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 14/4747 20130101;
A61P 35/04 20180101; A61P 35/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/00 20060101
C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2005 |
JP |
2005-073807 |
Mar 23, 2005 |
JP |
2005-084495 |
Claims
1. An apoptosis-inducing agent for prostate cancer, comprising, as
an active ingredient, the following REIC/Dkk-3 protein: (a) a
protein consisting of the amino acid sequence represented by SEQ ID
NO: 2; or (b) a protein consisting of an amino acid sequence
derived from the amino acid sequence represented by SEQ ID NO: 2 by
substitution, deletion, or addition of 1 or more amino acids and
having apoptosis activity.
2. A therapeutic agent for prostate cancer, comprising the
apoptosis-inducing agent according to claim 1.
3. An apoptosis-inducing agent for prostate cancer, comprising, as
an active ingredient, the following REIC/Dkk-3 DNA: (c) DNA
consisting of the nucleotide sequence represented by SEQ ID NO: 1;
or (d) DNA that hybridizes under stringent conditions to DNA
consisting of a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO: 1 and encodes a protein having
apoptosis activity.
4. A therapeutic agent for prostate cancer, comprising the
apoptosis-inducing agent according to claim 3.
5. An agent for inhibiting prostate cancer metastasis, comprising
the apoptosis-inducing agent according to claim 1.
6. An agent for inhibiting prostate cancer metastasis, comprising
the apoptosis-inducing agent according to claim 3.
7. The therapeutic agent for prostate cancer according to claim 2,
which is used in combination with hyperthermia.
8. The agent for inhibiting prostate cancer metastasis according to
claim 5, which is used in combination with hyperthermia.
9. The therapeutic agent for prostate cancer according to claim 4,
which is used in combination with hyperthermia.
10. The agent for inhibiting prostate cancer metastasis according
to claim 6 which is used in combination with hyperthermia.
Description
TECHNICAL FIELD
[0001] The present invention relates to apoptosis induction in
prostate cancer cells with the use of the REIC/Dkk-3 gene, which is
a tumor-inhibiting gene, an apoptosis-inducing agent for prostate
cancer cells, which comprises the REIC/Dkk-3 gene or an expression
product thereof, and a therapeutic agent for prostate cancer and an
agent for inhibiting prostate cancer metastasis, which comprise the
apoptosis-inducing agent.
BACKGROUND ART
[0002] Selective elimination of cancer cells is an important key
issue in treating cancer. During malignant conversion and
progression, various genetic changes occur in cells (Vogelstein, B.
et al., Trends Genet 9, 138-41 (1993)). Such mutations could be
targets of gene therapies for cancer.
[0003] Some types of genes exhibit a selective killing effect on
cancer cells when overexpressed. Representative examples of such
genes include p 53 (Chen, P. L. et al., Science 250, 1576-80
(1990); Fujiwara, T. et al., J Natl Cancer Inst 86, 1458-62 (1994);
and Nielsen, L. L. et al., Cancer Gene Ther 4, 129-38 (1997)) and
mda-7 (Fisher, P. B. et al., Cancer Biol Ther 2, S23-37 (2003)),
which have been known as antioncogenes.
[0004] Meanwhile, the REIC/Dkk-3 gene has been known as a gene
involved in cell immortalization. It has been reported that
expression of the gene is inhibited in cancer cells (see
WO01/038523, Tsuji, T. et al., BiochemBiophys Res Commun 268, 20-4
(2000), Tsuji, T. et al., BiochemBiophys Res Commun 289, 257-63
(2001), Nozaki, I. et al., Int J Oncol 19, 117-21 (2001) and
Kurose, K. et al., J Urol 171, 1314-8 (2004)).
[0005] The REIC/Dkk-3 gene is a member of the Dkk family and has
been known to interfere with Wnt signal transduction via Wnt
receptors (see Bafico, A. et al., Nat Cell Biol 3, 683-6 (2001) and
Hoang, B. H. et al., Cancer Res 64, 2734-9 (2004)). The Wnt genes
play pleiotropic roles in critical biological contexts, including
cell growth, differentiation, malignant transformation, and the
like (see Moon, R. T. et al., Science 296, 1644-6 (2002)).
Therefore, similarly, Dkk family members (including 4 genes that
have been known to exist in humans) are thought to fulfill
important functions regarding cell growth, differentiation, and
malignant transformation. However, most of their functions have not
yet been elucidated.
DISCLOSURE OF THE INVENTION
[0006] It is an objective of the present invention to provide an
apoptosis-inducing agent for prostate cancer cells, which comprises
REIC/Dkk-3 DNA or the REIC/Dkk-3 protein, and a therapeutic agent
for prostate cancer and an agent for inhibiting prostate cancer
metastasis, which comprise the apoptosis-inducing agent.
[0007] The present inventors have conducted intensive studies of
the previously reported relationship between the REIC/Dkk-3 gene
and cancer cells. It was discovered that the expression level of
the REIC/Dkk-3 gene is reduced in human cancer cell lines and
cancer tissue. The downregulation of the REIC/Dkk-3 gene was
partially caused by promoter hypermethylation. Overexpression of
the REIC/Dkk-3 gene with the use of vectors caused growth
inhibition in human osteosarcoma cells. These findings indicate
that the REIC/Dkk-3 gene could function as an antioncogene and thus
that the REIC/Dkk-3 gene could be a new therapeutic target for
human cancer.
[0008] In addition, the present inventors have found that the
expression level of the REIC/Dkk-3 gene is reduced in highly
malignant prostate cancer cells. Thus, they have conducted further
intensive studies of the use of the REIC/Dkk-3 gene as a
therapeutic agent for prostate cancer. Also, the present inventors
have found that it is possible to inhibit prostate cancer by
causing REIC/Dkk-3 gene expression in prostate cancer cells so as
to induce apoptosis in prostate cancer cells. This has led to the
completion of the present invention.
[0009] Further, the present inventors have examined effects of
prostate cancer metastasis inhibition by the REIC/Dkk-3 gene. As a
result, they have found that the REIC gene has
metastasis-inhibitory activity in mouse orthotopic transplantation
models of prostate cancer, in addition to its localized
tumor-inhibitory activity.
[0010] Furthermore, the present inventors have found that the
combined use of the REIC/Dkk-3 gene and hyperthermia results in the
improvement in the effects of prostate cancer treatment and
prostate cancer metastasis inhibition.
[0011] As a result, the present inventors have completed the
present invention.
[0012] Specifically, the present invention is described as follows.
[0013] [1] An apoptosis-inducing agent for prostate cancer,
comprising, as an active ingredient, the following REIC/Dkk-3
protein:
[0014] (a) a protein consisting of the amino acid sequence
represented by SEQ ID NO: 2; or
[0015] (b) a protein consisting of an amino acid sequence derived
from the amino acid sequence represented by SEQ ID NO: 2 by
substitution, deletion, or addition of 1 or more amino acids and
having apoptosis activity. [0016] [2] A therapeutic agent for
prostate cancer, comprising the apoptosis-inducing agent according
to [1]. [0017] [3] An apoptosis-inducing agent for prostate cancer,
comprising, as an active ingredient, the following REIC/Dkk-3
DNA:
[0018] (c) DNA consisting of the nucleotide sequence represented by
SEQ ID NO: 1; or
[0019] (d) DNA that hybridizes under stringent conditions to DNA
consisting of a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO: 1 and encodes a protein having
apoptosis activity. [0020] [4] A therapeutic agent for prostate
cancer, comprising the apoptosis-inducing agent according to [3].
[0021] [5] An agent for inhibiting prostate cancer metastasis,
comprising the apoptosis-inducing agent according to [1]. [0022]
[6] An agent for inhibiting prostate cancer metastasis, comprising
the apoptosis-inducing agent according to [3]. [0023] [7] The
therapeutic agent for prostate cancer according to [2] or [4],
which is used in combination with hyperthermia. [0024] [8] The
agent for inhibiting prostate cancer metastasis according to [5] or
[6], which is used in combination with hyperthermia.
[0025] This description includes part or all of the contents as
disclosed in the descriptions of Japanese Patent Application Nos.
2005-73807 and 2005-084495, which are priority documents of the
present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A shows an image of REIC/Dkk-3 expression in normal
cells and cancer cells. The image shows REIC protein levels in
normal human cells (OUMS-24: normal human fibroblast; PrSC:
prostate stromal cell; and PrEC: prostate epithelial cell) and
human cancer cell lines (PC3, DU145, and LNCaP: prostate cancer;
KpK-1: gastric cancer cell; HeLa: cervical cancer cell; SaOS-2:
osteosarcoma; and A-549: squamous carcinoma) obtained by the
Western blot method. Tubulin shown in the image was used as a
control.
[0027] FIG. 1B shows images of REIC/Dkk-3 expression in normal
cells and cancer cells. The images show immunostaining results for
the REIC/Dkk-3 protein (green) in OUMS-24 and prostate cancer cells
(PC3, DU145, and LNCaP). The nuclei were stained with propiodium
iodide (red).
[0028] FIG. 1C shows REIC/Dkk-3 expression in normal cells and
cancer cells. In the figure, REIC/Dkk-3 mRNA levels of normal human
fibroblasts and prostate cancer cell lines, which were analyzed by
real-time quantitative RT-PCR, are expressed as molar ratios to
those of GAPDH (p<0.05).
[0029] FIG. 1D shows images of the REIC/Dkk-3 expression in normal
cells and cancer cells. The images show immunostaining results for
REIC/Dkk-3 in a normal prostate, a benign hypertrophic prostate,
and prostate cancer tissue sections with Gleason scores of 4 and
9.
[0030] FIG. 1E shows REIC/Dkk-3 expression in normal cells and
cancer cells. The figure shows quantitative analysis results for
the REIC/Dkk-3 protein using a LandMark tissue Microarray. The
abbreviation "BPH" denotes benign hypertrophic prostate tissue and
the abbreviation "G" denotes a Gleason score (*p<0.05;
**p<0.01).
[0031] FIG. 1F shows REIC/Dkk-3 expression in normal cells and
cancer cells. The figure shows quantitative analysis results for
the REIC protein in fresh human prostate cancer tissue sections
with Gleason scores of 7 or higher and 7 or lower. The abbreviation
"BPH" denotes benign hypertrophic prostate tissue and the
abbreviation "G" denotes a Gleason score (*p<0.05;
**p<0.01).
[0032] FIG. 2A shows an image of apoptosis induction in human
prostate cancer cells caused by REIC/Dkk-3 overexpression. The
image shows REIC/Dkk-3 protein expression in prostate cancer cells
(PC3) 36 hours after REIC/Dkk-3 cDNA transfection using an
adenovirus vector. OUMS-24 was used as a positive control. The
abbreviation "Ad-lacZ" denotes an adenovirus vector carrying
lacZ.
[0033] FIG. 2B shows images of apoptosis induction in human
prostate cancer cells caused by REIC/Dkk-3 overexpression. The
images show TUNEL staining (green) results for normal human
prostate cell lines (OUMS-24, PrEC, and PrSC) and prostate cancer
cell lines (PC3, DU145, and LNCaP) 36 hours after transfection at
10 MOI. The right upper image shows results of Hoechst 33258
staining (blue).
[0034] FIG. 2C shows apoptosis induction in human prostate cancer
cells caused by REIC/Dkk-3 overexpression. The figure shows
percentages of TUNEL-staining-positive cells in normal human
prostate cell lines (OUMS-24, PrEC, and PrSC) and those in prostate
cancer cell lines (PC3, DU145, and LNCaP) 36 hours after
transfection at 10 MOI.
[0035] FIG. 2D shows an image of apoptosis induction in human
prostate cancer cells caused by REIC/Dkk-3 overexpression. The
image shows DNA fragments observed in PC3 cells transfected with
Ad-REIC at 1 MOI or higher.
[0036] FIG. 3A shows images of the involvement of Bax and JNK in
apoptosis induction in PC3 cells caused by Ad-REIC. The images show
TUNEL-staining results for PC3 cells 36 hours after transfection
with REIC or lacZ at 10 MOI. A peptide inhibitor (V5) for Bax was
added at 200 .mu.M to a medium 1 hour prior to transfection. The
scale bar corresponds to 200 .mu.m.
[0037] FIG. 3B shows the involvement of Bax and JNK in apoptosis
induction in PC3 cells caused by Ad-REIC. The figure shows effects
of apoptosis inhibition in PC3 cells caused by a Bax inhibitor V5.
The number of TUNEL-positive cells was determined under the same
conditions used in 3A.
[0038] FIG. 3C shows images of the involvement of Bax and JNK in
apoptosis induction in PC3 cells caused by Ad-REIC. The images
shows immunostaining results for the Bax protein in PC3 cells 36
hours after administration of a vector at 10 MOI. Intracellular
localization of mitochondria was visualized by staining using a
Mitotracker (Mt).
[0039] FIG. 3D shows images of the involvement of Bax and JNK in
apoptosis induction in PC3 cells caused by Ad-REIC. The images show
TUNEL-staining results for PC3 cells treated in the same manner as
was used for the examination of FIG. 3A. A JNK inhibitor SP600125
was used at 10 nM.
[0040] FIG. 3E shows the involvement of Bax and JNK in apoptosis
induction in PC3 cells caused by Ad-REIC. The figure shows effects
of inhibition of REIC/Dkk-3-induced apoptosis by the JNK inhibitor
SP600125. The number of TUNEL-positive cells was determined under
the same conditions used in 3C.
[0041] FIG. 3F shows an image of the involvement of Bax and JNK in
apoptosis induction in PC3 cells caused by Ad-REIC. The image shows
results of Western blot analysis of proteins in PC3 cells as
described in 3D. The abbreviation "P" denotes "phosphorylated," the
abbreviation "Cyto c" denotes cytochrome c, and the abbreviation
".beta.-cat" denotes .beta.-catenin.
[0042] FIG. 3G shows an image of the involvement of Bax and JNK in
apoptosis induction in PC3 cells caused by Ad-REIC. The image shows
results of Western blot analysis of proteins in cytoplasm and
mitochondria extracted from PC3 cells as described in FIG. 3D.
[0043] FIG. 4A shows images of effects of Ad-REIC on the growth of
PC3 cells in nude mice. The images show appearances of tumors at
the end of the observation period.
[0044] FIG. 4B shows effects of Ad-REIC on the growth of PC3 cells
in nude mice. The figure shows the mean volume of tumors of 5 nude
mice. The insert figure in FIG. 4B shows tumor volumes for mice
subjected to REIC injection. 30 days after virus injection, tumors
disappeared in 4 out of 5 mice.
[0045] FIG. 4C shows images of effects of Ad-REIC on the growth of
PC3 cells in nude mice. The images show TUNEL-staining results for
tumor tissue sections 30 days after virus injection. P1 was stained
with propiodium iodide for visualization of nuclei.
[0046] FIG. 5 shows images of localized tumor-inhibitory activity
of the REIC gene in orthotopic transplantation models of prostate
cancer.
[0047] FIG. 6 shows localized tumor-inhibitory activity of the REIC
gene in orthotopic transplantation models of prostate cancer.
[0048] FIG. 7A shows images of apoptosis-inducing effects of the
REIC/Dkk-3 gene.
[0049] FIG. 7B shows apoptosis-inducing effects of the REIC/Dkk-3
gene.
[0050] FIG. 8 shows effects of prostate cancer metastasis
inhibition by the REIC/Dkk-3 gene as expressed in metastasis
rates.
[0051] FIG. 9 shows effects of prostate cancer metastasis
inhibition by the REIC/Dkk-3 gene as expressed in total numbers of
metastatic sites.
[0052] FIG. 10 shows a zymographic image of effects of matrix
metalloproteinase activity inhibition by the REIC/Dkk-3 gene.
[0053] FIG. 11 shows effects of the REIC/Dkk-3 gene upon survival
rates of mice inoculated with prostate cancer cells.
[0054] FIG. 12 shows images of apoptosis induction in normal cells
caused by the combined use of the REIC/Dkk-3 gene and an HSP
inhibitor or an HSP inducer.
[0055] FIG. 13 shows images of apoptosis induction in prostate
cancer cells caused by the combined use of the REIC/Dkk-3 gene and
an HSP inhibitor or an HSP inducer.
[0056] FIG. 14 shows images of effects of the combined use of the
REIC/Dkk-3 gene and hyperthermic treatment in prostate cancer
cells.
BEST MODE FOR CARRYING OUT THE INVENTION
[0057] The apoptosis-inducing agent of the present invention
comprises, as an active ingredient, the REIC/Dkk-3 gene or a
protein encoded by the gene (REIC/Dkk-3 protein).
[0058] The nucleotide sequence of the REIC/Dkk-3 gene and the amino
acid sequence of the protein encoded by the gene are represented by
SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
[0059] As a protein encoded by the REIC/Dkk-3 gene and contained in
the apoptosis-inducing agent of the present invention, a protein
that has an amino acid sequence that is substantially identical to
the amino acid sequence represented by SEQ ID NO: 2 and apoptosis
inducing-activity in cancer cells can be used. Herein, examples of
such substantially identical amino acid sequence include: an amino
acid sequence derived from the amino acid sequence represented by
SEQ ID NO: 2 by substitution, deletion and/or addition of 1,
several, or more (1 to 10, preferably 1 to 5, and further
preferably 1 or 2) amino acids; and an amino acid sequence having a
homology of at least 85% or more, preferably 90% or more, further
preferably 95% or more, and particularly preferably 97% or more to
the aforementioned amino acid sequence upon calculation by BLAST
(Basic Local Alignment Search Tool at the National Center for
Biological Information, U.S.A.) or the like (with the use of a
default (default setting) parameter, for example).
[0060] In addition, examples of REIC/Dkk-3 DNA contained in the
apoptosis-inducing agent of the present invention include: DNA that
hybridizes under stringent conditions to DNA having a nucleotide
sequence complementary to the nucleotide sequence represented by
SEQ ID NO: 2; DNA having a homology of at least 85% or more,
preferably 90% or more, further preferably 95% or more, and
particularly preferably 97% or more to the nucleotide sequence
represented by SEQ ID NO: 2 upon calculation by BLAST (Basic Local
Alignment Search Tool at the National Center for Biological
Information, U.S.A.) or the like (with the use of a default
(default setting) parameter, for example); and DNA encoding a
protein comprising an amino acid sequence derived from the amino
acid sequence of a protein encoded by the aforementioned DNA by
substitution, deletion and/or addition of 1, several, or more (1 to
10, preferably 1 to 5, and further preferably 1 or 2) amino acids.
As long as such DNA encodes a protein having activity of inducing
cancer cell apoptosis, it can be contained in the
apoptosis-inducing agent of the present invention. The term
"stringent conditions" used herein refers to, for example,
stringent conditions (1XSSC, 0.1% SDS, 37.degree. C.) or conditions
comparable thereto, more stringent conditions (0.5XSSC, 0.1% SDS,
42.degree. C.) or conditions comparable thereto, or even more
stringent conditions (0.2XSSC, 0.1% SDS, 65.degree. C.) or
conditions comparable thereto. As such hybridization conditions
become more stringent, it can be expected that DNA having a high
homology to a probe sequence is more likely to be isolated. Note
that the above combinations of SSC, SDS, and temperature are mere
examples. Thus, a necessary level of stringency can be achieved
with the use of an adequate combination of probe concentration,
probe length, hybridization reaction time, and the like.
[0061] Further, an REIC/Dkk-3 protein and REIC/Dkk-3 DNA, which are
contained in the apoptosis-inducing agent, the therapeutic agent
for prostate cancer, and the agent for inhibiting prostate cancer
metastasis of the present invention, include: a peptide having
apoptosis-inducing activity, which is a fragment peptide comprising
a partial amino acid sequence of the amino acid sequence of the
protein; and a nucleotide encoding a peptide having
apoptosis-inducing activity, which is a fragment nucleotide
comprising a partial nucleotide sequence of the nucleotide sequence
of the DNA, respectively. Such fragment peptide or fragment
nucleotide can be readily obtained by cleaving a full-length
protein or full-length DNA at an adequate position and detecting
the presence or absence of apoptosis-inducing activity in the
resulting fragment.
[0062] The REIC/Dkk-3 protein of the present invention is
administered to a test subject and then incorporated into prostate
cancer cells so that the protein acts in the cancer cells and
induces apoptosis. The REIC/Dkk-3 protein can be incorporated into
prostate cancer cells in a manner such that the REIC/Dkk-3 protein
is allowed to bind to, for example, a cell-membrane-permeable
peptide such that the protein is administered to a prostate cancer
tissue site. A variety of cell-membrane-permeable peptides that
have been known to the public can be used. Examples of such
peptides include an HIV-1-TAT cell-membrane-permeable domain
(protein transduction domain), a cell-membrane-permeable domain of
the Drosophila homeobox protein antennapedia, a C-terminal
(267-300) peptide of VP22, an HIV-1/Rev (34-50) peptide, an
FHV/coat (35-49) peptide, and an N-terminal (7-22) hydrophobic
domain of K-FGF. In addition, it is also possible to allow the
REIC/Dkk-3 protein to bind to a compound that can specifically bind
to cancer cells for administration. In such case, such
protein-bound compound may be topically administered to a prostate
cancer tissue site or may be administered orally or via other
routes, such that the REIC/Dkk-3 protein can be delivered to
prostate cancer cells. Examples of such compound that can
specifically bind to cancer cells include a homing signal peptide,
which is a peptide that binds to a receptor expressed on the cancer
cell surface. Examples of such homing signal peptide include NGR
and RGD, which specifically bind to a homing signal peptide
receptor of an angiogenetic endothelial cell (Nat Med. 1999 Sep;
5(9): 1032-1038). For instance, in order to allow the above
cell-membrane-permeable peptide or homing signal peptide to bind to
the REIC/Dkk-3 protein, DNA encoding the REIC/Dkk-3 protein and DNA
encoding the above cell-membrane-permeable peptide or homing signal
peptide are ligated to each other in-frame such that a fusion
protein can be produced by conventional genetic engineering
techniques.
[0063] The REIC/Dkk-3 gene can be obtained from human cells, human
tissue, or the like based on sequence information of SEQ ID NO: 1.
In addition, it can be obtained according to WO01/038523.
[0064] Further, the present invention encompasses a vector
comprising REIC/Dkk-3 DNA. When a test subject is transfected with
such vector, the REIC/Dkk-3 protein is expressed in the test
subject such that apoptosis-inducing effects can be exerted. Such
transfection of a gene of interest into a test subject upon gene
therapy can be carried out in accordance with conventional methods.
Examples of a method for gene transfection into a test subject
include a method using a viral vector and a method using a nonviral
vector. A variety of such methods have been known to the public
(Experimental Medicine (additional volume), Basic Techniques for
Gene Therapy (Idenshi Chiryo no Kiso Gijutsu), Yodosha Co., Ltd.,
1996; Experimental Medicine (additional volume), Experimental
Methods for Gene Transfection & Expression Analysis (Idenshi
Donyu & Hatsugen Kaiseki Jikkenho), Yodosha Co., Ltd., 1997;
"Handbook for Gene Therapy Research and Development" (Idenshi
Chiryo Kaihatsu Kenkyu Handbook), edited by the Japan Society of
Gene Therapy, NTS Inc., 1999).
[0065] In typical methods, viral vectors used for gene transfection
such as adenovirus, adeno-associated virus, and retrovirus are
used. Gene transfection into cells can be carried out by
introducing a gene of interest into a DNA or RNA virus such as
detoxicated retrovirus, herpesvirus, vaccinia virus, poxvirus,
poliovirus, Sindbis virus, Sendai virus, SV40, or human
immunodeficiency virus (HIV) and allowing the obtained recombinant
virus to infect cells.
[0066] When the gene of the present invention is used for a gene
therapy using a virus, an adenovirus vector is preferably used. For
instance, adenovirus vectors are characterized in that:
[0067] (1) gene transfection can be carried out in a variety of
cells;
[0068] (2) gene transfection can be efficiently performed even in
cells in growth arrest;
[0069] (3) they can be concentrated by centrifugation such that a
virus at high titer (10 to 11 PFU/ml or more) can be obtained;
and
[0070] (4) they are appropriate for use for direct gene
transfection into in vivo tissue cells. As an adenovirus vector
used for gene therapy, the following vectors have been developed: a
second generation adenovirus vector (Lieber, A., et al., J. Virol.,
70, 8944, 1996; Mizuguchi, H. & Kay, M. A., Hum. Gene Ther.,
10, 2013, 1999) obtained by deleting the E2 or E4 domain in
addition to the E1/E3 domain from a first generation adenovirus
vector lacking the E1/E3 domain (Miyake, S., et al., Proc. Natl.
Acad. Sci. U.S.A., 93, 1320, 1996); and a third generation
adenovirus vector (Steinwaerder, D. S. et al., J. Virol., 73, 9303,
1999) almost completely lacking the adenovirus genome (GUTLESS).
However, for transfection of the gene of the present invention, any
adenovirus vector may be used without particular limitation.
Further, the gene of the present invention can be applied for
long-term gene expression using, for example, an adeno-AAV hybrid
vector having the ability to cause incorporation in the AAV
chromosome (Recchia, A. et al., Proc. Natl. Acad. Sci. U.S.A., 96,
2615, 1999) and an adenovirus vector that has acquired the ability
to cause incorporation in the chromosome with the use of a
transposon gene. In addition, by inserting a peptide sequence that
can be translocated to an H1 loop of an adenovirus fiber in a
tissue-specific manner, it is possible to impart tissue specificity
to an adenovirus vector (Mizuguchi, H. & Hayakawa, T., Nippon
Rinsho, 7, 1544, 2000).
[0071] Further, even without the use of the above viruses, it is
possible to transfect cells or tissue with a gene of interest with
the use of a recombinant expression vector comprising a gene
expression vector, such as a plasmid vector. For instance, gene
transfection into cells can be performed by a lipofection method, a
calcium phosphate coprecipitation method, a DEAE-dextran method,
and a DNA direct injection method using a micro glass tube.
Furthermore, it is possible to allow a recombinant expression
vector to be incorporated into cells by a gene transfection method
using internal liposomes, a gene transfection method using
electorostatic type liposomes, an HVJ-liposome method, a modified
HVJ-liposome method (HVJ-AVE liposome method), a method using an
HVJ-E (envelope) vector, a method of introducing a
receptor-mediated gene, a method of transferring DNA molecules into
cells with carriers (metallic particles) using a particle gun, a
method of direct transfection of naked DNA, a transfection method
using a variety of polymers, or the like. The expression vector
used in such case may be any expression vector as long as it can
cause in vivo expression of a gene of interest. Examples thereof
include expression vectors such as pCAGGS (Gene 108, 193-200
(1991)), pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen Corporation,
Stratagene), and pVAX1.
[0072] A vector comprising REIC/Dkk-3 DNA may adequately comprise a
promoter or enhancer for gene transcription, a polyA signal, a
marker gene used for labeling and/or selection of cells transfected
with a gene, or the like. In such case, examples of a promoter that
can be used include conventional promoters.
[0073] In order to introduce a pharmaceutical composition
comprising REIC/Dkk-3 DNA of the present invention into a test
subject, the following methods or the like may be used: an in vivo
method, wherein a gene therapeutic agent is directly introduced
into a living body; and an ex vivo method, wherein a specific cell
is collected from a human, a gene therapeutic agent is introduced
ex vivo into the cell, and the cell is introduced into the human
(Nikkei Science, 1994, 4, pp. 20-45; The Pharmaceuticals Monthly,
36(1), 23-48 (1994); Experimental Medicine (additional volume),
12(15), (1994); "Handbook for Gene Therapy Research and
Development" (Idenshi Chiryo Kaihatsu Kenkyu Handbook), edited by
the Japan Society of Gene Therapy, NTS Inc., 1999).
[0074] Prostate cancer is a therapeutic target of the present
invention. In particular, the present invention is especially
useful for highly malignant prostate cancer. The term "highly
malignant prostate cancer" used herein indicates, for example,
prostate cancer with a Gleason score of 8 or higher.
[0075] The REIC/Dkk-3 protein or REIC/Dkk-3 DNA of the present
invention can be further used for inhibition of prostate cancer
metastasis. That is, the present invention encompasses an agent for
inhibiting prostate cancer metastasis comprising, as an active
ingredient, the REIC/Dkk-3 protein or REIC/Dkk-3 DNA. The agent for
inhibiting prostate cancer metastasis can be used in the same
manner as an apoptosis-inducing agent for prostate cancer and a
therapeutic agent for prostate cancer. When administering such
agent to prostate cancer cells, it is possible to inhibit prostate
cancer metastasis.
[0076] Further, the REIC/Dkk-3 protein or REIC/Dkk-3 DNA can
enhance effects of hyperthermia for prostate cancer. Hyperthermia
is a cancer therapy utilizing the cancer cell characteristic of
being more heat-sensitive than normal cells. Apoptosis induction by
the REIC/Dkk-3 protein is associated with the expression of heat
shock protein (HSP) in cells and is affected by cell stress
responsiveness. That is, with the combined use of hyperthermia and
an apoptosis-inducing agent, cancer therapeutic agent, or agent for
inhibiting cancer metastasis that comprises, as an active
ingredient, an REIC/Dkk-3 protein or REIC/Dkk-3 DNA, apoptosis is
more likely to be induced so that it becomes possible to treat
cancer and to inhibit cancer metastasis more effectively.
Specifically, upon hyperthermic treatment of cancer regions, the
apoptosis-inducing agent, cancer therapeutic agent, or agent for
inhibiting cancer metastasis, which comprises, as an active
ingredient, the REIC/Dkk-3 protein or REIC/Dkk-3 DNA of the present
invention can be administered to the relevant regions. Any heating
method may be used for a hyperthermic treatment for hyperthermia as
long as it allows a prostate cancer region of a test subject to be
subjected to hyperthermic treatment. Either a localized heating
method or a whole-body heating method can be adopted. A heating
apparatus to be used may be based on any heating method such as an
external heating method, an intracavitary heating method, or a
tissue heating method. A specific example of a heating apparatus is
a heating apparatus equipped with a thermostatic bath and using
microwaves, ultrasonic waves, electromagnetic waves, RF waves
(radio-frequency waves), or the like. The temperature for
hyperthermic treatment is 41.degree. C. to 45.degree. C. and
preferably 43.degree. C. to 44.degree. C.
[0077] The apoptosis-inducing agent, cancer therapeutic agent, or
agent for inhibiting cancer metastasis, which comprises, as an
active ingredient, an REIC/Dkk-3 protein or REIC/Dkk-3 DNA of the
present invention may be administered prior to, during, or after
hyperthermia.
[0078] That is, the present invention encompasses a method of
treating prostate cancer or inhibiting prostate cancer metastasis,
comprising administering an apoptosis-inducing agent, cancer
therapeutic agent, or agent for inhibiting cancer metastasis, which
comprises, as an active ingredient, an REIC/Dkk-3 protein or
REIC/Dkk-3 DNA to a test subject upon hyperthermic treatment of a
prostate cancer region of the test subject. Further, the present
invention encompasses: an apoptosis-inducing agent, cancer
therapeutic agent, or agent for inhibiting cancer metastasis, which
comprises, as an active ingredient, an REIC/Dkk-3 protein or
REIC/Dkk-3 DNA to be used in combination with hyperthermia; and an
apoptosis-inducing agent, cancer therapeutic agent, or agent for
inhibiting cancer metastasis, which comprises, as an active
ingredient, an REIC/Dkk-3 protein or REIC/Dkk-3 DNA, and which is
administered prior to, during, or after application of
hyperthermia.
[0079] The pharmaceutical composition of the present invention
contains an REIC/Dkk-3 protein, a fusion protein comprising the
REIC/Dkk-3 protein and a cell-membrane-permeable peptide or a
homing signal peptide, REIC/Dkk-3 DNA, or a vector comprising the
DNA, together with a pharmaceutically acceptable carrier, diluent,
and/or excipient.
[0080] The pharmaceutical composition of the present invention can
be administered in various forms. For instance, it can be formed
into tablets, capsules, granules, powder, syrups, or the like for
oral administration. Alternatively, it can be formed into
injections, infusions, suppositories, sprays, eye drops, nasal
preparations, adhesive preparations, or the like for parenteral
administration.
[0081] The pharmaceutical composition of the present invention may
be topically administered. For instance, the effects thereof can be
exerted when the composition is administered to cancer regions by,
for example, injection.
[0082] The pharmaceutical composition of the present invention
contains a carrier, a diluent, and/or an excipient, which are
usually used in the pharmaceutical field. Examples of a carrier and
an excipient used for tablets include lactose and magnesium
stearate. Examples of aqueous liquid used for injection include
physiological saline and isotonic solutions containing glucose or
different adjuvants, which may be used in combination with adequate
solubilizers, including alcohol, polyalcohol such as propylene
glycol, and nonionic surfactants. Examples of oily liquid that can
be used include sesame oil and soybean oil. Examples of a
solubilizer that may be used in combination include benzyl benzoate
and benzyl alcohol.
[0083] The dose of the pharmaceutical composition of the present
invention is determined depending upon patient's symptoms, age, and
weight, and the like. Upon oral administration, the daily dose in
terms of an REIC/Dkk-3 protein is approximately 0.001 mg to 100 mg.
It may be administered in a single dose or multiple doses. In
addition, upon parenteral administration, it is possible to carry
out subcutaneous, intramuscular, or intravenous administration at
0.001 mg to 100 mg in a single dose. In addition, REIC/Dkk-3 DNA
that is inserted into an expression vector or the like so as to be
translated in a test subject may be subcutaneously,
intramuscularly, or intravenously administered at 0.001 mg to 100
mg in a single dose every several days, weeks, or months.
EXAMPLES
[0084] The present invention is hereafter described in greater
detail with reference to the following examples, although the
technical scope of the present invention is not limited
thereto.
Example 1
[0085] Prostate Cancer Cell Apoptosis Induction by REIC/Dkk-3
[0086] Materials used in the Examples of the present invention were
obtained as described below. The Examples were carried out by
methods described below.
Cells and Culture Method
[0087] Normal prostate epithelial cells (PrEC) and prostate stromal
cells (PrSC) were purchased from Cambrex (Baltimore, Md.). The
prostate cancer cell lines PC3, DU145, and LNCaP were provided by
ATCC (Rockville, Md.). OUMS-24 was provided by Dr. Masayoshi Nanba.
An HAM'S F-12 K medium, an RPMI 1640 medium, and modified MEM
medium (Nissui) were separately used with a supplement of 10% calf
serum for PC3, DU145 and LNCaP, and OUMS-24.
Human Prostate Tissue
[0088] A LandMark.TM. low-density prostate tissue microarray
(Ambion, Austin) was used for immunostaining of the REIC/Dkk-3
gene. Fresh prostate cancer tissue samples were obtained from 40
patients. Of them, 20 samples had a Gleason score of 8 or higher
and another 20 samples had a Gleason score of 7 or lower.
Immunological analyses
[0089] After fixation with 4% paraformaldehyde, the cells and the
tissue samples were immunostained with a primary antibody
(anti-human REIC/Dkk-3 antibody purified in the laboratory of the
present inventors) and a secondary antibody (FITC-conjugated
anti-rabbit immunoglobulin G antibody, Sigma, St. Louis). For Bax
and Bcl-2, an anti-human Bax antibody (Upstate Cell signaling
solutions, Lake Placid, N.Y.) and an anti-human Bcl-2 antibody (BD
Biosciences, San Diego) were used, respectively. A Vectashield
mounting medium with DAPI or with propiodium iodide (Vector
Laboratories, Burlingame, Calif.) was used for staining of cell
nuclei. The signal intensity of each stained sample was quantitated
using computer software. Western blot analysis was performed as
previously described. The antibodies used were as follows: a
purified rabbit anti-human REIC/Dkk-3 antibody; an Apoptosis
sampler I kit (BD Biosciences) for Apf-1, Bad, Bcl-2, Bcl-xL, and p
53; a rabbit anti-human Bax antibody (Upstate Cell signaling
solutions, Lake Placid, N.Y.); a rabbit anti-human Bax antibody (BD
Biosciences); a mouse anti-human Apaf-1 antibody (BD Biosciences);
and a mouse anti-human tubulin antibody (Sigma).
Real-Time Quantitative RT-PCR
[0090] Real-time PCR was performed under the conditions recommended
by the manufacturer using a LightCycler.TM. (Roche Diagnostic,
Lewes, UK). The primers used for real-time PCR were as follows:
TABLE-US-00001 REIC/Dkk-3-F' 5'-GTAAGTTCCCCTCTGGCTTG-3'; (SEQ ID
NO: 3) REIC/Dkk-3-R' 5'-AAGCACCAGACTGTGAAGCCT-3'; (SEQ ID NO: 4)
GAPDH-F' 5'-GGGTGTGAACCATGAGAAGTATGA-3'; (SEQ ID NO: 5) and
GAPDH-R' 5'-TGCTAAGCAGTTGGTGGTGC-3'. (SEQ ID NO: 6)
[0091] The obtained products were checked by melting point
analysis, electrophoresis, and direct sequencing. Standard curves
were created using the plasmids containing the respective inserts.
The results are shown as molar ratios of REIC/Dkk-3 to GAPDH.
Apoptosis Analyses
[0092] A DNA ladder method was performed under conventional
conditions. DNA was extracted after lysing cells with Triton X-100,
treated with RNase and proteinase K, and electrophoresed on 2%
agarose gel. An In Situ Cell Death Detection Kit, Fluorescein
(Roche) was used for TUNEL staining. A Bax-specific peptide
inhibitor V5 was purchased from Sigma Genosis (Woodlands,
Tex.).
Animal Experiments
[0093] PC3 cells (2.5.times.10.sup.6/PBS 50 .mu.l) were
subcutaneously injected into the right flanks of 8-week-old BALB/C
nude mice. One week later, when the tumor diameters had reached 5
mm, adenovirus vectors (2.5.times.10.sup.8 pfu) incorporating the
REIC gene (or LacZ gene) were injected intratumorally. The same
volume of PBS was injected for controls. Tumor sizes were measured
every 3 days up to 30 days after adenovirus vector injection. Tumor
volumes (V) were calculated using the following formula:
1/2.times.(the shorter diameter).sup.2.times.(the longer
diameter).
[0094] In this Example, results described below were obtained.
[0095] In order to find the potential usefulness of the REIC/Dkk-3
gene as a therapeutic target, a variety of cells were first
examined in terms of REIC/Dkk-3 gene expression. In human
fibroblasts (OUMS-24), prostate epithelial cells (PrEC), and
prostate stromal cells (PrSC), the REIC/Dkk-3 protein was detected
in two bands of 62 to 83 kDa (FIG. 1A). The observed size was
larger than the size estimated from the amino acid sequence (38.3
kDa) because of glycosylation. The REIC/Dkk-3 protein was barely
detected in cancer cell lines, excluding 3 representative prostate
cancer cell lines (PC3, LNCaP, and DU145) and 4 prostate cancer
cell lines. Lack of REIC/Dkk-3 protein in the 3 different prostate
cancer cell lines was confirmed by immunostaining (FIG. 1B).
Downregulation of REIC/Dkk-3 mRNA was also confirmed by
Quantitative RT-PCR (FIG. 1C).
[0096] The expression of the REIC/Dkk-3 gene was examined in human
prostate tissue by immunostaining (FIG. 1D). The REIC/Dkk-3 protein
was detected in epithelial cells and stromal cells in normal
prostates and benign hypertrophic prostates. In prostate cancer
with a Gleason score of 7 or lower, the REIC/Dkk-3 protein level
was reduced differently. In prostate cancer with a Gleason score of
8 to 10, the expression of REIC/Dkk-3 protein was not observed.
Signal intensity was quantitatively measured using computer
software (FIG. 1E). In both a commercially available prostate
tissue microarray (FIG. 1E, left) and fresh prostate cancer tissue
(FIG. 1E, right), the expression level of REIC/Dkk-3 protein was
reduced in proportion to the cancer grade. Upon immunostaining of
other normal human tissues (from the brain, heart, liver, pancreas,
kidney, mammary glands, and lymph nodes), the REIC/Dkk-3 protein
was expressed at different intensities.
[0097] In order to examine the possible use of the REIC/Dkk-3 gene
as a gene therapy tool, the REIC/Dkk-3 gene was overexpressed using
an adenovirus vector incorporating the REIC/Dkk-3 gene. The
REIC/Dkk-3 protein level in PC3 cells transfected with REIC/Dkk-3
with the use of vectors at 1 MOI was almost comparable to that of
OUMS-24 (FIG. 2A). Within a few days after transfection, most of
the prostate cancer cells had become detached from the bottom of
the culture vessel. In order to examine the cause, cells were
stained 36 hours after REIC/Dkk-3 gene transfection by the TUNEL
method. As shown in FIG. 2B, many prostate cancer cells (PC3,
DU145, and LNCaP). were TUNEL-positive while fewer normal cells
(OUMS-24, prostate stromal cells, and prostate epithelial cells)
were TUNEL-positive. The incidences of TUNEL-positive cells were
49%, 24%, and 41% in the cases of PC3, DU145, and LNCaP,
respectively. However, the incidence of TUNEL-positive cells was
less than 1% in the case of normal cells (FIG. 2C). DNAs of PC3
cells were analyzed 36 hours after vector transfection so as to
confirm apoptotic properties.
[0098] In the case of PC3 cells transfected with the REIC gene at 1
MOI or higher, different patterns of DNA bands were clearly
observed (FIG. 2D). As shown in FIG. 2D, specific DNA fragments
were observed in PC3 cells transfected with Ad-REIC at 1 MOI or
higher. One day after seeding of 5.times.10.sup.5 PC3 cells, viral
vectors were allowed to transfect the cells. The cells were
harvested 36 hours after transfection. The transfection efficiency
of Ad-REIC and the expression level of the REIC/Dkk-3 gene were
similar among the cells (data not shown). These results indicate
that overexpression of REIC/Dkk-3 selectively induces apoptosis in
prostate cancer cells that substantially lack endogenous REIC/Dkk-3
expression.
[0099] In order to discover the mechanism for tumor-specific
apoptosis caused by the REIC/Dkk-3 gene, expression levels of
different apoptosis/cell-cycle-regulation-related proteins in PC3
cells and OUMS-24 cells were compared. In PC3 cells transfected
with the RE1C/Dkk-3 gene, reduced levels of apoptosis-inhibiting
Bcl-2 and Bcl-XL proteins were observed (FIG. 3F). No significant
changes in levels of Bax, Bad, ApaF-1, p 53, p 21 (CIP1/WAF1), or p
16 (INK4a) were observed among cells (data not shown).
[0100] A caspase 8 inhibitor did not inhibit Ad-REIC-induced
apoptosis in PC3 cells. Meanwhile, a Bax inhibitor V5 (Sawada, M.
et al., Nat Cell Biol 5, 352-7 (2003)) completely inhibited
apoptosis (FIG. 3A). Translocation of Bax protein from cytoplasm to
mitochondria may trigger activation of a Bax-mediated apoptotic
pathway (Gross, A. et al., Embo J 17, 3878-85 (1998)). FIG. 3C
shows mitochondrial translocation of Bax protein by Ad-REIC and
inhibition of such translocation by V5. V5 showed no effects on the
protein levels of Bcl-2 and Bax.
[0101] It has been known that c-jun N-terminal kinase (JNK)
mediates a non-canonical pathway of Wnt signaling (Lei, K. et al.,
Mol Cell Biol 22, 4929-42 (2002); and Tsuruta, F. et al., Embo J
23, 1889-99 (2004)). Further, it has been reported that Bax plays
an important role in JNK-induced apoptosis and JNK promotes
mitochondrial translocation of Bax. When a JNK-specific inhibitor
SP600125 was added to PC3 cells, Ad-REIC-induced apoptosis was
inhibited in a concentration-dependent manner (FIGS. 3D and 3E).
Activation of JNK in PC3 cells infected with Ad-REIC was confirmed
using a phosphorylated JNK antibody (FIG. 3F). SP600125 inhibited
JNK kinase activity but not phosphorylation of JNK itself. Bax was
detected in cytoplasm of PC3 cells but clearly observed to be
translocated to mitochondria in Ad-REIC-infected cells (FIG. 3G).
The mitochondrial translocation of Bax was associated with the
release of cytochrome c into cytoplasm and was suppressed by a JNK
inhibitor SP600125. Phosphorylated JNK was also translocated by
Ad-REIC to mitochondria. These results suggest that overexpression
of REIC activates JNK, reduces the Bcl-2 protein level, promotes
mitochondrial translocation of Bax protein, releases cytochrome c
into cytoplasm, and finally induces apoptosis. Recently, Hsieh et
al. reported that overexpression of REIC/Dkk-3 gene induces
apoptosis in different human cancer cells through activation of
caspase 3, which is known as a major apoptosis executor downstream
of cytochrome c (Hsieh, S. Y. et al., Oncogene 23, 9183-9
(2004)).
[0102] At present, it is unclear whether Ad-REIC-induced JNK
activation directly or indirectly acts on Bcl-2 and Bax, although
Bcl-2 (Yamamoto, K. et al., Mol Cell Biol 19, 8469-78 (1999); and
Deng, X. et al., J Biol Chem 276, 23681-8 (2001)), Bcl-XL (Basu, A.
et al., FEBS Lett 538, 41-7 (2003)), and Bim (Lei, K. et al., Mol
Cell Biol 22, 4929-42 (2002)) have been identified as targets for
JNK. It was confirmed that the REIC/Dkk-3 protein was glycosylated
and secreted into a culture solution when overexpressed in PC3
cells by Ad-REIC. The amount of .beta.-catenin and the
intracellular localization thereof were not affected by Ad-REIC.
Thus, it is considered that secreted REIC/Dkk-3 is unlikely to act
through the Wnt canonical pathway (data not shown). JNK are thought
to be activated through the non-canonical pathway of Wnt signaling
or by an intracellular stress sensing system for the overexpressed
protein or unknown intracellular factors. In practice, an IL-10
family cytokine (intercellular signal transduction protein)
MDA-7/IL-24 (also referred to as MDA7/IL-24) efficiently induces
apoptosis in many types of human cancer cells (particularly human
prostate cancer cells) even when secretion of the cytokine is
blocked (Sauane, M. et al., Cancer Res 64, 2988-93 (2004)). Bax is
activated by p 53 (Han, J. et al., Genes Dev 10, 461-77 (1996)).
However, since PC3 cells are null in p 53, apoptosis induction by
the REIC gene is independent of the p 53 function (Arah, I. N. et
al., Anticancer Res 18, 1845-9 (1998)). MDA-7 also selectively
induces apoptosis by p53-independent activation of Bax (Su, z. z.
et al., Proc Natl Acd Sci U.S.A. 95, 14400-5 (1998)). Recently,
Hoang et al. reported that the overexpression of the REIC/Dkk-3
gene did not induce apoptosis in a human osteosarcoma cell line
Saos-2 but inhibited invasion and motility of the cells in in vitro
experiments (Hoang, B. H. et al., Cancer Res 64, 2734-9 (2004)).
REIC/Dkk-3 may exert its anticancer activity at different
points.
[0103] Since apoptosis induction by REIC/Dkk-3 gene transfection
was proved by in vitro studies, the effect of the REIC/Dkk-3 gene
upon PC3 cells was examined in vivo (in animal experiments). PC3
cells (2.5.times.10.sup.6) were subcutaneously injected into nude
mice. One week later, when the tumor volumes reached 30 to 100
cm.sup.3, 2.5.times.10.sup.8 pfu of REIC or lacZ was injected
intratumorally with the use of adenovectors. The same volume of
buffer (PBS) was injected into a control group. Tumor sizes were
measured every 3 days during the 27 days following injection.
Tumors in the control group and the lacZ group grew gradually
during the observation period of 1 month after injection (FIGS. 4A
and 4B). In contrast, tumors completely disappeared in 4 out of 5
mice in the REIC group (treatment group). Further, tumors did not
completely disappear in 1 out of 5 mice in the REIC group; instead,
they shrunk and then remained unchanged over the observation
period. The above tumors were resected after the end of the
observation period, followed by TUNEL staining (FIG. 4C). No
apoptosis was observed in the buffer group or the lacZ group.
Meanwhile, in the REIC group, many TUNEL-positive cells were
observed 1 month after injection. Selective apoptosis-inducing
activity in culture cells and in vivo tumor-inhibitory activity
that are exerted by the REIC/Dkk-3 gene strongly indicate that the
REIC/Dkk-3 gene can be a target for cancer therapies in addition to
p 53 and mda-7 (Sauane, M. et al., Cancer Res 64, 2988-93 (2004)).
Prostate cancer is one of the most common malignant diseases in
Western countries. Various therapeutic measures, including hormone
therapies, have been applied to prostate cancer. However, once
prostate cancers acquire hormone resistance at later stages, it is
difficult to control them by conventional therapies.
[0104] This Example indicates that REIC/Dkk-3 can be a new
molecular target to counteract highly malignant prostate
cancers.
Example 2
[0105] Effects of Prostate Cancer Metastasis Inhibition caused by
REIC/Dkk-3 Gene Transfection using Adenovirus Vectors
[0106] Mouse prostate cancer cells RM-9 (5.0.times.10.sup.3 cells)
were injected into prostates of C57BL/6 mice. One week later,
1.2.times.10.sup.8 pfu of REIC or lacZ (or PBS) was directly
injected intratumorally (mean tumor diameter: 60 mm3) with the use
of adenovectors. Tumor growth was significantly inhibited in the
REIC/Dkk-3 group (treatment group) compared with the control group
and the lacZ group (FIGS. 5 and 6). Tumor diameters were measured
every 3 days using a transrectal ultrasonography system previously
developed by the present inventors. Tumor volumes (V) were
calculated by the following formula: 1/2.times.(short
diameter).sup.2.times.(long diameter). FIG. 5 shows ultrasound
images and images of mouse intraperitoneal cavities taken with the
transrectal ultrasonography system. FIG. 6 shows time-dependent
changes in tumor volumes. As shown in FIGS. 5 and 6, stronger tumor
inhibition effects were observed in the REIC/Dkk-3 group than were
observed in the LacZ group.
[0107] The above tumors were resected on days 10 and 16 (3 and 9
days after adenovector injection, respectively), followed by TUNEL
staining for detection of apoptotic cells. As a result, a greater
number of cells were found to be TUNEL-positive in the REIC/Dkk-3
group compared with the buffer group and the LacZ group (FIGS. 7A
and 7B).
[0108] Subsequently, in order to analyze metastasis-inhibitory
activity of the REIC gene, each group was subjected to laparotomy
when prostate cancer volumes reached 750 mm.sup.3. Then,
intrapelvic lymph nodes were observed. In order to confirm
metastasis rates when volumes of primary tumor lesions reached the
same level, prostate cancer volumes were measured using transrectal
ultrasonography. When tumor diameters reached 750 mm.sup.3 in each
group, laparotomy was carried out so that intrapelvic lymph nodes
were collected. In the control group and the lacZ group, 80% to 85%
of mice were found to experience lymph node metastasis. Meanwhile,
in the REIC/Dkk-3 group, approximately 40% of mice were found to
experience such metastasis (PBS: n=10; Lacz: n=12; and REIC/Dkk-3:
n=12) (FIG. 8). Further, the number of lymph node metastasis
lesions was counted. The number of lymph node metastasis lesions
was 0 to 5 (mean value: 4) in the control group and the lacZ group;
however, it was 0 to 2 (mean value: 1) in the REIC/Dkk-3 group
(PBS: n=10; Lacz: n=12; and REIC/Dkk-3: n=12)(FIG. 9).
[0109] Subsequently, in order to examine the mechanism for
metastasis-inhibitory activity of the REIC/Dkk-3 gene, RM-9 was
transfected in vitro with REIC/Dkk-3 or lacZ with the use of
adenovectors. Then, matrix metalloproteinase (MMP) activity in a
culture solution was measured by zymography. The term "zymography"
used herein refers to a representative technique for detecting
activity of MMP (matrix metalloproteinase) which is a
collagen-degrading enzyme that exists in a basal membrane, such
activity serving as an index of the invasion/metastasis ability of
cancer cells. A culture supernatant or the like is added to gel
containing modified collagen (gelatin) that is a substrate for MMP,
followed by migration. Then, a reaction solution that activates MMP
is added thereto to stain the gel so that bands appear in
connection with the presence of enzymatic activity. MMP2 (72 kDa)
and MMP9 (92 kDa) are closely associated with basal membrane
invasion. RM-9 was transfected with LacZ, human REIC, or the mouse
REIC gene at 10 MOI with the use of adenovectors. 6 hours after
transfection, the culture solution used was exchanged and the newly
exchanged culture solution was collected 42 hours later, followed
by zymography. As shown in FIG. 10, significant signal reduction
was observed in the REIC/Dkk-3 group compared with the lacZ group.
The results indicate that the matrix protease activity was reduced.
Thus, it was suggested that such reduction in MMP activity is
involved in the metastasis inhibition effects of the REIC/Dkk-3
gene.
[0110] Regarding survival curves for orthotopic transplantation
models of prostate cancer, survival was significantly extended in
the REIC/Dkk-3 group compared with the control group and the LacZ
group. As a result of single-dose administration of the REIC/Dkk-3
gene, the improvement was observed in survival rates in addition to
effects of localized tumor inhibition and metastasis inhibition
(FIG. 11).
[0111] The longest survival time extension effect was obtained in
the above case compared with the results of gene therapy
experiments using the other genes (HSV-tk and IL-12) and the same
system (Median survival: control group=22 to 25 days; REIC/Dkk-3
treatment group=42 to 50 days; HSV-tk treatment group=26 days
(Prostate Cancer and Prostatic Diseases, 4: 44-55, 2001); and IL-12
treatment group=28 days (Gene Therapy 6: 338-349, 1999)).
Example 3
[0112] Relationship between HSP70 and Apoptosis Induction caused by
Forced Expression of REIC/Dkk-3
[0113] It was examined whether tumor selectivity in terms of
apoptosis expression caused by Ad-REIC would be associated with
HSP70 expression in normal cells and cancer cells.
[0114] Normal cells OUMS 24 (human immortalized fibroblast) and
prostate cancer cells PC3 were used for comparison. The cells were
infected with Ad-REIC at 20 MOI. 24 hours later, an HSP70 inhibitor
(heat shock protein inhibitor I, CALBIOCHEM, BIOCHEMICALS) and an
HSP70 inducer (Geranyl-Geranyl Acetone) (250 mM each) were
separately added to a culture solution. 48 hours later, TUNEL
staining was performed to determine influence upon apoptosis. FIGS.
12 and 13 show staining images for OUMS24 and of PC3, respectively.
In FIGS. 12 and 13, images on the left side indicate TUNEL-staining
results and images on the right side indicate DAPI-staining
results. As shown in FIG. 12, among normal cells OUMS24, a large
number of TUNEL-positive cells were obtained with the combined use
of the Ad-REIC and the HSP70 inhibitor. On the other hand, a small
number of TUNEL-positive cells were obtained with the combined use
of the Ad-REIC and the HSP inducer. Also, as shown in FIG. 13,
among prostate cancer cells PC3, the number of TUNEL-positive cells
obtained with the combined use of the Ad-REIC and the HSP70
inhibitor was greater than that obtained with the use of Ad-REIC
alone. On the other hand, the number of TUNEL-positive cells
obtained with the combined use of the Ad-REIC and the HSP inducer
was smaller than that obtained with the use of Ad-REIC alone. That
is, apoptosis was induced in normal cells with the use of the HSP70
inhibitor in combination. On the other hand, apoptosis expression
was inhibited in cancer cells with the use of the HSP70 inducer in
combination. Further, apoptosis induction was promoted with the use
of the HSP70 inhibitor in combination.
[0115] The above results revealed that apoptosis expression caused
by Ad-REIC significantly depends on cell stress responsiveness.
Example 4
[0116] PC3 cells and OUMS-24 cells were separately infected with
viruses (Ad-LacZ/Ad-REIC) at 20 MOI for 36 hours. Thereafter, the
cells were separately placed on media at 45.degree. C. and then the
media were placed in an incubator at 45.degree. C. for 6 hours,
followed by CBB staining. The cells subjected to heat treatment at
45.degree. C. belong to a heat (+) group (group subjected to
hyperthermia following infection). In addition, cells that had not
been heat-treated (heat (-) group) were infected with viruses for
42 hours, followed by staining.
[0117] Almost all PC3 cells in the group subjected to hyperthermia
following infection completely died (FIG. 14). Since cancer cells
are generally sensitive to hyperthermia, synergistic effects can be
obtained by a therapy involving combined use of cancer
hyperthermia, which has been widely examined in medical practice,
and the apoptosis- inducing agent of the present invention.
INDUSTRIAL APPLICABILITY
[0118] As shown in the Examples, the REIC/Dkk-3 expression level
was specifically reduced in highly malignant prostate cancer
tissue. In addition, apoptosis was selectively induced in cancer
cells by transfecting the REIC/Dkk-3 gene into prostate cancer
cells lacking REIC/Dkk-3 expression so as to cause REIC/Dkk-3
expression in such cells. Further, when nude mice into which human
prostate cancer cells had been implanted for tumor development were
transfected with the REIC/Dkk-3 gene, tumor inhibition was
observed. That is, the REIC/Dkk-3 gene of the present invention and
an expression product thereof can be used as an apoptosis-inducing
agent for prostate cancer cells and a therapeutic agent for
prostate cancer, which can cause apoptosis in prostate cancer
cells, resulting in prostate cancer inhibition. Furthermore, the
REIC/Dkk-3 gene of the present invention and an expression product
thereof can be used as agents for inhibiting prostate cancer
metastasis, which can inhibit prostate cancer metastasis. Moreover,
prostate cancer can be treated more effectively with the combined
use of hyperthermia and the REIC/Dkk-3 gene of the present
invention and an expression product thereof.
[0119] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
[0120] Free Text of Sequence Listing
[0121] SEQ ID NOS: 3 to 6 (primers)
Sequence CWU 1
1
611053DNAHomo sapiensCDS(1)..(1053) 1atg cag cgg ctt ggg gcc acc
ctg ctg tgc ctg cta ctg gcg gcg gcg 48Met Gln Arg Leu Gly Ala Thr
Leu Leu Cys Leu Leu Leu Ala Ala Ala1 5 10 15gtc ccc acg gcc ccc gcg
ccc gct ccg acg gcg acc tcg gct cca gtc 96Val Pro Thr Ala Pro Ala
Pro Ala Pro Thr Ala Thr Ser Ala Pro Val 20 25 30aag ccc ggc ccg gct
ctc agc tac ccg cag gag gag gcc acc ctc aat 144Lys Pro Gly Pro Ala
Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn 35 40 45gag atg ttc cgc
gag gtt gag gaa ctg gtg gag gac acg cag cac aaa 192Glu Met Phe Arg
Glu Val Glu Glu Leu Val Glu Asp Thr Gln His Lys 50 55 60ttg cgc agc
gcg gtg gaa gag atg gag gca gaa gaa gct gct gct aaa 240Leu Arg Ser
Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys65 70 75 80gca
tca tca gaa gtg aac ctg gca aac tta cct ccc agc tat cac aat 288Ala
Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn 85 90
95gag acc aac aca gac acg aag gtt gga aat aat acc atc cat gtg cac
336Glu Thr Asn Thr Asp Thr Lys Val Gly Asn Asn Thr Ile His Val His
100 105 110cga gaa att cac aag ata acc aac aac cag gct cga caa atg
gtc ttt 384Arg Glu Ile His Lys Ile Thr Asn Asn Gln Ala Arg Gln Met
Val Phe 115 120 125tca gag aca gtt atc aca tct gtg gga gac gaa gaa
ggc aga agg agc 432Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu
Gly Arg Arg Ser 130 135 140cac gag tgc atc atc gac gag gac tgt ggg
ccc agc atg tac tgc cag 480His Glu Cys Ile Ile Asp Glu Asp Cys Gly
Pro Ser Met Tyr Cys Gln145 150 155 160ttt gcc agc ttc cag tac acc
tgc cag cca tgc cgg ggc cag agg atg 528Phe Ala Ser Phe Gln Tyr Thr
Cys Gln Pro Cys Arg Gly Gln Arg Met 165 170 175ctc tgc acc cgg gac
agt gag tgc tgt gga gac cag ctg tgt gtc tgg 576Leu Cys Thr Arg Asp
Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp 180 185 190ggt cac tgc
acc aaa atg gcc acc agg ggc agc aat ggg acc atc tgt 624Gly His Cys
Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys 195 200 205gac
aac cag agg gac tgc cag ccg ggg ctg tgc tgt gcc ttc cag aga 672Asp
Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg 210 215
220ggc ctg ctg ttc cct gtg tgc ata ccc ctg ccc gtg gag ggc gag ctt
720Gly Leu Leu Phe Pro Val Cys Ile Pro Leu Pro Val Glu Gly Glu
Leu225 230 235 240tgc cat gac ccc gcc agc cgg ctt ctg gac ctc atc
acc tgg gag cta 768Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile
Thr Trp Glu Leu 245 250 255gag cct gat gga gcc ttg gac cga tgc cct
tgt gcc agt ggc ctc ctc 816Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro
Cys Ala Ser Gly Leu Leu 260 265 270tgc cag ccc cac agc cac agc ctg
gtg tat gtg tgc aag ccg acc ttc 864Cys Gln Pro His Ser His Ser Leu
Val Tyr Val Cys Lys Pro Thr Phe 275 280 285gtg ggg agc cgt gac caa
gat ggg gag atc ctg ctg ccc aga gag gtc 912Val Gly Ser Arg Asp Gln
Asp Gly Glu Ile Leu Leu Pro Arg Glu Val 290 295 300ccc gat gag tat
gaa gtt ggc agc ttc atg gag gag gtg cgc cag gag 960Pro Asp Glu Tyr
Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu305 310 315 320ctg
gag gac ctg gag agg agc ctg act gaa gag atg gcg ctg ggg gag 1008Leu
Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala Leu Gly Glu 325 330
335cct gcg gct gcc gcc gct gca ctg ctg gga ggg gaa gag att tag
1053Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Gly Glu Glu Ile 340 345
3502350PRTHomo sapiens 2Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu
Leu Leu Ala Ala Ala1 5 10 15Val Pro Thr Ala Pro Ala Pro Ala Pro Thr
Ala Thr Ser Ala Pro Val 20 25 30Lys Pro Gly Pro Ala Leu Ser Tyr Pro
Gln Glu Glu Ala Thr Leu Asn 35 40 45Glu Met Phe Arg Glu Val Glu Glu
Leu Val Glu Asp Thr Gln His Lys 50 55 60Leu Arg Ser Ala Val Glu Glu
Met Glu Ala Glu Glu Ala Ala Ala Lys65 70 75 80Ala Ser Ser Glu Val
Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn 85 90 95Glu Thr Asn Thr
Asp Thr Lys Val Gly Asn Asn Thr Ile His Val His 100 105 110Arg Glu
Ile His Lys Ile Thr Asn Asn Gln Ala Arg Gln Met Val Phe 115 120
125Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser
130 135 140His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr
Cys Gln145 150 155 160Phe Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys
Arg Gly Gln Arg Met 165 170 175Leu Cys Thr Arg Asp Ser Glu Cys Cys
Gly Asp Gln Leu Cys Val Trp 180 185 190Gly His Cys Thr Lys Met Ala
Thr Arg Gly Ser Asn Gly Thr Ile Cys 195 200 205Asp Asn Gln Arg Asp
Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg 210 215 220Gly Leu Leu
Phe Pro Val Cys Ile Pro Leu Pro Val Glu Gly Glu Leu225 230 235
240Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu
245 250 255Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly
Leu Leu 260 265 270Cys Gln Pro His Ser His Ser Leu Val Tyr Val Cys
Lys Pro Thr Phe 275 280 285Val Gly Ser Arg Asp Gln Asp Gly Glu Ile
Leu Leu Pro Arg Glu Val 290 295 300Pro Asp Glu Tyr Glu Val Gly Ser
Phe Met Glu Glu Val Arg Gln Glu305 310 315 320Leu Glu Asp Leu Glu
Arg Ser Leu Thr Glu Glu Met Ala Leu Gly Glu 325 330 335Pro Ala Ala
Ala Ala Ala Ala Leu Leu Gly Gly Glu Glu Ile 340 345
350320DNAArtificial SequenceDescription of Artificial Sequence
Primer 3gtaagttccc ctctggcttg 20421DNAArtificial
SequenceDescription of Artificial Sequence Primer 4aagcaccaga
ctgtgaagcc t 21524DNAArtificial SequenceDescription of Artificial
Sequence Primer 5gggtgtgaac catgagaagt atga 24620DNAArtificial
SequenceDescription of Artificial Sequence Primer 6tgctaagcag
ttggtggtgc 20
* * * * *