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Published OnlineFirst March 17, 2009; DOI: 10.1158/0008-5472.CAN-08-3530
Published Online First on March 17, 2009 as 10.1158/0008-5472.CAN-08-3530
Research Article
A Novel Human AlkB Homologue, ALKBH8, Contributes to
Human Bladder Cancer Progression
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Keiji Shimada, Mitsutoshi Nakamura, Satoshi Anai, Marco De Velasco, Motoyoshi Tanaka,
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Kazutake Tsujikawa, Yukiteru Ouji, and Noboru Konishi
Departments of 1Pathology, 2Urology, and 3Parasitology, Nara Medical University School of Medicine, Shijo-cho, Kashihara, Nara;
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Department of Urology, Kinki University School of Medicine, Sayama, Osaka; and 5Department of Immunology, Osaka University,
Graduate School and School of Pharmaceutical Sciences, Yamadaoka, Suita, Osaka, Japan
Reactive oxygen species (ROS) are implicated in both stimulation
and inhibition of cell proliferation, apoptosis, and cell senescence
(4, 5). ROS trigger genetic programs associated with transformation, resulting in alteration of genes by manipulating the cell cycle
and signal transduction (6). Similarly, oxygen radical generation is
increased in Ras-transformed fibroblasts (7) and antioxidants can
block DNA synthesis, suggesting that ROS are involved in
mitogenic signaling in the course of neoplastic transformation.
NADPH oxidase (NOX) is one of the major sources for cellular ROS
(8, 9). NOX enzymes are the structural homologues of phagocytic
NOX (gp91phox/NOX2) and consist of both single (NOX-1–NOX-5)
and dual oxidases (DuOX1 and DuOX2; ref. 10). Emerging evidence
continues to accumulate that implicates low levels of ROS
generated by NOX enzymes as mediators in inflammation,
apoptosis, cell growth, and angiogenesis in various types of human
cancers. Among NOX family members, the function of NOX-1, in
particular, has been focused on in human cancer. NOX-1 potentially
stimulates branching morphogenesis in the nontubulogenic
endothelial cell line NP 31 (11). de Carvalho and colleagues (12)
reported that 12-lipoxygenase signals downstream NOX-1 to
control cell proliferation in human colon cancer cells, whereas
another group showed that ROS induced by NOX-1 overexpression can modulate the c-fos–mediated growth factors, interleukin-8 and Cav-1, which participate specifically in prostate cancer
cell proliferation (13). It would thus seem that NOX and downstream
targets, including ROS, are necessary for tumor growth, angiogenesis, and potentially metastasis and, therefore, are attractive targets for therapeutic intervention in cancer development.
In the present study, we found ALKBH8 to be an upstream target
of NOX-1 and to be involved in intracellular ROS generation, and
we further determined that ALKBH8/NOX-1 signals function
mainly in the acquisition of the aggressive human urothelial
carcinoma phenotype. In addition, our results clearly showed that
transurethral injection of ALKBH8 small interfering RNA (siRNA)
offers promise as a new therapeutic strategy for bladder cancer.
Abstract
We recently identified a novel human AlkB homologue,
ALKBH8, which is expressed in various types of human
cancers including human urothelial carcinomas. In examining
the role and function of ALKBH8 in human bladder cancer
development in vitro, we found that silencing of ALKBH8
through small interfering RNA transfection reduced reactive
oxygen species (ROS) production via down-regulation of
NAD(P)H oxidase-1 (NOX-1) and induced apoptosis through
subsequent activation of c-jun NH2-terminal kinase (JNK) and
p38. However, we also found that JNK and p38 activation
resulted in phosphorylation of H2AX (;H2AX), a variant of
mammalian histone H2A, which contributes to the apoptosis
induced by silencing ALKBH8 and NOX-1. Silencing of ALKBH8
significantly suppressed invasion, angiogenesis, and growth of
bladder cancers in vivo as assessed both in the chorioallantoic
membrane assay and in an orthotopic mouse model using
green fluorescent protein–labeled KU7 human urothelial
carcinoma cells. Immunohistochemical examination showed
high expression of ALKBH8 and NOX-1 proteins in high-grade,
superficially and deeply invasive carcinomas (pT1 and >pT2)
as well as in carcinoma in situ, but not in low-grade and
noninvasive phenotypes (pTa). These findings indicate an
essential role for ALKBH8 in urothelial carcinoma cell survival
mediated by NOX-1–dependent ROS signals, further suggesting new therapeutic strategies in human bladder cancer by
inducing JNK/p38/;H2AX–mediated cell death by silencing of
ALKBH8. [Cancer Res 2009;69(7):OF1–8]
Introduction
In E. coli, AlkB protein is involved with repair of methylationinduced DNA damage by oxidative demethylation; in various types
of mammalian cells, AlkB is implicated in preventing mutation
and carcinogenesis (1, 2). Human homologues of the AlkB family
include ALKBH1, ALKBH2, and ALKBH3, and our laboratory has
recently identified some additional members, ALKBH4, ALKBH5,
ALKBH6, ALKBH7, and ALKBH8, and determined their expression
in human tissues (3). Immunohistochemical analysis has shown
that ALKBH8 is highly expressed in a variety of human cancers
including bladder cancer6; however, the physiologic role of ALKBH8
in cancer progression remains unclear.
Materials and Methods
Bladder cancer cell line and transfection. We purchased the human
urothelial carcinoma cell line UMUC2 from American Type Culture
Collection. KU7 was derived from human papillary bladder cancer (14).
We generated stable green fluorescent protein (GFP) clones as previously
described (15).
Chemicals, antibodies, and preparation of antisera. The c-jun NH2terminal kinase (JNK) inhibitor SP600125, JNK inhibitor cell-permeable
peptide, and the p38 inhibitor SB203580 were obtained from Calbiochem.
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Noboru Konishi, Department of Pathology, Nara Medical
University School of Medicine, Shijo-cho, Kashihara, Nara, 634-8521, Japan. Phone:
81-744-22-3051; Fax: 81-744-23-5687; E-mail: [email protected].
I2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-3530
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K. Shimada et al. unpublished data.
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Anti–poly(ADP-ribose) polymerase (PARP), anti–phosphorylated JNK,
anti–phosphorylated p38, and anti–basic fibroblast growth factor (bFGF)
antibodies were purchased from Cell Signaling; anti-actin, anti-JNK,
anti-p38, and anti–vascular endothelial growth factor (VEGF) antibodies
were purchased Santa Cruz Biotechnology, Inc. Antibodies to NOX-1,
platelet-derived growth factor (PDGF)-BB, and H2AX were supplied by
Abcam, whereas anti–phosphorylated H2AX (gH2AX) was purchased
from Upstate Biotechnology. Anti-ALKBH8 antisera were raised against
the synthetic peptide of ALKBH8 (NKQKSKYLRGNRNS) as an antigen. The
peptides (0.5 mg) were emulsified in an equal volume of Freund’s complete
adjuvant and then injected s.c. at several sites in each rabbit. Antiserum was
prepared and we evaluated the relative reactivity of the antisera against the
synthetic peptide by ELISA, and those antisera with high titers were affinity
purified using SulfoLink (Pierce Biotech).
Preparation of cell lysates and Western blotting analysis. We
resolved the cell lysates in SDS-polyacrylamide gels and transferred them
onto polyvinylidene difluoride membranes (Millipore, Ltd.), which were
then blocked in 5% skim milk at room temperature for 1 h. The membranes
were incubated with the indicated primary antibody for 1 h, and then
incubated with horseradish peroxidase–conjugated antimouse or antirabbit
IgG (Amersham Pharmacia Biotech). We detected peroxidase activity on
X-ray films using an enhanced chemiluminescence detection system.
Reverse transcription-PCR. Using the OneStep RT-PCR kit (Qiagen),
we extracted total RNA using Trizol reagent and subjected it to reverse
transcription-PCR (RT-PCR). PCR conditions were 95jC for 30 s, 55 to 60jC
for 30 s, and 72jC for 1 min through a total of 30 cycles. The PCR primer
sequences for ALKBH8 were 5¶-AGATGTACTGGTTCCCTGGC-3¶ (sense) and
5¶-CCTCACGGAACACATGGTAG-3¶(antisense). The primers for Bcl-2 were
5¶-TTGTGGCCTTCTTTGAGTTC-3¶ (sense) and 5¶-TCTTCAGAGACAGCCAGGAG-3¶ (antisense). NOX-1 primers were 5¶-CTTGCCTCCATTCTCTCCAG-3¶
(sense) and 5¶-CACTCCAGTGAGACCAGCAA-3¶ (antisense). For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the primers used were 5¶-ACCACAGTCCATGCCATCAC-3¶ (sense) and 5¶-TCCACCACCCTGTTGCTGTA-3¶
(antisense).
Measurement of H2O2 production. We assessed the production of
intracellular H2O2 with 5,6-chloromethyl-2¶,7¶-dichlorodihydrofluorescein
diacetate (CM-H2DCF-DA; Wako) using a method modified from Bae
and colleagues (16). Briefly, KU7 and UMUC2 cells were plated on
chambered slides, transfected with either control RNA or ALKBH8 siRNA,
and incubated for 60 h, or were treated with 1,500 units/mL of catalase for
30 min. Then, we incubated them with a 5 Amol/L CM-H2DCF-DA solution
for 20 min at 37jC. Excess CM-H2DCF-DA was washed, and H2O2 formation
in the cells was visualized using a Leica CTR6000 photomicroscope and
analyzed by measuring the fluorescence intensity of 50 to 100 randomly
selected cells (3).
siRNA transfection of ALKBH8, NOX-1, and H2AX. For our
transfection analyses, 106 cells from each bladder cancer cell line were
seeded in 6-cm dish plates and transfected either with 100 nmol/L of control
RNA (Santa Cruz Biotechnology) or with the siRNA of ALKBH8, NOX-1.
Transfections were carried out using the Lipofectamine system (Invitrogen) in
accordance with the manufacturer’s protocol. The ALKBH8, NOX-1 siRNA
duplexes, generated with 3¶-dTdT overhangs and prepared by Qiagen, were
chosen against the DNA target sequences as follows: 5¶-AAGCCAAAACCTTGTGGACAT-3¶ for ALKBH8; 5¶-AGCCTATCTCATGATGAGAAA-3¶, 5¶-CCACCTCTTGACAATGGGAAA-3¶, and 5¶-CCTGTTTAACTTTGACTGCTA-3¶ for NOX-1; 5¶-CCGGGACGAAGCACTTGGTAA-3¶, 5¶-CACGACTAGAACCTTAGGCAT-3¶, and 5¶-TGCCTCCTAGGAGGACATTTA-3¶ for H2AX.
Terminal deoxynucleotidyl transferase–mediated dUTP nick end
labeling assay. We detected DNA cleavage, a characteristic of apoptosis,
using the terminal deoxynucleotidyl transferase–mediated dUTP nick
end labeling (TUNEL) assay, as directed by the manufacturer (Roche
Diagnostics). Images of nuclear fluorescence typical of apoptotic cells
were collected by fluorescence microscopy as previously described,
examining at least 600 cells from three different fields in each experiment;
cell death was expressed as percentage of TUNEL-positive cells (17).
Tissue samples and immunohistochemistry. We obtained specimens
of human urinary bladder cancers diagnosed as urothelial carcinomas
Cancer Res 2009; 69: (7). April 1, 2009
(n = 68) from patients undergoing transurethral resection or radical
cystectomy, without previous radiation or chemotherapy, at Nara Medical
University Hospital. Clinicopathologic data of the present cases were
summarized in Supplementary Table S1. Informed consent was obtained
from all patients before the collection of specimens, as appropriate, and
tumor stage and grade were noted at the time of diagnosis. We followed the
same tissue fixation and processing procedure as described in a previous
report (18). Percentages of cells positive for ALKBH8 or NOX-1 were
expressed per z1,000 cells examined.
Cell cycle analysis. We performed cell cycle analyses by flow cytometry
as previously described (19); in cell cycle analysis, fragmented apoptotic
nuclei as well as necrotic cells or cell debris are recognized by their
subdiploid (sub-G1) DNA content. All experiments were done at least thrice
in duplicate.
Chorioallantoic membrane assay. Cells (106) from each cell line
transfected with pEGFP (Clontech) were seeded on the chorioallantoic
membranes (CAM) of 11-d-old chick embryos. After a 24-h cultivation,
we added control RNA or 10 Amol/L ALKBH8 siRNA combined with
atelocollagen (Atelogene, Koken Co., Ltd.) according to the manufacturer’s
protocol. Five days post-implantation, CAM samples were collected and
fixed, then stained with anti-GFP to allow the numbers of invading GFPpositive cells to be quantified in three or more randomly selected fields. The
depth of invasion from the CAM surface was defined as the leading front of
three or more invading cells in five randomly selected fields. We assessed
angiogenesis as the number of visible blood vessel branch points within a
defined area of the sample (20). At least three CAMs were used for each
experiment.
Orthotopic tumor implantation and intravesical treatment. The
method we used for the orthotopic instillation of tumor cells has been
described (15); briefly, we anesthetized 8-wk-old female athymic nude
mice and inserted a 24-gauge catheter transurethrally into the bladder.
Then, 1 107 GFP-tagged KU7 cells suspended in 100 AL medium were
instilled into the bladder lumen and the urethra was ligated for 2 to 3 h.
Fourteen days after the cell instillation, we transurethrally injected either
control RNA (n = 5) or 10 Amol/L of the ALKBH8 siRNA + atelocollagen
mixture (n = 4) previously described (Koken Co., Ltd.) into the bladder
(retain the RNA for 1 h).
In vivo fluorescence and image analyses. Twenty-eight days after the
instillation of tumor cells, digital images were captured under both
fluorescent and incandescent light. Image analysis was done with Image J
public domain software available through the NIH.7 All images were
spatially calibrated for area measurements. The mice were then sacrificed,
and the bladders were excised and fixed for histologic examination by
H&E and immunohistochemistry.
Gelatin zymography. The activity and expression of matrix metalloproteinase (MMP)-9 and MMP2 were analyzed by gelatin zymography.
Equal amounts of concentrated condition medium were mixed with sample
buffer [0.25 mol/L Tris-HCl (pH 6.8), 0.4% SDS, 40% glucerol, and
bromophenol blue] and loaded onto 7.5% SDS-PAGE containing 1 mg/mL
gelatin (Wako Pure Chemical Indusries, Ltd.). After electrophoresis, the gel
was stained with Coomassie blue solution.
Statistical analysis. Data were statistically analyzed using the Student t
test or, for nonparametric analysis, the Kruskal-Wallis test (18, 21). Results
were considered significant if P < 0.05.
Results
Silencing of ALKBH8 induced apoptosis in human urothelial
carcinoma. Two human urothelial carcinoma cell lines, KU7 and
UMUC2, were selected because these cells express ALKBH8 mRNA
and protein, exhibiting high cell growth, invasion, and angiogenesis
as shown in CAM assay. Both mRNA and protein expressions were
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ALKBH8 and Bladder Cancer
Figure 1. Down-regulation of ALKBH8 induced apoptosis in human urothelial carcinoma cells. A, KU7 and UMUC2 were transfected with 100 nmol/L of ALKBH8 siRNA
or control RNA. Expressions of both mRNA and protein of ALKBH8 or NOX-1, respectively, were examined by RT-PCR and Western blotting. B, transfected cells
were stained by propidium iodide, and flow cytometric analysis was done. C, transfected cells were stained by TUNEL (left ). Columns, mean; bars, SE. D, PARP
cleavage was examined by Western blotting. Actin and GAPDH were used as internal controls.
strongly reduced in both human urothelial carcinoma cell lines, KU7
and UMUC2, when transfected with 100 nmol/L of ALKBH8 siRNA
for up to72 hours (Fig. 1A). As can be seen in Fig. 1B and C, the
TUNEL and flow cytometric analyses showed a significant increase
in the percentage of TUNEL-positive cells with down-regulation
of ALKBH8 (KU7: 3.8% versus 35.3%; UMUC2: 2.8% versus 41.8%;
P < 0.01). In addition, the number of cells in the sub-G1 fraction was
also significantly increased by silencing ALKBH8. ALKBH8 silencing
also resulted in PARP cleavage (Fig. 1D).
JNK and p38 activation contributes to apoptosis induced
by ALKBH8 down-regulation. Because mitogen-activated protein
(MAP) kinases, including JNK and p38, are known to play
essential roles in apoptosis induced by various extrinsic stimuli
in cancer cells (22), we examined whether MAP kinase activation
contributes to cytotoxicity induced by silencing ALKBH8. As
assessed by TUNEL assay and flow cytometric analyses shown in
Fig. 2A, p38 and JNK were activated in both KU7 and UMUC2
cells in a time-dependent manner in response to transfection
with ALKBH8 siRNA, an effect significantly blocked by treatment
with specific inhibitors for JNK and p38, such as JNK inhibitory
peptide (JNKI), SP500125, and SB203580 (Fig. 2B and C).
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NOX-1–dependent ROS generation is a downstream effect of
ALKBH8 silencing. As assessed by fluorescence microscopy using
CM-H2DCF-DA, silencing ALKBH8 significantly suppressed intracellular levels of ROS in both KU7 and UMUC2 cells to a degree
almost similar to that seen with catalase treatment (Fig. 3A and
B). RT-PCR indicated that NOX-1, ubiquitously expressed in
both cell lines, was also decreased by ALKBH8 down-regulation,
and protein expression was similarly reduced (Fig. 3C). Other
isoforms of NOX gene were not significantly modified by ALKBH8
silencing (data not shown). Transfection with 100 nmol/L NOX-1
siRNA induced JNK and p38 activation and strongly suppressed
not only NOX-1 expression but also intracellular ROS levels in
KU7 and UMUC2 cells; apoptosis induced by NOX-1 silencing was
inhibited by treatments with JNK/p38–specific inhibitors (Fig. 3D;
Supplementary Fig. S1). The percentages of apoptotic cells
detected as a result of NOX-1 silencing and the degree of
suppression of apoptosis by JNK/p38 inhibitors were similar
to those resulting from silencing ALKBH8, indicating that
NOX-1–mediated signals downstream of ALKBH8 are key to
ROS generation and survival of urothelial carcinoma. JNKdependent phosphorylation of H2AX has recently been shown
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Figure 2. Both JNK and p38 were activated by ALKBH8 gene silencing. A, KU7 and UMUC2 cells were collected after transfection with ALKBH8 siRNA, and the
expression of phosphorylated p38 or JNK was examined by Western blotting. B and C, KU7 and UMUC2 cells were treated with 25 Amol/L of SP600125 (SP ), 1 Amol/L
of JNK inhibitory peptide (JNKI ), or 25 Amol/L of SB202190 (SB ) for 1 h before transfection with either control RNA (Cont. ) or ALKBH8 siRNA and subjected to
TUNEL staining. The percentages of TUNEL-positive cells and those in sub-G1 phase were determined by fluorescence microscopy and flow cytometric analysis,
respectively. Columns, mean; bars, SE.
modified by ALKBH8 silencing (Supplementary Fig. S3). The data
indicate that ALKBH8 silencing resulted in suppression of invasion
and angiogenesis mainly through apoptosis induction.
In vivo growth of urothelial carcinoma is suppressed
by ALKBH8 siRNA transfection in the orthotopic implant
mouse model. Figure 5A illustrates the urinary catheterization
procedure used in the murine orthotopic implant model. Using
a KU7 cell line stably overexpressing GFP, we have recently
established GFP image analysis in this system in which tumor
growth and treatment response can be quickly measured by
image analysis in lieu of slower histologic methods. As shown
in Fig. 5B and C, ALKBH8 down-regulation produced an f6-fold
decrease in tumor area. Conventional histologic evaluation showed
the same results: Control mice showed large tumors with cancer
cells deeply infiltrating into the bladder wall, whereas only a few
small foci of cancer cells were observed in mice administered
ALKBH8 siRNA in the presence of atelocollagen. Western blotting
further confirmed that ALKBH8 and NOX-1 expression was
strongly decreased; in contrast, phosphorylated JNK, p38, and
cleaved PARP were increased in vivo by this single injection of
ALKBH8 siRNA versus control RNA, using GFP expression as a
positive internal control (Fig. 5D). This model also proves the
efficacy of atelocollagen to successfully deliver ALKBH8 siRNA to
orthotopically implanted bladder cancer cells.
to directly affect apoptosis induction. H2AX was significantly
phosphorylated in KU7 cells when ALKBH8 and NOX-1 were
silenced, and this effect was canceled by addition of specific
inhibitors for JNK and p38 (Supplementary Fig. S2A). Moreover,
inhibition of H2AX phosphorylation by transfection with H2AX
siRNA suppressed apoptosis induced by knocking down ALKBH8
expression (Supplementary Fig. S2B). Taken together, phosphorylation of H2AX is apparently a downstream signal of JNK/p38
activation and, following silencing of the ALKBH8 and NOX-1
genes, is probably involved mainly in JNK/p38–dependent
apoptosis in human urothelial carcinoma.
Invasion and angiogenesis are suppressed by ALKBH8
down-regulation in urothelial carcinoma cells. Figure 4 shows
the significant suppression of angiogenesis and of both the
numbers of invasive cells and the depth of invasion by transfection
of ALKBH8 siRNA in KU7 and UMUC2 cells in the CAM assay.
The efficacy of this suppression was similar to the growth
inhibitory effect induced by down-regulating ALKBH8, suggesting
that ALKBH8 may enhance cancer invasion and angiogenesis
by promoting cell survival signals. To investigate this line of
reasoning, we examined several molecules involved in the
process of invasion and angiogenesis, including MMPs, PDGF,
bFGF, and VEGF; however, in our urothelial carcinoma cells, we
found that expression of these molecules was not significantly
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ALKBH8 and Bladder Cancer
in vitro and in vivo. Because ALKBH8 is a member of DNA repair
molecules, we speculated that ALKBH8 knockdown–induced
apoptosis might be mediated by DNA damage. It is well known
that JNK, p38, or gH2AX is activated in response to DNA damage and
closely associated with cell death induction. The present results
clearly showed that JNK, p38, and gH2AX were significantly activated
following ALKBH8 gene silencing. Then, we examined whether
ALKBH8 knockdown affected endogenous stress including free
radicals, resulting in apoptosis induction. Gene silencing experiments in human bladder cancer cells by means of siRNA transfection
definitively identified NOX-1 as a downstream target of ALKBH8
involved mainly in intracellular ROS generation and in resistance to
apoptosis. ROS generation induced through NOX-1 genes is known
to be essential for oncogenesis and tumor progression (23, 24).
Several other reports, however, suggest that NOX genes may regulate
signal transductions far more than ROS generation. We found that
down-regulation of NOX-1 strongly reduced generation of ROS, but
treating urothelial carcinoma cells with catalase did not induce MAP
kinase activation and apoptosis. This indicates the presence of a
ROS-dependent and a ROS-independent transduction pathway
operating in NOX-mediated cancer progression.
There have been conflicting reports about the function of ROS in
cancer cells. Overproduction of ROS contributes to cytotoxicity by
ALKBH8 and NOX-1 are expressed in human urothelial
carcinoma of the urinary bladder. We performed immunohistochemical analysis of ALKBH8 and NOX-1 in human bladder
cancer specimens to examine the correlation of expression to
tumor grade, invasiveness, and degree of angiogenesis. To ensure
the specificity of antibodies used in this study, we used Western
blotting on surgical specimens of low-grade/noninvasive, highgrade/invasive, and carcinoma in situ (CIS), all of which were
diagnosed by two urological pathologists. ALKBH8 and NOX-1
were observed as a single band, indicating that the antibodies
selected were of high specificity (Fig. 6B). The expression pattern of
these molecules was consistent with the immunohistochemical
analysis: As shown in Fig. 6A and B, the percentages of cells
immunopositive for ALKBH8 and NOX-1 were much higher in
high-grade (G3) urothelial carcinomas, including CIS, with both
minimal and wide invasion (pT1) than in low-grade (G1 and
G2) lesions with a noninvasive phenotype (pTa; Supplementary
Table S2).
Discussion
We have shown here for the first time that ALKBH8, a novel
member of the human AlkB family, plays important roles in the
survival and progression of human urothelial carcinoma cells both
Figure 3. The endogenous generation of hydrogen peroxide was suppressed by ALKBH8 siRNA transfection in urothelial carcinoma cells. A and B, KU7 and UMUC2
cells were either transfected with ALKBH8 siRNA or treated with 1,500 units/mL of catalase for 30 min before exposure to CM-H2DCF-DA. H2O2 formation was
analyzed as described in Materials and Methods. Columns, mean; bars, SE. C, after ALKBH8 siRNA transfection, cells were collected and subjected to RT-PCR and
Western blotting to detect the mRNA and protein expression of NOX-1, respectively. D, after transfection with 100 nmol/L of NOX-1 siRNA, KU7 and UMUC2 cells
were exposed to 5 Amol/L CM-H2DCF-DA. Cellular H2O2 formation was analyzed, and expression of NOX-1 was examined by Western blotting.
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Figure 4. Silencing ALKBH8 reduced angiogenesis and invasion of urothelial carcinoma cells in vivo in the CAM assay. A and B, KU7 and UMUC2 cells were
seeded on top of the CAMs of 11-d-old chicks and atelocollagen-control RNA or atelocollagen-ALKBH8 siRNA mixture was added, and then the cells were incubated
for 3 d. The macroscopic data are shown in A. Invaded cell number, invasion depth, and angiogenesis were analyzed in B. Columns, mean of at least three
experiments; bars, SE.
malignant potential (18), whereas strong transient activation
resulted in cell death, as shown in this present study. This
‘‘bipolar’’ characteristic of JNK is similar to that of p53; p53
participates in DNA repair following various mild DNA damage
yet mediates apoptosis induction with severe damage (29).
Both our in vivo and immunohistochemical analyses implicate
the ALKBH8 gene as a potential therapeutic target in human
bladder cancer. Silencing of ALKBH8 resulted in strong suppression
of cancer invasion and angiogenesis in the CAM assay. This is
probably due to induction of apoptosis because the apoptotic index
in vitro was similar to that resulting from ALKBH8 downregulation; further, the malignant potential of cancer cells was
suppressed without significant modification of the representative
molecules associated with migration and angiogenesis (Supplementary Fig. S3). Therefore, induction of cell death, particularly
apoptosis, might be a key mechanism by which ALKBH8 silencing
suppressed the malignant potential of urothelial carcinoma.
However, we do not deny any possibilities that there are other
critical mechanisms than apoptosis induction because the effects
of suppression by ALKBH8 down-regulation on invasion/angiogenesis were more prominent than on the percentages of cells
undergoing apoptosis.
In our orthotopic mouse model of implanted bladder cancer,
designed to assess the growth and response of GFP-labeled human
such extracellular stimuli as anticancer drugs, for instance, the
enhanced bystander effects on paclitaxel cytotoxicity mediated by
ROS, demonstrated by Alexandre and colleagues (25). The key point
in these discrepancies is the effect of differences in intracellular
ROS levels: Low endogenous levels of ROS generated mainly by
NOX are linked to increased aggressiveness of cancer cells, whereas
the explosive production of ROS stimulated by cytotoxic drugs
promotes cell death.
It is widely accepted that the phosphoinositide 3-kinase–related
protein kinase ataxia-telangiectasia mutated rapidly phosphorylates the COOH-terminal SQE motif of the mammalian histone
variant, H2A (H2AX), to generate ‘‘g-H2AX’’ over large chromatin
domains flanking double-strand breaks induced by DNA damage.
The resulting repair of oxidative DNA damage suppresses
genomic instability (26). Moreover, H2AX confers cellular
protection against alkylation-induced DNA damage (27). Lu and
colleagues (28) also reported that, at the sites of DNA doublestrand breaks induced by ionizing radiation, H2AX is phosphorylated by JNK and plays an important role in the induction of
apoptosis, suggesting that H2AX phosphorylation has biphasic
functions on cell growth. Our results suggest that the level of
expression (i.e., the degree of ALKBH8 regulation) determines
whether gH2AX promotes cell death or cell survival. We have
seen that mild but persistent activation of JNK promotes
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ALKBH8 and Bladder Cancer
Figure 5. Intravesical injection of ALKBH8
siRNA inhibits tumor growth in vivo in
the mouse orthotopic bladder cancer
implant model. A, the anatomic view
of a nude mouse during the urinary
catheterization procedure. B, fourteen
days after KU7/GFP cells were
transplanted into the mouse bladder,
atelocollagen with either ALKBH8 siRNA
or control RNA was transurethrally instilled
into the bladder lumen. Bladders were
resected 14 d post-instillation, and in situ
images captured under fluorescent
light (left ). Cancerous tissues (arrows )
were confirmed by corresponding H&E
histologic sections (right ). C, growth
suppression in lesions treated with
ALKBH8 siRNA/atelocollagen and control
RNA/atelocollagen was compared in
terms of tumor area measured by GFP
expression and histologic analysis.
Columns, mean; bars, SE. D, bladder
cancer samples were resected and lysed,
and expression of ALKBH8, NOX1,
phosphorylated JNK and p38, and PARP
was examined in Western blot analyses.
Expression of GFP was used as control.
bladder cancer cells to intravesical treatment, we found exciting
that a single injection of ALKBH8 siRNA, in the presence of
atelocollagen, reduced both ALKBH8 and NOX-1 protein expression and resulted in significant reduction of tumor volume. Nogawa
and colleagues also showed that intravesical injection of siRNA/
liposomes to silence PLK-1 successfully inhibited bladder cancer
growth. Intravesical injection of siRNA for target genes in the
presence of atelocollagen or liposome instillation could thus be
a useful therapeutic tool. The immunohistochemical analyses
we performed on human bladder cancer samples suggest the
contribution of signal activation to early development of urothelial
carcinoma in that increased expression of ALKBH8/NOX-1 is linked
Figure 6. The immunohistochemistry data for ALKBH8 and NOX1 in human urothelial carcinomas of the urinary bladder indicate a correlation with grade.
Immunohistochemistry in A for ALKBH8 and Nox-1 expression was done on representative samples of each group, and Western blotting in B was used to confirm
antibody specificity (pTa, n = 3; pT1, n = 1; RpT2, n = 1; CIS, n = 1). The percentage of immunopositive cells was calculated per 1,000 cells per high-power field. As can
be seen, there seems to be a positive correlation between the percentages of ALKBH8 or NOX-1 immunopositive cells and tumor grade. Each value is the
mean F SE.
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down-regulation of the gene and provide a new therapy in the
treatment of human bladder cancer (Supplementary Fig. S4).
to the change from a noninvasive to an invasive bladder cancer.
Other studies posit that immunoprofiling of ALKBH8 and NOX-1,
as well as of TP53, will provide more reliable information for
bladder cancer prognosis (30).
In summary, a novel member of the human AlkB family,
ALKBH8, plays an important role in cell survival by regulating
NOX-1–dependent signals and enhances cancer progression in
urothelial carcinoma of human bladder. Amplification of ALKBH8/
NOX-1 expression is closely associated with transformation from a
noninvasive to an invasive phenotype as determined by immunohistochemical analysis. Silencing ALKBH8 by siRNA transfection
strongly suppressed malignant potential in vitro and in vivo;
moreover, intravesical injection of ALKBH8 siRNA combined with
atelocollagen may be a good delivery system to achieve successful
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 9/18/08; revised 1/24/09; accepted 1/29/09; published OnlineFirst 3/17/09.
Grant support: Grant-in-Aid from the Ministry of Education, Culture, Sports,
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Research.
Published OnlineFirst March 17, 2009; DOI: 10.1158/0008-5472.CAN-08-3530
A Novel Human AlkB Homologue, ALKBH8, Contributes to
Human Bladder Cancer Progression
Keiji Shimada, Mitsutoshi Nakamura, Satoshi Anai, et al.
Cancer Res Published OnlineFirst March 17, 2009.
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