Abstract. Aberrant promoter hypermethylation (methylation)
is an epigenetic change that silences the expression of crucial
genes, thus inactivating the apoptotic pathway in various
cancers. Inactivation of the apoptotic pathway has been
considered to be associated with chemoresistance. The
objective of the present study was to clarify the effect of the
methylation of the apoptosis-related genes, Bcl-2/adenovirus
E1B 19 kDa-interacting protein 3 (BNIP3) and death-
associated protein kinase (DAPK), on the response to chemo-
therapy in metastatic or recurrent gastric cancers. Tumor
samples were obtained from 80 gastric cancer patients who
were treated with fluoropyrimidine-based chemotherapy for
distant metastatic or recurrent disease, after surgical resection
of the primary tumor. The methylation status of the apop-
tosis-related genes, BNIP3 and DAPK, was investigated by
methylation-specific PCR. Methylation in BNIP3 was
detected in 31 tumors (39%) and in DAPK in 33 tumors (41%).
There was no correlation between the methylation status of
BNIP3 and that of DAPK. The response rate was significantly
lower in patients with methylation of DAPK, than in those
without (21 vs. 49% p=0.012). Progression-free survival time
(PFS) was shorter in patients with methylation of DAPK than
in those without (p=0.007). The overall survival time (OS)
was shorter in patients with methylation of BNIP3 than in
those without (p=0.031). The response rate was significantly
lower in patients with methylation of either DAPK or BNIP3,
or both, than in those without methylation (p=0.003). PFS
and OS were significantly shorter in patients with methylation
of either or both of these genes than in those without (p=0.002,
p=0.001). The methylation of BNIP3 and DAPK can predict
lower response to chemotherapy and poor prognosis in gastric
Gastric cancer is the fourth most common cancer and the
second most frequent cause of cancer-related death world-
wide (1,2). Surgical treatment is the mainstay of therapy for
gastric cancer, although chemotherapy is required for
unresectable advanced disease (3). The survival time of
patients with distant metastases or recurrence has been
prolonged by the introduction of new regimens including oral
fluoropyrimi-dines, oxaliplatin, taxans and irinotecan (4,5).
However, the effectiveness of chemotherapy is insufficient for
gastric cancer patients. It would be useful to be able to select
patients whose tumor would be sensitive to chemotherapy.
However, there is no reliable marker that can predict
response to chemotherapy for gastric cancer.
Most chemotherapeutic agents, such as fluoropyrimidines
and irinotecan, damage DNA and induce apoptosis. The
inactivation of the apoptotic pathway has been considered to
be associated with chemoresistance (6,7). The dysfunction of
apoptosis-related genes could decrease apoptosis induced by
chemotherapy. The methylation of many apoptosis-related
genes, including Bcl-2/adenovirus E1B 19 kDa-interacting
protein 3 (BNIP3) and death-associated protein kinase
(DAPK), has been reported in various cancers. DNA methyl-
ation plays a key role in carcinogenesis, as well as in genome
mutation or deletion. Methylation is associated with the
transcriptional silencing of selected genes and affects cell
growth and differentiation in gastric cancer (8-11).
In the present study, the methylation of the apoptosis-
related genes, BNIP3 and DAPK, was examined in patients
who were treated with fluoropyrimidine-based chemotherapy
for metastatic or recurrent gastric cancer, in order to analyze
the association between the methylation of apoptosis-related
genes and the response to chemotherapy.
Materials and methods
A total of 80 patients who had undergone surgical resection
for primary gastric cancer at the Department of Surgical
ONCOLOGY REPORTS 25: 513-518, 2011
Methylation of BNIP3 and DAPK indicates lower response
to chemotherapy and poor prognosis in gastric cancer
HIROFUMI SUGITA1, SATORU IIDA1, MIKITO INOKUCHI1, KEIJI KATO1, MEGUMI ISHIGURO1,
TOSHIAKI ISHIKAWA2, YOKO TAKAGI2, MEGUMU ENJOJI1, HIROYUKI YAMADA1,
HIROYUKI UETAKE2, KAZUYUKI KOJIMA1and KENICHI SUGIHARA1
Departments of 1Surgical Oncology and 2Translational Oncology, Graduate School,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Received August 16, 2010; Accepted September 30, 2010
Correspondence to: Dr Hirofumi Sugita, Department of Surgical
Oncology, Graduate School, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Key words: Bcl-2/adenovirus E1B 19 kDa-interacting protein 3,
death-associated protein kinase, methylation, gastric cancer, chemo-
Oncology, Tokyo Medical and Dental University (Tokyo,
Japan), were included in the present study. Forty-four patients
had recurrent disease after radical resection, and 36 underwent
non-curative resection due to distant metastases. All patients
received oral or intravenous fluoropyrimidine-based chemo-
therapy. No patient received any other adjuvant therapy.
Overall, 41, 18, 15 and 6 patients were treated with S-1 +
irinotecan, S-1 + docetaxel, 5-fluorouracil (5-FU) + cisplatin,
and 5-FU + other agents, respectively.
All resected specimens were fixed in 10% pH-neutral
formalin and embedded in paraffin. In all cases, archival
H&E slides of the gastric tumors were retrieved and reviewed
in order to confirm the pathological features. Histological
findings and tumor staging were classified according to the
tumor-node-metastasis (TNM) classification advocated by
the International Union against Cancer. Response to treatment
was evaluated according to the Response Evaluation Criteria
in Solid Tumors. The resected gastric cancer samples and
clinical data were collected after obtaining appropriate
institutional review board approval and written informed
consent from all the patients.
DNA extraction and methylation-specific PCR. Tissue was
cut into 10-μm-thick sections from paraffin-embedded blocks.
The specimens were deparaffinized and washed, and then
using the H&E slide as a guide, tumor tissue was manually
dissected. As much as possible, equal amounts of tissue were
dissected for each case. Genomic DNA was extracted by
standard proteinase K (Invitrogen, Carlsbad, CA, USA)
digestion, phenol/chloroform extraction and ethanol precipi-
tation, as previously described (12). Bisulfite treatment was
performed using a CpGenome DNA modification kit (Oncor,
Gaithersburg, MD, USA) according to the manufacturer's
instructions. Treatment of genomic DNA with sodium bisulfite
converts unmethylated cytosine (but not methylated cytosine)
into uracil, which is then converted to thymidine during the
subsequent PCR step, thus resulting in sequence differences
between methylated and unmethylated DNA. The methylation
status of the BNIP3 and DAPK genes was determined by
methylation-specific PCR (13). The following primer
sequences were designed for the BNIP3 and DAPK genes:
SUGITA et al: BNIP3 AND DAPK METHYLATION IN GASTRIC CANCER
Table I. Clinicopathological features associated with BNIP3
and DAPK methylation.
Met, methylation; Unm, unmethylation; NS, not significant.
Figure 1. Response rate to chemotherapy with respect to methylation status. (A) The methylation of BNIP3 led to a lower response rate, although the
differences were not significant. (B) The methylation of DAPK is significantly associated with lower response rate, as opposed to unmethylation (p=0.012).
M, methylation; U, unmethylation; NS, not significant.
Methylated BNIP3 forward PCR primer, 5'-ATTCGTTTC
GCGTACGAGTC-3' and reverse, 5-GCGTCGCCCAT
TAACCGCGA-3'; unmethylated BNIP3 forward PCR
primer, 5'-ATTTGTTTTGTGTATGTGTTGTA-3' and
reverse, 5'-ACATCACCCATTAACCACAA-3'; methylated
DAPK forward PCR primer, 5'-GGATAGTCGGATCGAGT
TAACGTC-3' and reverse, 5'-CCCTCCCAAACGCCGA-3';
unmethylated DAPK forward PCR primer, 5'-GGAGGATA
GTTGGATTGAGTTAATGTT-3' and reverse, 5'-CA
AATCCCTCCCAAACACCAA-3'. PCR was performed in
25-μl reaction mixtures as follows: 95˚C for 2 min, followed
by 30 cycles of 95˚C for 1 min, 6˚C for 1 min, 72˚C for 1 min,
and a final 10-min extension at 72˚C. The PCR products
were loaded onto a 2% agarose gel, stained with 0.5 μg/ml
ethidium bromide, and visualized under ultraviolet illumi-
Statistical analysis. The correlations between BNIP3 or
DAPK methylation status and clinicopathological features
were statistically analyzed using the t-test (for age), Chi-
square test (for gender and histological type), and the Mann-
Whitney U test (for tumor depth, lymph node metastases and
stage). Anti-cancer drug receptivity according to methylation
status was analyzed using the Chi-square test.
Overall survival time (OS) was defined as the period
between the start of chemotherapy and the time of death.
Progression-free survival time (PFS) was calculated from the
first day of chemotherapy to the date on which progression of
disease was first observed. OS and PFS were assessed using
the Kaplan-Meier method, and differences between survival
curves were analyzed using the log-rank test. Values of
p<0.05 were considered to be significant. All analyses were
performed using the statistical software Dr. SPSS2 (SPSS
Japan Inc., an IBM company).
DNA methylation status of BNIP3 and DAPK in gastric
cancer. Methylation was detected in BNIP3 in 31 tumors
(39%) and in DAPK in 33 tumors (41%). There was no
correlation between the methylation status of BNIP3 and that
of DAPK. There was no relationship between the methylation
status of the two genes and clinicopathological findings,
including age, gender, histological type, tumor depth, lymph
node metastases and stage (Table I).
Correlation between response to chemotherapy and BNIP3
and DAPK methylation status. Of the 80 patients who
underwent fluoropyrimidine-based chemotherapy treatment,
30 patients responded (complete response and partial response)
and 50 did not (stable disease and progressive disease).
The response rate was lower in patients with methylation
than in those without. DAPK methylation was significantly
associated with the response rate to chemotherapy (BNIP3,
29 vs. 43%, p=0.210; DAPK, 21 vs. 49%, p=0.012, Fig. 1A
and B). PFS was significantly shorter in patients with DAPK
methylation than in those without (p=0.007, Fig. 2B). BNIP3
methylation was not related to PFS (Fig. 2A). OS was
significantly shorter in patients with BNIP3 methylation than
in those without (p=0.031, Fig. 3A). No significant correlation
was observed between OS and the DAPK methylation status
The response rate was significantly lower in patients with
methylation of either BNIP3 or DAPK, or both, compared to
that in patients with no methylation (p=0.003, Fig. 4). PFS
and OS were significantly shorter in patients with methylation
of either BNIP3 and DAPK, or both, than in patients without
methylation (p=0.002 and p=0.001, respectively, Fig. 5A
and B). The median survival was 15.8 months with methyl-
ation of either BNIP3 and DAPK, or both, and 6.9 months
without methylation. PFS and OS were not associated with
any other clinicopathological factors.
In the present study, the methylation of the apoptosis-related
genes, BNIP3 and DAPK, was a good genetic marker of
chemoresistance in gastric cancer. The results also demon-
strate that apoptosis-related genes play a crucial role in
response to chemotherapy in gastric cancer.
In gastric cancer, carcinogenesis and progression involve
genetic alterations of tumor-related genes, such as p53 and
PTEN. The mutation of p53 is one of the most prevalent
genetic alterations and is mainly associated with tumor
progression (14). The methylation of tumor-suppressor genes
also plays a crucial role in carcinogenesis and progression.
ONCOLOGY REPORTS 25: 513-518, 2011
Figure 2. PFS with respect to methylation status. (A) BNIP3 methylation was not related to PFS. (B) PFS of patients with methylation of DAPK was
significantly shorter than that of patients without methylation (p=0.007). NS, not significant.
The methylation of E-cadherin has been observed in >50%
of early stage undifferentiated gastric cancers and has also
been observed in surrounding non-cancerous epithelial cells
(15,16). E-cadherin methylation could thus play a major role
in the development of undifferentiated cancer of the stomach
(17). Furthermore, methylation could provide useful infor-
mation regarding prognosis. Yu et al reported that patients
with methylation of Dkk-3, a Wnt/b-Catenin signaling
antagonist, had a significantly shorter survival than patients
with no methylation, in a study of primary gastric cancers
(18). There have been many studies on the relationship
between the methylation of tumor-suppressor genes and the
response to chemotherapy in gastric cancer. Satoh et al
reported that CHFR gene expression was frequently silenced
by methylation, and that gastric cancer cells not expressing
CHFR, lacked a mitotic checkpoint and were highly
susceptible to microtubule inhibitors, such as taxans (9).
Napieralski et al reported the relationship between neo-
adjuvant chemotherapy and the methylation of six genes
(MGMT, LOX, p16, 14-3-3σ, E-cadherin, HPP1) in 54
gastric cancer patients. A trend toward shorter survival and
lower response rate was observed in patients with two or
more methylated genes than in those with no methylation, or
the methylation of one gene (19). Our previous study also
showed that the methylation of DAPK and TMS1 correlated
SUGITA et al: BNIP3 AND DAPK METHYLATION IN GASTRIC CANCER
Figure 5. Analysis of two genes together for PFS and OS. PFS (A) and OS (B) of patients with methylation of either, or both genes were significantly shorter
than those of patients with no methylation (p=0.002, p=0.001).
Figure 4. Analysis of two genes (BNIP3 and DAPK) together with respect to
response rate. The response rate with methylation of either, or both of the
two genes, was significantly lower than that with no methylation (p=0.003).
Figure 3. OS with respect to methylation status. (A) OS of patients with methylation of BNIP3 was shorter than that of patients without methylation
(p=0.031). (B) DAPK methylation was not related to OS. NS, not significant.
with lower response rate and shorter OS in 43 gastric cancer
patients treated with fluoropyrimidine-based therapy (20).
There have been many studies on demethylating agents in
various cancers (21,22). The combination of demethylating
agents and DNA-damaging agents, such as fluoropyrimidine
and irinotecan, has been shown to produce marked suppression
in tumor growth and good prognosis (7,23). From this
perspective, further evidence is required regarding the
association of the methylation of tumor-suppressor genes
with response to chemotherapy and prognosis in gastric
Apoptosis is the process of programmed cell death that
occurs in multicellular organisms, and it is important for the
cellular response to genotoxic drug-induced damage (24).
The major actions of chemotherapeutic drugs are exerted via
the activation of apoptosis (25). In cancer cells, the apoptotic
pathway has been disrupted at several points during the
carcinogenesis process (25,26). Considerable evidence has
accrued regarding the effect of genetic and epigenetic
alterations of these pathways on drug sensitivity.
BNIP3 is a pro-apoptotic member of the Bcl-2 family
(27). The expression of BNIP3 is induced by hypoxia, such
as that which occurs during cardiac ischemia and in the
hypoxic regions of tumors, and it acts against pro-survival
proteins, including Bcl-2 and Bcl-xl (28-31). The methylation
of BNIP3 has been reported in many cancers, such as
colorectal, breast and pancreatic cancers (32). Murai et al
also reported that the aberrant hypermethylation of BNIP3
was detected in 66% of primary colorectal and in 49% of
primary gastric cancers, but not in normal tissue. Further-
more, the BNIP3 methylation correlated with the down-
regulation of its expression (33). Akada et al reported that the
chemoresistance to gemcitabine was associated with the
decreased expression of BNIP3 in pancreatic cancer. The
reduced expression of BNIP3 increased resistance to
gemcitabine and 5-FU and significantly decreased patient
survival in pancreatic cancer (34). Our study also shows that
gastric cancer patients with BNIP3 methylation have lower
chemosensitivity and shorter prognosis compared to patients
DAPK, a Ca2+/calmodulin-dependent enzyme with
serine/threonine kinase activity, has been defined as a tumor
suppressor and has been shown to be part of a p53-dependent
apoptotic pathway that operates via p19ARF(35,36). DAPK
functions as a regulator of apoptosis and induces programmed
cell death. It has been demonstrated that the loss of DAPK
expression is caused by the methylation of its promoter in
many cancers (37). The methylation of DAPK has been
reported to be associated with poor disease-specific survival
rates in patients with stage I non-small cell lung carcinoma
(38). The study by Fischer et al showed that when combining
DAPK, RARß and RASSF1A methylation, patients with
double or triple methylations had a worse prognosis than the
group with only one or no methylated gene in malignant
In our study, the frequency of methylation of the two
apoptosis-related genes, BNIP3 and DAPK, was 39 and 42%,
respectively. The methylation status of BNIP3 or DAPK was
not associated with the clinicopathological features in gastric
cancer. However, DAPK was significantly associated with
the response rate and PFS, and BNIP3 was significantly
associated with OS. On the analysis of the two genes together,
patients with methylation of either gene had a lower response
to chemotherapy and poorer prognosis than patients with no
methylation. The combined impact of BNIP3 and DAPK is
greater than that of an individual gene alone in all factors.
These results suggest that the inactivation of the apoptotic
pathway caused by the methylation of apoptosis-related
genes interferes with the response to chemotherapy. More-
over, the chemoresistance produced by the methylation of
apoptosis-related genes, causes poor prognosis in gastric
Thus, the present study shows that the methylation of the
apoptosis-related genes causes resistance to chemotherapy
and worsens prognosis in patients with metastatic or recurrent
gastric cancer. The methylation of BNIP3 or DAPK can
predict chemosensitivity and prognosis in gastric cancer.
We thank Ms. Aiko Saito for her exceptional technical
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