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A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces cell autophagy and apoptosis in esophageal squamous cell carcinoma cells via up-regulation of BNIP3 and N-myc downstream-regulated gene-1.
[esophageal squamous cell carcinoma]
The
protocatechuic
acid
ethyl
ester
ethyl-
3
,
4
-
dihydroxybenzoate
is
an
antioxidant
found
in
the
testa
of
peanut
seeds
.
Previous
studies
have
shown
that
ethyl-
3
,
4
-
dihydroxybenzoate
can
effectively
reduce
breast
cancer
cell
metastasis
by
inhibiting
prolyl-hydroxylase
.
In
this
study
,
we
investigated
the
cytotoxic
effect
of
ethyl-
3
,
4
-
dihydroxybenzoate
on
esophageal
squamous
cell
carcinoma
cells
in
vitro
and
identified
key
regulators
of
ethyl-
3
,
4
-
dihydroxybenzoate-induced
esophageal
cancer
cell
death
through
transcription
expression
profiling
.
Using
flow
cytometry
analysis
,
we
found
that
ethyl-
3
,
4
-
dihydroxybenzoate
induced
S
phase
accumulation
,
a
loss
in
mitochondrial
membrane
permeabilization
,
and
caspase-dependent
apoptosis
.
Moreover
,
an
expression
profile
analysis
identified
46
up-
and
9
down-regulated
genes
in
esophageal
cancer
KYSE
170
cells
treated
with
ethyl-
3
,
4
-
dihydroxybenzoate
.
These
differentially
expressed
genes
are
involved
in
several
signaling
pathways
associated
with
cell
cycle
regulation
and
cellular
metabolism
.
Consistent
with
the
expression
profile
results
,
the
transcriptional
and
protein
expression
levels
of
candidate
genes
NDRG
1
,
BNIP
3
,
AKR
1
C
1
,
CCNG
2
and
VEGFA
were
found
to
be
significantly
increased
in
treated
KYSE
170
cells
by
reverse-transcription
PCR
and
western
blot
analysis
.
We
also
found
that
protein
levels
of
hypoxia-inducible
factor
-
1
α
,
BNIP
3
,
Beclin
and
NDRG
1
were
increased
and
that
enriched
expression
of
BNIP
3
and
Beclin
caused
autophagy
mediated
by
microtubule-associated
protein
1
light
chain
3
in
the
treated
cells
.
Autophagy
and
apoptosis
were
activated
together
in
esophageal
cancer
cells
after
exposed
to
ethyl-
3
,
4
-
dihydroxybenzoate
.
Furthermore
,
knock-down
of
NDRG
1
expression
by
siRNA
significantly
attenuated
apoptosis
in
the
cancer
cells
,
implying
that
NDRG
1
may
be
required
for
ethyl-
3
,
4
-
dihydroxybenzoate-induced
apoptosis
.
Together
,
these
results
suggest
that
the
cytotoxic
effects
of
ethyl-
3
,
4
-
dihydroxybenzoate
were
mediated
by
the
up-regulation
of
NDRG
1
,
BNIP
3
,
Beclin
and
hypoxia-inducible
factor
-
1
α
,
initiating
BNIP
3
and
Beclin
mediated
autophagy
at
an
early
stage
and
ultimately
resulting
in
esophageal
cancer
cell
apoptosis
.
Diseases
Validation
Diseases presenting
"and caspase-dependent apoptosis"
symptom
esophageal squamous cell carcinoma
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