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Experimental Therapeutics, Preclinical Pharmacology |
Laboratory of Experimental Therapeutics [S-H. P., Y. M. C., Y. D. Y.] and Department of Otolaryngology [Y-S. L.], Korea Cancer Center Hospital, Seoul 139-706; Department of Internal Medicine, Yonsei University College of Medicine, Seoul 135-720 [H. J. K.]; Department of Internal Medicine, Korea University Guro Hospital, Seoul 152-050 [J. S. K.]; and Department of Biology, Chonnam National University, Kwangju 500-757 [H. Z. C.], Korea
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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radiation. This
increased expression conferred radiation resistance to cancer cells
because overexpression of Prx II protected 11A cells from
radiation-induced cell death, suggesting that blocking Prx II
expression could enhance radiation sensitivity. Treatment of 11A cells
with a Prx II antisense decreased induction of Prx II, enhancing the
radiation sensitivity. From these results, we suggest that
stress-induced overexpression of Prx II increases radiation resistance
via protection of cancer cells from radiation-induced oxidative
cytolysis and that a Prx II antisense can be used as a radiosensitizer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammalian Prx5 family is divided into two groups, based on the amino acid sequences: one group in which two cysteines are conserved (Prx I, II, III, and IV); and one group in which one cysteine is conserved (Prx V and VI; Refs. 1, 2, 3 ). Member of the Prx family have previously been known as thioredoxin peroxidase, which reduces H2O2 using the electrons provided by thioredoxin (4 , 5) . However, it is not yet established whether all members of the Prx family found in a variety of species actually function as a peroxidase (4, 5, 6) . Some members of the Prx family do not require thioredoxin as an electron donor; therefore, they are not termed thioredoxin peroxidase (7) . Mammalian tissues express Prx isoforms, and their overexpression prevented intracellular accumulation of H2O2, inhibiting apoptosis (7 , 8) .
Prx II has also been known as NKEF-B (9 , 10) . NKEF, Mr 48,000, is composed of two subunits linked by disulfide bonds (9) . It was initially found in human RBCs and was known to play a role in enhancing cytotoxicity of natural killer cells. It was classified into two subgroups, NKEF-A and NKEF-B, which were later identified as Prx I and II, respectively; the primary sequences of Prx I and II are highly homologous to each other (10) . The cDNA sequence of Prx I is homologous to a human proliferation-associated gene (PAG), and it is known to be associated with cell proliferation and differentiation (11) . In contrast, the cDNA sequence of Prx II shows a homology to thiol-specific antioxidant proteins, which scavenge or suppress formation of protein thiol radicals (2) . Therefore, it has been proposed that Prx II functions as an antioxidant protein that protects cells from ROS or cellular oxidative damage (3 , 12) .
Kim et al. (13) observed that induction of Prx II mRNA was increased in human endothelial (ECV304) cells treated with H2O2. Moreover, overexpression of Prx II in ECV304 cells exposed to t-butylperoxide or methyl mercury reduced ROS generation, resulting in an increased protection from oxidative stress (14) . However, overexpression of Prx II did not protect the cells from oxidative damages by depletion of the intracellular GSH (13) . It was therefore proposed that the activity of Prx II was regulated by protein thiol-thiol exchange via the thioredoxin system, but not by GSH (15) .
Delays in G2 induced by basic fibroblast growth factor in HeLa cells have been proposed to be related to radiation resistance (16) , and in Burkitt’s lymphoma cells, ceramide signaling has been implicated in the radiation-resistance mechanism (17) . Although the mechanisms of chemo- and/or radiation resistance in cancer cells is not well understood, we propose that inhibition of the stress-activated gene induction could be promising in efforts to overcome chemo- and/or radiation resistance. In the present study, therefore, we examined a possible role of Prx II in cellular resistance to radiation treatments and investigated a Prx II antisense as a possible radiation sensitizer in head-and-neck cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Preparations and Northern Blot Analysis.
Tumor tissues were obtained from nine patients with head and neck
cancer undergoing surgical resections, rinsed with ice-cold saline
solution, quickly frozen in liquid nitrogen, and stored at -70°C
until needed for RNA isolation. The frozen tissues were minced with
liquid nitrogen, and RNA was isolated using the phenol-chloroform
method with TRIzol reagent (Life Technologies) according to the
manufacturer’s instructions. Ten µg of total RNA per lane were
fractionated on a 1% agarose-formaldehyde gel and transferred to a
nylon membrane (NEN, Boston, MA). The blot was probed with a 400-bp
32P-labeled ClaI-XbaI
fragment of human Prx II. Hybridization was carried out as described
previously (18)
.
Immunoblotting.
After treatment, cells were harvested and solubilized with RIPA buffer
[150 mM NaCl, 1.0% NP40, 0.5% deoxycholic acid, 0.1%
SDS, 50 mM Tris (pH 8.0)] containing protease inhibitors
(1 mM sodium orthovanadate, 30 mM NaF, 1
mM phenylmethylsulfonyl fluoride, and 30 mM
sodium pyrophosphate). Protein concentrations were determined by the
Bio-Rad protein assay (Bio-Rad, Hercules, CA). After separated on
SDS-polyacrylamide gel, the proteins were transferred to nitrocellulose
membranes, and the Prx II protein was detected using the anti-Prx II
polyclonal antibody (7)
.
Construction of Sense and Antisense Prx II Expression Vector and
Stable Cell Lines.
A NdeI-BamHI fragment from pETprxII
(7)
containing the entire coding region for Prx II was
subcloned into pcDNA3 (Invitrogen, the Netherlands) to generate
pPrxII/S and pPrxII/AS. pPrxII/AS contained an inverted
NdeI-BamHI fragment of the Prx II
gene. Prx II-overexpressing cell lines were constructed in 11A cells as
described previously (19)
. Three colonies were isolated
and cultured for the use of clonogenic assay and Western blotting.
Clonogenic Assay.
Exponentially growing cells were counted, diluted, and seeded in
triplicate at 300 cells per culture dish (100-mm dish). Cells were
incubated for 24 h in a humidified CO2
incubator at 37°C, irradiated to -rays with a
137Cs -ray source (Atomic Energy of Canada,
Ltd., Ontario, Canada) at a dose rate of 3.81 Gy/min. Colonies were
allowed to grow for 14 days and stained with 1% methylene blue in
methanol. Colonies larger than 200 µm in diameter were counted
with a colony counter (Imaging Products, Chantilly, VA).
Radiation Sensitization of Cells.
Exponentially growing cells (2 x 105
) were
transferred to 60-mm culture dishes and cultured for 24 h. After
being washed twice with serum-free medium, the cells were incubated
with a mixture containing 15 µg of cationic liposome (Life
Technologies) and various doses of the anti-sense Prx II expression
vector (pPrxII/AS) or antisense/sense oligomers (AS1,
5'-CGCGCGTTACCGGAGGCCAT-3'; AS2, 5'-ACGCCGTAATCCTCAGACAA-3'; S1,
5'-ATGGCCTCCGGTAACGCGCG-3') in 1 ml of serum-free medium for 4–6 h.
The cells were washed with serum-free medium, added to culture medium,
and then cultured for 16 h. Transfection was repeated one more
time. At 16 h after transfection, the cells were irradiated to
-rays with a 137Cs -ray source. After 14
days of incubation at 37°C, colonies larger than 200 µm in diameter
were counted with a colony counter.
Determination of Apoptotic Cell Death.
For DAPI staining, 11A cells were transfected with AS1 twice and
irradiated with 3 Gy. After 48 h, the cells were seeded into
100-mm tissue culture dishes. After the scheduled experiments, cells
were harvested and fixed with 3.7% formaldehyde on glass slides, which
were precoated with 1 mg/ml poly-L-lysine (Sigma), and
stained with 1 µg/ml DAPI in PBS for 30 min. After slides were washed
with PBS and mounted, the apoptotic cells were counted under a
fluorescence microscope (Axioplan2; Zeiss).
For 7-AAD staining, 7-AAD (Sigma) was diluted in PBS at a concentration of 1 mg/ml as a stock solution. 11A cells were treated with either oligomers or radiation and mixed with 7-AAD stock solution to give a final 7-AAD concentration of 10 µg/ml. The cells were incubated for 20 min at 4°C in the dark and analyzed using flow cytometry (Cellquest software; Becton Dickinson). Analysis was performed as described by Schmid et al. (20) to select cells undergoing apoptotic death.
Statistical Analysis.
Every assay was performed in triplicate and repeated at least three
times. Statistical analysis was performed using the Mann-Whitney
U test. The statistical significance of the difference
between control and treatment groups was evaluated using one-way ANOVA.
The criterion for statistical significance was taken as
P < 0.05. Means, SE, and Ps were calculated
using GraphPad PRISM, version 2.00 for Windows (GraphPad
Software, San Diego, CA).
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RESULTS |
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-radiation of the
surviving tumors after chemotherapy. The profiles of the patients are
listed in Table 1
. Fig. 1
shows the strong expression of the Prx II mRNA in tumor tissues from
all four patients whose tumors did not respond to combined
chemo-/radiotherapy, whereas expression was weak or not induced in five
of five patients whose tumors completely or partially regressed after
radiation or combined chemotherapy/radiation treatments. These results
suggest that increased expression of Prx II in head and neck tumors
might protect the cells from radiation treatments, thereby contributing
to cellular resistance to radiation treatments in cancers.
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-radiation.
Prx II expression in 11A cells gradually increased until 24 h
after 3 Gy irradiation (Fig. 2)
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50%
after a 2-Gy dose of radiation (Fig. 3
A). The expression profile of Prx II in the pooled
transfectants was detected by Western blot analysis (Fig. 3
B). From these data, we suggest that the induced Prx II
protein could protect the cells from radiation-induced cellular damage.
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B), antisense oligomers of Prx II were transfected into 11A
cells as described in "Materials and Methods," and we examined
whether down-regulation of Prx II sensitized the cells to radiation
in vitro. Fig. 4
A showed that with increasing
dosages of a Prx II antisense (AS1) in 11A cells, the more cell death
was observed. Treatment of a second antisense (AS2) targeting the other
open-reading frame sequence of the Prx II cDNA showed a result similar
to that for AS1 treatment (data not shown). Treatment of 11A cells with
a Prx II sense oligomer (S1) did not alter cell viability compared with
the control cells. These results demonstrated that a Prx II antisense
caused cell death, which was dependent on the concentration of a Prx II
antisense used.
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37% of 11A cells survived after 2 Gy of irradiation, and
90% survived after treatment with 0.1 µM Prx II
antisense (AS1), cell survival decreased to 13% after 2 Gy of
irradiation in the presence of 0.1 µM Prx II antisense
and to 8% with irradiation in the presence of 1.0
µM Prx II antisense (Fig. 4
A). At 1.0
µM, Prx II antisense itself caused cell death
in 48% of 11A cells.
To confirm whether down-regulation of Prx II enhanced cell death
after irradiation, 11A cells were transiently transfected with the
antisense Prx II expression vector containing the full-length antisense
Prx II cDNA at three different concentrations. When the cells were
irradiated with 2 Gy of -radiation in the presence of 0.1 µg/ml
pcDNA3, it increased cell death by 53% compared with no
-radiation. However, 0.1 µg/ml pPrxII/AS with 2 Gy of
-radiation increased cell death by 89% (Fig. 5)
. Thus, transfected cells with a Prx II antisense showed a synergistic
increase of cell death after radiation compared with parent cells
transfected with pcDNA3. It is apparent from these data that expression
of Prx II can protect cells from radiation-induced cell death and,
therefore, that radiation sensitivity can be increased by the
inhibition of Prx II expression.
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A shows that Prx II antisense alone could induce apoptosis.
Furthermore, increased apoptotic cell death was observed in the
irradiated 11A cells pretreated with a Prx II antisense. We also
examined apoptotic cell death by DAPI staining. The irradiated cells
pretreated with Prx II antisense demonstrated chromosome condensation,
which was indicative of apoptosis (Fig. 6
B).
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DISCUSSION |
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-radiation and
chemotherapeutic drugs have been widely used for the treatment of
numerous cancers, cells increasingly become resistant to consecutive
administration of chemotherapeutic drugs and radiation. Therefore,
studies involving strategies to overcome this resistance to
chemotherapy and radiation or to increase cellular sensitivity to
chemo- and radiation therapy contribute a critical step for better
cancer treatments. In this study, we showed enhanced expression of Prx
II in head-and-neck cancer cells that are chemotherapy- and/or
radiation-resistant and showed the possible use of Prx II antisense as
a radiosensitizer. Induction of the ROS-activated proteins could
protect cells from oxidative cytolysis and reduce cellular damage by
regulating cellular redox status (21, 22, 23, 24)
. Baker et
al. (25)
observed that the redox protein thioredoxin
was overexpressed in human primary lung and colon tumors and that it
prevented apoptosis through the antioxidant mechanism. We also observed
strong expression of Prx II in tumor tissues isolated from patients
with chemotherapy- and/or radiation therapy-resistant tumors,
whereas expression of Prx II was weak in the
chemotherapy-/radiation-sensitive tumor tissues, which regressed after
radiotherapy (Fig. 1)
. Similar to increased expression of Prx II in
irradiated 11A cells, the expression of Prx II in 11A cells was also
enhanced by chemotherapeutic drugs such as cisplatin (5 µg/ml) and
5-FU (2.5 µg/ml; data not shown). It is therefore likely that
intracellular ROS induced by cisplatin, 5-FU, and radiation increase
expression of Prx II in cancer cells, thereby endowing the cancer cells
with resistance. It should be noted here that overexpression of Prx II
has been observed to give more resistance to the chemotherapeutic drug
CT-2584 in ECV304 cells (12)
, and we also observed strong
expression of Prx II in cisplatin-resistant stomach cells compared with
parent
cells.6
From the above considerations, we propose that Prx II functions not
only as an antioxidant protein but also as a stress-activated protein. Prx II protein has been proposed to play a role as a thiol-specific antioxidative protein (15) that reacts with thiol-containing proteins such as thioredoxin. Because cellular thiol-specific proteins are considered to regulate the activity of other cellular proteins with thiols at regulatory domains (21 , 24) , inhibition of Prx II activity could possibly affect signaling pathway for cell growth and survival. Disruption of cellular redox status has been shown to activate the caspase 3 cascade, resulting in apoptosis (22) . Zhang et al. (8) showed that overexpression of Prx II inhibited apoptosis in Molt-4 leukemia cells and functioned similarly to Bcl-2. One difference in function between Prx II and Bcl-2 is that Prx II can prevent accumulation of H2O2 in cells, thereby protecting cells from H2O2-induced cell death. These observations could explain how treatment of 11A cells with a Prx II antisense induces apoptotic cell death. Inhibition of Prx II expression did not influence the intracellular concentration of GSH, an indicator of cellular redox status, in irradiated 11A cells treated with a Prx II antisense (data not shown). This observation was consistent with a previous result that activity of Prx II was not regulated by intracellular concentrations of GSH (13) . Therefore, blocking the expression of Prx II might disrupt total cellular redox homeostasis but not the GSH concentration, resulting in apoptosis (24) . It should again be emphasized that because overcoming radiation resistance is critical for better cancer treatment, inactivation of the stress-activated protein Prx II may be a promising approach to increased radiation sensitivity in head-and-neck cancer cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the National Nuclear
Research and Development Program of the Ministry of Science and
Technology of Korea. 
2 These authors contributed equally to this
work.
3 Present address: School of Medicine, Korea
University, 152-703 Seoul, Korea.
4 To whom requests for reprints should be
addressed, at Laboratory of Experimental Therapeutics, Korea Cancer
Center Hospital, Seoul 139-706, Korea. Phone: 82-2-970-1321; Fax:
82-2-977-0381; E-mail: ydy{at}mail.kcch.re.kr
5 The abbreviations used are: Prx II,
peroxiredoxin II; NKEF, natural killer-enhancing factor; ROS, reactive
oxygen species; GSH, glutathione; DAPI, 4',6-diamidino-2-phenylindole;
7-AAD, 7-amino-actinomycin D; 5-FU, 5-fluorouracil.
6 S-H. Park, Y. M. Chung, and D. Y.
Young, unpublished data.
Received 4/18/00; revised 9/26/00; accepted 10/ 3/00.
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REFERENCES |
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. J. Biol. Chem., 273: 6297-6302, 1998.This article has been cited by other articles:
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 I. Iraqui, G. Faye, S. Ragu, A. Masurel-Heneman, R. D. Kolodner, and M.-E. Huang Human Peroxiredoxin PrxI Is an Orthologue of Yeast Tsa1, Capable of Suppressing Genome Instability in Saccharomyces cerevisiae Cancer Res., February 15, 2008; 68(4): 1055 - 1063. [Abstract] [Full Text] [PDF]
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), 0.05 (
), and 0.1
µg/ml (
) pPrxII/AS, as described in "Materials and
Methods." The cells were then irradiated with 0.5, 1, 2, and 4 Gy.
Cell survival was measured by a clonogenic assay. As a control, the
cells were transfected with 0.05 µg/ml of sense Prx II expression
plasmid (pPrxII/S; •). Results shown are the means ± SE
(bars) of three independent experiments. Statistical
significance of the difference between control (pcDNA3; 
) and
pPrxII/AS (0.1 µg/ml; 