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Introduction
Human hepatocellular carcinoma (HCC) has been proven
to display high resistance to chemotherapeutic agents, such
as cisplatin, 5-fluorouracil (5-FU), and doxorubicin
et al[1_3]. This phenomenon is partially due to
defects in caspase activation, the execution phase of apoptosis. The inhibitor
of apoptosis proteins (IAP) is a family of caspase inhibitors
that bind and inhibit the activation of caspases-3, -7, and -9.
By inhibiting downstream caspases-3 and -7, IAP block the
convergence point of multiple caspase activation pathways
and thus inhibit apoptosis from multiple
stimuli[4_6]. IAP overexpression, the X-linked inhibitor of apoptosis protein
(XIAP) in particular, is associated with chemoresistance in a
variety of human cancers[7,8]. Therefore the removal of IAP
inhibition could be critical for sensitizing cancer cells to
various chemo-anticancer regiments. The second
mitochondria-derived activator of caspases (Smac)/Direct IAP Binding
Protein with Low PI (DIABLO), an IAP-binding
protein, is released from mitochondria and promotes caspase
activation by eliminating IAP function during
apoptosis[9_11]. Consequently, the overexpression of active Smac could be
proposed to render resistant tumor cells sensitive to
chemotherapeutic treatment.
ONYX-015, a tumor-targeting oncolytic adenoviral agent
that selectively replicates in tumor cells due
to E1B-55K gene deletion, has been widely investigated in
cancer therapy. This genetic design takes advantage of the fact that
adenovirus E1B 55K binds and inactivates the wild-type p53
protein that is essential to virus replication.
ONYX-015 has been well tolerated in phase I and II trials, but durable
objective responses were not achieved in
patients[12,13]. However, durable regressions were
subsequently achieved in combination with chemotherapy (such as cisplatin)
in head and neck cancer
patients[14]. Importantly, ONYX-015
and cisplatin do not have overlapping
toxicities[15]. Although the conjugation of ONYX-015 with chemo-agents has attained potent
antitumor activity with the reduction of drug resistance and
side-effects to some extent, further improvements could be
acquired by arming the single viro agent with therapeutic
genes.
In this study, we employed a gene-viro agent ZD55-Smac
to sensitize HCC cells to chemotherapy (cisplatin or 5-FU).
ZD55 was constructed based on adenovirus serotype 5
(Ad5), with E1B-55K gene deletion, but different from
ONYX-015 that was designed for the incorporation of therapeutic
genes. ZD55-Smac was produced by inserting the Smac gene
into the vector, driven by the human cytomegalovirus
immediate-early (CMV-IE) promoter and terminated by the simian
virus 40 polyadenylation signal. As an oncolytic transgenic
delivery system, ZD55 can not only specifically replicate
and lyse in tumor cells, but also restrict therapeutic gene
expression within a tumor
microenviroment[16_18]. Here, we have observed that ZD55-Smac could significantly enhance
the sensitivity of HCC cells to both cisplatin and 5-FU.
Furthermore, the toxic effects to normal cells are distinctly
abolished by utilizing the oncolytic vector and reducing the
drug dosage.
Materials and methods
Cell culture The normal human liver cell lines L-02 and
QSG-7701, human HCC cell lines BEL7404 (with p53 deletion),
SMMC7721, and Huh-7 were purchased from Shanghai Cell
Collection (Shanghai, China). HEK293 (a human embryonic
kidney cell) was obtained from Microbix Biosystems (Toronto,
ON, Canada). The cells were cultured in Dulbecco's
modified Eagle's medium (Gibco_BRL, Grand Island, NY, USA)
supplemented with 10% heat-inactivated fetal bovine serum
(Gibco_BRL, Grand Island, NY, USA) at 37 °C in a humidified
incubator with 95% air and 5% CO2.
Generation of recombinant viruses The ZD55 system
was constructed by deleting the E1B-55K gene based on
adenovirus serotype 5 and introducing a BglII clone site
for carrying foreign genes. The Smac gene was then cloned
into this site to generate oncolytic adenovirus
ZD55-Smac[17]. ONYX-015 and wild-type adenovirus (Ad_wt) were
previously stored in our laboratory. The large-scale purification
of adenoviruses was performed by ultracentrifugation with
cesium chloride. The titers were determined by plaque
formation assay on the HEK293 cells.
Conventional PCR for verification The viral genome
was purified from the ZD55-Smac recombinant oncolytic
adenovirus by using a virus DNA-QIAGEN kit (QIAGEN,
Hilden, Germany). The Smac codon sequence (720 bp) was
verified by conventional PCR using the following primer
(forward: 5'-ATGGCGGCTCTGAAGAGTTGGCTGT-3' and reverse: 5'-TCAATCCTCACGCAGGTAGGCCTCC-3'). The
amplification product was visualized by electrophoresis on
a 2% agarose gel containing ethidium bromide.
Cytopathic effect assay The HCC cell lines BEL7404
and Huh-7 and normal liver cell lines L-02 and QSG-7701
were cultured in 24-well plates and infected with Ad-wt,
ONYX-015, and ZD55-Smac, respectively, at different MOI
(Multiplicity of Infection=ratio of infectious virus particles
to cells). Four days after infection, the cells were
washed, paraformaldehyde-fixed, stained with crystal
violet, and scanned on a Bio Imagine System scanner (Syngene, San
Diego, CA, USA).
Western blot analysis Total proteins were separated
on 8%_12% polyacrylamide gels and transferred
onto 0.45 µm nitrocellulose in a buffer containing 25 mmol/L
Tris-HCl (pH 8.3)/192 mmol/L glycine/20% methanol and blocked with
Odyssey blocking buffer (Li-Cor, Lincoln, NE, USA) for at
least 1 h. The membranes were incubated with primary
antibodies, detected by the addition of antirabbit infrared
(IR) dye 700 or antimouse IR dye 800 (Li-Cor, Lincoln,
Nebraska, USA). The fluorescent signal was
revealed by using the Odyssey infrared imaging system (Li-Cor, Lincoln,
Nebraska, USA). The primary antibodies were from Santa
Cruz Biotechnology [Santa Cruz, CA, USA;
anti-poly-adenyl ribonuclease polymerase (PARP) , anti-E1A, and
anti-actin].
In vitro cell viability assay
The cells were plated in 96-well plates and treated with ZD55_Smac, cisplatin, 5-FU, or a
combination of viruses with drugs. After incubating for the
indicated intervals, cell viability was determined by the 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT, 0.5 mg/mL) assay.
Apoptotic cell staining Huh-7 HCC cells or L-02 normal
liver cells were seeded in 6-well plates. After being treated
for 48 h, the cells were incubated with Hoechst 33342 for 2 h
and observed under a fluorescence microscope.
Statistics Values are expressed as the mean±SD. The
significance of differences was determined using Student's
t-test, with significance set at P<0.05.
Results
Construction and characterization of
ZD55-Smac ZD55 is an oncolytic adenovirus, which can selectively replicate
and lyse in a great number of tumor cells due to the deletion
of the E1B-55K gene from Ad5 that is similar to the typical
viro agent ONYX-015. In this vector, we designed a BglII
clone site transferring exogenous genes, that is, the main
character different from ONYX-015. The Smac gene was
inserted into this site, driving by the human CMV-IE promoter.
To determine the selective cytopathic effect (CPE) of
ZD55-Smac, 2 HCC cell lines (BEL7404 and Huh-7) and 2
normal liver cell lines (L-02 and QSG-7701) were infected with
ZD55-Smac, ONYX-015, or Ad-wt at various MOI. The cells
were stained with crystal violet 4 d later. ZD55-Smac
performed robust cytotoxity in the HCC cells that was equal or
a little higher in comparison with either ONYX-015 or Ad-wt,
while a significant suppression of cell proliferation was not
observed in normal liver cells (Figure 1).
Consequently, the CPE assay indicated that the ZD55
system could specifically induce toxicity in HCC cancer cell
lines regarding the tumor selective replication capability, and
Smac displayed slight toxicity to Huh-7 cells that was
consistent with the results of Figure 2, but do not affect normal
cell viability. These observations suggest that ZD55 is an
ideal tumor-specific replicative adenovirus for Smac gene
delivery.
ZD55-Smac is superior to ONYX-015 in sensitizing
chemotherapy ONYX-015 has been extensively studied as
an effective viro agent for relieving chemoresistance both in
experimental and clinical cancer therapy. Here, we proposed
to evaluate the assistance capability of ZD55-Smac in
enhancing the tumor cytotoxicity of cisplatin- or 5-FU-treated
Huh-7 cells compared with ONYX-015. Significant cell growth
inhibition was detected in the groups treated with a
combination of ZD55-Smac (MOI=1) with cisplatin (1 µg/mL)/5-FU
(5 µg/mL), compared with the groups treated with a
combination of ONYX-015 (MOI=1) with cisplatin (1 µg/mL)/5-FU
(5 µg/mL) (MOI=1), or ZD55-Smac (MOI=1), ONYX-015
(MOI=1), and cisplatin (1 µg/mL)/5-FU (5 µg/mL) alone
(P<0.01; Figure 2). These results indicated that blocking the IAP
activity by Smac could further improve the enhanced
effects of ONYX-015 with cisplatin or 5-FU. Thus,
ZD55-Smac-based chemo-gene virotherapy is obviously superior to
conventional ONYX-015-based chemo virotherapy.
ZD55-Smac enhanced sensitivity of several HCC cell
lines to cisplatin or 5-FU To explore the enhanced
toxicity of ZD55-Smac with cisplatin or 5-FU, the HCC cells were
treated with cisplatin (1 µg/mL), 5-FU (5 µg/mL), ZD55-Smac
(MOI=1), and a combination of ZD55-Smac with cisplatin or
5-FU, respectively. According to the results of the MTT
assay, a significant decrease in cell viability was
observed in the cells treated with ZD55-Smac plus cisplatin or 5-FU (Figure
3). For instance, in the Huh-7 cells, the cell viability
significantly decreased 3 d post-treatment in the group of
ZD55-Smac plus cisplatin, compared with both the ZD55-Smac
alone and cisplatin alone groups (P<0.01; Figure 3). However,
the toxic effects are different from the HCC cell types. Using
SMMC7721 as an example, ZD55-Smac played an essential
role in killing cancer cells, but only promoted the effects of
the chemo drugs to a limited extent.
To further evaluate the enhanced cytotoxic effects, a fixed
viral titer (ZD55-Smac, MOI=1) was used in combination with
variable concentrations of drugs (cisplatin 1, 5, and 20 µg/mL; 5-FU 5, 20, and 50 µg/mL). The cytotoxic effects of
the groups using the combination were more apparent
compared with either virus or cisplatin/5-FU alone at each
concentration (P<0.01; Figure 4).
Although ZD55 is a tumor-selective replication
adenoviral vector and the Smac does not have a negative impact on
normal cells, extremely high dosages of ZD55_Smac could
still cause damage to normal cells. Similarly, the
combination of ZD55_Smac with cisplatin or 5-FU also resulted in
concentration-dependent cell death as assessed by MTT
assay (P<0.01; Figure 5). Importantly, the combination of
ZD55-Smac with chemotherapy does not show apparent
overlapping toxicities in normal cells.
Apoptosis induction by treatment with ZD55-Smac
or/and chemotherapy The expression levels of XIAP in HCC
cells were analyzed by immunoblot assay. A much higher
level of the XIAP protein was detected in Huh-7, BEL7404,
and SMMC7721 cells, compared with that in QSG-7701 and
L-02 normal liver cells. ZD55-Smac could efficiently mediate
Smac expressed in HCC cells as shown in our previous
study[17]. Here we verified the virus by conventional PCR and detected
adenoviral E1A protein expression in HCC cells by Western
blotting (Figure 6). To determine the underlying mechanism
by which ZD55-Smac, cisplatin, 5-FU alone, or a
combination of ZD55-Smac and cisplatin or 5-FU can induce apoptosis
in cancer cells, the activation of PARP in BEL7404 HCC cells
was studied by Western blot analysis. The results showed
that the cleavage of PARP was more apparent 2 d
post-infection with ZD55-Smac, compared with 1 d post-infection for
the control group. Furthermore, the combination of
ZD55-Smac with either cisplatin or 5-FU led to a slight increase of
PARP cleavage, rather than ZD55-Smac, cisplatin, or 5-FU
alone (Figure 6).
These observations were consistent with the
morphological features. Most cancer cells underwent apoptosis
during ZD55-Smac treatment in
combination with cisplatin or 5-FU as shown by DNA specific
fluorochrome staining in Huh-7 cells (Figure 7A). A higher dosage also led to
remarkable chromatin condensation and nuclear
fragmentation in normal L-02 cells (Figure 7B).
Discussion
Despite the recent introduction of new agents and
schedules for the treatment of cancer, chemotherapy still obtains
unsatisfactory overall response rates; rare complete
remissions; and responses of relatively short duration, mainly
due to chemoresistance and severe side-effects. Currently,
many studies have revealed the molecular machinery of
chemoresistance. Notable advances have been documented
in elucidating the mechanisms of drug resistance and
sensitization that represent a useful basis for further development
of strategies to circumvent chemoresistance in clinical
practice.
Apoptosis or programmed cell death is a genetically
encoded cell death program characterized by biochemical and
morphological changes[19], which has been shown to be the
principal mechanism of chemotherapy-induced
regression[20]. IAP, including XIAP, cellular inhibitor apoptosis (cIAP) 1
and 2, ronal apoptosis inhibitory protein (NIAP), and
survivin, are an evolutionarily conserved family of proteins
that prevent cell death by directly inhibiting
caspases[21]. XIAP is one of the most potent inhibitors in the family and
presents as a potential drug target overexpressed in various
human cancer cells[22,23]. In cultured cells, XIAP knock-out
directly induced apoptosis and sensitized resistant cells to
chemotherapy. In animal models, siRNA or antisense
oligonucleotides directed against XIAP delayed tumor growth in
human cancer xenografts[24_26]. Moreover, recent studies
have shown that Smac can act as an effective pro-apoptotic
molecule to bind to IAP[9,10], leading to the design and
synthesis of Smac or mimic that can sensitize cancer cells to
apoptotic induction by antagonizing
IAP[27,28]. Our previous work has also shown great success of attenuating IAP
function to enhance the tumor necrosis factor£related
apoptosis-inducing ligand (TRAIL)-induced apoptosis by
an oncolytic adenovirus (ZD55-Smac) expressing the Smac
protein[17]. Cisplatin and 5-FU are widely used anticancer
agents with a broad range of antitumor activities. Cisplatin,
a platinum-based chemotherapy drug, acts as a factor
crosslinking DNA, making it impossible for proper mitosis.
The damaged DNA sets off DNA repair mechanisms, which
finally activate apoptosis. Cisplatin has been used in many
cancers, especially in testicular cancer and epidermal
carcinomas[29]. In the case of HCC, cisplatin has been shown to
be more effective than other agents and more so in
combination with drugs, such as 5-FU, which induces synergistic
effects[30,31]. 5-FU is one of the first-line treatment options
for gastrointestinal tumors, but its effects on HCC have been
negative[32].
In the present study, we utilized ZD55-Smac to sensitize
HCC cells to chemotherapeutic agents cisplatin or 5-FU. Our
data indicated that the combination of ZD55-Smac and
chemotherapy demonstrated robust cytotoxicity, compared with
ZD55-Smac, cisplatin, or 5-FU alone as shown in both the
MTT assay and cell apoptotic staining. Thus, the dosage
could be reduced greatly in order to protect normal cells,
according to the increased tumor killing effect within the
cooperation of chemo agents and ZD55-Smac. In this study,
as shown by the MTT assay in L-02 normal cells, we
introduced the lowest dose (MOI=1, ZD55-Smac+1 µg/mL
cisplatin or 5 µg/mL 5-FU) to treat cells. These results
showed that there was slight damage to the normal cells at
such a low dose.
Furthermore, ZD55 is similar to ONYX-015 with
E1B-55K gene deletion that can selectively replicate and lyse in a
number of cancer cells approved by our previous
tests[17,18]. For example, in the case of ONYX-015, which works
synergistically with chemotherapy in certain human cancers, we
employed ZD55 to deliver Smac and then combined it with
chemo agents to treat the HCC cells. Therefore, ZD55 has
played triple roles: (1) as an oncolytic viro agent; (2) a
transgenic vector; and (3) a chemotherapeutic assistant. As
shown in Figure 2, ZD55_Smac was much more efficient in
enhancing the sensitivity of HCC cells to chemotherapy than
ONYX-015.
In conclusion, the ZD55 adenoviral vector could
efficiently transfer Smac into HCC cells. ZD55-Smac conjugated
with cisplatin or 5-FU performs enhanced sensitivity of
several HCC cell lines to chemotherapy, while evidently
relieving the negative toxicity in normal cells by applying the
tumor-selective replication vector and reducing the dosage.
Moreover, the current chemo-gene virotherapeutic (cisplatin
or 5-FU+ZD55-Smac) strategy seems superior to the
conventional chemo-gene or chemo-viro approach.
References
1 Nishiyama M, Yamamoto W, Park JS, Okamoto R, Hanaoka H,
Takano H, et al. Low-dose cisplatin and 5-fluorouracil in
combination can repress increased gene expression of cellular
resistance determinants to themselves. Clin Cancer Res 1999; 5:
2620_8.
2 Shimada M, Takenaka K, Kawahara N, Yamamoto K, Shirabe K,
Maehara Y, et al. Chemosensitivity in primary liver cancers:
evaluation of the correlation between chemosensitivity and
clinicopathological factors. Hepatogastroenterology 1996; 43:
1159_64.
3 Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R,
Burroughs AK, et al. EASL Panel of Experts on HCC. Clinical
management of hepatocellular carcinoma. Conclusions of the
Barcelona-2000 EASL conference. European Association for
the Study of the Liver. J Hepatol 2001; 35: 421_30.
4 Deveraux QL, Reed JC. IAP family proteins — suppressors of
apoptosis. Genes Dev 1999; 13: 239_52.
5 Chai J, Shiozaki E, Srinivasula SM, Wu Q, Dataa P, Alnemri ES,
et al. Structural basis of caspase-7 inhibition by XIAP. Cell
2001; 104: 769_80.
6 Suzuki Y, Nakabayashi Y, Nakata K, Reed JC, Takahashi R.
X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3
and -7 in distinct modes. J Biol Chem 2001; 276: 27 058_63.
7 Nakagawa Y, Abe S, Kurata M, Hasegawa M, Yamamoto K, Inoue
M, et al. IAP family protein expression correlates with poor
outcome of multiple myeloma patients in association with
chemotherapy-induced overexpression of multidrug resistance genes.
Am J Hematol 2006; 81: 824_31.
8 Nomura T, Yamasaki M, Nomura Y, Mimata H. Expression of
the inhibitors of apoptosis proteins in cisplatin-resistant
prostate cancer cells. Oncol Rep 2005; 14: 993_7.
9 Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X,
et al. Structural basis of IAP recognition by Smac/DIABLO. Nature 2000; 408:
1008_12.
10 Srinivasula S, Hegde R, Saleh A, Dattap, Shiozaki E, Chai J,
et al. A conserved XIAP-interaction motif in caspase-9 and
Smac/DIABLO regulates caspase activity and apoptosis. Nature 2001;
410: 112_6.
11 Chai J, Du C, Wu J, Kyin S, Wang X, Shi Y. Structural and
biochemical basis of apoptotic activation by Smac/DIABLO.
Nature 2000; 406: 855_62.
12 Chiocca EA, Abbed KM, Tatter S, Louis DN, Hochberg FH, Barker
F, et al. A phase I open-label, dose-escalation,
multi-institutional trial of injection with an E1B-attenuated adenovirus,
ONYX-015, into the peritumoral region of recurrent malignant
gliomas, in the adjuvant setting. Mol Ther 2004; 10: 958_66.
13 Kirn D, Hermiston T, McCormick F. ONYX-015. Clinical data
are encouraging. Nat Med 1998; 4: 1341_2.
14 Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel
L, et al. A controlled trial of intratumoral ONYX-015, a
selectively-replicating adenovirus, in combination with cisplatin and
5-fluorouracil in patients with recurrent head and neck cancer. Nat Med
2000; 6: 879_85.
15 Heise C, Sampson-Johannes A, Williams A, McCormick F, von
Hoff DD, Kirn DH. ONYX-015, an E1B gene-attenuated
adenovirus, causes tumor-specific cytolysis and antitumoral
efficacy that can be augmented by standard chemotherapeutic agents.
Nat Med 1997; 3: 639_45.
16 Wang J, Gu JF, Yang SY, Xiao T, Qi R, Sun LY,
et al. Security of dual cancer-specific targeting vector and its cytotoxic effect
when harbored. Ai Zheng 2006; 25: 385_92.
17 Pei Z, Chu L, Zou W, Zhang Z, Qiu S, Qi R,
et al. An oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL
against HCC in human cells and in mice. Hepatology 2004; 39:
1371_81.
18 Zhao L, Gu J, Dong A, Zhang Y, Zhong L, He L,
et al. Potent antitumor activity of oncolytic adenovirus expressing
mda-7/IL-24 for colorectal cancer. Hum Gene Ther 2005; 16: 845_58.
19 Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological
phenomenon with wide-ranging implications in tissue kinetics.
Br J Cancer 1972; 26: 239_57.
20 Fisher DE. Apoptosis in cancer therapy: Crossing the threshold.
Cell 1994; 78: 539_42.
21 Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC. The
c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific
caspases. EMBO J 1997; 16: 6914_25.
22 Deveraux QL, Takahashi R, Salvesen GS, Reed JC. X-linked IAP
is a direct inhibitor of cell-death proteases. Nature 1997; 388:
300_4.
23 Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS, Reed
JC. Cleavage of human inhibitor of apoptosis protein XIAP
results in fragments with distinct specificities for caspases. EMBO
J 1999; 18: 5242_51.
24 Hu Y, Cherton-Horvat G, Dragowska V, Baird S, Korneluk RG,
Durkin JP, et al. Antisense oligonucleotides targeting XIAP
induce apoptosis and enhance chemotherapeutic activity against
human lung cancer cells in vitro and in
vivo. Clin Cancer Res 2003; 9: 2826_36.
25 Tu SP, Jiang XH, Lin MC, Cui JT, Yang Y, Lum CT,
et al. Suppression of survivin expression
inhibits in vivo tumorigenicity and angiogenesis in gastric cancer. Cancer Res 2003; 63:
7724_32.
26 Chen J, Xiao XQ, Deng CM, Su XS, Li GY. Downregulation of
XIAP expression by small interfering RNA inhibits cellular
viability and increases chemosensitivity to methotrexate in
human hepatoma cell line HepG2. J Chemother 2006; 18: 525_31.
27 Zobel K, Wang L, Varfolomeev E, Franklin MC, Elliott LO,
Wallweber HJ, et al. Design, synthesis, and biological activity of
a potent Smac mimetic that sensitizes cancer cells to apoptosis
by antagonizing IAPs. ACS Chem Biol 2006; 1: 525_33.
28 Galluzzi L, Larochette N, Zamzami N, Kroemer G.
Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene 2006;
25: 4812_30.
29 Wang D, Lippard SJ. Cellular processing of platinum anticancer
drugs. Nat Rev Drug Discov 2005; 4: 307_20.
30 Okamura M, Hashimoto K, Shimada J, Sakagami H.
Apoptosis-inducing activity of cisplatin (CDDP) against human hepatoma
and oral squamous cell carcinoma cell lines. Anticancer Res 2004;
24: 655_61.
31 Tanioka H, Tsuji A, Morita S, Horimi T, Takamatsu M, Shirasaka
T, et al. Combination chemotherapy with continuous
5-fluorouracil and low-dose cisplatin infusion for advanced hepatocellular
carcinoma. Anticancer Res 2003; 23: 1891_7.
32 Li YX, Lin ZB, Tan HR. Wild type p53 increased
chemosensitivity of drug-resistant human hepatocellular carcinoma
Bel7402/5-FU cells. Acta Pharmacol Sin 2004; 25: 76_82.
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