Therapeutic effect of arsenic trioxide (As2O3) on cervical cancer in vitro and in vivo through apoptosis induction.

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e1 Cancer Biology & Therapy 2007; Vol. 6 Issue 4
Research Paper
Therapeutic Effect of Arsenic Trioxide (As2O3) on Cervical Cancer
In Vitro and In Vivo through Apoptosis Induction
Jing Yu
Haili Qian
Yunfeng Li
Yang Wang
Xueyan Zhang
Xiao Liang
Ming Fu
Chen Lin*
State Key Laboratory of Molecular Oncology; Cancer Institute/Cancer Hospital;
Chinese Academy of Medical Sciences; Peking Union Medical College; Beijing
China
*Correspondence to: Chen Lin; Cancer Institute/Cancer Hospital; Chinese Academy
of Medical Sciences; Peking Union Medical College; State Key Laboratory of
Molecular Oncology; No.17 Panjiayuan Nanli; Chao Yang District; Beijing
100021 China; Tel.: +8610.67723793; Fax: 8610.87714054; Email: clinwk@
yahoo.com.cn
Original manuscript submitted: 11/28/06
Manuscript accepted: 01/19/07
This manuscript has been published online, prior to printing for Cancer Biology &
Therapy, Volume 6, Issue 4. Definitive page numbers have not been assigned. The
current citation is: Cancer Biol Ther 2007; 6(4):
http://www.landesbioscience.com/journals/cc/abstract.php?id=3887
Once the issue is complete and page numbers have been assigned, the citation
will change accordingly.
KeY Words
arsenic trioxide, apoptosis, cervical cancer,
therapy, Bax, Bcl-2, mitochondrion
AbbreviAtions
As2O3 Arsenic trioxide
HPV human papillomavirus
APL Acute promyelocytic leukemia
VDAC Voltage-dependent anion channel
Gadd 45 Growth arrest and DNA damage 45
MTT 3-(4,5-dimethyl-thiazol-2-yl)-
2,5-Diphenyl-tetrazolium bromide
DSS Disuccinimidyl suberate
s.c. subcutaneously
i.p. intraperitoneally
ACKnoWLedgeMents
We thank Dr. T.-C. Wu (Professor of
Pathology,The Johns Hopkins University) for
providing TC-1 cells, and Dr. You-Ping Deng
for HeLa-Bcl-2 cell establishment.
AbstrACt
Arsenic trioxide (As2O3) induces apoptosis in certain types of cancer cells. But the
detailed mechanisms of As2O3 efficacy are not completely known. Here we demonstrate
that As2O3 has a therapeutic effect on cervical cancer in vitro and in vivo. We investi-
gated the As2O3-induced apoptosis in various cervical cancer cells. The apoptosis was
triggered by mitochondrial pathway and associated with dissociation of Bcl-2 from Bax
and VDAC, then the release of cytochrome c from Bax and VDAC channel, resulting in
the activation of caspase-9 and caspase-3. The overexpression of Bcl-2 counteracted
the As2O3-mediated apoptosis. The As2O3 treatment also resulted in an increased M
phase cell cycle distribution by inducing microtubule polymerization. Two independent
death-signaling pathways in cervical cancer cells were activated, one dominated by
JNK/p38/GADD45 and one by p53 signals. Further investigation involving assessment
of As2O3 on tumor cell growth in mice indicated that As2O3 also inhibited in vivo tumor
growth. As2O3 as an inhibitor of cervical cancer proliferation both in vitro and in vivo
suggests a potential clinical application in cervical cancer therapies.
introduCtion
Worldwide, cervical cancer is second only to breast cancer as the most common
malignancy in both incidence and mortality.1 Over 90% of human cervical carcinoma are
associated with high-risk human papillomavirus (HPV), mainly the serotypes16 and 18.2
A satisfying therapeutic reagent should eliminate the continuous effect of HPV, as well as
correcting the defective gene expression in cancer cell.
As2O3 is a naturally occurring substance that has been used as a medicinal agent for
more than 2,400 years, to treat a variety of medical conditions ranging from infectious
disease to cancer.3 As2O3 has a long history of application in the treatment of leukemia,
and was approved by the Food and Drug Administration for usage in the treatment of
relapsed/refractory acute promyelocytic leukemia (APL). The detailed mechanisms of
As2O3 cytotoxicity are not completely known, but the mechanisms of action were shown
to be associated with the induction of apoptosis and differentiation.4 In particular, As2O3
inhibited growth and induced apoptosis with a decrease of HPV E6/E7 in the HCE
16/3 immortalized cells.5 Since E6-mediated p53 degradation may play a crucial role for
HPV-associated carcinogenesis, in the present study, we investigated whether As2O3 has
an effective treatment of cervical cancer. We also aimed at obtaining the mechanism of
apoptosis induction in cervical cancer cells by As2O3. Here we provide a better under-
standing of the molecular mechanisms by which apoptosis of cervical cancer cells was
induced by As2O3.
MAteriALs And MetHods
Materials. As2O3, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-Diphenyl-tetrazolium bromide
(MTT), Giemsa stain and Disuccinimidyl suberate (DSS) were purchased from Sigma
Chemical Co.(St Louis, MO, USA). The following antibodies were used in the experi-
ments: cytochrome c (sc-13561), Bcl-2(sc-509), Bax (sc-493), VDAC1 (sc-8828),
caspase-9 (sc-8355), caspase-3 (sc-7148), PARP (sc-8007), caspase-8 (sc-7890), p21
(sc-6246), b-tubulin (sc-23949), JNK (sc-1648), p38 (sc-7972), Gadd45a (sc-792)
and actin (sc-8432) were commercially provided by Santa Cruz Biotechnology (Santa
Cruz, CA). p53 (554147) was from BD Biosciences Pharmingen (San Diego, CA).
Puma (No. PC686) was purchased from Merck Biosciences represents EMD Biosciences
(Calbiochem, Cat).
[Cancer Biology & Therapy 6:4, e1-e7, EPUB Ahead of Print: http://www.landesbioscience.com/journals/cbt/abstract.php?id=3887; April 2007]; ©2007 Landes Bioscience

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Therapeutic Effect of Arsenic Trioxide on Cervical Cancer
Cell culture, cell transfection and treatment. The human cervical
cell line HeLa, SiHa, Caski or C33A and TC-1 cells (C57BL/6
mouse lung endothelial cells transformed with the HPV16 E6 and E7
oncogenes and activated H-ras) were grown in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin,
and 100 mg/ml streptomycin. All cell lines were maintained at 37˚C
in a humidified atmosphere of 5% carbon dioxide.
We have established HeLa cells overexpressing Bcl-2 (HeLa-Bcl-2)
by transfecting pSFFV-neo-bcl-2 plasmid into HeLa cells. The
transfected clones were selected with 600-800 mg/ml G418 (Life
Technologies Inc., Paisley, Scotland) cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, and 100 mg/ml streptomycin.
As2O3 powder was dissolved in phosphate buffered saline (PBS)
as 1 mM stock solution and diluted in the medium at the indicated
concentrations.
MTT assay. The effect of As2O3 on cell survival was determined
using the MTT assay, as described previously.6
Cell cycle analysis by flow‑cytometry. After treated with 5 mM
As2O3 for 24 or 72-hr, cells were trypsinized, washed once with PBS
and fixed in ice-cold 70% ethanol overnight at 4˚C.Fixed cells were
pelleted, resuspended in 0.5 ml PBS containing 75 U/ml of RNaseA
at 37˚C for 30 min and stained with propidium iodide (PI, Sigma).
The DNA content of cells was analyzed by a flow cytometer. Data
were generated from samples with at least 10000 cells and analyzed
by the DNA MultiCycle program.
Reverse transcription‑polymerase chain reaction (RT‑PCR)
Analysis. Total RNA was isolated from cancer cells and RT-PCR
was performed according to the manufacturer’s instruction to detect
the gene expression. The primers used for amplification were as
follows: GAPDH (forward primer: 5'-ACCACAGTCCATGCCAT-
CAC-3', reverse primer: 5'-TCCACCACCCTGTTGCTGTA-3');
HPV18 E6 (forward primer: 5'-CAGACTCTGTGTATGGAGAC-3
', reverse primer: 5'-TTGTGTTTCTCTGCGTCGTT-3'); HPV18
E7 (forward primer: 5'-GACCTTCTATGTCACGAGCA-3', reverse
primer: 5'-C ACGGAACACAAAGG ACAG-3'); HPV16 E6(forward
primer: 5'-ACCCAGAAAGTTACCACAGT-3', reverse primer: 5'-G-
CAACAAGACATACATCGAC-3'); HPV16 E7 (forward primer:
5'-TACCACAGTTATGCACAGAG-3’, reverse primer: 5'-TGT-
TCTTGATGATCTGCAAC-3'). The cycling conditions were as
follows: initial denaturation at 94˚C for 5 min, followed by 28 cycles
at 94˚C for 30 s, 56˚C for 40 s, and 72˚C for 30 s.
Western blot assay, and immunoprecipitation. Immunoblotting
analysis and immunoprecipitation were prepared as described
previously.7
Crosslinking of Bax and VDAC protein. After As2O3 treat-
ment, cells were collected at the indicated time points and subjected
to treatment with 2mM DSS (cross-linking agent), as described
previously.8 Bax or VDAC was detected by Western blotting.
Isolation of mitochondria and mitochondrion‑free cytosolic
protein. Mitochondria and mitochondrion-free cytosolic protein
were prepared as described previously.7
Isolation of nuclear and cytosolic protein. Cells were solubilized
in Buffer 1(1mM KCl, 5 mM NaCl, 3 mM MgCl2, 50 mM HEPES,
1 mM DTT, 0.5 mg/ml Leupeptin, 20 mM PMSF PH7.4), thaw and
frozen, and centrifuged at 5000 rpm for 5 min at 4˚C. Supernatants
were spun at 14000 rpm for 10 min. The final supernatants were
cytosolic protein.
The pellets spun dowm at 5000 rpm in Buffer 1 were resuspended
in Buffer 2 (10 mM HEPES pH7.9, 10 mM KCL, 0.2 mM EDTA,
1 mM DTT, 0.5 mM PMSF, 1%NP-40) and centrifuged for 10 min
at 14000 rpm. Pellets were resuspended in Buffer 3 (20 mM HEPES
pH7.9, 400 mM KCL, 2 mM EDTA, 1 mM DTT, 1 mM PMSF)
on ice for 40 min and spun down at 14000 rpm for 10 min at 4˚C.
The final pelleted were nuclear protein.
Immunofluorescent staining. Cells were fixed with cold methanol
at -20˚C, and incubated with monoclonal anti-b-tubulin antibody
(1:100) at room temperature. After washing three times with PBS
solution, cells were reincubated with FITC-conjugated second anti-
body (1:500) in the dark room for 30 min. Cellular microtubules were
observed with a Leica TCS SP2 fluorescent confocal microscopy.
In vivo mouse studies. The in vivo therapeutic effect of As2O3
was evaluated in a cervical cancer mouse model. In this model,
C57BL/6 female mice were subcutaneously (s.c.) inoculated with
105 TC-1 cells on day 0. Mice were divided into three subgroups
according to the daily dose: Group 1 (n = 8), PBS alone intraperi-
toneally (i.p.); Group 2 (n = 8), 2 mg.kg-1.day-1As2O3 i.p.; Group 3
(n = 8), 5 mg.kg-1.day-1 As2O3 i.p. The drug was administrated
one day after TC-1 cells inoculation and continued daily for three
weeks. The volumes of tumor were recorded twice a week by the
formula: tumor volume = 0.52 (width x width x length). At 21 days
after inoculation, the mice were killed and the tumor weight of
different group was measured.
Statistical analysis. Statistical analysis was performed by using
a standard Student’s t-test analysis, with p-values <0.05 considered
significant.
resuLts
As2O3 treatment results in growth inhibition and apoptosis in
cervical cancer cells. Significant dose-dependent inhibition of cell
growth was observed in all of the cell lines used in MTT assay following
the treatment of As2O3 for 72 hr. The IC50 are 2.35 mM, 4.01 mM,
14.29 mM, 14.37 mM respectively for C33A cells (HPV-negative),
HeLa cells (HPV18-positive), Siha and Caski cells (HPV18-positive)
(Fig. 1A). Treatment of cervical cancer cells with As2O3 caused phase
G2/M arrest, also, apoptosis was induced in C33A cells after being
treated with 5 mM As2O3 for 48 hours by flow-cytometric analysis
(Fig. 1B). Giemsa stain confirmed that growth of cervical cancer cells
was inhibited in mitotic phase (Fig. 1C). Therefore, we used Western
blot assay to detect the As2O3 caused pro-apoptotic protein cleavages
of PARP and caspase-3 in SiHa cells (Fig. 1D). Thus, As2O3 induced
growth suppression in cervical cancer cells may mainly correlate with
As2O3 activated cell apoptosis.
As2O3 induces caspase‑9 activity, cytosolic cytochrome c accu‑
mulation and apoptotic mitochondrial events. To determine which
signaling pathways were mainly involved in As2O3 induced apop-
tosis, the activation of caspase-9 (mitochondria pathway) and
caspase-8 (death receptor pathway) were examined in cervical cancer
cells (Fig. 1D). Caspase-9, but not caspase-8, was cleaved starting at
4 hr, suggesting that the mitochondrion mediated pathway is acti-
vated by As2O3.The levels of cytochrome c were evidently elevated in
mitochondrion-free cytosol protein (Fig. 2A), and this finding went
along with the activation of caspase-9. Thus mitochondria pathway
was mainly involved in As2O3 induced apoptosis.
As2O3 induces dissociation of Bcl‑2 from Bax and VDAC, Bax
and VDAC oligomerization. Bax and Voltage-dependent anion
channel (VDAC) are very attractive candidates for the pathway for
cytochrome c release. In addition, the ability of Bcl-2 proteins to
regulate the state of Bax and VDAC could thus account for their

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ability to be either anti- or pro-apoptotic. We then examined the
physical interaction of Bcl-2 with Bax and VDAC protein in mito-
chondria. As shown in Figure 2B, after 8 hr of As2O3 treatment,
VDAC and Bax proteins decreased in Bcl-2-associated complexes.
The oligomerization of Bax has previously been reported to only
occur in apoptotic cells, possibly playing a role in mediating cyto-
chrome c release.9 In Figure 2C, multimerization of Bax was
detectable after As2O3 treated in HeLa cells.
VDAC, an abundant protein in the outer mitochondrial
membrane, plays a significant role in cytochrome c-mediated cell
death.10 One possible mechanism for VDAC-mediated cytochrome
c release is the formation of a channel through oligomerization of
VDAC. We found that As2O3 induced the homodimerization of
VDAC in As2O3 treated HeLa cells (Fig. 2C). We also examined
the physical interaction of VDAC with Bax and Bcl-2 protein
in mitochondria with immunoprecipitation. After 8hr of As2O3
treatment, Bcl-2 and Bax proteins decreased in VDAC-associated
immunocomplexes (Fig. 2B). These results suggested that oligomer-
ized Bax and homodimerized VDAC could respectively form pores
capable of releasing cytochrome c following As2O3 treatment in
HeLa cells.
We also examined protein levels of antiapoptotic Bcl-2 and
pro-apoptotic Puma in the mitochondrion apoptotic pathway.
Western blot showed increased expression of Puma after 2 hr
of As2O3 addition, decreased expression of Bcl-2 after 24 hr of
As2O3 addition. Interestingly, cell fractionation results showed Bcl-2
protein translocated from cytosol to nucleus in As2O3 treated HeLa
cells (Fig. 2D).
We have established HeLa cells overexpressing Bcl-2 (HeLa-Bcl-2)
by transfecting pSFFV-neo-bcl-2 plasmid into HeLa cells using lipo-
Figure 1. Effects of As2O3 on the growth of cervical cancer cells and As2O3 treatment induces apoptosis of cervical cancer cells. (A) Effect of As2O3 on
the cell proliferation were determined by MTT assay. The results show the mean ± S.D. of three independent experiments. (B) Flow cytometry showed that
growth of cervical cancer cells was inhibited in G2/M phase following exposure to 5 mM As2O3. Apoptosis was induced in C33A cells after being treated
with 5 mM As2O3 for 48 hours. (C) Giemsa stain showed that growth of cervical cancer cells was inhibited in M phase following exposure to 5 mM As2O3.
After being treated with 5 mM As2O3, (D) SiHa cells were analyzed by Western blot for caspase-8, activation of caspase-9, and cleavages of PARP and
caspase-3. Actin was used as a protein loading control.

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Therapeutic Effect of Arsenic Trioxide on Cervical Cancer
fectin. The protein levels of Bcl-2 in both HeLa and HeLa-Bcl-2
cells were analyzed by immunoblot (Fig. 2F). Using MTT assay,
contrast to HeLa cells, HeLa-Bcl-2 cells (IC50 was 33.09mM)
were more resistant to As2O3 (Fig. 2E). In addition, no appreciable
Bcl-2 protein translocation from cytosol to nucleus or activation of
caspase-9 was observed after As2O3 treatment in HeLa-Bcl-2 cells
(Fig. 2G). These results further confirm that Bcl-2 translocation
from cytosol to nucleus is an early event of cytochrome c/caspase-9
mediated apoptosis.
Effect of As2O3 on cellular microtubules. Because of mitotic
arrest and apoptosis effect, we tested whether As2O3 affected the
cellular microtubule network. Western blot showed increased protein
level of b-tubulin starting at 12 hr of As2O3 addition in HeLa cells
(Fig. 3A). Further, the microtubule network was visualized in HeLa
cells by immunofluoresent staining. The microtubule network
in control cells exhibited normal arrangement and organization.
Treatment with As2O3 resulted in microtubule polymerization with
an increase in the density of cellular microtubules (Fig. 3B). All
results indicated that the microtubule is the intracellular target for
As2O3 accounting for the subsequent mitotic arrest and apoptosis.
The JNK/p38 and p53‑dependent pathways are the major
regulators of As2O3 induced apoptosis. HPV E6 may promote
genetic instability by inactivating the tumor suppressor and cell cycle
checkpoint protein p53.11 RT-PCR confirmed that 5 mM As2O3
could inhibit expression of the HPV E6/E7 gene in HPV-positive
cells starting at 12 hr treatment (Fig. 4A–C). We suspected that p53
pathway might be induced by As2O3. We determined the expres-
sion levels of p53 and then its responsive genes in As2O3 treated
HPV-positive cells. There was significant upregulation of p53 and
its downstream target gene p21, coincidentally with the decrease of
HPV E6. PUMA, a recently identified p53-upregulated modulator
of the apoptosis gene, and Bax oligomers were found upregulated
before the increase of p53 (Fig. 2 and Fig. 2D). We suspected that
other pathway in addition to p53 might be induced by As2O3.
Stimulations of MAPKs are generally known to promote cell prolif-
eration, but occasionally lead to apoptosis of severely effected cells by
a number of physical and chemical stresses.4,12 Here we examined
whether As2O3 could stimulate activities of members of MAPK
family. When HeLa and SiHa cells were treated with As2O3, remark-
able stimulations of JNK1/2 and p38, members of MAPK family
were observed from immunoblots (Fig. 4D and E). The growth arrest
and DNA damage 45 (gadd 45) gene is a downstream target for
p5313 and has also been previously reported to physically associate
with activated MTK1/MEKK4, an upstream activator of JNK/p38
kinase pathways, which may be involved in induction of apoptosis.14
The findings presented here that stress protein Gadd45a increased
after stimulations of JNK1/2 and p38 but preceding to the increase
Figure 2. As2O3 treatment induces apoptosis of HeLa cells, targeting the
mitochondrial cell death patheway. (A) HeLa cells were exposed to 5 mM
As2O3 and subjected to subcellular fractionation. Cytochrome c release was
measured in mitochondrial and mitochondrion-free cytosol by immunoblot.
(B) After exposed to 5 mM As2O3, HeLa cells were collected at the indicated
time points. Mitochondrial lysates were prepared to examine the interactions
between Bax or VDAC and Bcl-2. (C) The oligomerization of Bax and VDAC
were assessed by crosslinking with DSS followed by immunoblot analysis.
(D) After exposed to 5 mM As2O3, HeLa cells were collected for preparation of
whole-cell lysates or nuclear protein. The protein levels of Bcl-2 in both whole
lysates and nucleus were analyzed by immunoblot. In addition, proapoptotic
protein Puma was examined in whole lysates by immunoblot. Actin was used
as a protein loading control. (E) Effects of As2O3 on the cell proliferation
of HeLa cells and HeLa-Bcl-2 cells were determined by MTT assay. (F) The
protein levels of Bcl-2 in both HeLa cells and HeLa-Bcl-2 cells were analyzed
by immunoblot. (G) HeLa-Bcl-2 cells were collected and analyzed by Western
blot for caspase-9. In addition, the protein levels of Bcl-2 in both whole
lysates and nucleus were analyzed by immunoblot in HeLa-Bcl-2 cells.

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of p53 (Fig. 4D and E). The data thus suggested that As2O3 activated
two independent death-signaling pathways in HPV-positive cells, one
dominated by JNK/p38/GADD45 signals and one by p53 signals.
In vivo tumor growth inhibition in a mouse model of cervical
cancer. The in vivo therapeutic efficacy of As2O3 against cervical
cancer was assessed by a mouse model. One day after inoculation
of TC-1 cells to C57BL/6 female mice, As2O3 treatment continued
daily for three weeks. Dose-dependent inhibition of cervical cancer
growth by As2O3 was statistically significant (Fig. 4F and G). Neither
severe toxicity nor body weight loss occurred during the treatment
period (data not shown). These findings indicate that, at a thera-
peutic dosage, As2O3 induces in vivo tumor growth inhibition in the
mouse model of cervical cancer without severe signs of toxicity.
disCussion
As2O3, a reagent successfully employed in the treatment of APL
and recently also in solid tumors, induces growth inhibition and
apoptosis. However, the molecular bases are not yet fully defined in
solid tumor cells, further molecular investigations should be required
for better clinical application of As2O3. In this regard, detailed inves-
tigations were undertaken here to define the mechanism by which
As2O3 plays a role in the induction of apoptosis. Using C33A cells,
HeLa cells, SiHa cells and Caski cells, well studied cervical cancer
cell lines, we demonstrated that As2O3 resulted in apoptosis in
cervical cancer cells. Furthermore, the findings in the in vitro assay
were supported by the in vivo experiments. As2O3 induces in vivo
tumor growth inhibition in the cervical cancer with no severe signs
of toxicity.
In the present study, we showed that As2O3 induced cell apoptosis
through the mitochondrial pathway since As2O3 induction caused
caspase-9, but not caspase-8, activation and cytochrome c release
from mitochondria to cytoplasm (Fig. 1 and Fig. 2). As2O3 induction
of apoptosis was likely mediated through Puma accumulation and
Bcl-2 decrease. As2O3-activated apoptosis was coupled with Bcl-2
translocation from cytosol to nucleus, dissociating from Bax / Bcl-2
and VDAC / Bcl-2 complexes, and formation of Bax and VDAC
oligomerization (Fig. 2). An interesting model proposes that
VDAC could interact with Bax to form a hybrid channel to
mediate cytochrome c release.15 However, our results differ
from this model due to the observation that at least in HeLa
cells, As2O3 induced dissociation of Bax and VDAC, forma-
tion of Bax tetramers and VDAC dimers. The exact protein
composition of these oligomers constituting the cytochrome
c-conducting channel in mitochondria now remains to be
determined. Further work is needed to more closely examine
the dynamic interaction of Bcl-2 family members and VDAC
members, which could govern the release of cytochrome c. It
is also of interest to note that Bcl-2, which is mainly localized
at the intracellular membranes (mitochondria and ER), could
prevent intracellular free radical generation and glutathione
depletion, which are key factors involved in protecting intra-
cellular redox environments against diverse death stimuli.16,17
This is the first report in our results that Bcl-2 translocated
from cytosol to nucleus, dissociating from Bax/Bcl-2 and
VDAC / Bcl-2 complexes as assayed by immunoprecipitation
in the cells undergoing apoptosis (Fig. 2). Interestingly, we
also found that in Bcl-2-overexpressing cells that are resistant
to As2O3-induced apoptosis (Fig. 2). But Bcl-2 translocation
from cytosol to nucleus preceding to the Bax and VDAC
releasing from Bcl-2-bound complexes in mitochondria suggests that
some different pool of Bcl-2 may also be antiapoptotic. Our findings
could offer an explanation for As2O3-induced apoptosis, since release
of cytochrome c is causally linked to Bcl-2 translocation from cytosol
to nucleus, dissociating from Bax/Bcl-2 and VDAC/Bcl-2 complexs,
and Bax tetramers and VDAC dimers formation.
Several lines of evidence suggest that As2O3 has a particular affinity
for the cytoskeleton, which contains proteins with a higher sulfhy-
dryl content.18 Tubulin is the main component of the cytoskeleton
and is essential to cell division. Disorganized microtubule formation
prevents cell cycle progression and is a potent signal for apoptosis. We
observed that As2O3 treatment was associated with accumulation of
cells at M phase (Fig. 1B and C). Interestingly, in our observation,
As2O3 stabilized tubulin polymerization (Fig. 3), also supported by
Ling,18 which suggested a mechanism of As2O3 induced mitotic
arrest. As mitochondria are regarded as new targets for anti-cancer
agents,19 the existence of mitochondrial tubulin and its association
with apoptosis could extend the range of stimuli that can be orches-
trated by mitochondria. We also found that mitochondrial tubulin
was specifically associated with Bcl-2 and VDAC by immunopre-
cipitation (data not shown). Such an association might have close
relationship with the release of cytochrome c. Mitochondrial tubulin
may participate in a pathway from the cytoskeleton to the mitochon-
dria in the apoptotic mechanism.
Cervical cancer cells have a unique environment, because most of
carcinoma cells contain human papillomavirus as a major etiologic
agent for carcinogenesis. Viral E6 oncoprotein has been shown to
induce the ubiquitin-dependent degradation of p53 and to inhibit
the activity of p53 as a transcription factor.20 This suggested that
E6-mediated inhibition of p53 induction may play a crucial role for
HPV-associated carcinogenesis. Then we aimed at obtaining evidence
whether repression of E6/E7 in HPV-positive cervical cancer cells by
As2O3 is functionally competent to activate p53 gene. In the present
study, we found that As2O3 decreased E6/E7 expression resulting in
significant upregulation of p53 and its important downstream target
p21 in HPV-positive cells (Fig. 4A–C). This could be explained that
a pathway strictly depending on functional p53 is inducible by As2O3
Figure 3. As2O3 treatment induces b-tubulin upregulation and microtubule polymer-
ization. (A) HeLa cells were collected and analyzed by Western blot for b-tubulin.
(B) With immunofluorescent staining, the cellular microtubules were observed by
fluorescent confocal microscopy. The long polymerized microtubule bundles were
found in As2O3-treated HeLa cells.