ARTICLE
Auteur(s) : Rongrong Li1, Yufeng
Cheng2, Chengjin
Gao3
1Department of Radiology, Jinshan Hospital, Fudan
University, 200540 Shanghai, China
2Department of Radiology, Qilu Hospital, Shandong
University, 250012 Jinan, China
3Department of Burn and Plastic, Jinshan Hospital,
China
Uterocervical cancer, of which the incidence and mortality rate are
very high, has become more dangerous to married women. Besides
routine therapies to uterocervical cancer, radiotherapy is
effective and widely accepted. However, the neoplastic bed of
uterocervical cancer is most composed of fibrae, most tolerant of
radiations. A lower radio-dosage to uterocervical cancer may result
in a recurrence, while a higher radio-dosage harms the peripheral
normal tissues around tumors, and depressed the life quality. Then
it is most stringent to search for a new strategy to enhance the
radiosensitivity of tumor cells while lower the side-effects of
radiations. More and more investigations suggested that there was a
significant relationship between apoptosis rate and
radiosensitivity of tumor cells, and apoptosis-inhibiting genes,
such as bcl2 could reduce the radiosensitivity of tumor cells. The
application to blocking some oncogenes or apoptosis-inhibiting
genes to improve the radiosensitivity by inducing tumor cells
apoptosis is intriguing, and our aim is to investigate whether
combining transfection of bcl2 and c-myc ASODNs followed
radiotherapy to Hela cells was a new way to treat malignant tumors.
Materials and methods
Cell culture and oligodeoxynucleotide synthesis and cell
treatment
Hela cells were obtained from the laboratory of QiLu Hospital,
Shandong University (Jinan, China) and cultured in RPMI 1640
(GIBCO, USA) containing 10% calf serum (NBS), incubated in 5%
CO2 incubator at 37°C. Confluent monolayers were
passaged using 0.25% pancregenase, then plated in 24-well plates
supplemented with 2.5 μm ASDON, 7.5 μm
OligofectamineTM Reagent (Invitrogen, USA) liposome. The
antisense and phosphorothioate oligodeoxynucleotides (ASODNs) of
bcl2 and c-myc were synthesized (Shenggong Co, China) and were
added directly to the culture medium at a concentration of
10 μM for 4 h. Sequences used were as follows: antisense
for bcl2: 5′-CAG CGT GCG CCA TCC TTC CC-3′; and c-myc: 5′-CTC CAT
CAC CAC CTC-3′.
Experimental design
Cultured cells were first divided into four groups: bcl2 ASODN
treated group, c-myc ASODN treated group, bcl2 and c-myc ASODNs
combined treated group, and control group. According to the fact
whether X irradiation was performed to cultured cells or not, four
subgroups emerged: bcl2 ASODN combined irradiation treated group,
c-myc ASODN combined irradiation treated group, bcl2 and c-myc
ASODNs combined irradiation combined treated group, and irradiation
treated group.
ASODNs effect assessment
After 4h ASODNs treatment, IHC staining and FCM for bcl2 and c-myc
protein detection was carried out using fluorescein
isothiocyanate-labeled anti-mouse BCL2 and C-MYC monoclonal
antibody (Sigma, USA). On the other hand, after incubated, followed
by a supplementation with 10 μl MTT (5 μg/ml) and
100 μl dimethyl suphoxide (DMSO) to every well (1 ×
105cells/ml) and a quantification by UV
spectrophotometer, cell survival rate (CSR) was obtained by MTT
colorimetry performance using a ratio calculation between every
ASODN treated group and control group with an equation of CSR = A
treated group/A control group.
Apoptosis assay
For apoptosis analysis post-transfection with ASODNs, cells treated
with 0.5 ml propidium iodide (PI) staining solution
(Yuetai Co, China) for 30 minutes, then a FACscan (Becton
Dickinson, USA)was used to determine the apoptotic cells and
apoptosis index (AI). DNA ladder also illustrated apoptosis. Cells
were incubated for 12 h at 37 °C in lysis buffer
(10 mM Tris·HCl (pH 8.0)/10 mM NaCl/10 mM
EDTA/0.5% SDS/100 μg/ml proteinase K). DNA was isolated with
chloroform extraction and treated with 50 μg/ml RNaseA (Sigma,
USA) for 1 h at room temperature. DNA samples (20 μg)
were separated by electrophoresis in a 1.5% agarose gel. The gel
was stained with SYBR Green I nucleic acid gel stain (molecular
probes) after electrophoresis, and the DNA was visualized under UV
light.
Radiosensitivity assay
Clonony formation analyses
For the detection of apoptosis, 1 x 104 cells in every
well were cultured as normal for 12 h, harvested by
centrifugation, washed in phosphate-buffered saline(PBS) and X
irradiated (2 Gy), then planted into RPMI 1640 for
7-14 days culture until cell clones brought into forms. PBS
Washed, methanol fixed, Giemsa stained, clones number counted, then
plantation efficiency (PE) and survival fraction (SF) were
calculated.
Cell survival rate curve
To quantify survival Hela cells post-irradiation, cell culture was
performed as described previously, then X irradiation (0, 2, 4, 6,
8, 10 Gy respectively) was put into practice upon every group. SF
and lnSF calculated, cell survival rate curve was derived by using
the equation of SF = -αD-βD2 (D represents dose) ,
subsequently by using the softwares of Origin 6.0 and SPSS11.0.
Statistical analysis
All results are presented as mean value ± SEM. Differences among
categorical variables were analyzed with analysis of variance
(Anova). Comparisons between control group and ASODN treated group
were analyzed with Dunnet-t test. Statistical comparisons among
ASODN treated group were performed by a 2-tailed unpaired Student’s
t test. A probability value < 0.05 was considered statistically
significant.
Results
ASODNs effectively downregulated the expressions of BCL2 and
C-MYC
The potential role of ASODNs in Hela cells apoptosis progress has
been studied in this work. We immunohistologically assayed the
expressions of BCL2 and C-MYC, which were lower in every ASODN
treated group in comparison with control group ( (figure 1) ). By FCM
analysis ( (figure 2) ) we found
that the expression of BCL2 of ASODNs treated groups was low
compared with control group (p < 0.05), and the C-MYC expression
of c-myc ASODN treated group is 90% lower than that of control
group. Comparisons among the c-myc ASODN treated group, bcl2 +
c-myc ASODNs treated group and control group also showed the same
results (p < 0.05). All these results suggest liposome
functioned effectively as an carrier in the transfection of ASODNs
to Hela cells and ASODNs played a key role for in the regulation of
bcl2 and c-myc.
ASODNs accelerated Hela cells apoptosis
Under microscope, cells of control group which is blooming
clustered together closely and clung firmly to the culture dish
wall with clear figures, while a great deal of ASODNs treated cells
in ill growth with anamorphic forms were encircled by disseminated
cell fragments around them ( (figure 3) ) and
apoptotic bodies could be detected by Giemsa staining ( (figure 4) ). After
transfected with ASODNs, DNALadder showed isometric strips which
were ruptured DNA fragments on the gel ( (figure 5) ). MTT
analyses ( (figure 6) ) indicated
that the CSRs of bcl2 ASODN treated group, c-myc ASODN treated
group, and bcl2 + c-myc ASODNs treated group are all lower than
that of control group (p < 0.01 respectively), and the CSR of
bcl2 + c-myc ASODNs treated group is lower than both bcl2 ASODN
treated group and c-myc ASODN treated group (p < 0.01
respectively). On the other hand, FCM analyses (figures 7 and
8) suggested that the AIs of bcl2 + c-myc ASODNs treated group,
bcl2 ASODN treated group and c-myc ASODN treated group are 11.83 ±
0.57 %, 6.60 ± 0.70% and 10.29 ± 0.66 % respectively,
which were all higher than that of control group (p < 0.01
respectively). Compared the AI of bcl2 + c-myc ASODNs treated
group, each AI of the other two ASODN treated groups is more higher
(p < 0.01 respectively). These results indicated ASODNs (bcl2
and c-myc) might play a key role in the modulation of cell
apoptosis by blocking the gene expressions of bcl2 and c-myc, and
combining transfection could effectively induce the Hela cell
apoptosis by gene interference.
Transfection of ASODNs enhanced the radiosensitivity of Hela
cells
On completion of clonal formation assay, by Giemsa staining, the
bcl2 + c-myc ASODNs combined irradiation treated group was short of
clonal formations which were little and sparse, while the clones of
control group were big and dense ( (figure 9) ), and
there were different size and different numbers of clones in the
other six groups. Compared the SFs of any two groups, a significant
difference (p < 0.05) could be obtained except the comparison (p
> 0.05) between the bcl2 ASODN treated group and the c-myc ASODN
treated group and the comparison (p > 0.05) between the
irradiation combined bcl2 ASODN treated group and the irradiation
combined c-myc ASODN treated group ( (figure 10) ). Then it
could be learned that transfection of ASODN(bcl2/c-myc), especially
combining ASODN(bcl2 + c-myc) transfection to Hela cells could make
them more sensitive to X-rays ( (figure 11) ),
furthermore, ASODN transfection to cervical tumor cells prior to
irradiation might be an alluring means to restrain them from
proliferating excessively.
Sensitivity enhancement ratio(SER) analysis
SER, an indirect index symbolizing the radiosensitivity of Hela
cells, is a dose ratio between the irradiation group and the
irradiation combined sensitized reagent group, while the bioeffects
of the two groups were identical. After the accomplishment of X
irradiation (0, 2, 4, 6, 8, 10Gy respectively) to every group, a
cell survival rate curve was deduced ( (figure 11) ). From (
figure 11
), we could see that the sensitivity of every ASODN combined
irradiation treated group is higher than that of irradiation group,
and the SER of the bcl2 ASODN combined irradiation treated group,
the c-myc ASODN combined irradiation treated group and the bcl2 +
c-myc ASODNs combined irradiation treated group which could be
obtained by calculation was 1.20, 1.53, and 2.18 respectively. From
these results, a hypothesis could be identified that combining
transfection of bcl2 and c-myc ASODNs could more effectively
increase the radiosensitivity of Hela cells than single gene ASODN
transfection, which has been identified effective to augment the
sensitivity of Hela cells to irradiation.
Discussion
The genes of the bcl2 family have emerged as key regulators of
apoptosis [1,2,3–5], and appear to be dysregulated in a number of
tumors, including cervical cancer. Several members of the bcl2
family, including bcl2, Bcl-XL, Mcl-1, and A1/Bfl-1,
suppress apoptosis [1, 5]. The extent of apoptosis is inversely
associated with bcl2 expression in cervical cancer. Expression of
bcl2 would change from high levels in early or low-grade tumors,
characterized by low AIs, to low levels in advanced or high-grade
tumors, characterized by high AIs.
A few evidences suggest that c-myc is a basic region
helix-loop-helix leucine zipper (bHLHZip) transcription factor
which regulates cell proliferation [6], but the amino terminus of
c-myc has both repression activities and transcriptional
activation. It means in addition to gene repression activation,
c-myc also induce gene activation. Furthermore, several lines of
evidence suggest that there is no absolute correlation between
transcriptional activation by c-myc and its function in growth
regulation [7-10], but in our investigation, an important
consideration for studies of candidate target genes is the
observation of inducible apoptosis in cells which were transfected
with c-myc ASODN. The most straightforward interpretation of this
observation is that c-myc downregulation establishes a state of
sensitization to apoptotic triggers, which include irradiation
rays.
Radiation therapy is used extensively in the management of
patients with cervical cancer. Despite the widespread use of
radiation therapy for the treatment of cervical cancer, the
uniformly side-effects of radiations remains a critical problem in
the management of these patients, then enhancing the
radiosensitivity of this malignant tumor while lower the
side-effects of radiations is most stringent, so identifying gene
targets for radiosensitization is an important strategy in
improving anticancer treatments.
X-irradiation induces apoptosis through DNA damage and
generation of free radicals in many types of cells [11, 12].
Apoptosis is regulated by a delicate balance in signal transduction
pathways between apoptosis-activating factors, such as p53 and
caspases, and antiapoptotic factors, such as the bcl2 and the
inhibitor of apoptosis protein (IAP) family [13-15]. It has been
demonstrated that overexpression of bcl2 resulted in faster cell
proliferation via a decrease in the duration of the G0/G1 phase and
an increase in the S phase through its antagonism with bax, bak and
caspases family [16, 17]. Malignant cells exposed to radiation die
via both apoptosis and necrosis, and inhibition of
apoptosis-inactivating factors contributed to sensitivity to
radiotherapy. Many studies have demonstrated that bcl2, c-myc and
p53 which regulate apoptosis of human tumor cells, are important in
many responses to irradiation. In this study we investigated
whether sensitivity to X-irradiation could be affected by bcl2 and
c-myc expression using Hela cells transduced with ASODN(bcl2 or
c-myc, or bcl2 + c-myc), and the first evidence was obtained that
ASODN(bcl2 or c-myc, or bcl2 + c-myc) can augment sensitivity to
X-irradiation via acceleration apoptosis of Hela cells, and
combining transfection could more effectively result in Hela cell
apoptosis than single transfection. So, we could say modulation of
a limited number of target genes by encoding apoptosis-related
proteins would therefore have multiple beneficial effects on
apoptosis.
From all the facts we have detected, a certain conclusion could
be drawn that downregulation of bcl2 on tumor cells combined with
c-myc, an important oncogene modulating apoptosis, would enhance
the radiosensitivity of tumor cells to irradiation and render the
cytotoxicity to the peripheral normal tissues of tumors induced by
radiation retreat. With the increasing recognition of the molecular
basis of the apoptotic pathway, and the description of several of
its components acting as oncogenes or apoptosis inhibiting genes,
gene therapy has thus emerged as a rational strategy for the
modulation of apoptosis of tumor cells.
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