Effect of three Small Moleculer chemotherapeutic agents on the

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23 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

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Effect of
three Small M
olecule
r

chemotherapeutic agents
o
n

t
he
Expression
of Bloom H
elicase

in
A549
lung cancer
cells
1

Huihui MENG
1,
2
,

Heng LUO
1,
2
, Hou
-
qiang XU
1,
2
*
, Kun LI
1,2
,Jinhe LIU
1,
2

1
.
Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of
Education, College of animal science, Guizhou University, Guiyang, 550025, China; 2
.
Guizhou Key Laboratory of
Animal Genetics, Breeding and Re
production, College of animal science, Guizhou University, Guiyang 550025,
China


Email: MHH0118@163.com

Abstract:
Bloom

(BLM) helicase
plays an
important

role

in the processes of DNA metabolism
including DNA replication, repair, transcription and recombination
, which indicated that BLM
helicase may be as a potent target of anticancer.
The study was aim to investigating the
anti
-
proliferation activity of

adriamy
cin

(ADM), Dihydromyricetin

(DMY) and Oleanolic Acid

(OA)

to
A549 cells and to
analyze

the effect of ADM, DMY and OA on the expression extent of
B
LM
helicase

at protein and mRNA level
, which will provide the relate material for studying the
efficiency of D
NA helicase as a target of anticancer
.
The

human lung cancer A549 cells were
treated by
ADM
,
DMY

and
OA
;
the
proliferation and survival
of cells was measured by MTT
,
Hoechst/PI and
DNA agarose gel electrophoresis
;

the expression of B
LM

helicase

w
as detected at
mRNA and protein

level by quantitative RT
-
PCR and
immunocytochemistry
.
The results
indicated that the proliferation of A549 cells
was

inhibited by ADM, DMY and OA remarkably.

IC
50

of 72hours were
0.34μg/mL

13.16μg/mL

80.45μg/mL,
respectively.

Morphological changes
were observed through fluorescence microscopy

after Hoechst/PI.
The typical DNA ladder on
agarose gel electrophoresis for analysis of cellular apoptosis appeared significantly. The results of
quantitative RT
-
PCR and immu
nocytochemistry revealed that the expression of Bloom
helicase at

mRNA and protein level was inhibited by ADM, DMY and OA significantly
.

Conclusion:

T
he
proliferation of A549 cells
was
inhibit
ed
effectively

by

ADM
,
DMY

and
OA
;
the
expression of
B
LM

helicase

was
down
-
regulated
at
transcription

and

translation

level, and the
apoptosis of
the
cell may be related to t
he expression of B
LM

helicase
.

Key words:

C
hemotherapeutic agents;

Bloom helicase;

A
nti
-
proliferation
;

E
xpression




1
*
Supported by National Basic Research Program of China (No.2010CB534912), the Doctoral Program of the
Ministry of Education
(No.200806570003), the Governor of Guizhou Province Talents Fund (No.200822), and
International Cooperative Projects of Guizhou Province (No. qian ke he wai G zi [2011]7008).



I
NTRODUCTION

As an
important member of highly conserved DNA helicases from bacteria to humans

[
1
]
,
RecQ

helicase plays a significant role in cellular processes
in

genetic recombination, transcription,
DNA replication, DNA repair

and

maintenance of telomere stability

[
2
-
5
]
.
The
reports
suggested

that RecQ helicase can restrain altered recombination

and maintain the stability of genome
through stopping the synthesis of non
-
exactly
-
matching DNA double stran
d
s
[6
-
7]
.
Genomic
instability is a characteristic of most cancers. In he
reditary
and
sporadic (non
-
hereditary) cancers,

genomic instability results from mutations in DNA repair genes and drives cancer

development.
RecQ helicase gene a
s
the important DNA repair gene
, the
change
of
biological function of
RecQ
helicase may leads
to genome unstability

that involvs in carcinogenic.
There are five RecQ
helicase
s
including
BLM, WRN, RecQ1, RecQ4 and RecQ5
, and
BLM gene defects can result in
Bloom syndrome (
cancer predisposition
)
[8
-
9]
. Bloom syndrome patients are predisposed to
various
cancers such as lung cancer,

colon cancer, breast cancer, leukemia,
lymphoma
,
sarcoma
,
unusual pediatric oncology

[10]
.

Lung cancer is one of the most common malignancies

[11]
,

the
morbidity

and

mortality

of
it
are

higher
in recent years. At present, chemotherapy is the main
measure of
treatment
to lung

cancer
by

contro
l
ing

the
proliferation of
cancer cells through blocking DNA replication or cell
division

[12]
. However, the resistance of many drugs

becomes more and more serious
[13]
,
which
make us
develop more available biological measures to improve the therapeutic of cancer. R
ecQ

family
helicase
s

take part in many cellular processes and play
the
key role in the stability of
genome, the defects of
their
can cause human disease

predispose
d

to cancer
, suggesting that
RecQ
helicase
may be as

a potential target
of anti
cancer
drugs

[14
-
15]
.

ADM is a common broad spectrum
antineoplastic

drug in the clinic and can cause
tumour

cell

apoptosis through inducing DNA damage and disturbing DNA met
abolism processes [16]. It’s
reported that ADM can intercalated the intramolecular G4 structure to restrain
telomere
’s
extension [17
-
18]. DMY as the drug of anti
-
cancer, antioxidation and regulating blood sugar, the
study sh
owed that it may restrain
telomerase

activity

through stabilizing G4 structure [19]. OA
served as natural inhibitors of human DNA ligase I that can affect the activity of ligase through
allosteric effects. There are the activities of anti
-
cancer, anti
-
mutation and inhibiting canceration
in the experi
ment of pharmacology

of OA. In addition, OA restrains DNA synthesis and blocks
tumor cells to S period [20].
ADM, DMY and OA can all suppress DNA synthesis in cells.
However, BLM helicase is the

essential for the replication of DNA, and its function defect
s can
prevent the DNA synthesis and further affect the proliferation of cells.

At present,
little is known about the expression of BLM helicase at transcription and
translation levels in cancers cells treated with the anticancer drugs.

In the study,
the
p
roliferation
and survival rates, and the
expression extent of
BLM
helicase

at protein and mRNA level

were
assayed by different methods in

human
lung

cancer

A549

cells

subjected with

ADM, DMY and
OA
.
The

results would be
provide
d

a
new
insight
for
determining of the mechanism of DNA
helicase as target of
anti
-
cancer drug
s

[12]
.

R
ESULTS

Effect of
ADM, DMY and OA

on the viability of A549 cells

Effect of ADM, DMY and
OA on A549 cells for 72h
, the p
ercentage
s

of cell survival was
significantly decreased compared with the
control

group

(
P

< 0.01
). The result indicated that the
s
mall molecule
chemotherapeutic agents

have inhibitory effect on
proliferation of lung cancer cell
and the inhibition was obvious dose
-
dependent (figure 1). The IC
50

value were 0.34μg/mL

13.16μg/mL

80.45μg/mL, respectively. ADM, DMY and OA with final concentration (0.3μg/ mL,
15μg/mL and 90μg/mL, respectively) were used

to treat lung cancer cell
s for different time (24h,
48h
and 72h). The result showed that the

s
mall molecule
chemotherapeutic agents

have inhibitory
effect on lung cancer cell proliferation, and the inhibition was obvious time
-
dependent (figure 2).

Compari
ng of the IC
50

values for A549 cells exposured to
s
mall molecule
chemotherapeutic
agents

for 48 h
,
t
he inhibiting effect of

s
mall molecule
chemotherapeutic agents
: ADM > OA >
DMY (Table
2
).
More
o
ver,
cell morphological change
s

were more clearly observed by
fluorescence microscope after the treatments of
ADM, DMY and
OA

(
figure 3
).

According to the
above data, ADM, DMY, OA with selected concentrations (0.3μg/mL, 15μg/mL, 90μg/mL,
respectively) were used to treat A549 cells for 48
h.







Figure 1.

Effect

of
small molecule chemotherapeutic agents

on the viability

in
A549 cells
.
Effect of ADM,

DMY and OA on A549 cells for 72h,

respectively

,

The
higher of the concentration is,
the better of the inhibitory
effect. The
inhibitory effect of

s
mall molecule
chemotherapeutic agents

has in dose
-
dependence.


Figure
2. Time
-
dependent effect of three
s
mall molecule chemotherapeutic agents

i
n A549 cells
.
The IC
50

values of ADM,

DMY and
OA on A549 cells
for 24 h, 48
h, 72 h,
respectively
,

the proliferation of cells was
inhibited
in a

time
-
dependence manner.

Cell viability was determined by MTT assay.


Small molecule chemotherapeutic agents IC
50
(μg/mL)

† † † † † † † †
A a M ㌴ ⸰ 㞱 〮 ㄲ ㌪ ⨠

† † † † † † † †
a M v 㐶 ⸵ ㎱ 〮 〹 㠪 ⨠

† † † † † † † †
l A 㐰 ⸰ 㦱 〮 ㈷ 㤪⨠

Ta b l e
2
.

T h e I C
5 0

v a l u e s f o r A5 4 9 c e l l s

e x p o s u r e t
o

t h r e e
s ma l l mo l e c u l e c h e mo t h e r a p e u t i c a g e n t s
.
Comparing of the IC
50

values for

A549 cells exposure to three small molecule

chemotherapeutic agents

for 48 h.
the optical density was measured at 49
0 nm, v
alues are expressed

as mean ± SD of three independent experiments.
**
P

< 0.01, *
P

< 0.05 compared with the control
(
untreated
).


Figure 3.
M
orphological

characteristic

of a
poptotic

of
A549 cells
.

U
nder

the

inverted microscope (10
×
20)

A549 cells we
re spindle
-
shaped or
polygonal, A
fter the treatments of

ADM,

DMY and
OA
,
cells

became elongated,
and shrunken
.


Effect of ADM, DMY and OA on the apoptosis of A549 cells

Hoechst 33342 / PI staining

When cell apoptosis occurs, chromatin pyknosis will appear. Hoechst 33342 can pe
netrate
the cell membrane, after dyed fluorescent of apoptotic cells were stronger than normal cells.
Propyl iodide organism (PI) can not penetrate the cell membrane and normal cells and apoptosis
cells with complete cell membrane can not be dyed. However,

propyl iodide organism (PI) can
stain dead cells which lost membrane integrity. After double staining, normal cells were detected
weak red and blue fluorescent under fluorescent microscope and apoptotic cells took on weak red
fluorescence and strong blue
fluorescence. Besides, dead cells were strong red fluorescence and
strong blue fluorescence. The results showed that the numbers of the living cells of the drugs
treatment were less than that of control, significantly.


Figure 4
.
Hoechst 33342
/
PI
staining of A549 cells
.

Fluorescence photomicrographs of cells stained with Hoechst
33342/PI at (40×) magnification.

Cells treated with
0.3μg/mL ADM, 15μg/mL DMY, and 90μg/mL OA,
for 48 h,
respectively, Control (untreated) A549 cells showing
weak red + blu
e fluorescent, apoptotic cells took on weak red
fluorescence + strong blue fluorescence.
necrotic cells
were strong red fluorescence + strong blue fluorescence.

DNA ladder assay

DNA strands of

apoptotic ce
lls were cut off between nucleosome by nuclease whose
activation dependent on
Mg
2+
,
Ca
2+
. About 180
-
200 bp DNA fragment or its multiples formed and
DNA
could

form "Ladder" in gel electrophoresis [22]. However, DNA of normal cells was a
whole band close to
the well in gel electrophoresis.

A
s shown in Figure 5, following 48 h
exposure, all of small molecule compounds used significantly increased the apoptotic DNA
fragmentation in the extracted DNA. Thus, these results demonstrate that the small
m
oleculer
chem
otherapeutic agents in
duced

fragmentation of chromosomal DNA leading to apoptosis.


Figure 5
.
DNA

ladder of A549 cells.
A549
cells were treated with 0.3μg/mL ADM, 15μg/mL

DMY

and
90μg/mL OA, f
or 48 h,

respectively.
1
-
3: ADM, DMY, OA.

Effect of
ADM, DMY
and OA on the expression extent of B
LM

helicase at
mRNA level in A549 cells

After
treated

by
ADM, DMY and OA

for 48 h,

the expression of Bloom helicase at mRNA
level in A549 cells significantly decreased with the relative expression was 0.846745±0.032,
0.90125±0.024, and 0.806642±0.017, respectively

(
2
-
△△
Ct

represents the relative
expression of
genes in the PCR
,
△△
Ct

= me
dication

Ct

untreated

Ct)
.
As shown in
Figure

6
, there was
an extremely remarkable difference (
P
< 0.01) in the percents of the expression of BLM helicase
by quantitative analysis (Figure 6C).

The result showed that

ADM, DMY and OA

inhibited

the
mRNA expression of B
LM

helicase
in A549 cells and
inhibited extent of the expression of BLM

helicase was the most obvious under the treatment of ADM.







Figure 6.
E
ffect of ADM, DMY and OA
on the mRNA expression of BLM helicase in A549 cells.
(A)

The total
RNA was extracted and assayed by1% (w/v) agarose gel electrophoresis. 1
-
4: control group, ADM, DMY and OA.
Respectively, There were three bands of RNA in every sample and were 28 S, 18 S a
nd 5S, suggesting that the
quality of extracted RNA could be used for
Quantitative

RT
-
PCR.

(B)

Quantitative

RT
-
PCR results showed that the
melt curves had a unique peaks which indicated that the primers had the good specificity that were appropriate for
am
plification of BLM gene. (C)The mRNA expression of B
LM

helicase in A549 cells by ADM, DMY and OA (2
-
△△
Ct represents the relative expression of genes in the PCR results). (D)PCR amplification products were
assayed by1% (w/v) agarose gel electrophoresis.

Eff
ect of
ADM, DMY and OA on the expression extent of B
LM

helicase at
protein

level in A549 cells

Immunocyto
chemistry

start
s

with

the combination of

enzyme labeled antibody and cell,

and
then add the enzyme substrate to
generate

colored insoluble product or

particles with a range of
electron density
. Finally,
positioning research

on the cell surface and intracell
ular antigen
composition is carried out

through

light microscopy or electron microscopy.

The

protein

expression

of
B
LM

helicase

in

A549

cells is
inside the cell nucleus

and the appearance of
brownish
-
yellow granules
in nuclei

after
DAB staining

is taken

as positive.
A
fter

treated by
ADM,

DMY

and
OA

for 48h, the
relative proteins expression
of
B
LM

helicase in lung

cancer

A549 cells

are

11±0.56
,
17±1
.02

and
13±0.88
, respectively
. Besides, the
relative proteins

expression of

positive control
is
23±0.88
.
The sequences of inhibited extent of the expression of BLM helicase
in the treatment of

three small molecule compounds
: ADM > OA >DMY .
The

p
rotein
expression

of BLM

helicase in A549

cells decreased significantly (
P

< 0.01
).




Figure 7
.
Effect of
ADM, DMY and OA

on
the protein expression of BLM helicase in A549 cells
.
(A)

A549

Cells

(with

different

treatments

for

48

h)

observed

u
nder

optical

microscopy

(10×40)

,Nucleuses

in

A54
9

Ce
lls

were

not

dyed

in

negative

treatment

group.

However,

the

nucleuses

were

all

dyed

in

the

positive

treatm
-
ent

gro
up

and

three

medication

groups.

(B)
Values are expressed as mean ± SD of three independen
t experiments. **
P

<
0.01, *
P

< 0.05 compared with the control
(
untreated
).

M
ATERIALS AND METHODS

Reagent
s


ADM, DMY, OA,
3
-
(4, 5
-
dimethylthiazol
-
2
-
yl)
-
2, 5
-
diphenyltetrazolium bromide (MTT) were
purchased from Sigma
(Sigma Chemical Co.). Dulbecco’s
modified Eagle’s medium (DMEM)

and fetal bovine serum (FBS) were purchased
from Invitrogen
(Carlsbad, CA, USA).
Apoptosis
and Necrosis Assay Kit were from Beyotime Institute of Biotechnology (Beyotime,
Co, China
).
RNApure Tissue Kit,
SYBR Premix Ex Taq™

Ki
t (Perfect Real Time) was obtained from Takara
Bio Inc. (Kyoto, Japan).β
-
actin antibody rabbit polyclonal antibody and peroxidase
-
conjugated
goat anti
-
rabbit IgG were purchased
from

Affinity BioReagents
(Golden, Co, USA

).
Anti
-
Blooms
Syndrome Protein Blm
antibody (
Abcam, Cambridge, MA, USA
)

Cell culture

Human lung cancer cell line A549 was obtained from the Type Culture Collection of Chinese
Academy of Sciences (Shanghai, China).

A549 cells were grown in DMEM, supplemented
with
10% heat
-
inactivated

FBS, 100U penicillin and 100
μg
/mL streptomycin in disp
osable plastic
bottles at 37

with 5% CO
2
.

Methye Thiazolye Telrazlium assay (MTT assay)

MTT assay was

used to

assess

the
growth inhibition
effect of
ADM
,
DMY

and OA on

human
lung
cancer

cell
s,
respectively
.

Distribute

A549 cells

(
5
×
10
3
/well) of logarithmic phase in 96
-
well
plate
s.
Different
concentrations of ADM

(
0.2
,
0.3
,
0.4
,
0.5
,
0.6μg/m
L
),

DMY

(
1
,
5
,
10
,
15
,

20μg/m
L
)

and
OA

(10
,
50
,
100
,
150
,
200μg/m
L
)

were cultured
in a humidified incubator

at 37°C
under 5% CO
2

in air
for different time (24, 48, 72 h). Cell samples were incubated with MTT

(
5 mg/m
l, 20
μL
)
for 4 h

before

removal of the MTT solution. The
n 150
µ
L
/well dimethyl
sulfoxide was added and the formed crystals were dissolved gently b
y pipetting 2 to 3 times
slowly.
At

the same time
,

set zero well (DMEM
,
MTT

and DMSO
)
,
controlled well

(cells
,
DMEM
,
MTT

and DMSO
)
.
The absorbance was measured at a wavelength of 490 nm. Growth inhibition
rate was calculated as follows.
Results were
expressed as the mean±SD of percent inhibition of
cell viability.

Experiments were repeated at least three times.

Growth inhibition=
1
-
A
490

of treated cells

A
490

ofcontrol cells

×100%


Hoechst 33342 / PI staining

Distribute

A549 cells

(4
x10
4
/well) of logarithmic phase
in 6
well plates.

Cells were treated
with
ADM

(
0.3μg/m
L
)
,
DMY

(
15μg/m
L
)

and
OA

(
90μg/m
L
) and control

(without drug)
,

then
incubated
in a humidified incubator at 37°C under 5% CO2 in air

for 48 h
. A
dd 1 mL cells
staining buffer,
5
μ
L Hoechst staining fluid, 5
μ
L

PI dye

into the well and

incubated
at
4


for 30
minutes.
Wash the cells using

phosphate
-
buffered saline

(PBS)

solution

after staining and
observe

them

under fluorescence microscope.

DNA ladder assay

A549 cells (2x10
4
/well)
of logarithmic phase

were
seeded

in 6
well plates.
Cells were treated
with ADM
(
0.3μg/mL
),
DMY
(
15μg/mL
)
and
OA
(
90μg/mL
) for 48h,

control

(without drug)

incubated
in a humidified incubator at 37°C under 5% CO
2

in air
.
Then harvested by
centrifugation and
washed twice in ice
-
cold PBS, respectively. An Apoptotic DNA Laddering Kit
(Beyotime Institute of Biotechnology, China) was used to analyze DNA fragmentation according
to the manufacturer’s protocol, and then the DNA fragments in the samples were separated

by
electrophoresis in 2% (w/v) agarose gel.

Quantitative

real
-
time

(
RT
-
PCR) assays

In brief,

A549

cells plated as described in
DNA ladder assay
were incubated

at 37°C under
5% CO
2

in air

for 48 h with
ADM
(
0.3μg/mL
),
DMY
(
15μg/mL
)
,
OA
(
90μg/mL
) and
blank
control

(without drug)

before being harvested, centrifuged.

Then

washed twice in ice
-
cold PBS, total RNA
was extracted using

Trizol reagent (Nippon Gene, Tokyo, Japan)

according to the manufacturer’s
instructions. Use
Ultra SYBR

Two Step qRT
-
PCR Kit follow
ing the manufacturer’s conditions
(Invitro
-
gen).
RT
-
PCR was performed as follows: after an initial denaturation at 95
°C

for 10 min,
the amplification was done by denaturation at 95
°C

for 10 s, annealing at 58
°C

for 15 s, and
extension at 72
°C

for 15 s for 39 cycles.
The number of amplification cycles was optimized in
preliminary experiments to ensure that the PCR did not reach a plateau.

Sequences

of BLM and
β
-
actin were amplified using the following.

T
able 1:

the sequences of primer of BLM an
d

-
actin genes

GENE

P
rimer


S
e q u e n c e

L e n g t h

B L M

P r i me r F

5 ’

G G AT C C T G G T T C C G T C C GC
-
3 ’

P r i me r R

5 ’
-
C C T C A G T C A A AT C TAT T T GC T C G
-
3 ’


-
a c t i n

P r i me r F

5 ’

C G G A G T C A A C G G AT'T T G G T C G T AT
-
3 ’

P r i me r R

5 ’
-

A G C C T T C T C G AT G GT GG T G A AG A C
-
3 ’


BLM a n d β
-
a
c t i o n

p r i me r s s e q u e n c e s d e s i g n b y b i o l o g y s o f t w a r e p r i me r p r e mi e r 5.0 a n d
s y n t h e s i s b y S h e n g Go n g ( S h a n g h a i, Co, Ch i n a )
.
PCR products were subjected to a 1% (w/v)
agarose gel electrophoresis
, and analyzed by ChemiDoc XRS (Bio
-
Rad, Hercules, CA).
PCR
results
using
2
-
△△
Ct

represents the relative expression of genes [21],
△△
Ct
= medication


Ct

untreate
d

Ct
,

e
xperiments were repeated at least three times.


I
mmunocytochemistry

assay
s

A459 cells (2
×
10
4
/well) were seeded on cover slips in 24 well plates and then treated with

ADM
(0.3μg/mL),
DMY
(15μg/mL) and
OA
(90μg/mL)
. After 48h incubation, the cells were
washed twice with PBS

solution

and fixed with a 4% buffered paraformaldehyde for 30 min
and
en
dogenous peroxidase

was blocked for

3% H
2
O
2

incubation for 5
-
10 minutes,

after that cells
were incubated with a 1:100 dilution of
BLM
(Abcam, Cambridge, MA, USA) primary antibodies
maintained overnight at 4°C in a humid chamber, then, cells were washed in
PBS and incubated
for 1 h with

HRP conjugated secondary antibodies at 1:1000 dilutions.

After that

the coverslips
were washed (3 times) with PBS
.
Blots were developed using the DAB according to the
manufacturer’s instructions. I
mage analysis
was quantified

using
Image
-
Pro Plus

Software and
take the result

that

brownish
-
yellow granules

appeared

in nuclei

after
DAB staining

as positive
.

708bp


306bp

Statistical analysis

The data were analyzed using the SPSS 18.0 and data are reported as mean±SD of the
number of experiments indicated. For all the measurements, one
-
way ANOVA followed by a
Student’s t
-
test was used to assess the statistical significance of difference between

each group.
LSD method is used to assess the statistical significance of difference between every two group.
A
statistically significant difference
was considered at the level of
P

< 0.05.

D
ISSCUSSION

Lung cancer
is one of the world’s highest morbidity
and mortality of malignant tumors. The
incidence and mortality of lung cancer are rapidly rising all over the world, especially in the
developed countries. The treatment of lung cancer mainly includes surgery, chemotherapy and
radiotherapy clinically
. Howe
ver,
many problems appear,
On the one hand, the side effects of
these methods are serious; On the other hand, these treatments also can cause severe damage to
the body's own immune function and the patients’ immunity to decline. With the continuous
develop
ment of molecular biology and biotechnology, looking for working site and small
antineoplastic drugs of high specificity, little side effects is becoming a hot spot in the basic and
clinical research on tumors.

Data presented in this paper

demonstrated tha
t

ADM, DMY

and
OA

can all inhibit the
proliferation of A549 cell and present a certain time and concentration dependence. IC
50

of
ADM,
DMY
and
OA
concentration were 0.34
μ
g/ml, 13.16
μ
g/ml, 80.
45
μ
g/ml, respectively.
And there
was significant difference betwe
en the experiment group and the control group (
P
< 0.01).
Cells
were tr
eated in their concentration of
IC
50

for 48h to compare inhibition. The result showed that
inhibition effect of ADM was the best with the apoptosis cells were shriveled, turned round
and
floated when observed under inverted fluorescence microscopy. After Hoechst/PI staining, the
apoptotic cells

appeared condensed chromatin, karyorrhexis.

Besides, apoptosis morphology
appeared such as chromatin pyknosis.

Furthermore,
DNA Ladder detectio
n showed ladder
electrophoresis bands of lung
cancer
A549 apoptosis cell fragments which were in interval of
180
-
200 bp by integer times. However,
untreated
A549 cell show a whole belt.
The
Hoechst/PI
staining

and
DNA Ladde
r
indicate
d ADM, DMY

and
OA induc
e cells
apoptosis
.
B
LM

helicase
mRNA and protein expression in A549 cells were detected through
RT
-
PCR

and

Immunocyto
chemistry

assays
. The results show that
ADM, DMY and OA

inhibited B
LM

helicase
of A549 cells
at
mRNA and protein expression

level
,

and

the inhibitory effect of

ADM was the
best, too.

Research has

shown that human BLM

helicase can unwind G4 structure

[23
-
24]
.
G4 DNA
can not only prevent telomerase from identifying telomere DNA but also inhibit the expression of
telomerase activity

[25].

Telomerase can stable telomere length and make cancer cells
immortalized

[26
-
27]
.

ADM
,
DMY
can both be applied to act on G4 and cut B
LM

gene
expression. However, the apoptosis
hypothesis

that through inhibiting
B
LM

gene expression, and
then losing the unwinding of G4, destabilizing the telomerase, thereby promoting apoptosis is still
needed further verification.

Ellis et al [28] reported
B
LM

genetic mutation resulted in the lack of RecQ DNA spiral
enzyme activity in B
LM

mutant protein which could not repair the abnormal DNA structure
appeared in the process of DNA replication. Thereby cytogenetic characteristics of chromosome
instability synd
romes such as chromosome break, translocation, and sister chromatid exchange
(SCEs), and so on appeared.
Cells from patients with BS and normal cells lack of B
LM

function
are very sensitive to various inhibitors (chemicals, radiation and reactive oxygen sp
ecies) which
cause DNA damage and blocking DNA replication
[29]
. O
A

can damage DNA to inhibit B
LM

gene expression and then reduce RecQ DNA spiral enzyme activity of the cell so as to result in
cell apoptosis.

Cell apoptos
is is mostly related to p53 gene
[3
0]
.
p
53 gene is one of the most important genes
in all the tumor suppressor genes, which widely participate in the development of tumor. One of
the most important functions of p53 is to be able to activate apoptosis

[31]
. p
53 protein can play a
role only i
n the nucleus. Related drugs can induce cell apoptosis by up
-
regulating tumor
suppressor gene p53.
Kaneko

et al

[32]
pointed out that the reduced expression of B
LM

helicase
protein can up
-
regulate the p53 protein so as to promote cell apoptosis. According to the literature
[33
-
34]
,
ADM
can induce cell apoptosis through p53 gene’s activation of cell apoptosis process.
OA induced apoptosis of melanoma B16F and the exp
ression of p53 increased, both of which may
be correlated. As a result, detection by RT
-
PCR and Western blot
assays
can be used to confirm
whether

ADM
,
DMY
and

OA

affect the expression of p53 protein in tumor cells. If the three
drugs can influence the exp
ression of p53 protein in tumor cells, it may suggest
ADM
,
DMY
and

OA

that activated cell apoptosis through down
-
regulating the expression of B
LM

helicase and
raising the expression of p53 protein.

This study s
howed that
ADM
,
DMY
and

OA

can all inhibited
t
he
expression

of BLM
helicase

at
mRNA and protein

level

and promote
d

the apoptosis of lung cancer cells. Besides, our
study provide important basis for treating cancer at the molecular level targeting DNA helicase to
design and screen anticancer drugs.






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